Redefining the Evolution of Pulpal Pathology: Inflammasomes as Central Integrative Hubs and Biological Switches Governing the Transition From Reversible to Irreversible Pulpal Inflammation Beyond Symptom‐Based Classification

ABSTRACT

Background

The transition from reversible to irreversible pulpitis remains inadequately defined at the molecular level. Current classifications rely largely on clinical symptoms rather than objective biological determinants, limiting diagnostic precision and therapeutic decision‐making.

Objectives

To integrate current experimental and translational evidence positioning inflammasomes as central molecular decision‐making platforms in pulpal inflammation, and to propose a biologically grounded model in which inflammasome activation and supramolecular assembly define the threshold separating adaptive, reversible inflammation from irreversible pulpal damage, with direct clinical implications.

Methods

A structured narrative review was conducted following a comprehensive literature search of PubMed/MEDLINE, Scopus, and Web of Science from inception to December 2025. Evidence derived from in vitro systems, in vivo animal models, and analyses of human inflamed pulpal tissues was synthesised to characterise inflammasome sensors, adaptor and effector components, regulatory checkpoints, downstream inflammatory outputs, and their relevance to pulpal disease progression.

Results

Dental pulp cells express functional inflammasome sensors, including NLRP3, AIM2, and NLRP6, which respond to inflammatory, metabolic, and damage‐associated signals. Inflammasome activation follows a stepwise process of transcriptional priming and threshold‐dependent assembly. While NF‐κB– and type I interferon–mediated priming is adaptive and potentially reversible, ASC speck formation represents a molecular commitment point leading to caspase‐1 activation, gasdermin D cleavage, IL‐1β/IL‐18 maturation, and pyroptotic cell death. Mitochondrial dysfunction, cytosolic mtDNA release, sustained ion fluxes, P2X7 signalling, and loss of endogenous inhibitory regulators further amplify inflammasome activity, promoting self‐sustaining inflammatory circuits and progressive tissue breakdown.

Conclusions

Pulpal inflammation should be understood not as a linear increase in microbial burden, but as a biological transition driven by inflammasome checkpoint failure. Irreversible ASC speck formation functions as a commitment switch that sustains caspase‐1 activation, gasdermin D–mediated pyroptosis, and self‐amplifying IL‐1β/IL‐18 signalling. Within the confined pulp environment, this feed‐forward inflammatory circuitry promotes neurovascular dysregulation and severe pain, ultimately becoming uncoupled from the initiating insult and incompatible with tissue recovery. Recognising Inflammasome activation (ASC speck formation) as a point of no return may refine diagnostic paradigms and help redefine the biological limits of vital pulp therapy.

1. Introduction

Pulpal inflammation constitutes a dynamic continuum of immune and cellular responses initiated by diverse noxious stimuli, including microbial challenge, mechanical injury and chemical or thermal insults (Wang et al. 2025; Duncan and Cooper 2020). Rather than reflecting a simple quantitative escalation of inflammatory mediators, disease progression involves qualitatively distinct stages characterised by shifts in cellular stress responses, innate immune activation and progressive disruption of tissue architecture (Wang et al. 2025; Duncan and Cooper 2020). This trajectory is critically shaped by the anatomical confinement of the dental pulp within rigid mineralised walls and its intrinsically limited vascular outflow (Heyeraas and Berggreen 1999). Inflammatory oedema rapidly elevates interstitial pressure, impairing microvascular perfusion and inducing local hypoxia, thereby exacerbating tissue injury through ischaemic and metabolic stress and destabilising pulpal homeostasis (Wang et al. 2024; Jiang et al. 2022; Heyeraas and Berggreen 1999).

Existing models of pulpitis evolution predominantly emphasise bacterial aetiology and host inflammatory responses, yet remain limited to provide integrative frameworks explaining how diverse noxious signals—encompassing pathogen‐associated molecular patterns (PAMPs) and damage‐associated molecular patterns (DAMPs)—converge on common effector pathways that drive sustained tissue injury (al Natour et al. 2021). Although prostaglandin‐mediated inflammation, matrix metalloproteinase‐dependent extracellular matrix degradation and adaptive immune cell infiltration are well‐established features of advanced pulpitis (Accorsi‐Mendonça et al. 2013; Duncan and Cooper 2020; Rechenberg et al. 2016; Kritikou et al. 2021), these processes largely represent downstream consequences rather than primary regulatory checkpoints in disease progression.

In contrast, inflammasome assembly constitutes a critical biological commitment step that translates inflammatory sensing into sustained inflammatory amplification and structural damage (al Natour et al. 2021, 2023; Jiang et al. 2015; Sun et al. 2023). Notably, the inflammasome‐dependent molecular machinery governing interleukin‐1β (IL‐1β) and interleukin‐18 (IL‐18) maturation—both synthesised as inactive precursors yet consistently elevated in inflamed pulp tissue—has received disproportionately limited attention within endodontic research (al Natour et al. 2021, 2023; Jiang et al. 2015; Sun et al. 2023). In parallel, pyroptosis, a lytic and highly pro‐inflammatory form of programmed cell death distinct from apoptosis and necroptosis, remains underexplored despite its capacity to amplify inflammation and propagate tissue injury beyond the initial insult (Wei et al. 2025; Gu et al. 2025; Zhang et al. 2021).

Inflammasomes are cytosolic multiprotein oligomeric platforms that function as central pattern‐recognition hubs, integrating diverse danger signals to activate caspase‐1, the cysteine protease responsible for the proteolytic maturation of pro‐IL‐1β and pro‐IL‐18 into their bioactive forms (Yang et al. 2014; Jiang et al. 2015). Beyond cytokine processing, caspase‐1 cleaves gasdermin D (GSDMD), generating N‐terminal pore‐forming fragments (GSDMD‐N) that execute pyroptotic cell death, thereby directly coupling inflammatory signalling to structural tissue injury (Martinon et al. 2002; Jiang et al. 2015; Sun et al. 2023; al Natour et al. 2021).

Canonical inflammasomes comprise a sensor protein—most commonly a nucleotide‐binding oligomerisation domain‐like receptor (NLR) such as NLRP3, NLRP1 or NLRC4, or a HIN‐200 family member such as AIM2—an adaptor protein, apoptosis‐associated speck‐like protein (ASC) containing a caspase recruitment domain (CARD), and the effector protease pro‐caspase‐1. Upon sensor engagement, ASC nucleates a supramolecular platform through homotypic pyrin domain (PYD)–PYD and CARD–CARD interactions, facilitating proximity‐induced autocatalytic cleavage of pro‐caspase‐1 into enzymatically active p20/p10 heterotetramers capable of processing multiple downstream substrates (Martinon et al. 2002; Jiang et al. 2015; Sun et al. 2023; al Natour et al. 2021).

Non‐canonical inflammasome activation, mediated by caspase‐4 and caspase‐5 in humans (and caspase‐11 in mice) in response to cytosolic lipopolysaccharide (LPS), converges on GSDMD cleavage and pyroptosis, with GSDMD‐N inducing potassium efflux that secondarily activates NLRP3 and establishes feed‐forward amplification loops sustaining inflammatory signalling beyond the initiating trigger (Rühl and Broz 2015; Gu et al. 2025).

Mechanistically, inflammasome activation proceeds through a tightly regulated, threshold‐dependent two‐step process comprising priming and activation. Priming, mediated by NF‐κB–dependent transcriptional upregulation of NLRP3, pro‐IL‐1β, and pro‐IL‐18, establishes inflammasome competence, whereas activation is triggered by cumulative cellular stress signals, including extracellular Adenosine triphosphate (ATP), mitochondrial reactive oxygen species (mtROS), and ionic perturbations (Jiang et al. 2015; al Natour et al. 2021; Sun et al. 2023; Yang et al. 2014).

Activation drives sensor oligomerisation, ASC recruitment and caspase‐1 engagement, culminating in full inflammasome assembly with subsequent proteolytic maturation of IL‐1β and IL‐18 and cleavage of GSDMD, thereby linking cytokine activation to pyroptotic pore formation (Jiang et al. 2015; al Natour et al. 2021; Sun et al. 2023; Yang et al. 2014). This molecular checkpoint enables the translation of escalating danger signals into irreversible inflammatory outcomes. Consistent with this framework, NLRP3 protein expression is markedly elevated in pulp tissue affected by irreversible pulpitis compared with normal or reversible conditions. In parallel, caspase‐1 is detected in its active, cleaved form exclusively in irreversible pulpitis, directly linking inflammasome activation to the irreversible phase of pulpal inflammation (Jiang et al. 2015).

Once activated, inflammasomes intrinsically amplify inflammation through IL‐1β– and IL‐18–dependent feedback loops and pyroptosis‐mediated release of DAMPSs, sustaining inflammatory cascades even when microbial burden is controlled (Yang et al. 2014; Jiang et al. 2015; al Natour et al. 2021; Sun et al. 2023). Clinically, this self‐perpetuating signalling axis aligns with persistent pain, microvascular dysfunction, reduced pulpal resilience and loss of reparative capacity observed in advanced pulpitis. Unlike downstream cytokines that propagate inflammation without directly inducing cell death, inflammasome activation precipitates cellular destruction and architectural breakdown (Yang et al. 2014; Jiang et al. 2015; al Natour et al. 2021; Sun et al. 2023) (Figure 1).

FIGURE 1.

Overview of NLRP3 inflammasome activation in pulpal inflammation. The NLRP3 sensor protein comprises an N‐terminal pyrin domain (PYD), a central NACHT domain responsible for ATP‐dependent oligomerisation, and C‐terminal leucine‐rich repeats (LRRs) that mediate the recognition of danger signals. Upon sensing pathogen‐associated or damage‐associated molecular patterns, NLRP3 undergoes conformational activation and oligomerises, facilitating recruitment of the adaptor protein ASC (apoptosis‐associated speck‐like protein containing a CARD). ASC harbours both a PYD and a caspase recruitment domain (CARD), enabling homotypic PYD–PYD interactions with NLRP3 and CARD–CARD interactions with pro‐caspase‐1. The multimeric assembly of NLRP3 and ASC generates a supramolecular inflammasome platform, promoting proximity‐induced autocatalytic cleavage of pro‐caspase‐1 into its active p20/p10 subunits. Active caspase‐1 then processes the inactive precursors pro‐IL‐1β and pro‐IL‐18 into their mature, bioactive forms, IL‐1β and IL‐18, which are secreted to propagate inflammatory signalling. Concurrently, caspase‐1 cleaves gasdermin D (GSDMD), releasing the N‐terminal pore‐forming fragment (GSDMD‐N), which inserts into the plasma membrane to form lytic pores. These pores facilitate cytokine release, ionic fluxes, and ultimately pyroptotic cell death, thereby directly linking inflammasome activation to inflammatory tissue injury. Created in https://BioRender.com.

Although inflammasome components are present in normal dental pulp and overexpressed in pulpitis, no existing mechanistic framework explains how inflammatory sensing commits tissue to irreversible pulpal breakdown. This review synthesises current evidence positioning inflammasomes as central molecular decision‐making hubs in pulpal pathology and proposes an integrative mechanistic model that redefines traditional views on pulpitis progression.

Specifically, we examine: (i) the cellular distribution and activation dynamics of inflammasome components within pulp tissue; (ii) molecular pathways by which PAMPs, DAMPs, and metabolic stress converge to drive inflammasome assembly and caspase‐1 activation; (iii) downstream effects of IL‐1β and IL‐18 maturation, alongside pyroptotic cell death; and (iv) endogenous regulatory mechanisms whose failure leads to maladaptive inflammasome activation.

By integrating these molecular insights with clinical phenotypes, this review reframes pulpitis as a checkpoint‐governed disease. In this model, the transition from reversible to irreversible states is not simply a function of inflammatory intensity, but a consequence of inflammasome‐driven molecular commitment to structural breakdown and biological irreversibility.

2. Methods

2.1. Study Design

This study was conducted as an integrative review underpinned by a systematic literature search, with the aim of synthesising current evidence on the cellular and molecular roles of inflammasomes in the biological evolution of dental pulp pathology. Given the mechanistic nature of the research question and the substantial heterogeneity of experimental models, study designs, and outcome measures, a formal quantitative systematic review or meta‐analysis was not appropriate. Instead, evidence was analysed and integrated through a structured, theme‐driven narrative synthesis, prioritising biological plausibility and mechanistic consistency across models. By synthesising molecular and cellular findings, this work underscores the central role of inflammasomes in driving the evolution of pulp pathology.

2.2. Focused Review Question

The review was guided by the following focused question:

What cellular and molecular mechanisms define the role of inflammasomes in the progression of dental pulp pathology from reversible inflammation to irreversible injury and necrosis?

2.3. Inclusion Criteria

Studies were included if they met any of the following criteria:

• Experimental in vitro investigations involving human or animal dental pulp cells, odontoblast‐like cells, or related cell types that examined inflammasome components or associated signalling pathways.

• In vivo animal models of pulpitis or pulpal injury provide molecular or cellular evidence of inflammasome activation.

• Human studies analysing pulpal tissue specimens using molecular, biochemical, or immunohistochemical approaches to assess inflammasome‐related markers.

• Narrative or systematic reviews offering mechanistic insights into inflammasome biology relevant to dental pulp pathology.

Studies examining upstream priming signals, inflammasome assembly, caspase activation, cytokine maturation (including IL‐1β and IL‐18), pyroptotic cell death, and their contribution to pulpal tissue injury were considered particularly relevant.

2.4. Exclusion Criteria

Studies were excluded if they met any of the following conditions:

• Limited to case reports, purely clinical descriptions, or epidemiological analyses lacking molecular or cellular data;

• Focused exclusively on microbiological profiling without assessment of host inflammasome‐related mechanisms:

• Addressed general inflammatory responses without specific reference to inflammasomes or inflammasome‐dependent pathways:

• Concerned non‐pulpal tissues without clear mechanistic relevance or extrapolation to dental pulp biology

2.5. Information Sources

A comprehensive literature search was performed in PubMed/MEDLINE, Scopus, and Web of Science (Core Collection) from database inception to 30 March 2026, without applying any restrictions on language or study design. The search strategy combined controlled vocabulary and free‐text keywords related to dental pulp pathology and inflammasome biology. To maximise literature coverage and ensure thematic saturation, additional sources beyond the primary electronic databases were explored, including thesis repositories, conference proceedings, OpenGrey, Google Scholar, and ClinicalTrials.gov. Furthermore, the reference lists of included articles and relevant reviews were manually screened to identify pertinent studies not retrieved through database searches. Search strategies were iteratively refined to maximise sensitivity for mechanistic studies on inflammasome biology while maintaining specificity and relevance to dental pulp pathology.

2.6. Search Strategy

2.6.1. MEDLINE (PubMed)

(“Dental Pulp”[MeSH] OR “Pulpitis”[MeSH] OR “Dental Pulp Necrosis”[MeSH] OR dental pulp*[tiab] OR pulpitis[tiab] OR pulpal inflammation[tiab] OR pulp necrosis[tiab]) AND (“Inflammasomes”[MeSH] OR inflammasome* OR “inflammasome complex” OR NLRP3 OR NLRP1 OR NLRC4 OR AIM2 OR “NLR family” OR ASC OR “Caspase‐1”[MeSH] OR caspase‐1 OR caspase‐4 OR caspase‐5 OR caspase‐11 OR “Interleukin‐1beta”[MeSH] OR IL‐1β OR IL‐1 beta OR “Interleukin‐18”[MeSH] OR IL‐18 OR pyroptosis OR “Gasdermin D”[tiab] OR GSDMD).

2.6.2. Scopus

TITLE‐ABS‐KEY (dental pulp* OR pulpitis OR pulpal inflammation OR pulp necrosis) AND TITLE‐ABS‐KEY (inflammasome* OR “inflammasome complex” OR NLRP3 OR NLRP1 OR NLRC4 OR AIM2 OR ASC OR caspase‐1 OR caspase‐4 OR caspase‐5 OR caspase‐11 OR IL‐1beta OR IL‐1 beta OR IL‐18 OR pyroptosis OR “Gasdermin D” OR GSDMD).

2.6.3. Web of Science

TS = (dental pulp* OR pulpitis OR pulpal inflammation OR pulp necrosis) AND TS = (inflammasome* OR “inflammasome complex” OR NLRP3 OR NLRP1 OR NLRC4 OR AIM2 OR ASC OR caspase‐1 OR caspase‐4 OR caspase‐5 OR caspase‐11 OR IL‐1beta OR IL‐1 beta OR IL‐18 OR pyroptosis OR “Gasdermin D” OR GSDMD).

2.7. Study Selection and Data Extraction

The literature search was conducted in accordance with the PRISMA flow diagram (Page et al. 2021), adapted for a narrative review with systematic search. Two independent reviewers systematically screened the retrieved records, first by titles and abstracts, followed by an in‐depth full‐text assessment against the pre‐specified inclusion and exclusion criteria to determine study eligibility. Data were extracted qualitatively from included studies, with particular attention to: (i) the inflammasome sensor(s) involved (e.g., NLRP3, AIM2); (ii) priming and activation signals; (iii) cellular sources of inflammasome activation within the pulp; (iv) downstream effects including cytokine release, pyroptosis, hypoxia‐related responses, and tissue injury; and (v) the experimental model and methodological context.

3. Results

3.1. Study Selection

Initially, 565 articles were retrieved based on the described search strategy, with an additional 4 records identified through other sources. A preliminary screening of titles and abstracts was performed (n = 569), after which 129 full‐text articles were assessed for eligibility according to predetermined inclusion and exclusion criteria. Twenty‐one articles (n = 22) were excluded for specific reasons, leaving 107 studies that met the inclusion criteria for narrative analysis and synthesis (Figure 2).

FIGURE 2.

Flowchart of literature search and selection based on PRISMA guidelines.

4. Inflammasome Sensor Diversity in Dental Pulp

The dental pulp expresses a diverse repertoire of inflammasome sensors that orchestrate stage‐ and pathogen‐specific innate immune responses. Among these, NLRP3, AIM2, and NLRP6 have been functionally characterised in pulpal tissue. While all recruit ASC and activate caspase‐1, they differ in molecular structure, ligand specificity, priming requirements, and downstream effects, enabling the pulp to tailor inflammasome signalling to the microbial context and inflammatory stage (Table 1).

TABLE 1.

Comparative characteristics of NLRP3, AIM2 and NLRP6 inflammasomes in dental pulp.

