Bidirectional crosstalk between TRP channels and Toll-like receptors in inflammation and host defense

Abstract

Integrating physicochemical environmental cues with pathogen detection is essential for an effective and balanced host defense. While Toll-like receptors (TLRs) detect pathogen-associated molecular patterns (PAMPs), transient receptor potential (TRP) ion channels detect specific physicochemical shifts characteristic of inflamed tissues, including extracellular acidosis, hyperosmolarity, oxidative stress, and mechanical perturbations of the lipid bilayer. Emerging evidence indicates that these two sensory systems interact via a bidirectional signaling axis. This review summarizes recent advances in the molecular mechanisms of TRP-TLR crosstalk, focusing on the three principal modes: direct physical coupling, indirect functional coupling via second messengers, and spatial co-organization within membrane microdomains. We examine how TLR signaling regulates TRP channel expression and post-translational modification. Reciprocally, we detail how TRP-mediated ionic fluxes modulate TLR ligand recognition, signal amplification, and effector functions, including phagocytosis and cytokine secretion. Crucially, we illustrate how the dysregulation of this subcellular crosstalk scales up to drive tissue pathologies, including neuropathic pain sensitization, central neuroinflammation, and barrier organ dysfunction. The TRP-TLR axis represents a convergence of immune and sensory signaling. Understanding this interaction provides a basis for identifying novel therapeutic targets for infectious and inflammatory diseases.

Introduction

Innate immunity serves as the first line of defense against microbial pathogens, playing a crucial role in activating adaptive immune responses and facilitating tissue repair [1]. The host defense system faces the challenge of sensing pathogens or tissue damage while simultaneously assessing the fluctuating physicochemical parameters that define the inflammatory milieu, such as local temperature spikes, drops in interstitial pH, alterations in extracellular matrix stiffness, and the accumulation of endogenous electrophiles. This dual sensing capability is necessary to mount an appropriate response [2]. To meet this challenge, cells have evolved a sophisticated network of sensors. Among these, the Toll-like receptor (TLR) family serves as the primary class of pattern recognition receptors (PRRs) that identify conserved PAMPs and damage-associated molecular patterns (DAMPs) [1, 3, 4]. In parallel, the transient receptor potential (TRP) family of ion channels monitors the local microenvironment for physical and chemical changes, including temperature, osmolarity, mechanical stress, and irritants [5, 6]. Functional categorization is not absolute. Certain TRP channels, such as TRPA1, function as TLR-independent sensory modalities that detect microbial products through unique biophysical mechanisms rather than classical ligand-receptor interactions [7]. Collectively, they provide critical input regarding the magnitude of the insult and its spatial localization.

These two sensory systems have conventionally been investigated separately within the disciplines of immunology and neurobiology [8, 9]. However, a growing body of evidence reveals a substantial functional crosstalk between them. Indeed, TRP channels are widely expressed across both immune cells and sensory neurons, and their function is tightly modulated by TLR signaling pathways [10]. Recent studies suggest that the TRP-TLR axis functions as a critical signaling node, allowing the neuro-immune network to integrate information regarding pathogenic threats and the physical environment [11]. This integration ensures the precise, context-dependent regulation of the magnitude, nature, and duration