Characteristic	NLRP3	AIM2	NLRP6	References
Basal expression in healthy pulp	Constitutive; localised predominantly to odontoblast layer; present at lower levels in stromal and immune cells	Mainly confined to odontoblasts in healthy pulp	Not prominently expressed in health; minimal basal evidence; primarily upregulated during inflammation	Jiang et al. (2015); al Natour et al. (2023); Huang et al. (2018); Tian et al. (2021)
Predominance in pulpitis	Predominant and most extensively characterised inflammasome in pulpitis; consistent expression in human pulp; inhibitor validation	Expanded expression in pulpitis and periapical lesions	Specialised inflammasome axis activated during advanced pulp inflammation	Jiang et al. (2015); al Natour et al. (2023); Sun et al. (2023); Huang et al. (2018); Tian et al. (2021); Zhao et al. (2020)
Expansion in irreversible pulpitis	Diffuse parenchymal expression with accumulation of cleaved caspase‐1	Expanded to neutrophils, macrophages, monocytes, plasma cells, fibroblasts, DPSCs	Elevated NLRP6, caspase‐4, caspase‐1 and cleaved caspase‐1 in irreversible pulpitis	Jiang et al. (2015); al Natour et al. (2023); Zanini et al. (2024); Tian et al. (2021); Zhao et al. (2020)
Primary ligands sensed	LPS; LTA; MDP; extracellular ATP; mtROS; mtDNA; reduced cytoplasmic K+	Cytosolic double‐stranded DNA (bacterial or mitochondrial)	Cytosolic lipoteichoic acid (LTA), particularly from S. mutans	Jiang et al. (2015); Aral et al. (2020); Lee et al. (2015); Huang et al. (2018); Ji et al. (2022); Tian et al. (2021)
Priming pathway (Signal 1)	TLR4/TLR2 → MyD88 → NF‐κB → ↑NLRP3, ↑pro‐IL‐1β, ↑pro‐IL‐18	IFN‐γ enhances AIM2 expression; dsDNA recognition sequence‐independent	LPS → IFN‐β → IFNAR1 → ↑NLRP6, ↑CASP4, ↑CASP1; NF‐κB regulates pro‐IL‐1β	Zhang et al. (2015); Huang et al. (2018); Tian et al. (2021); Wei et al. (2025)
Activation mechanism (Signal 2)	ATP–P2X7R → K+ efflux; pannexin‐1 pore formation; mtROS; ASC assembly	dsDNA binding → ASC recruitment → caspase‐1 cleavage	Cytosolic LTA → NLRP6 assembly → CASP4 → CASP1	Jiang et al. (2015); Zhang et al. (2021); Huang et al. (2018); Tian et al. (2021)
Effector caspases	Caspase‐1	Caspase‐1	Caspase‐4 → caspase‐1	Shi et al. (2015); Huang et al. (2018); Tian et al. (2021)
Main cytokines matured	IL‐1β; IL‐18	IL‐1β	IL‐1β; IL‐18	Jiang et al. (2015); Huang et al. (2018); Tian et al. (2021)
Additional biological outputs	GSDMD‐mediated pyroptosis; COX‐2/PGE2; NO; TNF‐α; IL‐6; IL‐8; hBD2	CXCL10; IFN‐β; STING‐dependent signalling; pyroptosis	Strong IL‐1β/IL‐18 release during Gram‐positive infection	Lee et al. (2015); Zhang et al. (2021); Ji et al. (2022); Tian et al. (2021)
Pharmacological/genetic validation	Suppressed by high extracellular K+, glibenclamide, NAC; inhibited by MCC950	AIM2 or ASC silencing abolishes IL‐1β; unaffected by MCC950	MCC950 ineffective; NLRP6 or CASP4 silencing abolishes IL‐1β; IFNAR1 blockade prevents priming	Jiang et al. (2015); Ma (2023); Tian et al. (2021)
Unique mechanistic feature	Broad ligand promiscuity; integrates multiple DAMP/PAMP signals; modulates macrophage phenotype independently of IL‐1β	Sequence‐independent dsDNA sensing; compensatory mtDNA–GSDMD–STING axis	Interferon‐dependent dual‐checkpoint licensing; NLRP3‐independent Gram‐positive sensing	Honda et al. (2023); Zhang et al. (2021); Tian et al. (2021)

Abbreviations: AIM2, absent in melanoma 2; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; ATP, adenosine triphosphate; CASP1, caspase‐1; CASP4, caspase‐4; COX‐2, cyclooxygenase‐2; DAMPs, damage‐associated molecular patterns; DPSCs, dental pulp stem cells; dsDNA, double‐stranded DNA; GSDMD, gasdermin D; hBD2, human β‐defensin 2; IFN‐β, interferon beta; IFN‐γ, interferon gamma; IFNAR1, interferon alpha/beta receptor 1; IL‐18, interleukin‐18; IL‐1β, interleukin‐1 beta; IL‐6, interleukin‐6; IL‐8, interleukin‐8; K⁺, potassium; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MCC950, selective NLRP3 inhibitor; MDP, muramyl dipeptide; mtDNA, mitochondrial DNA; mtROS, mitochondrial reactive oxygen species; MyD88, myeloid differentiation primary response protein 88; NAC, N‐acetylcysteine; NF‐κB, nuclear factor kappa B; NLRP3, NOD‐like receptor family pyrin domain‐containing 3; NLRP6, NOD‐like receptor family pyrin domain‐containing 6; NO, nitric oxide; P2X7R, purinergic receptor P2X7; PAMPs, pathogen‐associated molecular patterns; PGE2, prostaglandin E2; STING, stimulator of interferon genes; TLR2, Toll‐like receptor 2; TLR4, Toll‐like receptor 4; TNF‐α, tumour necrosis factor alpha.

4.1. The NLRP3 Sensor

NLRP3 is the predominant and most extensively characterised inflammasome sensor in the dental pulp, being expressed under basal conditions and further upregulated in pulpitis, as evidenced by its consistent detection in human pulp tissues, robust activation by cariogenic bacteria, and functional validation in inhibitor studies (Jiang et al. 2015; al Natour et al. 2023; Sun et al. 2023).

At baseline, NLRP3 is constitutively expressed across multiple resident pulp cell populations, with pronounced cell‐type‐specific functional implications. In odontoblasts, expression is concentrated at the pulp–dentine interface, reflecting their epithelial‐like barrier role (Jiang et al. 2015; al Natour et al. 2021, 2023). This strategic localisation positions odontoblasts as primary sentinels, capable of detecting microbial products diffusing through dentinal tubules and initiating early inflammatory signalling prior to deeper pulpal involvement (Jiang et al. 2015; al Natour et al. 2021, 2023).

Immunofluorescence analyses of human dental pulp tissue show that NLRP3 is predominantly localised to the organised odontoblast layer in healthy pulp. In irreversible pulpitis, NLRP3 expression expands throughout the pulp parenchyma, accompanied by accumulation of cleaved caspase‐1 (Jiang et al. 2015; al Natour et al. 2023). This spatial redistribution parallels the clinical transition from reversible to irreversible pulpitis and suggests a shift from localised barrier defence toward widespread inflammasome‐driven tissue injury (Figure 3).

FIGURE 3.

Conceptual representation of NLRP3 inflammasome activation across pulpal inflammatory stages. Schematic immunofluorescence‐style illustration depicting the spatial distribution of NLRP3 (green), nuclei (DAPI, blue), and cleaved (active) caspase‐1 (magenta) in different pulpal conditions. (A) Healthy dental pulp: NLRP3 expression is confined predominantly to the odontoblast layer, which remains structurally organised and intact. Cleaved caspase‐1 is absent, indicating lack of inflammasome activation under physiological conditions. (B) Reversible pulpitis: Increased NLRP3 expression is observed within odontoblasts, reflecting inflammatory priming. The odontoblastic layer shows early structural alteration; however, cleaved caspase‐1 remains minimal or undetectable, suggesting that full inflammasome activation has not yet occurred. (C) Irreversible pulpitis: Marked disruption of the odontoblast layer is evident, with diffuse NLRP3 expression extending throughout the pulp tissue. Prominent detection of cleaved caspase‐1 (magenta puncta) indicates active inflammasome assembly and caspase‐1 activation, consistent with amplified IL‐1β processing and sustained inflammatory signalling. This figure is a conceptual illustration created for explanatory purposes based on the findings reported by Jiang et al. (2015) and does not represent original experimental data.

In vitro studies with functional validation using human dental pulp fibroblasts (hDPFs) have demonstrated that these predominant stromal cells constitutively express NLRP3 mRNA and protein; however, basal expression levels are insufficient to support inflammasome assembly in the absence of priming signals, reflecting a surveillance‐ready but activation‐restricted phenotype (Wang et al. 2021; Wang et al. 2024; Jiang et al. 2015; Jiang et al. 2022). This pattern indicates that hDPFs require transcriptional upregulation—typically via toll‐like receptor (TLR)–NF‐κB signalling—to reach the threshold for NLRP3 oligomerisation and downstream effector activation (Zhang et al. 2015). In this context, noxious stimuli such as LPS engage the TLR4/MyD88/NF‐κB axis to enhance NLRP3 and pro‐IL‐1β expression, while extracellular ATP provides the second signal necessary for caspase‐1 activation and IL‐1β secretion (Zhang et al. 2015).

Dental pulp stem cells (DPSCs) exhibit context‐dependent inflammasome activity that integrates immune sensing with tissue regeneration (Wei et al. 2025; Gu et al. 2025). DPSCs express NLRP3 constitutively, but its levels are dynamically regulated by differentiation status and microenvironmental cues. Inflammasome activation in DPSCs extends beyond cytokine maturation, influencing lineage commitment and regenerative behaviour, thereby linking innate immune sensing to pulp repair capacity (Li et al. 2023; Zanini et al. 2024; Wei et al. 2025). In vitro Transwell co‐culture studies have demonstrated that DPSCs modulate fibroblast inflammatory responses through selective AIM2 inflammasome upregulation rather than NLRP3. This cross‐talk drives differential cytokine regulation: IL‐1β and IL‐6 are enhanced, while Tumour necrosis factor‐alpha (TNF‐α) secretion is suppressed, and pro‐fibrotic markers such as α‐SMA and fibronectin are reduced at the protein level (Zanini et al. 2024). Concurrently, DPSCs promote matrix metalloproteinase‐9 (MMP‐9) expression in fibroblasts exposed to TNF‐α/IL‐1β, facilitating extracellular matrix remodelling and fibrosis resolution, while preserving the fibroblasts' pericyte‐like phenotype (PDGFRβ) and upregulating Programmed Death‐Ligand 1 (PD‐L1) immune checkpoint signalling (Zanini et al. 2024). Together, these context‐dependent anti‐fibrotic and immunomodulatory mechanisms position DPSCs as dynamic sentinel cells, capable of orchestrating tissue‐protective responses. By integrating inflammasome signalling with regenerative and immune‐regulatory functions, DPSCs help coordinate inflammation resolution with pulpal tissue remodelling.

Pulp‐resident macrophages and dendritic cells display higher basal NLRP3 expression than stromal cell populations, reflecting their specialised roles in pathogen surveillance, inflammasome competence, and the orchestration of inflammatory responses (Wei et al. 2025; Zhang et al. 2020; Pohl et al. 2024). In these mononuclear phagocytes, NLRP3 functions not only as a canonical inflammasome sensor driving caspase‐1–dependent IL‐1β maturation, but also as a broader regulator of cellular phenotype and immune function. In macrophages, NLRP3 activity modulates inflammatory versus reparative programming independently of IL‐1β, whereas in dendritic cells it facilitates inflammasome‐dependent hyperactivation, IL‐1β release, and effective T‐cell priming, underscoring their central role in coordinating innate and adaptive immune responses (Honda et al. 2023; Wei et al. 2025; Zhang et al. 2020; Pohl et al. 2024).

NLRP3 exhibits broad ligand promiscuity, recognising multiple danger signals relevant to pulpitis. These include bacterial components such as LPS from Gram‐negative species, including P. gingivalis , which mediates TLR4‐dependent priming and induces NLRP3 and AIM2 activation in pulp cells (Jiang et al. 2015; Aral et al. 2020; Ji et al. 2022); lipoteichoic acid (LTA) from Gram‐positive bacteria via TLR2 (al Natour et al. 2023; Yang et al. 2014); and muramyl dipeptide (MDP), a peptidoglycan‐derived ligand present in both Gram‐positive and Gram‐negative bacteria, which activates NLRP3 through intracellular NOD2 engagement with TLR2/TLR4 amplification (Lee et al. 2015). In vitro studies with functional validation in human dental pulp cells (HDPCs)—a term denoting a heterogeneous population of pulp‐derived cells, as isolation and characterisation methods across studies do not selectively purify a specific lineage—have demonstrated that stimulation with MDP upregulates NLRP3, ASC, caspase‐1, and IL‐1β and drives production of inflammatory mediators including cyclooxygenase‐2 (COX‐2)–derived Prostaglandin E2 (PGE2), nitric oxide (NO), TNF‐α, IL‐6, IL‐8, and human beta‐defensin 2 (hBD2) (Lee et al. 2015).

Beyond microbial ligands, NLRP3 also responds to sterile danger signals. Extracellular ATP released from dying cells engages P2X7 receptors (P2X7R), inducing K⁺ efflux and direct inflammasome activation (Jiang et al. 2015; Sun et al. 2023; Zhang et al. 2015). Mitochondrial stress signals, including mtROS and cytosolic mitochondrial DNA (mtDNA) following outer membrane permeabilisation, further promote activation (Zhang et al. 2021; Wang et al. 2023). Notably, reduced cytoplasmic K⁺ acts as a convergent threshold signal integrating multiple upstream triggers (Jiang et al. 2015).

Mechanistically, these inputs converge along a canonical two‐signal pathway. Signal 1 (priming) is initiated by PAMPs/DAMPs–TLR4–NF‐κB signalling, leading to transcriptional upregulation of NLRP3 and the cytokine precursors pro‐IL‐1β and pro‐IL‐18. Signal 2 (activation) is triggered by extracellular ATP acting on P2X7 receptors, inducing K⁺ efflux, pannexin‐1 pore formation and mtROS generation, which collectively promote NLRP3 oligomerisation, ASC speck formation and caspase‐1 activation, resulting in IL‐1β and IL‐18 maturation and GSDMD‐mediated pyroptotic cell death (Jiang et al. 2015; Zhang et al. 2021; Shi et al. 2015). Pharmacological interventions substantiate this mechanism: elevated extracellular K⁺ preventing efflux, glibenclamide‐mediated K⁺ channel blockade, or ROS scavenging with N‐acetylcysteine each markedly suppress IL‐1β secretion (Jiang et al. 2015) (Table 1).

4.2. The AIM2 Sensor

Absent in melanoma 2 (AIM2) is a structurally distinct inflammasome sensor that selectively recognises cytosolic double‐stranded DNA (dsDNA) in a sequence‐independent manner (Huang et al. 2018; Ji et al. 2022). In HDPCs, cytosolic dsDNA triggers AIM2 inflammasome activation, resulting in ASC recruitment, caspase‐1 cleavage, and subsequent IL‐1β secretion. Notably, co‐stimulation with interferon‐γ (IFN‐ γ) and the synthetic AIM2 agonist poly(dA:dT) markedly upregulates the expression of AIM2, ASC, active caspase‐1, and pro‐IL‐1β, thereby confirming a pivotal role for AIM2 in dental pulp inflammatory signalling (Huang et al. 2018). Importantly, AIM2 activation extends beyond exogenous dsDNA, as endogenous mtDNA released during cellular stress can also engage this sensor, thereby establishing a conceptual link between AIM2 signalling and mitochondrial distress–mediated inflammatory responses (Zhang et al. 2021; Ji et al. 2022).

AIM2 expression exhibits a marked tissue‐ and disease‐specific distribution within pulpal and periapical tissues. In healthy dental pulp, it is largely confined to odontoblasts, whereas in pulpitis and periapical lesions it extends to inflammatory infiltrates, including neutrophils, macrophages, monocytes, plasma cells, fibroblasts (Wang et al. 2020).

DPSCs, characterised by PDGFRβ⁺ expression, engage in dynamic bidirectional crosstalk with immune cells during pulpal inflammation. In vitro co‐culture experiments show that, under stimulation with LPS or TNF‐α/IL‐1β, DPSCs markedly enhance AIM2 inflammasome activation, caspase‐1 expression, and IL‐1β and IL‐6 production compared with stimulated fibroblasts alone (Zanini et al. 2024). These findings indicate that DPSCs actively amplify early inflammatory responses, facilitating pathogen sensing and immune cell recruitment during acute injury. Notably, the same inflammatory milieu induces PD‐L1 upregulation in DPSCs following TNF‐α/IL‐1β exposure, suggesting concurrent acquisition of immunomodulatory properties that may restrain excessive immune activation (Zanini et al. 2024) (Table 1).

Functionally, cytosolic dsDNA robustly activates the AIM2 inflammasome in HDPCs, inducing caspase‐1‐dependent IL‐1β release. Gain‐ and loss‐of‐function experiments further support this mechanism: AIM2 overexpression enhances caspase‐1 activity and IL‐1β secretion, whereas AIM2 knockdown markedly suppresses both responses. Bacterial stimuli differentially modulate AIM2 activation. Gram‐negative periodontopathogens—including Porphyromonas gingivalis , Fusobacterium nucleatum , and Tannerella forsythia —as well as Streptococcus mutans , strongly induce AIM2 expression in macrophages, HDPCs, and THP‐1 cells (Song et al. 2018; Aral et al. 2020; Huang et al. 2018; Ji et al. 2022; Park et al. 2014). In contrast, Enterococcus faecalis fails to significantly upregulate AIM2, likely reflecting its limited capacity to deliver bacterial DNA into the macrophage cytosol (Ran et al. 2021; Ji et al. 2022).

Parallel activation of NLRP3—primarily sensing bacterial cell wall components and extracellular ATP—and AIM2—detecting cytosolic nucleic acids—ensures complementary innate immune surveillance across distinct microbial strategies. Consistently, small interfering RNA (siRNA)‐mediated silencing of AIM2 or its adaptor ASC abolishes AIM2‐dependent cytokine release (Fernandes‐Alnemri et al. 2009), whereas the selective NLRP3 inhibitor MCC950 suppresses NLRP3 activation without affecting AIM2‐driven IL‐1β secretion, underscoring the functional independence of the AIM2 pathway (Coll et al. 2015; Ma 2023) (Figure 4).

FIGURE 4.

AIM2 inflammasome activation in dental pulp. Bacterial invasion and cellular injury lead to the release of cytosolic double‐stranded DNA (dsDNA) derived from invading microorganisms and damaged host cells. Concurrent mitochondrial damage promotes reactive oxygen species (ROS) production and the release of mitochondrial DNA, further amplifying cytosolic DNA sensing. Recognition of dsDNA by AIM2 triggers inflammasome assembly through ASC recruitment and subsequent caspase‐1 activation. Activated caspase‐1 mediates the cleavage of pro–IL‐1β into mature IL‐1β and processes gasdermin D (GSDMD) to generate its N‐terminal pore‐forming fragment (N‐GSDMD). Membrane pore formation results in pyroptotic cell death and the extracellular release of pro‐inflammatory cytokines, thereby amplifying pulpal inflammation.

4.3. The NLRP6 Sensor

NLRP6 constitutes a functionally specialised inflammasome axis in the dental pulp, preferentially responding to Gram‐positive bacterial components via a non‐canonical, type I interferon–dependent priming mechanism (Tian et al. 2021; Zhao et al. 2020; Wei et al. 2025; al Natour et al. 2023). Unlike the classical NLRP3 paradigm, which relies on TLR4/NF‐κB‐driven priming, NLRP6 activation in HDPCs follows a bifurcated regulatory programme. PAMPSs (e.g., LPS) rapidly induce IFN‐β production (≈3 h), which signals via interferon alpha/beta receptor 1 (IFNAR1) to upregulate NLRP6, caspase‐4 and caspase‐1, while pro‐IL‐1β transcription remains under canonical NF‐κB control (Tian et al. 2021; Wei et al. 2025; al Natour et al. 2023).

Combined human tissue and in vitro studies with functional validation have demonstrated that functional blockade of IFNAR1 abrogates LPS‐induced NLRP6 and caspase expression, whereas exogenous IFN‐β alone is sufficient to reproduce this priming effect in the absence of direct TLR4 engagement, indicating a dominant interferon‐driven licensing step for the NLRP6 inflammasome machinery (Tian et al. 2021). This division of labour—NF‐κB regulating cytokine substrate availability and IFNAR1 controlling inflammasome competence—constitutes a dual‐checkpoint system that constrains the amplitude and timing of inflammatory signalling in pulp cells.

Following this interferon‐dependent priming phase (LPS → IFN‐β → IFNAR1 → ↑NLRP6/CASP4/CASP1), cytosolic sensing of Streptococcus mutans –derived LTA initiates inflammasome activation, leading to NLRP6 assembly, caspase‐4 engagement, secondary caspase‐1 activation, and subsequent maturation and release of IL‐1β and IL‐18 (LTA → NLRP6 → CASP4 → CASP1 → IL‐1β/IL‐18) (Tian et al. 2021). Notably, the biological effect of LTA depends on its cellular localisation. Cytosolic LTA, following LPS priming, preferentially activates NLRP6. In contrast, extracellular LTA acts mainly as a TLR2‐dependent priming stimulus in dental pulp cells, promoting NF‐κB‐driven upregulation of NLRP3 and pro‐IL‐1β, and in some settings may even exceed the priming capacity of LPS (al Natour et al. 2023).

Consistent with these in vitro findings, human irreversible pulpitis specimens show markedly increased expression of NLRP6, caspase‐4, caspase‐1, and cleaved caspase‐1 compared with healthy pulp, confirming the in vivo relevance of this signalling programme during advanced pulpal inflammation (Tian et al. 2021; Zhao et al. 2020).

Pharmacological and genetic evidence indicates that this pathway operates independently of NLRP3. The selective NLRP3 inhibitor MCC950 fails to suppress LTA‐induced cytokine secretion, whereas broad caspase inhibition or targeted silencing of NLRP6 or caspase‐4 completely abolishes IL‐1β release, establishing the critical role of the NLRP6–caspase‐4 axis (Tian et al. 2021).

During late‐stage carious progression—when Gram‐positive bacteria such as S. mutans access the pulp—interferon‐primed pulp cells preferentially activate NLRP6 in response to cytosolic LTA. Concurrently, NLRP3 (e.g., LPS, ATP, mtDNA) and AIM2 (cytosolic bacterial DNA) provide complementary sensing, demonstrating that the dental pulp deploys pathogen‐specific inflammasome programmes dynamically tailored to microbial composition and disease stage (Tian et al. 2021) (Table 1 and Figure 5).

FIGURE 5.

NLRP6 inflammasome activation pathway in dental pulp. Microbial challenge initiates type I interferon (IFN‐α) signalling through IFNAR, promoting transcriptional priming of NLRP6 expression. Subsequent stimulation by lipoteichoic acid (LTA) triggers NLRP6 inflammasome assembly via recruitment of the adaptor protein ASC and activation of caspase‐1 and/or caspase‐4. Activated caspases mediate cleavage of pro–IL‐1β and pro–IL‐18 into their mature forms and process gasdermin D (GSDMD) to generate its N‐terminal pore‐forming fragment (N‐GSDMD). Membrane pore formation culminates in pyroptotic cell death and extracellular release of IL‐1β and IL‐18, thereby amplifying inflammatory responses within the pulp tissue.

4.4. NLRP1 and NLRC4 Sensors

Earlier evidence suggested a limited contribution of NLRP1 and NLRC4 to pulpal inflammation. No significant differences in the mRNA expression of these sensors between healthy and inflamed human dental pulp have been reported, leading to the assumption that IL‐1β and IL‐18 production in pulpitis was predominantly mediated by NLRP3, AIM2, and NLRP6 (Yaghooti Khorasani et al. 2019).

However, emerging evidence has refined this perspective. Wu et al. (2026) recently identified NLRP1 as a novel biomarker of pyroptosis in irreversible pulpitis, demonstrating its significant upregulation in human pulpal tissues alongside activation of caspase‐1 and increased IL‐1β expression. Through an integrated methodological approach encompassing transcriptomic analysis, immunohistochemistry, immunofluorescence, and both in vitro and in vivo experimental models, the study provided compelling evidence that NLRP1 participates in a coordinated inflammasome network (Wu et al. 2026). Notably, cytoplasmic co‐localisation of NLRP1 with NLRP3 suggests functional cooperation between these sensors, contributing to the amplification and perpetuation of the inflammatory response (Wu et al. 2026). The association of NLRP1 activation with gasdermin‐mediated pyroptosis further supports its role in driving tissue damage and reinforcing the concept of biological irreversibility in advanced pulpal disease.

Collectively, these findings indicate that, while NLRC4 continues to show limited involvement in pulp inflammation, NLRP1 should no longer be considered negligible. Instead, it appears to play a context‐dependent role, particularly in the late stages of irreversible pulpitis, complementing the established functions of NLRP3, AIM2, and NLRP6 in shaping the pulpal inflammatory milieu.

5. The Adaptor Protein ASC and the Enzymatic Effector Caspase‐1

The adaptor protein ASC and the effector caspase‐1 serve as central hubs in dental pulp inflammasome signalling, translating microbial and endogenous danger signals into IL‐1β/IL‐18 maturation and pyroptotic cell death.

ASC possesses an N‐terminal PYD and a C‐terminal CARD, enabling it to connect sensor proteins lacking CARD domains, such as NLRP3 and AIM2, to pro‐caspase‐1 via homotypic PYD–PYD and CARD–CARD interactions (Huang et al. 2018; Erdag et al. 2023). Upon activation, ASC oligomerises into perinuclear “specks,” scaffolding multiple pro‐caspase‐1 molecules to facilitate proximity‐induced autocatalytic cleavage into the active p20/p10 heterotetramer, which subsequently processes pro‐IL‐1β and pro‐IL‐18 and cleaves GSDMD to induce pyroptosis (Huang et al. 2018; al Natour et al. 2023; Erdag et al. 2023; Schieffer et al. 2022).

In vitro studies with functional validation have demonstrated that stimulation of HDPCs with poly(dA:dT) in the presence of IFN‐γ strongly enhances ASC‐dependent caspase‐1 activation and IL‐1β maturation. Knockdown of ASC using siRNA significantly inhibits these processes without affecting pro‐IL‐1β transcription, confirming the critical adaptor role of ASC in the AIM2 inflammasome pathway (Huang et al. 2018).

In addition, exposure of HDPCs to periodontal pathogens such as Fusobacterium nucleatum and Porphyromonas gingivalis results in differential regulation of ASC expression and oligomerisation, depending on the microbial stimulus and ATP co‐stimulation. These findings indicate pathogen‐specific modulation of inflammasome activation in pulp cells (Aral et al. 2020).

Structural and docking analyses further demonstrate that ASC bridges activated NLRP3 to pro‐caspase‐1, enabling inflammasome assembly and downstream caspase‐1 activity (Erdag et al. 2023). Consistently, pharmacological inhibition of ASC oligomerisation blocks IL‐1β maturation and pyroptosis. Moreover, selective caspase‐1 inhibitors—including VX‐765, Z‐YVAD‐FMK, Ac‐YVAD‐CMK, and Boc‐D‐FMK—effectively suppress caspase‐1 activity. VX‐765 in particular reduces IL‐1β, MCP‐1, IL‐6, and IL‐8 production through targeted inhibition of caspase‐1 activation (Wei et al. 2025).

ASC and caspase‐1 function cooperatively: ASC nucleates inflammasome assembly, whereas caspase‐1 cleaves pro‐IL‐1β, pro‐IL‐18, and GSDMD (Kesavardhana and Kanneganti 2017). This partnership converts danger signals from NLRP3 or AIM2 into coordinated cytokine release and pyroptosis, amplifying local inflammation and triggering secondary signals such as extracellular ATP in neighbouring cells (al Natour et al. 2023; Schieffer et al. 2022). As such, ASC and caspase‐1 form the core effector machinery of pulp inflammasomes and represent a potential therapeutic target.

5.1. Non‐Canonical Inflammasome Components: Caspase‐4/5/11

Non‐canonical inflammasome activation, mediated by caspase‐4 and caspase‐5 in humans (caspase‐11 in mice), represents an alternative innate immune pathway responding specifically to LPS derived from Gram‐negative bacteria. Unlike canonical inflammasomes, these inflammatory caspases function as intracellular LPS sensors, directly binding cytoplasmic LPS through their CARD domains and undergoing oligomerisation and autocatalytic activation independently of classical sensor–ASC platforms (Kayagaki et al. 2013; Shi et al. 2014).

Caspase‐4/5 expression in inflamed dental pulp is cell‐type‐specific: resident immune cells exhibit high constitutive and inducible levels, whereas fibroblasts and odontoblasts retain inducible expression. Both populations are upregulated via type I interferon signalling during bacterial challenge. The non‐canonical NLRP6–caspase‐4 inflammasome is strongly elevated in irreversible pulpitis, with cytosolic S. mutans LTA (after LPS priming) directly triggering inflammasome activation and pro‐inflammatory cytokine release, confirming its in vivo pathological relevance and identifying caspase‐4 as a central driver of non‐canonical pyroptotic inflammation (Tian et al. 2021; Wei et al. 2025).

Functionally, cytoplasmic LPS from Gram‐negative bacteria engages this non‐canonical pathway by inducing autoproteolytic cleavage of caspase‐4 at Asp289, producing active fragments that cleave GSDMD and release its N‐terminal pore‐forming domain, thereby inducing noncanonical pyroptosis. Guanylate‐binding proteins facilitate this process by enhancing LPS–caspase interactions (Gu et al. 2025). Although caspases‐4/5/11 efficiently mediate GSDMD‐dependent pyroptosis, they cannot directly process pro‐IL‐1β, necessitating canonical inflammasome activation for full cytokine maturation. Notably, human caspases‐4 and‐5—but not murine caspase‐11—cleave pro‐IL‐18, highlighting species‐specific inflammatory responses relevant to pulp pathology (Gu et al. 2025). GSDMD pore formation mediated by caspase‐4/5 induces potassium efflux, a critical secondary signal that activates NLRP3 in neighbouring pulp cells. This establishes a hierarchical cascade in which initial non‐canonical sensing of Gram‐negative bacterial components amplifies into NLRP3‐dependent caspase‐1 activation and IL‐1β maturation (Shi et al. 2015; Kayagaki et al. 2013; Zhang et al. 2015), helping to explain the pronounced IL‐1β responses observed in pulp tissue exposed to Gram‐negative infection (Jiang et al. 2015; Aral et al. 2020).

Overall, canonical and non‐canonical inflammasomes in the dental pulp function as an integrated, context‐dependent network influenced by bacterial composition, cytosolic access of microbial products, and disease stage. Non‐canonical caspase‐4/5–GSDMD sensing amplifies NLRP3–ASC–caspase‐1 activation, driving the progression from controlled inflammation to self‐perpetuating pulpitis. This hierarchical architecture lowers the threshold for pyroptosis, promotes widespread DAMP release, and renders the inflammatory process increasingly independent of bacterial load. These insights suggest that therapeutic strategies should target convergent downstream effectors, such as GSDMD pores and secondary NLRP3 activation, rather than isolated inflammasome components.

6. Inflammasome Priming, Activation and Assembly: Molecular Checkpoints Governing Irreversibility in Dental Pulp Inflammation

Inflammasome signalling in dental pulp inflammation follows a temporally stratified cascade rather than a binary on–off response. Distinct checkpoints—priming, activation, assembly and effector execution—exhibit different activation thresholds and degrees of reversibility, giving rise to divergent inflammatory trajectories (Jiang et al. 2015; Wang et al. 2021; Zhang et al. 2021). This temporal organisation underpins the clinical heterogeneity of pulpitis and delineates discrete therapeutic windows, with checkpoint progression dictating whether inflammation remains reversible or evolves into a self‐sustaining, destructive state characteristic of irreversible tissue injury (Lee et al. 2015; Tian et al. 2021).

6.1. Priming (Signal 1): Licensing the Inflammasome Machinery

Inflammasome activation in dental pulp tissue is governed by a tightly regulated two‐signal sequence, in which the priming signal establishes the molecular prerequisites for downstream inflammatory amplification (Jiang et al. 2015; Lee et al. 2015). Importantly, priming alone does not elicit cytokine release or inflammatory cell death; instead, it acts as a licensing step that calibrates both the capacity and magnitude of inflammasome responses to subsequent cellular stress (Tian et al. 2021). This temporal uncoupling of licensing and activation offers a mechanistic basis for the clinical distinction between reversible and irreversible pulpitis, as primed yet unactivated inflammasomes may allow tissue recovery once noxious stimuli are removed (Wang et al. 2021; Jiang et al. 2015).

6.1.1. TLR‐Mediated Priming Pathways

Combined human tissue analyses and in vitro studies with hDPFs have demonstrated that PAMPs initiate inflammasome priming through engagement of TLRs, with TLR2 recognising peptidoglycan and LTA from Gram‐positive bacteria, and TLR4 detecting LPS from Gram‐negative species (Jiang et al. 2015). LPS–TLR signalling activates predominantly MyD88‐dependent cascades that converge on the IκB kinase (IKK) complex, leading to phosphorylation and proteasomal degradation of IκBα. This releases canonical NF‐κB dimers, most commonly p65/p50, allowing their nuclear translocation and transcriptional induction of inflammasome‐related genes (Jiang et al. 2015; Blevins et al. 2022; Wei et al. 2025). Nuclear NF‐κB binds κB response elements in promoter regions of multiple inflammasome‐related genes, driving transcriptional upregulation of NLRP3, pro‐IL‐1β, and pro‐IL‐18—none of which are expressed at sufficient levels under basal conditions to support robust inflammatory responses (Jiang et al. 2015; Wei et al. 2025; Blevins et al. 2022). Similarly, mechanistic in vitro studies in HDPCs have shown that MDP, sensed by NOD2, enhances NLRP3, pro–IL‐1β, and pro–IL‐18 expression via NF‐κB, while upregulating TLR2 and TLR4 in a feed‐forward loop and inducing hBD2, linking inflammasome licensing with antimicrobial peptide production (Lee et al. 2015).

Beyond transcriptional effects, priming induces post‐translational modifications of NLRP3 including deubiquitination by BRCC3, which removes inhibitory ubiquitin chains, and phosphorylation events that regulate subcellular localisation and oligomerisation capacity (Xia et al. 2023). These modifications lower the activation threshold, enabling subsequent Signal 2 stimuli to trigger assembly more efficiently.

In parallel with NLRP3 and pro–IL‐1β induction, priming also increases the availability of the adaptor protein ASC through NF‐κB‐dependent upregulation of the PYCARD gene, thereby establishing a permissive molecular scaffold for subsequent inflammasome assembly without triggering caspase‐1 activation or cytokine release (Schroder and Tschopp 2010).

The complexity of TLR‐mediated priming extends beyond canonical LPS and peptidoglycan sensing to encompass nucleic acid recognition pathways. Endosomal TLRs in odontoblasts and hDPFs detect bacterial nucleic acids, with TLR9 recognising CpG DNA via MyD88/IRAK to induce NF‐κB‐dependent cytokines, and TLR3 sensing double‐stranded RNA (dsRNA) to enhance chemokine expression and dendritic cell recruitment. Compartmentalization within endosomes prevents inappropriate activation by self‐nucleic acids while permitting detection of phagocytosed bacterial material. Notably, TLR3, TLR8, and TLR9 are upregulated in inflamed pulp, reflecting progressive sensitization to nucleic acid PAMPs (Ji et al. 2022). Collectively, this nucleic acid‐sensing cascade establishes a molecular basis for endodontic disease progression, wherein initial bacterial invasion triggers TLR upregulation that amplifies inflammatory responses.

6.1.2. Type I Interferon‐Dependent Priming of NLRP6‐Caspase 4 Axis

In HDPCs, NLRP6–caspase‐4 inflammasome priming is strictly type I interferon–dependent (Tian et al. 2021). Unlike NLRP3, which relies on TLR–NF‐κB signalling, NLRP6 transcription requires IFN‐β: P. gingivalis LPS induces IFN‐β, which via IFNAR1 upregulates NLRP6, caspase‐4, and caspase‐1. IFNAR1 blockade prevents this induction, whereas IFN‐β alone is sufficient, establishing its necessity and sufficiency. Once primed, NLRP6 senses S. mutans LTA, triggering inflammasome assembly, secondary caspase‐1 activation, and IL‐1β maturation (Tian et al. 2021).

Upstream of this interferon‐dependent priming axis, cytosolic sensing of bacterial and mtDNA through the cyclic GMP‐AMP synthase (cGAS) – stimulator of interferon genes (STING) pathway constitutes an important source of type I interferon in inflamed dental pulp. Activation of cGAS by cytosolic DNA leads to STING engagement and downstream TBK1–IRF3 signalling, resulting in IFN‐β production and concurrent NF‐κB activation (Wei et al. 2025; Ji et al. 2022). In this context, cGAS–STING does not function as an inflammasome activator but rather as an interferon‐generating licensing module that creates a permissive transcriptional environment required for NLRP6 inflammasome priming in dental pulp cells.

6.2. Cell Type‐Specific Priming Heterogeneity

Inflammasome priming displays marked cell‐type‐specific heterogeneity within the dental pulp (Ji et al. 2022). Odontoblasts constitutively express TLR3 and TLR9 and mount rapid priming responses following microbial challenge (Ji et al. 2022). In contrast, hDPFs express a broader TLR repertoire (TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9), with receptor expression dynamically modulated by inflammatory cytokines (Ji et al. 2022; Lee et al. 2015). DPSCs exhibit dynamic TLR regulation, with IL‐1β, type I interferons, and TNF‐α upregulating TLR3 and TLR8 whilst downregulating TLR7 and TLR9, suggesting that inflammatory mediators reshape pattern recognition receptor profiles to alter PAMP sensitivity (Ji et al. 2022). This cell type‐specific priming heterogeneity implies that different pulp cell populations may become inflammasome‐competent at distinct stages of inflammatory progression, with odontoblast priming occurring during early bacterial challenge and fibroblast/DPSC priming emerging as inflammation advances into deeper pulp tissue.

Beyond cellular heterogeneity, inflammasome priming is also quantitatively regulated by bacterial load. HDPCs exhibit dose‐dependent activation of NOD2, TLR, and NLRP3‐related pathways in response to bacterial peptidoglycan, with maximal immune engagement observed at higher MDP concentrations, defining a molecular threshold between controlled antimicrobial responses and overt inflammatory activation (Lee et al. 2015). This graded response provides a mechanistic framework linking bacterial burden to the clinical transition from reversible to irreversible pulpitis.

Priming increases the cellular availability of inflammasome components, sustaining NLRP3 and pro‐IL‐1β expression beyond the initial bacterial insult and thereby lowering the activation threshold for subsequent inflammasome assembly (Schroder and Tschopp 2010). Human tissue‐based analyses of dental pulp have shown that, in reversible pulpitis, this response is largely restricted to transcriptional priming without caspase‐1 activation, thereby preserving the potential for tissue recovery following removal of the noxious stimulus (Jiang et al. 2015; Wang et al. 2021). By contrast, if activation signals occur before priming resolves, the expanded pool of inflammasome precursors enables rapid and amplified inflammatory responses, promoting progression to irreversible pulpitis.

Priming is not dictated solely by transcriptional induction but is further constrained by post‐transcriptional regulation. In healthy pulp, microRNA‐223 (miR‐223) limits NLRP3 protein accumulation by repressing its translation, thereby restraining inflammasome abundance (Wang et al. 2021). During disease progression, miR‐223 downregulation parallels increased NLRP3 expression and heightened inflammasome activation, identifying loss of this regulatory brake as a critical driver of pathological inflammatory amplification (Wang et al. 2021).

Inflammasome priming establishes a transcriptionally licensed yet functionally restrained inflammatory state in dental pulp tissue. During this phase, NLRP3, NLRP6, ASC and pro–IL‐1β are upregulated in the absence of caspase‐1 cleavage, GSDMD activation or cytokine release, thereby preserving cellular viability and tissue integrity. In contrast, pro–IL‐18 is constitutively expressed in dental pulp cells and shows limited inducibility during priming. Thus, NF‐κB‐dependent licensing primarily governs the availability of NLRP3, ASC and pro–IL‐1β, defining the rate‐limiting components for subsequent inflammasome assembly (Schroder and Tschopp 2010) (Table 2).

TABLE 2.

Molecular architecture of inflammasome priming (signal 1) in dental pulp.

Regulatory level	Priming axis/mechanism	Upstream trigger	Core signalling pathway	Licensed components	Functional impact	References
I. Transcriptional Priming (Canonical NF‐κB axis)	TLR2/TLR4‐mediated priming	LTA, PGN (Gram+); LPS (Gram–)	MyD88 → IKK → IκBα degradation → NF‐κB (p65/p50) nuclear translocation	↑ NLRP3, ↑ pro–IL‐1β, ↑ pro–IL‐18, ↑ ASC (PYCARD)	Establishes inflammasome competence without caspase‐1 activation	Jiang et al. (2015); Blevins et al. (2022); Wei et al. (2025); Schroder and Tschopp (2010)
	NOD2‐mediated priming	MDP	NF‐κB activation; feed‐forward ↑TLR2/TLR4	↑ NLRP3, ↑ pro–IL‐1β, ↑ pro–IL‐18; ↑ hBD2	Links antimicrobial response to inflammasome licensing	Lee et al. (2015)
	Endosomal nucleic acid sensing	CpG DNA (TLR9); dsRNA (TLR3)	MyD88/IRAK (TLR9); TRIF pathways (TLR3) → NF‐κB	Indirect ↑ cytokine and PRR expression	Progressive sensitization during pulp inflammation	Ji et al. (2022)
II. Interferon‐Dependent Licensing (NLRP6 axis)	IFN‐β–dependent priming	P. gingivalis LPS	IFN‐β → IFNAR1 → JAK/STAT	↑ NLRP6, ↑ caspase‐4, ↑ caspase‐1	Establishes NLRP6–caspase‐4 inflammasome competence	Tian et al. (2021)
	cGAS–STING module	Cytosolic bacterial DNA; mtDNA	cGAS → STING → TBK1 → IRF3 → IFN‐β (+ NF‐κB)	Indirect licensing of NLRP6 pathway	Couples mitochondrial damage to interferon priming	Ji et al. (2022)
III. Post‐Translational Regulation	NLRP3 deubiquitination (BRCC3)	Priming‐dependent modification	Removal of inhibitory ubiquitin chains	Lowers activation threshold	Sensitises cells to Signal 2	Xia et al. (2023)
	NLRP3 phosphorylation	Inflammatory signalling	Modulates localisation and oligomerisation capacity	Facilitates assembly readiness	Fine‐tunes activation efficiency	Xia et al. (2023)
IV. Post‐Transcriptional Regulation	miR‐223 repression	Homeostatic regulation	Translational inhibition of NLRP3	Limits NLRP3 protein accumulation	Loss associated with irreversible pulpitis	Wang et al. (2021)
V. Cell‐Type Specific Priming Heterogeneity	Odontoblast priming	Early bacterial challenge	Constitutive TLR3, TLR9 expression	Rapid inflammasome competence	Early‐stage defensive barrier	Ji et al. (2022)
	HDPCs/DPSCs dynamic regulation	Cytokine milieu (IL‐1β, IFN‐I, TNF‐α)	Modulated TLR2–9 repertoire	Stage‐dependent licensing	Deep pulp involvement during advanced inflammation	Ji et al. (2022); Lee et al. (2015)
VI. Clinical Correlation	Reversible pulpitis	Priming without activation	NF‐κB–dependent transcription only	No caspase‐1 cleavage; no GSDMD activation	Potential tissue recovery	Jiang et al. (2015); Wang et al. (2021)
	Irreversible pulpitis	Persistent priming + activation signals	Amplified inflammasome assembly	Rapid cytokine release and pyroptosis	Disease progression	Jiang et al. (2015); Wang et al. (2021)

Abbreviations: ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; BRCC3, BRCA1/BRCA2‐containing complex subunit 3; cGAS, cyclic GMP–AMP synthase; CpG, cytosine–phosphate–guanine motifs; DPSCs, dental pulp stem cells; dsRNA, double‐stranded RNA; GSDMD, gasdermin D; hBD2, human β‐defensin 2; hDPFs, human dental pulp fibroblasts; IFNAR1, interferon alpha/beta receptor 1; IFN‐I, type I interferon; IFN‐β, interferon beta; IKK, IκB kinase; IL‐18, interleukin‐18; IL‐1β, interleukin‐1 beta; IRAK, interleukin‐1 receptor‐associated kinase; IRF3, interferon regulatory factor 3; IκBα, inhibitor of nuclear factor kappa B alpha; JAK/STAT, Janus kinase/signal transducer and activator of transcription; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MDP, muramyl dipeptide; miR‐223, microRNA‐223; mtDNA, mitochondrial DNA; MyD88, myeloid differentiation primary response protein 88; NF‐κB, nuclear factor kappa B; NLRP3, NOD‐like receptor family pyrin domain‐containing 3; NLRP6, NOD‐like receptor family pyrin domain‐containing 6; NOD2, nucleotide‐binding oligomerisation domain‐containing protein 2; PGN, peptidoglycan; PRR, pattern‐recognition receptor; STING, stimulator of interferon genes; TBK1, TANK‐binding kinase 1; TLR2, Toll‐like receptor 2; TLR3, Toll‐like receptor 3; TLR4, Toll‐like receptor 4; TLR9, Toll‐like receptor 9; TNF‐α, tumour necrosis factor alpha; TRIF, TIR‐domain‐containing adapter‐inducing interferon‐β; ↑, upregulation or increased expression; ↓, downregulation or decreased expression.

Priming represents the most reversible phase of inflammasome signalling (Wang et al. 2021; Lee et al. 2015). In reversible pulpitis, NLRP3 expression increases compared to healthy pulp tissue, yet remains significantly lower than in irreversible pulpitis, and occurs in the absence of caspase‐1 cleavage, indicating incomplete inflammasome activation (Jiang et al. 2015; Wang et al. 2021). The progressive downregulation of miR‐223 during the transition from reversible to irreversible pulpitis weakens endogenous suppression of NLRP3, facilitating complete inflammasome assembly upon secondary danger signal exposure such as ATP (Wang et al. 2021; Jiang et al. 2015). Chronic bacterial exposure, as occurs in progressive caries, may sustain elevated NLRP3 expression by continuous activation of TLR2/TLR4/NOD2 pathways whilst suppressing miR‐223, maintaining a state of inflammatory readiness (Lee et al. 2015; Wang et al. 2021). This temporal pattern explains why chronic low‐grade bacterial exposure—as occurs with slowly progressing caries—can maintain primed states for extended periods, increasing vulnerability to activation signals (Lee et al. 2015; Wang et al. 2021).

6.3. Activation (Signal 2): Stress Integration and Inflammasome Threshold Crossing

Whilst priming establishes inflammasome competence, activation constitutes the decisive threshold‐crossing event that commits dental pulp cells to caspase‐1 activation and inflammatory cytokine release (Jiang et al. 2015). Unlike priming, Signal 2 does not rely on a single molecular ligand but integrates multiple indicators of cellular stress—including membrane damage, ionic disequilibrium and mitochondrial dysfunction—whose cumulative intensity governs inflammasome assembly (Jiang et al. 2015; Zhang et al. 2021). This integrative sensing capacity allows pulp cells to discriminate transient, potentially reversible stress from sustained pathological injury requiring inflammatory amplification.

6.3.1. Purinergic Danger Signalling and Ionic Threshold Sensing

Extracellular ATP acts as a universal DAMP signalling cellular stress, injury and death across inflammatory contexts (Jiang et al. 2015; Sun et al. 2023; Zhang et al. 2015; Yang et al. 2014). Under physiological conditions, extracellular ATP is maintained at low nanomolar levels by ectonucleotidase activity. During pulpitis, however, dying odontoblasts and injured pulp cells release millimolar cytosolic ATP via pannexin‐1 hemichannels, membrane disruption or regulated exocytosis, generating steep extracellular ATP gradients (Jiang et al. 2015; Yang et al. 2014). Combined human tissue analyses and mechanistic in vitro studies have shown that ATP surges activate P2X7 receptors—ligand‐gated ion channels expressed by dental pulp fibroblasts, odontoblasts and immune cells—thereby initiating downstream inflammatory signalling (Jiang et al. 2015; Sun et al. 2023; Zhang et al. 2015).

P2X7 activation induces rapid K⁺ efflux, Ca²⁺ influx, and pannexin‐1 recruitment to plasma membranes, forming large pores permitting DAMPs/PAMPSs entry into the cytoplasm where it directly engages NLRP3 (Jiang et al. 2015; An et al. 2022). The resulting cytosolic K⁺ efflux constitutes a critical threshold signal for inflammasome activation, as reduced intracellular K⁺ levels promote reactive oxygen species (ROS) generation, thereby triggering NLRP3 inflammasome assembly (Jiang et al. 2015).

Pharmacological evidence supports this mechanism. Elevation of extracellular K⁺, which prevents K⁺ efflux, or treatment with glibenclamide or potassium chloride (KCl) effectively blocks K⁺ efflux, inhibits NLRP3 activation, and markedly suppresses LPS + ATP–induced IL‐1β production. Likewise, scavenging ROS with N‐acetyl‐L‐cysteine (NAC) inhibits NLRP3 inflammasome assembly, while P2X7 receptor antagonists significantly attenuate inflammasome activation (Jiang et al. 2015; Yang et al. 2014). In in vivo animal models of experimental pulpitis, complemented by ex vivo tissue analyses, P2X7 receptor expression is upregulated in the trigeminal ganglia and medulla and correlates with pain‐related behaviour; conversely, P2X7 inhibition increases pain thresholds and reduces NLRP3, IL‐1β and IL‐18 expression (Sun et al. 2023). Together, these findings establish P2X7–NLRP3 signalling as a direct contributor to pulpitis‐associated pain through nociceptor sensitisation.

Mechanistic in vitro studies in human macrophage cell lines (THP‐1) have shown that Enterococcus faecalis induces ATP‐dependent NLRP3 inflammasome activation, leading to caspase‐1 activation, IL‐1β secretion, and pyroptotic cell death; these effects are attenuated by the P2X7 antagonist oxATP (Yang et al. 2014). The temporal sequence—rapid ATP release preceding inflammasome activation—confirms extracellular ATP as an upstream activation signal. This process rapidly evolves into a self‐amplifying feed‐forward loop: initial cell damage → ATP release → P2X7 receptor activation → NLRP3 inflammasome assembly → pyroptotic cell death → secondary ATP release → recruitment and activation of neighbouring cells (Zhang et al. 2015; Sun et al. 2023; Jiang et al. 2015; Yang et al. 2014). Clinically, this ATP‐driven amplification loop helps explain how initially localised pulp injury can escalate into widespread inflammatory activation, intensifying IL‐1β production, expanding pyroptotic cell death, and accelerating the progression toward irreversible pulpitis.

Notably, in hDPFs, ATP alone does not induce robust IL‐1β secretion; rather, concurrent exposure to ATP and LPS is required to achieve caspase‐1 activation and cytokine release (Jiang et al. 2015; Wang et al. 2021). This cooperative requirement reinforces the two‐signal paradigm within the pulp microenvironment, indicating that sterile danger signalling may remain insufficient to trigger full inflammasome activation unless accompanied by microbial or sustained inflammatory priming. Clinically, this suggests that while sterile injuries such as trauma may still lead to pulpal necrosis through vascular compromise or ischaemic damage, sustained inflammasome‐driven cytokine amplification and IL‐1β–mediated inflammatory progression are more strongly associated with persistent microbial stimulation.

6.3.2. Calcium Mobilisation as an Upstream Inflammasome Amplifier

Calcium mobilisation functions as a critical upstream amplifier of NLRP3 inflammasome activation by integrating extracellular Ca²⁺ influx with release from intracellular stores. PLC‐dependent inositol‐1,4,5‐trisphosphate (InsP₃) generation triggers endoplasmic reticulum Ca²⁺ release, followed by store‐operated Ca²⁺ entry (SOCE), sustaining elevated cytosolic Ca²⁺ levels. Increased intracellular Ca²⁺ promotes NLRP3–ASC assembly and caspase‐1 activation, while concurrently inducing mitochondrial dysfunction, ROS production and mtDNA release—permissive signals for inflammasome engagement (An et al. 2022).

Consistently, pharmacological inhibition of PLC, InsP₃ receptors or SOCE abrogates inflammasome assembly and IL‐1β secretion, identifying Ca²⁺ signalling as an indispensable amplifier of NLRP3‐driven inflammation (Murakami et al. 2012; Lee et al. 2012). Although direct evidence in pulpal inflammasome models is currently limited, human dental pulp cells express functional calcium‐sensing receptors (CaSR), and modulation of CaSR–PI3K–AKT signalling alters IL‐1β and TNF‐α production in LPS‐stimulated pulp cells (An et al. 2022). This supports the biological plausibility that Ca²⁺‐dependent signalling contributes to inflammasome activation downstream of ATP–P2X7 signalling in pulpal inflammation.

6.3.3. Mitochondrial Distress and mtDNA‐Driven Inflammasome Engagement

mtDNA has emerged as a pivotal endogenous danger signal linking cellular stress to robust innate immune activation in dental pulp pathology. Upon release into the cytosol, mtDNA can directly engage pattern‐recognition receptors, including TLR9 and inflammasome sensors, thereby amplifying sterile inflammatory responses (Zhang et al. 2021). In odontoblasts, LPS induces mitochondrial dysfunction characterised by excessive ROS generation and structural damage. Mechanistically, this stress triggers the translocation of Bcl‐2‐associated X protein (BAX) from the cytosol to mitochondrial membranes, promoting mitochondrial outer membrane permeabilisation and facilitating the release of mtDNA into the cytosol (Zhang et al. 2021). Beyond its role in inflammasome activation, cytosolic mtDNA also contributes to GSDMD‐mediated pyroptosis, leading to the production of pro‐inflammatory mediators such as CXCL10 and IFN‐β. Notably, silencing of GSDMD does not completely abrogate cytokine expression, owing to compensatory signalling through the STING pathway, thereby highlighting an integrated mtDNA–GSDMD–STING axis governing odontoblast immune responses (Zhang et al. 2021; Ji et al. 2022).

Once released, mtDNA functions as a potent DAMP through two parallel pathways: (i) NLRP3 activation—cytosolic mtDNA promotes inflammasome assembly via mitochondrial ROS generation and direct NLRP3 engagement; and (ii) AIM2 activation—as a cytosolic dsDNA sensor, AIM2 directly binds leaked mtDNA, triggering inflammasome assembly independently of NLRP3 (Huang et al. 2018; Ji et al. 2022).

Mechanistic in vitro studies with functional validation in odontoblast‐like (mDPC6T) cells demonstrate that inhibition of mtDNA replication using ethidium bromide markedly reduces LPS‐induced cytosolic mtDNA accumulation, accompanied by decreased expression of NLRP3, cleaved caspase‐1, cleaved GSDMD, and IL‐1β, thereby supporting a causal role for mtDNA in inflammasome activation (Zhang et al. 2021). Conversely, direct transfection of purified mtDNA into mDPC6T cells is sufficient to trigger robust inflammasome activation, characterised by caspase‐1 cleavage, IL‐1β release and pyroptotic morphology, including cell swelling, in the absence of additional bacterial stimuli, confirming mtDNA sufficiency (Zhang et al. 2021) (Table 3 and Figure 6).

TABLE 3.

Molecular architecture of inflammasome activation (signal 2) in dental pulp.

Regulatory level	Activation axis/mechanism	Upstream trigger	Core molecular events	Inflammasome pathway engaged	Functional consequence	References
I. Purinergic Danger Signalling (Ionic Threshold Sensing)	ATP–P2X7 receptor axis	Extracellular ATP released from dying pulp cells	P2X7 activation → K+ efflux, Ca2+ influx, pannexin‐1 pore formation	Canonical NLRP3–caspase‐1	Caspase‐1 activation; IL‐1β/IL‐18 maturation; pyroptosis	Jiang et al. (2015); Sun et al. (2023); Yang et al. (2014); Zhang et al. (2015)
	Potassium efflux as threshold signal	Reduced intracellular K+	ROS generation; conformational activation of NLRP3	NLRP3	Assembly threshold crossing	Jiang et al. (2015)
	Pharmacological validation	High extracellular K+; glibenclamide; P2X7 antagonists	Blockade of K+ efflux or receptor activation	NLRP3 inhibition	Suppressed IL‐1β production; reduced pain signalling	Jiang et al. (2015); Yang et al. (2014); Sun et al. (2023)
II. Calcium‐Dependent Amplification	PLC–InsP3–SOCE signalling	Ca2+ influx and ER Ca2+ release	Sustained cytosolic Ca2+ elevation; mitochondrial dysfunction; mtROS generation	NLRP3	Amplified inflammasome assembly	Murakami et al. (2012); Lee et al. (2012)
	CaSR–PI3K–AKT modulation	LPS stimulation in pulp cells	Altered IL‐1β and TNF‐α production	Likely NLRP3 modulation	Supports Ca2+‐dependent inflammasome plausibility	An et al. (2022)
III. Mitochondrial Distress Signalling	mtROS production	LPS‐induced mitochondrial dysfunction	Oxidative stress; NLRP3 sensitisation	NLRP3	Activation amplification	Zhang et al. (2021)
	mtDNA cytosolic leakage	BAX‐mediated mitochondrial permeabilisation	Direct engagement of cytosolic sensors	NLRP3 and AIM2	Caspase‐1 cleavage; IL‐1β release; pyroptosis	Zhang et al. (2021); Huang et al. (2018); Ji et al. (2022)
	Functional validation of mtDNA	Ethidium bromide (mtDNA depletion); mtDNA transfection	Reduced or induced inflammasome activation	NLRP3/AIM2	Demonstrates mtDNA necessity and sufficiency	Zhang et al. (2021)
IV. Non‐Canonical Activation (Gram‐Positive Sensing)	NLRP6–caspase‐4 axis	Cytosolic LTA from S. mutans	Caspase‐4 activation → secondary caspase‐1 engagement	NLRP6–caspase‐4	IL‐1β and IL‐18 maturation; GSDMD cleavage	Tian et al. (2021)
	Independence from NLRP3	MCC950‐resistant activation	Silencing NLRP6 or caspase‐4 abolishes response	NLRP6‐specific	Parallel inflammasome redundancy	Tian et al. (2021)
V. Feed‐Forward Amplification Loops	ATP–pyroptosis loop	Initial cell damage	ATP release → P2X7 activation → secondary pyroptosis	NLRP3	Self‐sustaining inflammatory amplification	Zhang et al. (2015); Sun et al. (2023); Jiang et al. (2015)
	mtDNA propagation loop	Mitochondrial rupture during pyroptosis	Secondary mtDNA release to neighbouring cells	NLRP3/AIM2	Spatial inflammatory spread	Zhang et al. (2021)
	GSDMD pore reinforcement	Caspase‐1 cleavage of GSDMD	Sustained K+ efflux; cytokine release	Canonical & non‐canonical	Amplifies inflammasome signalling	Zhang et al. (2021)
VI. Spatial Expansion in Pulp Tissue	Odontoblast frontline activation	Bacterial penetration via dentinal tubules	Early NLRP3 activation in odontoblast layer	NLRP3 predominant	Barrier breach; DAMP release	Jiang et al. (2015); al Natour et al. (2021)
	Necroptosis–pyroptosis synergy	RIPK3‐mediated necroptosis; ROS	DAMP release; macrophage pyroptosis	NLRP3 amplification	Accelerated irreversible damage	Casey et al. (2025); Wang et al. (2025)
	PANoptosis ( E. faecalis )	Combined death pathways	Pyroptosis + necroptosis + apoptosis	Multiple inflammasomes	Exacerbated tissue injury	Chi et al. (2021)
VII. Inflammasome Assembly (SMOC Formation)	ASC speck formation	Sensor oligomerisation	PYD–PYD polymerisation; CARD–CARD recruitment of pro‐caspase‐1	NLRP3, AIM2, NLRP6	Digital caspase‐1 activation	Huang et al. (2018); Lee et al. (2015); Jiang et al. (2015)
	Binary threshold behaviour	Subthreshold vs. stable speck formation	Cooperative oligomerisation	All inflammasomes	Molecular point of no return	Liu et al. (2014); Huang et al. (2018)
	Extracellular ASC specks	Post‐pyroptotic release	Prion‐like inflammatory propagation	Persistent inflammasome signalling	Chronic inflammatory amplification	Bertheloot et al. (2022)

Abbreviations: AIM2, absent in melanoma 2; AKT, protein kinase B; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; ATP, adenosine triphosphate; BAX, Bcl‐2‐associated X protein; Ca²⁺, calcium; CARD, caspase recruitment domain; CaSR, calcium‐sensing receptor; DAMP, damage‐associated molecular pattern; ER, endoplasmic reticulum; GSDMD, gasdermin D; IL‐18, interleukin‐18; IL‐1β, interleukin‐1 beta; InsP₃, inositol 1,4,5‐trisphosphate; K⁺, potassium; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MCC950, selective NLRP3 inhibitor; mtDNA, mitochondrial DNA; mtROS, mitochondrial reactive oxygen species; NLRP3, NOD‐like receptor family pyrin domain‐containing 3; NLRP6, NOD‐like receptor family pyrin domain‐containing 6; P2X7, purinergic receptor P2X7; PANoptosis, coordinated pyroptosis–apoptosis–necroptosis cell death pathway; PI3K, phosphoinositide 3‐kinase; PLC, phospholipase C; PYD, pyrin domain; RIPK3, receptor‐interacting protein kinase 3; ROS, reactive oxygen species; SMOC, supramolecular organising centre; SOCE, store‐operated calcium entry; TNF‐α, tumour necrosis factor alpha; ↑, increased expression or activation; ↓, decreased expression or inhibition.

FIGURE 6.

Activation (signal 2). Stress integration and inflammasome threshold crossing. Extracellular ATP acts as an NLRP3 agonist by stimulating the purinergic P2X7 ATP‐gated ion channel, inducing K⁺ efflux and promoting pannexin‐1 hemichannel formation, thereby facilitating the cytosolic entry of danger‐associated or pathogen‐associated molecular patterns (DAMPs/PAMPs) that directly engage NLRP3. The efflux of K⁺ reduces intracellular potassium concentration, a critical permissive signal for NLRP3 activation. A decreased intracellular K⁺ concentration has also been proposed to enhance reactive oxygen species (ROS) generation, further amplifying NLRP3 activation. Concomitantly, increased intracellular Ca²⁺ promotes NLRP3–ASC complex assembly and caspase‐1 activation, while also inducing mitochondrial dysfunction, ROS production, and mitochondrial DNA (mtDNA) release—additional signals that facilitate inflammasome engagement. Activated caspase‐1 cleaves pro‐IL‐1β into mature IL‐1β and processes gasdermin D (GSDMD), generating the N‐terminal GSDMD fragment that mediates pyroptotic membrane pore formation. Cytosolic double‐stranded DNA (dsDNA) may additionally activate the AIM2 inflammasome, converging on caspase‐1 activation and IL‐1β maturation. Irreversible pulpitis.

6.4. Feed‐Forward Loops and Inflammatory Amplification

The clinical relevance becomes evident during disease progression: initial bacterial challenge → mitochondrial damage → mtDNA leakage → inflammasome activation → pyroptotic cell death → secondary mtDNA release → activation of neighbouring cells → progressive inflammatory amplification (Zhang et al. 2021).

This feed‐forward loop explains how pulpal inflammation may persist even after bacterial load is reduced. Therapeutically, strategies that preserve mitochondrial integrity or inhibit mtDNA sensing pathways (e.g., NLRP3 or AIM2) may interrupt this cycle. Notably, combined human tissue analyses and functionally validated mechanistic in vitro studies have shown that bone marrow–derived mesenchymal stem cells (BMSCs) can transfer functional mitochondria to odontoblasts via tunnelling nanotubes or extracellular vesicles, thereby restoring mitochondrial function and attenuating NLRP3 inflammasome activation (Wang et al. 2023). This mitochondrial transfer reduces pyroptosis and highlights a regenerative approach to limiting inflammasome‐driven inflammatory amplification.

Concurrently, pyroptotic cell death promotes extracellular ATP release from injured pulp cells, generating a secondary danger signal that activates purinergic P2X7 receptors on neighbouring cells and facilitates subsequent inflammasome activation (Jiang et al. 2015). Together, these processes mechanistically integrate mtDNA‐driven feed‐forward signalling with purinergic danger pathways, reinforcing inflammatory amplification in dental pulp tissue.

Stress summation and threshold integration underpin the logic of inflammasome activation: sub‐threshold stimuli such as low‐intensity ATP, mtDNA, ROS, or ionic perturbations individually may fail to trigger activation, but their combination exceeds the critical threshold, leading to robust caspase‐1 activation, IL‐1β secretion, and pyroptosis (Jiang et al. 2015; Zhang et al. 2021). Mitochondrial damage provides both direct (mtDNA) and amplifying (mtROS) signals that synergize with other stressors to ensure threshold crossing.

Beyond metabolic stress sensing, cytosolic nucleic acid sensing provides DNA‐driven activation pathways that complement metabolic stress detection. The AIM2 inflammasome directly recognises cytosolic dsDNA in a sequence‐independent manner, inducing robust caspase‐1 activation and IL‐1β release in interferon‐primed dental pulp cells (Huang et al. 2018).

Collectively, these mechanisms define a self‐sustaining inflammatory circuit in which mitochondrial distress, purinergic signalling, and nucleic acid sensing converge to amplify inflammasome activation. This integrated model provides a mechanistic basis for persistent pulpal inflammation beyond the initial microbial trigger.

6.5. Non‐Canonical Inflammasome Activation: The NLRP6–Caspase‐4 Axis

The NLRP6–caspase‐4 inflammasome constitutes a non‐canonical, NLRP3‐independent activation pathway that directly senses cytosolic LTA from Gram‐positive bacteria (Tian et al. 2021). Functionally validated mechanistic in vitro studies in HDPCs show that priming with Porphyromonas gingivalis LPS activates type I interferon signalling, leading to upregulation of NLRP6 and caspase‐4. This licensing step enables subsequent responsiveness to intracellular LTA. Upon cytosolic delivery of Streptococcus mutans LTA, HDPCs exhibit robust IL‐1β and IL‐18 secretion. This response is abrogated by silencing NLRP6 or caspase‐4, but remains unaffected by the NLRP3‐specific inhibitor MCC950, confirming pathway specificity and independence from NLRP3 signalling (Tian et al. 2021). This mechanism facilitates selective detection of Gram‐positive cariogenic bacteria, which predominate in early carious lesions. Although initiated by distinct upstream sensors, the canonical NLRP3–caspase‐1 and non‐canonical NLRP6–caspase‐4 pathways converge on GSDMD cleavage and IL‐1β maturation, establishing functional inflammasome redundancy (Tian et al. 2021; Zhang et al. 2021). In irreversible pulpitis, the simultaneous upregulation of NLRP3, NLRP6, and cleaved caspase‐1 reflects concurrent activation of parallel pathways, implying that inhibition of a single inflammasome axis may be insufficient to attenuate pulpal inflammation.

GSDMD pores act as secondary amplifiers of inflammasome signalling by sustaining K⁺ efflux and facilitating cytokine release independently of vesicular pathways (Zhang et al. 2021). In odontoblast‐like cells, LPS‐induced mitochondrial damage promotes cytosolic mtDNA leakage, activating the NLRP3 inflammasome and leading to caspase‐1‐mediated GSDMD cleavage and pore formation. Active GSDMD pores enable IL‐1β release and the dissemination of mtDNA to adjacent cells, establishing a feed‐forward inflammatory loop. Importantly, during dominant inflammasome activation, GSDMD‐mediated pore formation induces local ionic dysregulation that functionally restrains cGAS–STING signalling, thereby temporarily prioritising canonical NLRP3‐driven inflammatory responses over interferon‐mediated pathways (NLRP6‐caspase 4 axis) (Zhang et al. 2021).

6.6. Progressive Spatial Expansion of Inflammasome Activation

Odontoblasts express high levels of NLRP3, positioning inflammasome sensors at the frontline of bacterial invasion (Zhang et al. 2021; Jiang et al. 2015; al Natour et al. 2021). Bacterial products penetrating dentinal tubules encounter odontoblasts first, and their death—via pyroptosis, necroptosis, or apoptosis—has multiple consequences: (i) barrier breach, permitting unimpeded bacterial entry into the pulp; (ii) DAMP release, including ATP, HMGB1, S100 proteins, and mitochondrial components that activate inflammasomes in fibroblasts and immune cells; and (iii) amplification signalling, whereby DAMPs from necroptotic pulp cells engage TLR9 and the receptor for advanced glycation end‐products (RAGE) on neighbouring immune cells, promoting IL‐6 and CXCL8 secretion and further neutrophil recruitment (Mahmoudi et al. 2017; Wang et al. 2025).

Immune infiltration amplifies mitochondrial and cytosolic ROS, sustaining necroptotic signalling and providing a critical upstream trigger for NLRP3 inflammasome activation (Ziehr and MacDonald 2024). Inflammasome engagement induces caspase‐1 activation, macrophage pyroptosis, and IL‐1β release, intensifying the inflammatory milieu. The convergence of necroptosis‐derived DAMPs and inflammasome‐driven pyroptosis establishes a feed‐forward loop that accelerates irreversible pulp tissue damage (Casey et al. 2025). Within the hypoxic milieu, impaired mitochondrial respiration enhances glycolytic flux, resulting in lactate accumulation and tissue acidification (Solaini et al. 2010). This metabolic shift further destabilises mitochondrial homeostasis, lowers the activation threshold of the NLRP3 inflammasome, and favours progression from reversible pulpal inflammation to irreversible inflammation and pulp necrosis (Solaini et al. 2010).

Functionally validated mechanistic in vitro studies show that odontoblast death‐conditioned medium activates NLRP3 inflammasomes in dental pulp cells, enhancing proliferation and odontogenic differentiation in surviving cells—a compensatory regenerative response (al Natour et al. 2021). Excessive odontoblast loss, however, compromises this regenerative capacity. Immunofluorescence of irreversible pulpitis tissues reveals disrupted odontoblast layers with diffuse NLRP3 and caspase‐1 expression throughout the pulp parenchyma, contrasting with the organised, odontoblast‐restricted pattern in healthy pulp (Jiang et al. 2015). This shift from confined to diffuse activation marks the transition from reversible to irreversible pulpitis.

Furthermore, different pathogens elicit distinct odontoblast death mechanisms: Streptococcus infection triggers RIPK3‐mediated necroptosis and mitochondrial dysfunction, with mitochondrial ROS activating NLRP3 inflammasomes, inducing macrophage pyroptosis and amplifying IL‐1 secretion, thereby exacerbating inflammation (Casey et al. 2025). The synergy between pyroptosis and necroptosis accelerates irreversible pulp tissue damage (Wang et al. 2025; Huang et al. 2021). Additionally, E. faecalis infection triggers PANoptosis, concurrently activating pyroptosis, necroptosis, and apoptosis pathways (Chi et al. 2021). While this redundancy aids bacterial containment, excessive cell death exacerbates tissue injury when it surpasses regenerative capacity.

Importantly, inflammasome activation occurs along a graded continuum rather than as a binary process. Transient or low‐intensity signals—brief ATP release, limited potassium efflux, or low‐level ROS—may induce partial NLRP3 oligomerisation without reaching the cooperative threshold necessary for stable ASC speck formation (Jiang et al. 2015; Zhang et al. 2021). At this stage, inflammatory signalling may remain biologically reversible, consistent with cases clinically diagnosed as reversible pulpitis in which vital pulp therapy can succeed. However, sustained signalling that permits stable inflammasome assembly and caspase‐1 activation may represent a molecular commitment point beyond which inflammation becomes self‐propagating, reducing the likelihood of maintaining long‐term pulp vitality (Table 3).

Collectively, inflammasome activation (inflammasome assembly/ASC speck formation) might be considered as a biological point of no return at the cellular and microenvironmental level in pulpal inflammation. Importantly, this irreversible transition is not uniform across the entire pulp but emerges in spatially restricted compartments, along a corono–apical axis, where activation thresholds are exceeded. Within these microdomains, inflammasome‐driven pyroptosis, mitochondrial dysfunction, and secondary danger signal release establish self‐sustaining feed‐forward loops that locally commit tissue to irreversible injury, while adjacent regions may transiently retain regulatory capacity. Progressive expansion of these irreversible compartments ultimately underlies the clinical transition from reversible to irreversible pulpitis.

6.7. Inflammasome Assembly/ASC Speck Formation: The Molecular Point of no Return in Pulpal Inflammation

Inflammasome activation culminates in supramolecular organising centres (SMOCs), in which sensor, adaptor, and effector proteins assemble into micron‐scale signalling platforms forming the functional inflammasome complex (Kagan et al. 2014). Unlike graded receptor‐mediated cascades, inflammasome assembly is a discrete, binary transition (priming/activation) from inactive monomers to oligomeric structures, establishing a molecular point of no return in pulpal inflammation (Jiang et al. 2015; Tian et al. 2021; Lee et al. 2015; Zhang et al. 2021; Wang et al. 2021).

Recently, Wu et al. (2026), provide particularly compelling evidence supporting the concept of biological irreversibility in irreversible pulpitis. In their study, NLRP1 was identified as a novel biomarker of pyroptosis through an integrated methodological approach combining transcriptomic analysis, human dental pulp tissue examination, and both in vitro and in vivo experimental models. Immunohistochemical and immunofluorescence analyses demonstrated marked upregulation and cytoplasmic localisation of NLRP1 in irreversible pulpitis, accompanied by activation of caspase‐1 and increased expression of IL‐1β (Wu et al. 2026). Furthermore, evidence of gasdermin‐mediated membrane pore formation indicated the occurrence of pyroptotic cell death, a process inherently associated with amplification and perpetuation of inflammation. The observed co‐localisation of NLRP1 and NLRP3 suggests the engagement of a coordinated inflammasome network, reinforcing a self‐sustaining inflammatory microenvironment (Wu et al. 2026). Importantly, the authors explicitly relate these findings to the pathophysiology of irreversible pulpitis, thereby providing strong mechanistic support for the concept of tissue‐level irreversibility. Although ASC speck formation was not directly visualised, the activation of caspase‐1 implies the assembly of functional inflammasome complexes, in which ASC oligomerisation serves as the essential supramolecular platform for signal propagation.

Similarly, Jiang et al. (2015), provide molecular, functional, and spatial evidence supporting a biologically irreversible inflammatory state in irreversible pulpitis. Their analysis of human dental pulp tissues using qRT‐PCR and Western blotting demonstrated that NLRP3 mRNA levels were significantly increased in both reversible and irreversible pulpitis compared with healthy pulp, indicating an initial priming of the inflammasome pathway. However, only irreversible pulpitis exhibited a significant increase in NLRP3 protein expression, together with elevated mRNA levels of caspase‐1 and IL‐1β and the exclusive detection of cleaved caspase‐1 (p20), a definitive marker of functional inflammasome activation. This finding implies the assembly of the NLRP3 inflammasome complex, a process that critically depends on ASC speck formation as a supramolecular organising centre for caspase‐1 activation. The dissociation between transcriptional upregulation and protein‐level activation in reversible pulpitis suggests a regulated and potentially reversible inflammatory state, whereas the coordinated upregulation of inflammasome components and downstream effector cytokines in irreversible pulpitis reflects a transition to a self‐amplifying and biologically committed inflammatory response (Jiang et al. 2015). Complementary immunofluorescence analyses further demonstrated that, whereas NLRP3 and caspase‐1 expression in healthy and reversibly inflamed pulps was primarily confined to the odontoblast layer—consistent with a sentinel innate immune function at the dentine–pulp interface—irreversible pulpitis was characterised by disruption of this barrier and diffuse cytoplasmic distribution throughout the pulpal stroma. This spatial expansion indicates the propagation of inflammasome activation across multiple pulpal cell populations, signifying a shift from a compartmentalised defensive response to a disseminated and self‐sustaining inflammatory microenvironment (Jiang et al. 2015). Collectively, these findings support the concept of tissue‐level irreversibility in irreversible pulpitis, although direct structural evidence confirming the permanence of ASC speck formation in dental pulp cells remains limited.

Inflammasome assembly is initiated by oligomerisation of NLRP3, NLRP6, and AIM2 (Jiang et al. 2015; Lee et al. 2015; Huang et al. 2018; Tian et al. 2021). In resting cells, these sensors exist as auto‐inhibited monomers, with intramolecular interactions preventing spontaneous assembly (Jiang et al. 2015; Lee et al. 2015; Huang et al. 2018; Tian et al. 2021). Activation signals—K⁺ efflux and oxidative stress for NLRP3, cytosolic lipoteichoic acid for NLRP6, and cytosolic dsDNA for AIM2—induce conformational changes that relieve auto‐inhibition and expose oligomerisation interfaces. For AIM2, ligand‐templated oligomerisation along DNA scaffolds generates cooperative clustering, exposing PYDs for adaptor recruitment and ensuring that inflammasome assembly scales with intracellular danger signal burden (Huang et al. 2018).

Following sensor oligomerisation, exposed pyrin domains recruit the adaptor ASC via homotypic PYD–PYD interactions (Lee et al. 2015; Huang et al. 2018; Jiang et al. 2015). ASC undergoes prion‐like polymerisation into filaments that coalesce into a single macroscopic ASC speck per cell, serving as the central signalling hub of the assembled inflammasome (Lee et al. 2015; Huang et al. 2018). Speck formation exhibits switch‐like, all‐or‐none kinetics: once nucleation begins, polymerisation proceeds rapidly and irreversibly, recruiting most of the cellular ASC pool into a stable platform (Huang et al. 2018; Lee et al. 2015). In HDPCs, cytosolic dsDNA stimulation after IFN‐γ priming induces ASC upregulation and redistribution from diffuse cytoplasmic localisation to discrete specks, while ASC knockdown abolishes caspase‐1 activation and IL‐1β secretion across multiple inflammasome pathways, confirming ASC as an obligate hub for inflammasome signalling (Huang et al. 2018; Lee et al. 2015).

ASC specks recruit pro‐caspase‐1 via homotypic CARD–CARD interactions, concentrating the zymogen into supramolecular complexes that drive proximity‐induced dimerisation and autocatalytic processing into active p20/p10 heterotetramers (Yang et al. 2014; Jiang et al. 2015; Huang et al. 2018). In hDPFs, LPS priming followed by ATP stimulation induces caspase‐1 cleavage detectable exclusively in culture supernatants, indicating both enzymatic activation and extracellular release (Jiang et al. 2015). Likewise, Enterococcus faecalis elicits dose‐dependent caspase‐1 activation in macrophages, demonstrating that inflammasome assembly and protease activation scale with microbial burden (Yang et al. 2014). Once initiated, active caspase‐1 amplifies its own activation through a feed‐forward mechanism that accelerates neighbouring pro‐caspase‐1 cleavage, rapidly generating high local enzymatic concentrations. Pharmacological inhibition of caspase‐1 abolishes IL‐1β secretion, confirming its indispensable role in cytokine maturation downstream of inflammasome assembly (Yang et al. 2014).

Inflammasome assembly displays digital, threshold‐dependent signalling. Subthreshold priming leads to accumulation of pro‐IL‐1β and inflammasome components without caspase‐1 activation, whereas surpassing the activation threshold triggers rapid, cooperative assembly and robust caspase‐1 cleavage (Liu et al. 2014; Jiang et al. 2015; Huang et al. 2018). In irreversible pulpitis, diffuse ASC expression and caspase‐1 cleavage beyond the odontoblast layer indicate widespread, tissue‐level inflammasome assembly, which cannot be disassembled due to the amyloid‐like stability of ASC filaments (Huang et al. 2018; Tian et al. 2021; Zhang et al. 2021).

Inflammasome assembly and robust IL‐1β secretion precede overt cell pyroptosis and tissue necrosis, creating a temporal window wherein inflammation is biochemically irreversible but anatomical tissue destruction remains incomplete (Conos et al. 2016; Zhang et al. 2021; Yang et al. 2014). Following pyroptosis, ASC specks are released extracellularly, where their polymeric, structurally stable nature allows persistence beyond the initial trigger and propagation of inflammation through pro‐inflammatory, prion‐like activity, sustaining inflammasome signalling and potentially contributing to chronic inflammatory states such as advanced pulpitis and potentially extending the inflammatory response into more apical regions of the pulp (Jiang et al. 2015; Tian et al. 2021; Bertheloot et al. 2022).

Collectively, inflammasome assembly marks the molecular transition from reversible to irreversible pulpal inflammation. Once ASC specks form, inflammasomes act as autonomous cytokine‐processing platforms, persisting independently of upstream priming or activation, which explains why removal of bacterial stimuli or inhibition of NF‐κB‐ or P2X7‐dependent pathways fails to reverse advanced disease (Huang et al. 2018; Jiang et al. 2015; Tian et al. 2021; Wang et al. 2021). Accordingly, tissue affected by irreversible inflammation must be removed to prevent propagation of the inflammatory process into more apical regions of the pulp, providing a biological rationale for the principles of vital pulp therapy (VPT) (Table 3).

Irreversibility in pulpitis does not arise simply from increasing cytokine levels, but from the structural stabilisation of inflammasome platforms within a confined anatomical environment. Once ASC oligomerisation consolidates supramolecular complexes, inflammatory signalling shifts from inducible to self‐sustaining. In the rigid dentinal enclosure, this architectural transition amplifies caspase‐1 activity, perpetuates IL‐1β maturation, and promotes gasdermin D–mediated membrane permeabilisation, progressively uncoupling inflammation from its initiating trigger.

6.8. Proposed Cell Type–Specific Spatiotemporal Activation Model

The progression of pulpitis can be conceptualised as a coordinated, cell type–specific and corono–apically organised sequence of inflammasome activation that delineates the transition from a localised defensive response to a disseminated and biologically committed inflammatory state. In this context, we propose a cell type–specific spatiotemporal framework that integrates spatial localisation, temporal dynamics and functional specialisation of distinct pulpal cell populations, thereby providing a mechanistic basis for understanding disease progression and informing cell‐targeted therapeutic strategies.

6.8.1. Early Sentinel Phase—Odontoblast‐Led Initiation

Frontline odontoblasts represent the earliest cellular responders to microbial and sterile danger signals diffusing through dentinal tubules. Immunofluorescence and molecular analyses have demonstrated that NLRP3 expression in healthy pulp is largely confined to the odontoblastic layer, whereas irreversible pulpitis is characterised by its diffuse distribution throughout the pulpal stroma, accompanied by the exclusive detection of cleaved caspase‐1 (p20), indicating functional inflammasome activation (Jiang et al. 2015). The assembly of the inflammasome complex inherently involves ASC speck formation, which acts as a supramolecular organising centre for caspase‐1 activation and subsequent pyroptotic signalling (Shi et al. 2015). Pyroptotic death of odontoblasts results in disruption of the odontoblastic barrier and the release of DAMPs, thereby facilitating the propagation of inflammation into deeper pulpal compartments. Collectively, these events mark the initiation of a tissue‐level “molecular point of no return” (Jiang et al. 2015; al Natour et al. 2023).

6.8.2. Transitional Immunomodulatory Phase—DPSC‐Mediated Braking Capacity

DPSCs, predominantly located within perivascular niches, retain the greatest early “braking” capacity during the initial stages of inflammation. DPSCs constitutively express inflammasome components but display context‐dependent activation profiles that integrate immune sensing with regenerative functions (Li et al. 2023; Wei et al. 2025). As demonstrated by Zanini et al. (2024), DPSCs selectively modulate fibroblast inflammatory responses through preferential activation of the AIM2 inflammasome rather than NLRP3. This selective engagement results in a finely balanced cytokine milieu characterised by increased IL‐1β and IL‐6 production alongside suppression of TNF‐α secretion. Additionally, DPSCs exert anti‐fibrotic effects by reducing α‐SMA and fibronectin expression, promoting MMP‐9–mediated extracellular matrix remodelling, preserving their PDGFRβ⁺ pericyte‐like phenotype, and upregulating PD‐L1, thereby contributing to immune checkpoint–mediated regulation (Zanini et al. 2024). These coordinated mechanisms enable DPSCs to contain early inflammation while preserving the regenerative capacity of the pulp.

6.8.3. Failure of the Protective DPSC Effect

The immunoregulatory and regenerative functions of DPSCs progressively deteriorate as the inflammatory burden intensifies. Sustained exposure to pro‐inflammatory cytokines, mitochondrial dysfunction and persistent inflammasome signalling leads to functional exhaustion, senescence or pyroptotic cell death of DPSCs. This transition is further exacerbated by the depletion of endogenous inhibitory mechanisms, including microRNA‐mediated repression of NLRP3—particularly miR‐223—and the reduction of odontogenic matrix proteins such as osteomodulin (OMD), both of which contribute to the loss of inflammasome regulation and the establishment of a self‐amplifying inflammatory microenvironment (Zhang et al. 2020; Wei et al. 2025).

6.8.4. Late Amplification Phase—Stromal and Immune Cell Dominance

Following disruption of the odontoblastic barrier and failure of DPSC‐mediated containment, inflammasome activation propagates to stromal fibroblasts and pulp‐resident immune cells, including macrophages and dendritic cells. Fibroblasts, initially existing in a surveillance‐ready state, undergo TLR–NF‐κB–dependent priming, enabling subsequent NLRP3 oligomerisation and cytokine secretion (Zhang et al. 2015; Wang et al. 2021). Mononuclear phagocytes, which possess inherently higher basal inflammasome competence, become the principal drivers of inflammatory amplification, orchestrating robust secretion of IL‐1β and IL‐18 and sustaining the inflammatory milieu (Wei et al. 2025; Pohl et al. 2024). In advanced stages, the engagement of additional inflammasome sensors, including NLRP1 and NLRP6, further reinforces this inflammatory network and contributes to irreversible tissue damage (Tian et al. 2021; Zhao et al. 2020) (Figure 7).

FIGURE 7.

Proposed cell type–specific spatiotemporal model of inflammasome activation during pulpitis progression. The diagram depicts a progressive (corono–apically) organised sequence of events beginning with odontoblast‐led NLRP3 activation and ASC speck–mediated pyroptosis (Phase 1), followed by a transitional phase in which dental pulp stem cells (DPSCs) exert immunomodulatory and regenerative “braking” functions through preferential AIM2 activation (Phase 2). Persistent inflammatory stress leads to failure of this protective mechanism (Phase 3), enabling stromal fibroblast priming and dominance of mononuclear phagocytes that sustain IL‐1β and IL‐18 secretion (Phase 4). Additional engagement of NLRP1 and NLRP6 culminates in irreversible pulpal tissue damage, providing a biological foundation for cell‐targeted therapeutic strategies.

7. Pyroptosis: Gasdermin d‐Mediated Inflammatory Cell Death

7.1. Molecular Mechanisms: From Caspase‐1 Activation to Membrane Rupture

Pyroptosis is a lytic, inflammatory form of programmed cell death, distinct from apoptosis and necrosis, characterised by rapid membrane permeabilisation, cellular swelling, and release of pro‐inflammatory intracellular contents (Wei et al. 2025; Gu et al. 2025; Zhang et al. 2021). While early pyroptotic responses may support antimicrobial defence, sustained or hyperactivated pyroptosis correlates with progression to irreversible pulpitis (Wei et al. 2025).

In the canonical pathway, caspase‐1 executes pyroptosis through cleavage of GSDMD at Asp275 in humans, releasing its N‐terminal pore‐forming fragment (Liu et al. 2016). GSDMD is likewise processed by non‐canonical caspases‐4 and‐5 in humans and caspase‐11 in mice (Gu et al. 2025). However, overt pyroptotic cell death typically ensues when inflammasome signalling surpasses a critical intracellular threshold or is reinforced by secondary danger cues, including extracellular ATP, K⁺ efflux, ROS generation, and mitochondrial dysfunction (Evavold and Kagan 2019).

Following cleavage, GSDMD‐N oligomerises and inserts into the plasma membrane to form large transmembrane pores (Wei et al. 2025; Zhang et al. 2021; Liu et al. 2016). These pores collapse ionic gradients, promoting K⁺ efflux, Na⁺ and Ca²⁺ influx, osmotic swelling, and eventual membrane rupture. Concurrently, they enable the extracellular release of leaderless cytokines such as IL‐1β and IL‐18 (Wei et al. 2025; Liu et al. 2016).

Combined human tissue analysis and functionally validated mechanistic in vitro study in mDPC6T cells, LPS stimulation induces BAX translocation to mitochondria, facilitating cytosolic mtDNA release. mtDNA overexpression triggers pyroptotic death and increases CXCL10 and IFN‐β secretion, implicating mitochondrial damage as a driver of GSDMD‐dependent inflammatory activation (Zhang et al. 2021). Notably, mtDNA‐induced CXCL10 and IFN‐β production persists despite GSDMD knockdown, revealing a parallel STING‐dependent pathway that amplifies immune signalling independently of pyroptotic pore formation. Together, these findings delineate an mtDNA–GSDMD–STING axis linking mitochondrial injury to pulp inflammatory amplification (Zhang et al. 2021). Consistent with this pathological relevance, NLRP3‐mediated pyroptosis negatively correlates with nerve cell survival in dental pulp tissue, suggesting that elevated NLRP3 expression associates with reduced neuronal density (Sampoerno et al. 2025).

Functionally, propidium iodide uptake and lactate dehydrogenase release confirm membrane permeabilisation, while caspase‐1 inhibition blocks both GSDMD cleavage and pyroptotic death (Yang et al. 2014; Zhang et al. 2021), establishing a direct causal cascade: inflammasome activation → caspase‐1 → GSDMD cleavage → membrane rupture.

Therapeutically, several GSDMD inhibitors show translational potential. Necrosulfonamide covalently modifies GSDMD Cys86, disulfiram inhibits pore formation, and GI‐Y1 blocks GSDMD‐N membrane insertion (Wei et al. 2025). Importantly, these agents prevent membrane rupture without suppressing upstream caspase‐1 activation or IL‐1β processing, potentially preserving antimicrobial competence while limiting tissue injury (Wei et al. 2025) (Tables 4 and 5).

TABLE 4.

Inflammasome pathways regulating pyroptotic and cytokine responses in pulpal inflammation.

Inflammasome sensor	Signalling category	Cellular context	Triggering stimulus	Effector caspase(s)	Gasdermin involvement	Downstream inflammatory mediators	Biological implication	References
AIM2	Canonical DNA‐sensing	HDPCs	IFN‐γ + cytoplasmic DNA	Caspase‐1	N/A	IL‐1β	DNA‐driven innate immune activation	Huang et al. (2015); Wei et al. (2025)
NLRP6	Non‐canonical (Gram‐positive response)	HDPCs	LPS and LTA	Caspase‐1/4	N/A	IL‐1β, IL‐18	Amplification of Gram‐positive–induced inflammation	Tian et al. (2021); Wei et al. (2025)
NLRP3	Canonical	THP‐1 cells	LPS	Caspase‐1	N/A	IL‐1β	Monocytic inflammasome activation	Ran et al. (2017); Wei et al. (2025)
NLRP3	Canonical	HDPCs	Pg + ATP	N/A	N/A	IL‐1β	Bacteria‐induced priming with secondary activation	Aral et al. (2020); Wei et al. (2025)
NLRP3	Canonical	HDPCs	LTA	Caspase‐1	N/A	IL‐1β	Gram‐positive–mediated activation	al Natour et al. (2023); Wei et al. (2025)
NLRP3	Canonical with metabolic co‐stimulation	THP‐1 cells	Butyrate + E. faecalis LTA	Caspase‐1/4	GSDMD	IL‐1β	Metabolic enhancement of pyroptosis	Park et al. (2023); Wei et al. (2025)
—	Non‐canonical (cytosolic LPS sensing)	PDLFs/HDPCs	Cytoplasmic LPS	Caspase‐4	GSDMD	N/A	Direct intracellular endotoxin sensing	Gu et al. (2023); Wei et al. (2025)
NLRP6	Non‐canonical	HPDLCs	LPS	N/A	N/A	IL‐6, TNF‐α	Alternative inflammasome‐driven cytokine output	Lu et al. (2019); Wei et al. (2025)
NLRP3	Canonical	hDPFs	LPS + ATP	Caspase‐1	N/A	IL‐1β	Classical two‐signal activation	Zhang et al. (2015); Wei et al. (2025)
NLRP3	Canonical (mtDNA‐mediated)	mDPC6T	mtDNA	Caspase‐1	GSDMD	IL‐1β, CXCL10, IFN‐β	Mitochondrial damage–associated activation	Zhang et al. (2021); Wei et al. (2025)
NLRP3	Canonical (oxidative stress–enhanced)	mDPC6T	LPS + ATP + mitochondrial oxidative stress	Caspase‐1	N/A	IL‐1β, IL‐6, CXCL10	ROS‐amplified inflammasome signalling	Wang et al. (2023); Wei et al. (2025)
NLRP3	Canonical	THP‐1 cells	ONCL	Caspase‐1	N/A	IL‐1β, IL‐6, IL‐8, TNF‐α, IFN‐γ, CCL2	Broad pro‐inflammatory amplification	al Natour et al. (2021); Wei et al. (2025)
NLRP3	Canonical (material‐induced)	PBMCs	TEGDMA	N/A	N/A	IL‐18, IL‐1β	Biomaterial‐triggered inflammasome response	Alizadehgharib et al. (2017); Wei et al. (2025)
NLRP3	Canonical	HDPSCs	LPS + ATP	Caspase‐1	N/A	IL‐1β	Stem cell inflammasome activation	Lan et al. (2023); Wei et al. (2025)

Abbreviations: AIM2, absent in melanoma 2; ATP, adenosine triphosphate; CCL2, C‐C motif chemokine ligand 2; CXCL10, C‐X‐C motif chemokine ligand 10; GSDMD, gasdermin D; HDPCs, human dental pulp cells; hDPFs, human dental pulp fibroblasts; HDPSCs, human dental pulp stem cells; HPDLCs, human periodontal ligament cells; IFN‐β, interferon beta; IFN‐γ, interferon gamma; IL‐18, interleukin‐18; IL‐1β, interleukin‐1 beta; IL‐6, interleukin‐6; IL‐8, interleukin‐8; LPS, lipopolysaccharide; LTA, lipoteichoic acid; mDPC6T, murine dental pulp cell line 6 T; mtDNA, mitochondrial DNA; N/A, not available or not reported; NLRP3, NOD‐like receptor family pyrin domain‐containing 3; NLRP6, NOD‐like receptor family pyrin domain‐containing 6; ONCL, odontogenic necrotic cell lysate; PBMCs, peripheral blood mononuclear cells; PDLFs, periodontal ligament fibroblasts; Pg, Porphyromonas gingivalis ; ROS, reactive oxygen species; TEGDMA, triethylene glycol dimethacrylate; THP‐1, human monocytic cell line.

Source: Adapted from Wei et al. (2025).

TABLE 5.

Molecular interplay between pyroptosis, necroptosis, apoptosis, and panoptosis in pulpal inflammation.

Cell death modality	Core molecular axis	Key triggers in pulp	Mechanistic features	Biological consequence in pulpitis	References
Pyroptosis	Inflammasome → caspase‐1 → GSDMD	LPS, ATP, K+ efflux, ROS, mitochondrial dysfunction	GSDMD cleavage (Asp275) → pore formation → IL‐1β/IL‐18 release → membrane rupture	Antimicrobial defence (early); hyperactivation correlates with irreversible pulpitis; negative correlation with nerve survival	Wei et al. (2025); Gu et al. (2025); Liu et al. (2016); Evavold and Kagan (2019); Sampoerno et al. (2025); Zhang et al. (2021)
Necroptosis	RIPK1–RIPK3–MLKL axis	TLR4/TLR2 activation, TNFR1 signalling, caspase‐8 inhibition	MLKL oligomerisation → membrane permeabilisation → DAMP release; PGAM5–Drp1‐mediated mitochondrial fission; ROS amplification	Sustained DAMP release; inflammasome priming; oxidative escalation; progression to irreversible pulpitis and pulp necrosis	Wang et al. (2025); Murphy and Vince (2015); Tummers and Green (2017); Feltham and Silke (2017); Deragon et al. (2020)
Apoptosis	FADD–caspase‐8 pathway	Homeostatic signalling	Caspase‐dependent, membrane integrity preserved, phagocytic clearance	Immunologically silent; limits inflammasome activation; restrains excessive inflammation	Murphy and Vince (2015); Tummers and Green (2017)
PANoptosis	Integrated platform (RIPK1 hub) activating pyroptosis + apoptosis + necroptosis	High bacterial load; virulent E. faecalis strains	Concurrent NLRP3/caspase‐1 activation, caspase‐3 activation, RIPK3/MLKL phosphorylation	Maximal host defence; catastrophic tissue destruction in irreversible pulpitis	Chi et al. (2021); Wang et al. (2025); Wei et al. (2025)

7.2. Cell Type‐Specific Vulnerability and Functional Consequences of Pyroptosis in Dental Pulp

Different dental pulp cell populations exhibit distinct susceptibilities to pyroptosis and generate divergent functional outcomes (Gu et al. 2025; Wei et al. 2025).

Pyroptotic odontoblast death releases DAMPs that activate inner pulp immune cells, enhancing antimicrobial defence, yet simultaneously abolishes the odontoblastic barrier and reparative dentine‐forming capacity, thereby facilitating bacterial penetration (al Natour et al. 2021; Gu et al. 2025).

HDPCs, as major cytokine‐producing and extracellular matrix–maintaining cells, undergo LPS‐induced pyroptosis that correlates with pulpitis severity. Their loss disrupts collagen synthesis, extracellular matrix integrity, and overall tissue architecture (Gu et al. 2025; Wei et al. 2025; DeLeon‐Pennell et al. 2020).

Macrophages further amplify inflammation through robust NLRP3 activation and IL‐1β production. Under sustained inflammasome signalling, M1 macrophages may themselves undergo pyroptotic death, intensifying local inflammatory circuits. Irreversible pulpitis is characterised by M1 predominance, whereas resolution involves a phenotypic shift toward reparative M2 macrophages (Wei et al. 2025; Zhang et al. 2020; Pohl et al. 2024).

DPSCs in perivascular niches provide regenerative capacity; NLRP3‐ and AIM2‐mediated pyroptosis depletes these reserves, impairing odontoblastic differentiation and accelerating progression to irreversible pulpitis, while released cytokines amplify inflammation and induce senescence in neighbouring progenitors (Liu et al. 2025). Conversely, DPSCs secrete immunomodulatory factors that suppress inflammasome activation in fibroblasts, offering endogenous anti‐pyroptotic activity (Zanini et al. 2024). Thus, preservation of DPSC viability or enhancement of their suppressive capacity may represent a regenerative therapeutic strategy.

7.3. Necroptosis: RIPK3/MLKL‐Mediated Programmed Necrosis

Necroptosis is a tightly regulated, caspase‐independent form of programmed cell death orchestrated by the coordinated activation of receptor‐interacting protein kinase 1 (RIPK1), RIPK3, and the executioner pseudokinase mixed‐lineage kinase domain‐like protein (MLKL) (Dhuriya and Sharma 2018). In pulpitis, bacterial‐derived ligands may activate necroptotic signalling through the TLR4–RIPK3 axis, leading to MLKL oligomerisation, plasma membrane permeabilisation, and the subsequent release of DAMPs. These DAMPs act as potent endogenous danger signals that amplify innate immune activation and directly promote inflammasome priming and activation, while necroptosis‐associated mitochondrial dysfunction and excessive ROS generation further reinforce inflammasome signalling (Wang et al. 2025; Dhuriya and Sharma 2018). Together, these processes exacerbate oxidative stress and sustain a pro‐inflammatory pulpal microenvironment, thereby promoting irreversible tissue injury characteristic of irreversible pulpitis.

In contrast to necroptosis, apoptosis preserves plasma membrane integrity and is immunologically silent, with efficient phagocytic clearance of apoptotic bodies and minimal DAMP release (Murphy and Vince 2015; Wang et al. 2025). Apoptosis, largely driven by caspase‐8, acts as a homeostatic mechanism that limits inflammasome activation and curbs excessive inflammation. When caspase‐8 is inhibited, necroptosis becomes dominant, causing MLKL‐dependent membrane rupture and triggering a strong pro‐inflammatory response through the RIPK1–RIPK3–MLKL pathway (Murphy and Vince 2015; Wang et al. 2025).

A tightly regulated cross‐talk exists between apoptosis and necroptosis, with caspase‐8 acting as a central checkpoint. Active caspase‐8 suppresses necroptosis by cleaving RIPK1 and RIPK3, thereby preventing necrosome assembly; conversely, impaired caspase‐8 activity permits RIPK1–RIPK3 complex formation and necroptotic execution (Tummers and Green 2017; Wang et al. 2025). RIPK1 operates as a molecular switch within this axis: under apoptotic conditions, it signals through the FADD–caspase‐8 pathway to promote immunologically silent cell clearance, whereas alterations in RIPK1 ubiquitination favour its interaction with RIPK3, driving MLKL activation, DAMP release, inflammasome amplification, and sustained inflammatory signalling (Feltham and Silke 2017; Wang et al. 2025). Thus, the RIPK1–caspase‐8 checkpoint governs the transition between homeostatic apoptosis and pro‐inflammatory necroptosis, shaping the magnitude and persistence of pulp inflammation.

Mitochondria exert context‐dependent regulatory roles across these cell death pathways. In apoptosis, mitochondrial outer membrane permeabilisation leads to cytochrome c release and orderly caspase activation without inflammasome engagement. In contrast, necroptosis is characterised by mitochondrial dysfunction and excessive ROS production, which promote NLRP3 activation and reinforce inflammatory signalling through positive feedback mechanisms (Deragon et al. 2020; Wang et al. 2025). This divergence defines a functional continuum in pulpal pathology: apoptosis favours controlled resolution, whereas necroptosis amplifies inflammasome‐driven inflammation and accelerates progression from reversible to irreversible pulpitis.

Wang et al. (2025) provide the first comprehensive framework positioning necroptosis as a central pathogenic driver in pulpitis, integrating bacterial infection, immune dysregulation, and oxidative stress into a unified model. The process unfolds through interconnected phases.

Phase 1 (Bacterial initiation): PAMPs such as LPS and peptidoglycan activate TLR4/TLR2 signalling, engaging the TRIF–RIPK3 axis or inducing TNF‐α production, which promotes Tumour Necrosis Factor Receptor 1 (TNFR1)‐mediated necrosome assembly, initiating necroptosis and subsequent DAMP release (e.g., HMGB1, ATP, mtDNA).

Phase 2 (Immune amplification): Released DAMPs activate TLRs on immune cells, driving M1 macrophage polarisation and sustained TNF‐α production. Failure of the M1‐to‐M2 transition perpetuates TNFR1 signalling and reinforces necroptotic activity.

Phase 3 (Oxidative escalation): MLKL‐mediated mitochondrial disruption promotes ROS accumulation, which further activates RIPK3 in a feed‐forward loop. Concurrently, RIPK3‐dependent phosphorylation of PGAM5 induces Drp1‐mediated mitochondrial fission, amplifying oxidative stress and ATP depletion.

Phase 4 (Irreversibility): Excessive necroptosis results in massive DAMP release, odontoblast loss, barrier breakdown, chronic inflammation, and ultimately pulp necrosis.

Importantly, these necroptotic processes are not functionally isolated but are tightly coupled to inflammasome signalling. DAMPs released during necroptosis, including ATP and mtDNA, act as potent activators of NLRP3 and AIM2 inflammasomes, while RIPK3‐driven mitochondrial dysfunction and reactive oxygen species generation further facilitate inflammasome activation. In this context, necroptosis operates as an upstream amplifier that lowers the activation threshold and sustains inflammasome‐driven inflammation (Wang et al. 2025).

Key crosstalk mechanisms functionally integrate necroptosis and inflammasome signalling. RIPK3 directly promotes NLRP3 activation, enabling secondary pyroptosis, while necroptotic release of ATP and mtDNA further stimulates inflammasomes in neighbouring cells (Wang et al. 2025; Wei et al. 2025; Zhang et al. 2021). In parallel, IL‐1β derived from pyroptotic cells enhances TNF‐α production, reinforcing TNFR1‐dependent necroptotic signalling (Wei et al. 2025; Wang et al. 2025).

At the execution level, RIPK3‐phosphorylated MLKL induces odontoblast membrane permeabilisation and mitochondrial fragmentation via the PGAM5–Drp1 axis, amplifying DAMP release and inflammatory propagation (Wang et al. 2025).

Together, these reciprocal interactions establish a self‐reinforcing necroptosis–inflammasome circuit that escalates tissue damage and drives the transition from reversible to irreversible pulpitis, positioning MLKL oligomerisation as a potential therapeutic checkpoint (Table 5).

7.4. PANoptosis: Integrated Activation of Multiple Death Pathways

PANoptosis is a form of regulated cell death in which pyroptosis, apoptosis, and necroptosis are simultaneously activated through shared molecular platforms. This process can be triggered by high bacterial loads or particularly virulent strains (Chi et al. 2021).

Mechanistic in vitro infection studies in RAW264.7 macrophages indicate that clinical Enterococcus faecalis isolates expressing multiple virulence factors (aggregation substance, gelatinase, cytolysin) induce a PANoptotic cell death programme, characterised by concurrent activation of NLRP3/caspase‐1 (pyroptosis), caspase‐3 (apoptosis), and RIPK3/MLKL signalling (necroptosis), with accelerated cell death kinetics compared to individual pathways (Chi et al. 2021). Crosstalk is mediated by RIPK1, which can engage necroptosis (via RIPK3), apoptosis (via caspase‐8), or pyroptosis (via NLRP3), depending on cellular context (Wang et al. 2025; Wei et al. 2025). Caspase‐8 inhibition shifts the balance from apoptosis toward necroptosis and pyroptosis (Wang et al. 2025; Wei et al. 2025).

PANoptosis represents maximal host defence, ensuring pathogen elimination through redundancy—if one death pathway fails, others compensate. However, this comes at the cost of severe tissue damage. In irreversible pulpitis, PANoptosis may constitute a terminal phase, in which simultaneous activation of multiple death pathways drives catastrophic tissue destruction exceeding regenerative capacity (Chi et al. 2021; Wei et al. 2025) (Table 5 and Figure 8).

FIGURE 8.

Integrated crosstalk between inflammasome activation and regulated cell death in pulpal inflammation. Schematic overview of inflammasome pathways and their interaction with pyroptosis, apoptosis, necroptosis, and PANoptosis. Bacterial PAMPs activate TLR2/TLR4 signalling, triggering inflammasome‐driven pyroptosis (GSDMD pore formation) and converging on RIPK1 as the central molecular switch. Caspase‐8 status dictates cell fate: When active, it cleaves RIPK1/RIPK3 promoting immunologically silent apoptosis; when inhibited, it permits necrosome assembly and necroptosis (MLKL‐dependent). Released DAMPs establish an autocatalytic circuit amplifying both pathways. Under high bacterial load, simultaneous engagement of all three programmes constitutes PANoptosis, driving irreversible pulpitis and pulp necrosis.

8. Mitochondrial Dysfunction as Upstream Regulator of Inflammatory Cell Death Pathways

Mitochondrial damage functions as a central upstream driver linking bacterial infection to both necroptosis and pyroptosis (Wang et al. 2025; Zhang et al. 2021). LPS‐induced mitochondrial oxidative stress triggers BAX translocation and membrane permeabilization, releasing mtDNA (Zhang et al. 2021). This mitochondrial disruption activates multiple pathways: (i) mtDNA → NLRP3/AIM2 inflammasomes → pyroptosis; (ii) mitochondrial ROS → RIPK3 activation → necroptosis; (iii) ATP depletion → cellular energy crisis (Wang et al. 2025; Zhang et al. 2021). Collectively, these mitochondrial perturbations act as a unifying upstream signal that amplifies inflammasome activity and integrates multiple inflammatory cell death pathways.

9. Inflammasome‐Driven Microenvironmental Collapse: Vascular, Neural and Metabolic Consequences

9.1. Anatomical Constraint and Vascular Consequences: Oedema, and Ischaemia

The anatomical confinement of the dental pulp within the rigid, mineralised dentine profoundly influences the tissue's response to inflammation (Wang et al. 2024; Jiang et al. 2022). Unlike other tissues where inflammatory processes may remain self‐limiting, this spatial restriction transforms pulp inflammation into a self‐amplifying pathological cascade.

IL‐1β and TNF‐α enhance vascular permeability and immune cell recruitment, resulting in tissue oedema driven by neuropeptide release, thereby triggering the onset of neurogenic inflammation (Dejana et al. 2008; Fahey and Doyle 2019; Caviedes‐Bucheli et al. 2008). Furthermore, nuclear NF‐κB induces transcription of hundreds of inflammatory genes including COX‐2, which converts arachidonic acid to prostaglandin H2, the precursor for PGE2 synthesis by terminal prostaglandin E synthases (Kang et al. 2007; Chang et al. 2024). PGE2 binds E‐prostanoid‐receptors on vascular smooth muscle and endothelium, causing vasodilation and increased vascular permeability that contribute to pulpal hyperaemia and oedema (Bradbury et al. 2005; Petrini et al. 2012). Collectively, these processes amplify the inflammatory response and propagate it along the corono‐apical axis of the pulp, providing a mechanistic basis for the progressive nature of pulpitis.

In unconstrained tissues, oedema dissipates through lymphatic drainage and tissue expansion. However, dental pulp cannot expand due to surrounding dentine walls, causing elevated interstitial pressure that compresses blood vessels, reduces perfusion, and induces hypoxia (Wang et al. 2024; Jiang et al. 2022). This creates positive feedback amplification: inflammation → edema → vascular compression → hypoxia → HIF‐1α stabilisation → enhanced inflammatory gene transcription → amplified inflammation → worsening oedema (Wang et al. 2024; Jiang et al. 2022).

Hypoxia‐inducible factor 1α (HIF‐1α) functions as a master transcriptional regulator responding to reduced oxygen availability (Basheeruddin and Qausain 2024; Wang et al. 2024; Jiang et al. 2022). Under normoxic conditions, prolyl hydroxylases (PHDs) hydroxylate HIF‐1α, targeting it for von Hippel–Lindau (VHL)‐mediated ubiquitination and proteasomal degradation. Hypoxia inhibits PHD activity, resulting in HIF‐1α stabilisation, nuclear translocation, heterodimerisation with HIF‐1β, and activation of hypoxia‐response element (HRE)‐containing genes (Marxsen et al. 2004). In vitro studies in hDPFs cultured under hypoxic conditions (1% O₂, 5% CO₂, 94% N₂), which recapitulate the hypoxic microenvironment of inflamed dental pulp, HIF‐1α accumulates and activates NF‐κB signalling through enhanced IKK activity, increased p65 phosphorylation, and nuclear translocation, thereby inducing transcriptional priming of NLRP3, pro‐IL‐1β, and pro‐IL‐18 (Wang et al. 2024; Jiang et al. 2022).

Functionally, activation of this HIF‐1α–NF‐κB–NLRP3 axis drives the progression from reversible to irreversible pulpitis in hDPFs. Hypoxia markedly induces NLRP3 expression, caspase‐1 activation, and IL‐1β secretion, effects that are abolished by HIF‐1α downregulation, while HIF‐1α overexpression further amplifies NLRP3/CASP1 inflammasome activation (Wang et al. 2024; Jiang et al. 2022). Collectively, these findings establish a causal hypoxia‐driven inflammatory cascade—hypoxia → HIF‐1α → NF‐κB → NLRP3 → inflammasome activation—that provides mechanistic insight into how the hypoxic pulpal microenvironment promotes irreversible inflammatory progression during pulpitis (Wang et al. 2024).

9.2. IL‐1β Signalling and Feed‐Forward Inflammatory Loops

Mature IL‐1β, released via GSDMD pores during pyroptosis, engages interleukin‐1 receptor type 1 (IL‐1R1), which is ubiquitously expressed on pulp fibroblasts, endothelial cells, immune cells, and odontoblasts (Dinarello 2009; Yang et al. 2025; Horst et al. 2011), thereby initiating signalling cascades that amplify and sustain inflammatory responses. Ligand binding to IL‐1R1 recruits the adaptor protein MyD88, which oligomerises and facilitates the recruitment of IRAK kinases (IRAK1, IRAK4), culminating in TRAF6 activation and subsequent TAK1‐dependent activation of both IKK, promoting NF‐κB signalling, and MKK3/6, driving p38 MAPK signalling (Muroi and Tanamoto 2008; Wesche et al. 1997; Kawai and Akira 2007). This IL‐1β–IL‐1R1 axis establishes a paracrine priming mechanism, whereby IL‐1β enhances the transcriptional upregulation of pro‐IL‐1β, NLRP3, and other inflammasome components in neighbouring pulp cells, preparing them for subsequent inflammasome activation (Swanton et al. 2018; Dinarello 2009; Wan et al. 2022), a molecular mechanism that may underlie the clinical observation that inflammasome‐mediated symptomatic pulpitis tends to progress rather than resolve spontaneously.

IL‐1β and TNF‐α induce endothelial upregulation of E‐selectin, ICAM‐1, and VCAM‐1, thereby facilitating leukocyte rolling, adhesion, and transmigration (Chang et al. 2012; Zhang et al. 2002). IL‐1β also stimulates the production of chemokines, including CXCL8 (IL‐8), CCL2 (MCP‐1), and CCL5 (RANTES), establishing chemotactic gradients that recruit neutrophils, monocytes, and T cells from the pulp vasculature into the tissue (Chang et al. 2016; Kritikou et al. 2021; Lin et al. 2018). The ensuing immune cell influx amplifies local inflammation, exacerbating tissue edema and hypoxia within the confined pulp environment and further promoting inflammasome activation and pyroptotic cell death.

IL‐1β also induces the expression of MMPs, including MMP‐1, MMP‐2, MMP‐8, and MMP‐9, which degrade extracellular matrix components such as collagen, proteoglycans, and basement membrane proteins (Kritikou et al. 2021). Concurrently, IL‐1β suppresses the synthesis of critical structural and functional proteins, including alkaline phosphatase and collagen, both of which are essential for extracellular matrix deposition and tissue homeostasis (Kritikou et al. 2021). While limited MMP activity may facilitate immune cell migration and physiological tissue remodelling, excessive activation leads to structural collapse and contributes to pulp necrosis.

9.3. IL‐18 and Th1/Th17 Immune Polarisation

IL‐18, another key substrate of caspase‐1, signals through the IL‐18 receptor α/β heterodimer expressed on T cells, NK cells, macrophages, and non‐immune cells, thereby linking inflammasome activation to adaptive immune responses in chronic pulpal inflammation (Dinarello 2007; Dinarello et al. 2013; al Natour et al. 2023; Wei et al. 2025). Like IL‐1β, IL‐18 is synthesised as an inactive precursor that requires caspase‐1‐mediated cleavage following inflammasome assembly, and its elevated levels have been documented in inflamed dental pulp and periapical tissues (Zhang et al. 2020; Gleeson et al. 2024).

IL‐12 synergizes with IL‐18 and IL‐1β to induce IFN‐γ production from human T cells, promoting Th1 differentiation (Tominaga et al. 2000; Nakanishi 2018). In line with this mechanism, elevated IFN‐γ has been reported in chronically inflamed dental pulp and periapical lesions, reflecting a Th1‐skewed immune microenvironment in advanced pulpal disease (He et al. 2017; Chehimi and Trinchieri 1994; Rissoan et al. 1999). IFN‐γ activates macrophages, enhancing microbicidal activity via reactive nitrogen and oxygen species through inducible nitric oxide synthase (iNOS), while concurrently potentiating inflammasome priming through transcriptional upregulation of NLRP3 and pro‐IL‐1β (Tötemeyer et al. 2006; Xie et al. 2019). Experimental models further demonstrate increased NLRP3 and AIM2 expression, along with elevated IL‐1β and IL‐18, supporting the role of IFN‐γ‐mediated priming in sustaining inflammasome activity within pulp tissue (al Natour et al. 2021; Huang et al. 2018), establishing a feed‐forward loop in which IL‐18‐driven IFN‐γ enhances inflammasome competence (Guarda et al. 2011; Zhang et al. 2021).

In the presence of IL‐23 and TGF‐β, IL‐18 can alternatively promote Th17 differentiation, characterised by IL‐17 production. IL‐23 amplifies Th17 responses by sustaining the effector function of committed Th17 cells and by enhancing IL‐17 transcription in cooperation with IL‐18 (Stritesky et al. 2008). Th17‐associated cytokines, particularly IL‐17, have been consistently detected in inflamed dental pulp and periapical lesions, especially in acute or suppurative conditions (Carlos et al. 2023). IL‐17 induces neutrophil‐recruiting chemokines, including CXCL1, CXCL2, and CXCL8, as well as granulopoietic cytokines such as G‐CSF, driving neutrophilic inflammation characteristic of advanced pulpitis and periapical abscesses (Puerta‐Arias et al. 2020). It also stimulates IL‐6 and IL‐8 production in human dental pulp fibroblasts, further amplifying neutrophil recruitment and tissue injury (Xiong et al. 2015).

Collectively, IL‐18 serves as a central inflammasome‐derived cytokine linking innate immune activation to adaptive immune polarisation in pulpal and periapical tissues. By orchestrating both Th1/IFN‐γ and Th17/IL‐17 responses, IL‐18 sustains a self‐amplifying inflammatory circuit that drives macrophage activation, neutrophil recruitment, and progressive tissue damage in chronic and advanced pulpal pathology.

9.4. Neural Consequences: Nociceptor Sensitisation and Pain

Inflammasome‐mediated inflammation profoundly alters nociceptor function within the dental pulp, providing a robust mechanistic framework for the intense pain characteristic of symptomatic pulpitis. Activation of the NLRP3 inflammasome in both pulp‐resident cells and infiltrating immune populations drives the maturation and release of IL‐1β, a cytokine strongly implicated in nociceptor sensitisation across inflammatory pain states (Jiang et al. 2015; al Natour et al. 2023; Alam et al. 2026). IL‐1β enhances the excitability of Aδ‐ and C‐fibre afferents through coordinated modulation of both transducer and voltage‐gated ion channels, thereby promoting peripheral sensitisation (Starobova et al. 2020; Chantadul et al. 2025). At the mechanistic level, IL‐1β signals via p38 mitogen‐activated protein kinase (MAPK) pathways to relieve resting slow inactivation of tetrodotoxin‐resistant sodium channels and to augment persistent sodium currents near the firing threshold, culminating in sustained neuronal hyperexcitability (Binshtok et al. 2008).

Downstream of inflammasome activation, cyclo‐oxygenase–dependent production of PGE₂ further amplifies inflammatory hyperalgesia by increasing sodium current density and potentiating transducer‐driven depolarisation in peripheral nociceptors (Binshtok et al. 2008; Starobova et al. 2020). Binding of IL‐1β to its cognate receptor, IL‐1R1, on nociceptor terminals initiates intracellular signalling cascades that sensitise transient receptor potential channels, particularly TRPV1, thereby lowering its thermal activation threshold from approximately 43°C to near‐physiological temperatures (33°C–38°C) and allowing normally innocuous thermal stimuli to elicit pain (Ji et al. 2002; Mailhot et al. 2020). In parallel, PGE₂ signals through EP receptors coupled to protein kinase A (PKA) and protein kinase C (PKC), leading to phosphorylation of voltage‐gated sodium channels (Nav1.7, Nav1.8 and Nav1.9), enhanced channel activation, and reduced inactivation, collectively resulting in heightened nociceptor excitability (Ji et al. 2002; Mailhot et al. 2020).

Beyond acute ion channel modulation, inflammatory mediators such as IL‐1β and TNF‐α induce nerve growth factor (NGF) expression in pulp fibroblasts and infiltrating immune cells. NGF binding to tropomyosin receptor kinase A (TrkA) on nociceptors drives transcriptional up‐regulation of neuropeptides, including substance P and calcitonin gene‐related peptide, as well as pro‐nociceptive ion channels such as TRPV1 and Nav1.8, thereby establishing long‐term sensitisation (Woolf and Ma 2007; Chung et al. 2011; Chiu et al. 2012). Substance P and CGRP released from sensitised nociceptors promote vasodilation, plasma extravasation and immune modulation characteristic of neurogenic inflammation, while NGF additionally induces increased innervation density and ectopic nociceptor activity in pulpitis (Caviedes‐Bucheli et al. 2008; Zhan et al. 2021).

In addition to cytokine‐mediated sensitisation, extracellular ATP released from mechanically stressed or necrotic pulp cells functions as both a nociceptive mediator and an inflammasome activator. By engaging P2X3 receptors on nociceptor terminals, ATP generates rapid depolarising currents that initiate acute nociceptive signalling, while concomitantly promoting NLRP3 activation through P2X7‐dependent potassium efflux in immune cells (Toulme et al. 2010; Gombault et al. 2013).

Collectively, these converging mechanisms establish a self‐sustaining neuro‐immune inflammatory loop within the dental pulp, providing a coherent biological explanation for the intensity, persistence, and spontaneous nature of pain in irreversible pulpitis.

10. Endogenous Negative Regulators

10.1. MicroRNA‐22 as Negative Regulator of HIF‐1α/NLRP3

MicroRNA‐22 (miR‐22) has emerged as a critical endogenous regulator of the hypoxia–inflammasome axis (Jiang et al. 2022). Under physiological conditions, high miR‐22 expression in healthy pulp maintains HIF‐1α and NLRP3 at low baseline levels by directly targeting the 3′UTRs of their mRNAs, thereby preventing spontaneous inflammasome activation. Bioinformatic prediction and luciferase reporter assays confirmed this dual post‐transcriptional repression (Jiang et al. 2022).

During pulpitis progression, however, miR‐22 expression progressively declines: whereas normal pulp exhibits robust expression, reversible pulpitis shows a modest reduction, and irreversible pulpitis is characterised by marked loss (Jiang et al. 2022). This decrease correlates inversely with rising HIF‐1α and NLRP3 protein levels, suggesting loss of a key endogenous inhibitory checkpoint.

Concomitantly, hypoxic stress stabilises HIF‐1α, which transcriptionally upregulates NLRP3 expression. Thus, pulp hypoxia generates a dual pro‐inflammasome signal: enhanced transcriptional drive via HIF‐1α and removal of translational repression due to miR‐22 down‐regulation. The convergence of these mechanisms promotes synergistic NLRP3 accumulation and heightened inflammasome competence (Jiang et al. 2022; Tannahill et al. 2013).

Functionally, miR‐22 mimics suppress hypoxia‐induced NLRP3 activation, caspase‐1 cleavage, and IL‐1β secretion by approximately 50%–60%, whereas miR‐22 inhibition amplifies these responses, confirming its role as a critical negative regulator. Therapeutically, restoration of miR‐22 expression may therefore provide dual‐mechanism suppression of the hypoxia‐inflammasome axis by simultaneously targeting HIF‐1α and NLRP3, offering a biologically coherent strategy for modulating inflammasome activity in hypoxic pulpitis (Jiang et al. 2022) (Table 6).

TABLE 6.

Endogenous negative regulators of inflammasome signalling in pulpitis.

Regulator	Molecular target(s)	Mechanism of action	Evidence in pulpitis	Functional impact	Therapeutic implication	References
miR‐22	HIF‐1α, NLRP3 (3′UTR)	Direct binding to 3′UTRs represses translation of HIF‐1α and NLRP3; hypoxia reduces miR‐22 expression, removing translational repression	Progressive decline from healthy pulp → reversible → irreversible pulpitis; inverse correlation with HIF‐1α and NLRP3 protein levels	miR‐22 mimic reduces hypoxia‐induced NLRP3 activation, caspase‐1 cleavage, and IL‐1β secretion (50%–60%); inhibitor enhances inflammasome activation	Dual targeting of HIF‐1α and NLRP3 suggests miR‐22 mimics as strategy for hypoxia‐driven inflammasome suppression	Jiang et al. (2022)
miR‐223	NLRP3 (3′UTR)	Post‐transcriptional repression validated by luciferase assay; specific binding to wild‐type NLRP3 3′UTR	miR‐223 progressively decreases with pulpitis severity; inversely correlated with NLRP3 mRNA; reduced by ATP + LPS in hDPFs	miR‐223 mimic suppresses NLRP3 protein, caspase‐1 (p20), IL‐1β and IL‐18; inhibitor enhances inflammasome activation; rescue experiments confirm regulatory axis	Restoration of miR‐223 may modulate NLRP3‐mediated inflammation in dental pulp	Wang et al. (2021)
Osteomodulin (OMD)	IL1R1/NF‐κB pathway	(1) Transcriptional repression of IL1R1 (~68% reduction under LPS); (2) Direct binding to IL1R1 extracellular domain (hydrogen bonds + hydrophobic contacts), inhibiting NF‐κB activation	High in healthy pulp; markedly reduced in inflamed regions; OMD loss correlates with TNFα‐positive infiltration	OMD overexpression reduces p65 phosphorylation and cytokine production; Omd knockout mice show exacerbated inflammation; IL‐1R antagonist rescues phenotype	Recombinant OMD or IL‐1R targeting strategies may restore immunomodulatory balance in pulp tissue	Yang et al. (2025)

Abbreviations: 3′UTR, three‐prime untranslated region; ATP, adenosine triphosphate; hDPFs, human dental pulp fibroblasts; HIF‐1α, hypoxia‐inducible factor 1 alpha; IL‐18, interleukin‐18; IL‐1R, interleukin‐1 receptor; IL1R1, interleukin‐1 receptor type 1; IL‐1β, interleukin‐1 beta; LPS, lipopolysaccharide; miR‐22, microRNA‐22; miR‐223, microRNA‐223; NF‐κB, nuclear factor kappa B; NLRP3, NOD‐like receptor family pyrin domain‐containing 3; OMD, osteomodulin; p20, cleaved caspase‐1 subunit; p65, NF‐κB RelA subunit; TNF‐α, tumour necrosis factor alpha; ↑, increased expression; ↓, decreased expression.

10.2. MicroRNA‐223: Post‐Transcriptional NLRP3 Suppressor

MicroRNA‐223 (miR‐223) functions as a post‐transcriptional negative regulator of the NLRP3 inflammasome in human dental pulp fibroblasts (Wang et al. 2021; Bauernfeind et al. 2012; Haneklaus et al. 2012). Although miR‐223 is preferentially expressed in haematopoietic and myeloid cells, where it plays established roles in immune regulation, it is also detectable in dental pulp tissue (Bauernfeind et al. 2012; Wang et al. 2021).

Computational prediction identified a conserved miR‐223 binding site within the human NLRP3 3′UTR, a finding experimentally validated by luciferase reporter assays: co‐transfection with miR‐223 mimic significantly reduced luciferase activity in wild‐type NLRP3 constructs, whereas mutation of the binding site abrogated this effect, confirming direct and specific targeting (Wang et al. 2021).

Clinically, miR‐223 expression progressively declines during the transition from healthy pulp to reversible and irreversible pulpitis, inversely correlating with increasing NLRP3 mRNA levels (Wang et al. 2021). In vitro, ATP + LPS stimulation reduces miR‐223 expression in human dental pulp fibroblasts, concomitant with elevated NLRP3 protein, caspase‐1 activation (p20), and increased IL‐1β and IL‐18 secretion (Wang et al. 2021). However, the upstream mechanisms responsible for miR‐223 down‐regulation during pulpitis remain undefined.

Functional studies confirm the regulatory axis: miR‐223 inhibition enhances ATP + LPS‐induced NLRP3 expression and downstream cytokine release, whereas miR‐223 overexpression suppresses inflammasome activation. Rescue experiments further demonstrate causality, as NLRP3 overexpression‐induced cytokine secretion is partially attenuated by miR‐223 mimic, while NLRP3 silencing effects are partially reversed by miR‐223 inhibition (Wang et al. 2021).

Collectively, these findings position miR‐223 as an endogenous brake on NLRP3 inflammasome activation in dental pulp, suggesting that restoration of miR‐223 expression may represent a biologically coherent strategy to modulate pulp inflammatory responses (Table 6).

10.3. Osteomodulin: Extracellular Matrix Protein Suppressing IL1R1/NF‐κB

OMD, a small leucine‐rich proteoglycan, emerged as a novel anti‐inflammatory mediator negatively regulating IL‐1 receptor signalling in dental pulp (Yang et al. 2025). Immunofluorescence revealed OMD is abundantly expressed in healthy pulp but markedly reduced in inflamed areas of pulpitis tissues, particularly in TNF‐α‐positive inflammatory infiltration regions (Yang et al. 2025). This inflammatory downregulation suggests OMD loss contributes to pathological IL‐1β amplification.

Mechanistically, OMD suppresses inflammation through dual mechanisms: (1) Transcriptional repression: OMD overexpression significantly reduces IL1R1 (IL‐1 receptor type 1) expression; specifically, OMD administration decreased IL1R1 protein to approximately 32% of LPS‐stimulated levels (representing ~68% reduction), diminishing cellular sensitivity to IL‐1β (Yang et al. 2025; Weber et al. 2010). RNA‐sequencing confirmed NF‐κB and TNF signalling pathway enrichment among downregulated genes. (2) Direct protein interaction: Co‐immunoprecipitation demonstrated OMD physically interacts with IL1R1 in cells, and molecular docking analysis predicted OMD binding to the IL1R1 extracellular domain through three hydrogen bonds (ARG‐336/TYR‐306, GLU‐241/HIS‐308, HIS‐124/ASP‐268) and multiple hydrophobic contacts (Yang et al. 2025). This interaction inhibits IL1R1‐mediated NF‐κB activation, though the precise molecular mechanism of IL‐1β signalling interference remains to be elucidated.

Functional validation employed genetic manipulation: (i) OMD overexpression in LPS‐stimulated human dental pulp cells significantly reduced p65 phosphorylation at 12 and 24 h and substantially decreased downstream cytokine production (IL‐1β and TNF‐α expression significantly suppressed in a dose‐dependent manner) (Yang et al. 2025). (ii) Conditional Omd knockout mice (OC‐Cre; Omd^flox/flox^) exhibited exacerbated pulpal inflammation following LPS challenge: inflammatory infiltration with localised tissue necrosis and disrupted odontoblast layers, with IL‐1β and TNF‐α immunofluorescence intensity markedly elevated compared to wild‐type controls (Yang et al. 2025). In vitro OMD knockdown increased IL‐1β expression 2.5‐fold and TNF‐α 2.4‐fold under LPS stimulation (Yang et al. 2025). (iii) IL1R antagonist rescue: Treating OMD‐deficient mice with Raleukin (recombinant IL‐1R antagonist) significantly attenuated the excessive inflammatory phenotype, reducing cytokine expression and preserving pulp tissue architecture to levels comparable with recombinant OMD administration (Yang et al. 2025). These experiments establish causal necessity (OMD loss → excessive inflammation) and confirm mechanistic targeting (IL1R blockade compensates for OMD deficiency). The study suggests OMD may serve as a promising target for regenerative endodontics, potentially through recombinant OMD administration to optimise immunomodulatory functions in dental pulp cells (Yang et al. 2025) (Table 6 and Figure 9).

FIGURE 9.

Progressive loss of endogenous negative regulators driving inflammasome hyperactivation in pulpitis. Schematic representation of the miR‐22, miR‐223, and osteomodulin (OMD) regulatory axes under physiological and inflammatory conditions. In healthy pulp, three endogenous regulators maintain inflammasome homeostasis: MiR‐22 post‐transcriptionally suppresses both HIF‐1α and NLRP3 mRNAs via 3'UTR targeting; miR‐223 (haematopoietic origin) specifically targets NLRP3 3'UTR reducing caspase‐1 activation; Osteomodulin (OMD) suppresses IL‐1 signalling through dual mechanisms—physical interaction with IL1R1 extracellular domain and transcriptional repression of IL1R1 expression, blocking NF‐κB activation. During pulpitis progression, all three regulators decline: MiR‐22 and miR‐223 loss permits HIF‐1α/NLRP3 de‐repression creating dual transcriptional and translational amplification; OMD loss removes IL1R1 inhibition, hyperactivating NF‐κB signalling. This convergence drives inflammasome hyperactivation.

10.4. Upstream Mechanisms and Therapeutic Window: From Correlation to Causality

While the three negative regulators demonstrate clear downregulation during pulpitis progression, understanding their upstream regulatory mechanisms and reversibility potential is crucial for therapeutic intervention. Recent experimental evidence has begun to elucidate the molecular pathways governing their decline and the critical therapeutic window for intervention.

10.4.1. miR‐22: Hypoxia‐Driven Transcriptional Repression

The decline of miR‐22 during pulpitis involves a temporally regulated, hypoxia‐dependent mechanism. Experimental evidence demonstrates that miR‐22 expression exhibits biphasic kinetics under hypoxic stress (1% O₂): significant reduction at 6 h followed by partial recovery through 24 h, suggesting initial transcriptional repression with subsequent adaptive responses (Jiang et al. 2022). ATP plus LPS stimulation directly reduces miR‐22 expression in hDPFs, indicating that inflammatory mediators actively suppress its transcription. The mechanistic basis involves a pathological positive feedback loop: hypoxia stabilises HIF‐1α, which may function as a transcriptional repressor of miR‐22, while reduced miR‐22 permits further HIF‐1α accumulation, perpetuating hypoxic signalling and inflammasome priming (Jiang et al. 2022). This creates a self‐reinforcing cycle where initial hypoxic stress progressively diminishes endogenous inflammatory control mechanisms.

10.4.2. miR‐223: Cell‐Type Specific Inflammatory Silencing

miR‐223 downregulation presents a paradox: while ATP plus LPS reduces miR‐223 expression in hDPFs, this contradicts myeloid cell literature where LPS typically induces miR‐223 expression (Wang et al. 2021). This cell‐type specificity suggests distinct transcriptional control mechanisms in hDPFs. The dual stimulation environment (ATP + LPS) likely activates specific transcriptional repressors that override classical inflammatory induction pathways. Potential mechanisms include competition for transcriptional factors, cell‐lineage‐specific epigenetic modifications, or unique signalling crosstalk in the pulp microenvironment (Wang et al. 2021). The progressive decline from healthy pulp through reversible to irreversible pulpitis indicates sustained transcriptional suppression rather than transient inflammatory modulation.

10.4.3. OMD Dual Mechanism of Cell Death and Transcriptional Suppression

OMD decline involves both cellular and molecular mechanisms. Immunofluorescence analysis reveals spatial specificity: OMD expression is selectively lost in TNF‐α‐positive inflammatory infiltration areas while preserved in adjacent healthy regions (Yang et al. 2025). LPS stimulation reduces OMD mRNA and protein expression in a dose‐dependent manner, with significant suppression observed at concentrations ≥ 1 μg/mL. The decline mechanism is dual: (1) Direct cell death: apoptosis and necrosis of OMD‐producing cells (odontoblasts and pulp fibroblasts) in inflamed areas, evidenced by disrupted odontoblast layers in irreversible pulpitis; (2) Active transcriptional repression: pro‐inflammatory cytokines (IL‐1β, TNF‐α) likely repress OMD promoter activity via NF‐κB signalling, as RNA‐sequencing data demonstrate significant enrichment of NF‐κB pathway genes among downregulated targets in OMD‐overexpressing cells (Yang et al. 2025).

11. Future Directions and Research Priorities

Current evidence predominantly provides cross‐sectional snapshots of inflammasome activation in inflamed pulp, yet the temporal sequence of molecular commitment remains insufficiently characterised. Longitudinal mapping of disease progression is required to define the precise transition points between reversible and irreversible pulpitis, including the earliest detectable appearance of cleaved caspase‐1 and the stage at which regulatory mechanisms such as miR‐223 suppression become biologically irreversible (Jiang et al. 2015; Wang et al. 2021; Neha et al. 2025). Identifying these molecular inflection points would clarify therapeutic windows and refine biological staging beyond symptom‐based classifications.

Equally unresolved is the issue of cellular heterogeneity. Single‐cell RNA sequencing across defined stages of pulpal inflammation would enable resolution of cell‐specific contributions to inflammasome activation, clarifying the relative roles of odontoblasts, fibroblasts, discrete immune subsets, dental pulp stem cells, and endothelial populations (Gu et al. 2025; Wei et al. 2025). Such approaches would provide the mechanistic foundation necessary for cell‐targeted interventions rather than indiscriminate inflammatory suppression.

At the pathway level, further molecular dissection is required to elucidate the crosstalk between NLRP3, AIM2, and NLRP6 signalling platforms, the convergence of pyroptosis and necroptosis within PANoptotic frameworks (Yang et al. 2014; Wei et al. 2025; Wang et al. 2025; Chi et al. 2021). Clarifying these intersections may reveal previously unrecognised leverage points capable of interrupting inflammatory self‐amplification.

The mechanisms underlying the suppression of key regulatory molecules—including miR‐223, miR‐22, OMD—remain incompletely understood. Whether their downregulation reflects transcriptional repression, post‐transcriptional modification, or epigenetic remodelling requires systematic investigation (Wang et al. 2021; Jiang et al. 2022; Yang et al. 2025). Understanding these mechanisms would enable rational restoration strategies aimed at reinstating endogenous inflammatory control.

Recent clinical evidence has strengthened the translational relevance of NLRP3 signalling in endodontic disease. A nonrandomised matched comparative study involving 15 patients with symptomatic irreversible pulpitis and 15 healthy controls demonstrated significantly elevated gingival crevicular fluid (GCF) NLRP3 levels in affected teeth (4.33 ± 0.98 ng/mL) compared with controls (1.34 ± 0.45 ng/mL; p < 0.001). Following nonsurgical endodontic therapy, NLRP3 concentrations decreased markedly, with all post‐treatment samples falling below the proposed diagnostic threshold of 3.21 ng/mL, and this reduction correlated with pain resolution (r = 0.686) (Neha et al. 2025). Notably, a significant post‐treatment decrease was also observed in adjacent teeth, suggesting that pulpal inflammation may extend beyond the primary diseased tooth. This phenomenon can be explained by the theory of neurogenic inflammation, whereby collateral C‐fibre innervation facilitates the spread of inflammatory signals through antidromic reflexes, leading to the release of neuropeptides. Consequently, resolution of inflammation in the treated tooth may contribute to a concomitant reduction in inflammatory activity in neighbouring teeth, underscoring the presence of an interconnected pulpal–periapical inflammatory response mediated by inflammasome signalling (Neha et al. 2025). Compared with existing diagnostic methods—such as cold and heat tests or electric pulp testing—GCF‐NLRP3 provides complementary molecular insight into the underlying inflammatory status of the pulp–periapical complex. Accordingly, its most plausible clinical application at present is as a non‐invasive adjunctive biomarker for diagnosis and, particularly, for treatment monitoring, rather than as a standalone screening or prognostic tool. However, the limited sample size and the absence of comprehensive diagnostic accuracy metrics, including sensitivity and specificity, indicate that this approach should currently be regarded as a preliminary proof‐of‐concept practice (Neha et al. 2025). Furthermore, important questions remain regarding longitudinal stability, threshold standardisation, inter‐assay reproducibility, and predictive value across diverse populations. Consequently, large‐scale, prospective, multicentre studies with serial sampling are essential to validate its diagnostic performance and to determine whether GCF‐based inflammasome monitoring can reliably inform biological staging and therapeutic decision‐making in routine clinical practice (Neha et al. 2025).

Complementing these findings, Nawal et al. (2024) provided robust clinical validation for inflammasome‐associated biomarkers by demonstrating that pulpal blood levels of interleukin‐8 (IL‐8) and tumour necrosis factor‐α (TNF‐α) exhibit excellent discriminatory accuracy in differentiating reversible from irreversible pulpitis. In this prospective cross‐sectional study of 72 patients, IL‐8 showed outstanding diagnostic performance (AUC = 0.997; sensitivity 95.5% and specificity 99.76%), while TNF‐α demonstrated good accuracy (AUC = 0.812), with both cytokines remaining undetectable in normal pulp samples. Although IL‐8 and TNF‐α are not direct products of inflammasome activation, their expression is closely linked to the NF‐κB–mediated priming phase required for NLRP3 inflammasome assembly and to the amplification of downstream inflammatory cascades. TNF‐α enhances NLRP3 transcription through positive feedback mechanisms, whereas IL‐8 promotes neutrophil recruitment and the generation of secondary activation signals, such as reactive oxygen species. Collectively, these findings support the concept that molecular inflammatory mediators can serve as biologically plausible surrogate indicators of inflammasome‐driven activity, reinforcing the potential of biomarker‐based strategies to improve diagnostic precision and guide therapeutic decision‐making in vital pulp therapy (Nawal et al. 2024).

Finally, integrating these mechanistic and biomarker‐driven insights into VPT paradigms represents a logical translational progression. Rather than relying solely on symptomatology and intraoperative bleeding characteristics, future VPT protocols may benefit from molecular stratification of pulpal inflammation, enabling biologically informed case selection and timing of intervention. Defining quantitative inflammasome activation thresholds compatible with pulp preservation, and identifying stages at which endogenous regulatory mechanisms remain biologically reversible, could transform VPT from a predominantly empirical procedure into a precision‐guided therapeutic strategy. Such integration would align regenerative objectives with controlled modulation of inflammatory pathways, ultimately refining treatment predictability and long‐term pulpal survival.

12. Conclusions

This comprehensive narrative review establishes inflammasomes as central integrative hubs orchestrating dental pulp inflammatory responses, linking bacterial sensing, cellular stress detection, and regulated cell death execution into coordinated outputs determining disease trajectories. Multiple inflammasome sensors—NLRP3 (responding to bacterial cell walls, ATP, ROS, mtDNA), AIM2 (cytosolic DNA), and NLRP6 (lipoteichoic acid via interferon priming)—provide overlapping surveillance ensuring robust pathogen detection, while NLRP1 and NLRC4 contribute minimally to cariogenic pulpitis due to ligand unavailability.

The molecular transition from reversible to irreversible pulpitis represents crossing activation thresholds: reversible disease exhibits inflammasome priming (NLRP3 transcription, pro‐IL‐1β accumulation) without activation (pro‐caspase‐1 remains uncleaved), whereas irreversible pulpitis demonstrates supra‐threshold activation (abundant cleaved caspase‐1, robust IL‐1β secretion, pyroptotic/necroptotic cell death). This molecular distinction identifies precision intervention targets.

Critical amplification mechanisms perpetuate inflammation beyond initial bacterial insult: mitochondrial DNA leakage creates endogenous DAMP‐driven feed‐forward loops; anatomical constraint induces hypoxia that activates HIF‐1α/NF‐κB pathways, transcriptionally upregulating NLRP3; ATP and mtDNA from dying cells activate neighbouring cells, spatially propagating inflammation; and parallel necroptosis pathways (RIPK3/MLKL) converge with pyroptosis in PANoptotic cell death, ensuring tissue destruction through pathway redundancy.

Critically, endogenous regulatory mechanisms—microRNA‐223 suppressing NLRP3 translation, osteomodulin blocking IL‐1β receptor signalling and DPSC‐derived immunomodulatory factors—normally maintain homeostasis. The progressive loss of these regulatory brakes during disease progression removes negative feedback, permitting uncontrolled inflammasome activation. This regulatory failure, rather than mere bacterial burden, may determine irreversibility.

Therapeutic translation is advancing rapidly across multiple mechanistic levels: direct inflammasome inhibition (MCC950, VX‐765, GSDMD inhibitors), upstream pathway modulation (TLR antagonists, P2X7 inhibitors, NF‐κB inhibitors), regulatory molecule restoration (miR‐223/miR‐22 mimics, recombinant osteomodulin), necroptosis targeting (RIPK3/MLKL inhibitors), mitochondrial protection (antioxidants, mitochondrial transfer), and downstream cytokine neutralisation (anakinra). Stage‐specific intervention strategies—emphasising preservation in reversible pulpitis, controlled inflammation during treatment in irreversible cases, and regenerative support throughout—offer personalised precision approaches.

Collectively, these insights support a conceptual shift from symptom‐based classification toward mechanism‐informed molecular staging. Inflammasomes emerge not merely as inflammatory mediators but as integrative decision platforms that translate diverse danger signals—including microbial PAMPs, endogenous DAMPs, metabolic stress, and hypoxia—into coordinated biological outcomes determining tissue preservation or programmed destruction. Understanding these molecular decision points enables rational therapeutic design targeting specific pathways while preserving beneficial antimicrobial immunity.

13. Limitations

This narrative review has several inherent limitations. First, the synthesis relies on mechanistically heterogeneous experimental models and narrative integration rather than quantitative meta‐analysis, limiting direct comparability and effect estimation. Most evidence derives from in vitro studies or animal models, which may not fully replicate the complex architecture, cellular heterogeneity, and microenvironment of human pulpitis. Longitudinal validation in human disease is limited, and clinical translation of inflammasome‐targeted biomarkers and therapies remains largely unexplored. Mechanistically, critical uncertainties remain regarding temporal dynamics of inflammasome activation, the interplay of canonical and non‐canonical pathways, and the regulation of endogenous inhibitory mechanisms. Finally, this review focused on inflammasome‐mediated pathways and did not comprehensively address other inflammatory, regenerative, or adaptive immune processes that contribute to pulpitis.

Author Contributions

Néstor Ríos‐Osorio: conceptualisation, formal analysis, investigation, resources, methodology, project administration, supervision, validation, data curation, writing, writing – original draft preparation, writing – review and editing. Rafael Fernández‐Grisales: formal analysis, investigation, resources, project administration, supervision, validation, data curation. Tatiana M. Botero: formal analysis, supervision, validation, data curation. Nessrin Taha: formal analysis, supervision, validation, data curation. James L. Gutmann: formal analysis, supervision, validation, data curation. Mario Guerrero‐Torres: formal analysis, validation. Emmanuel J. N. L. Silva: methodology, supervision, validation, data curation. Javier Caviedes‐Bucheli: methodology, project administration, supervision, validation, data curation.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.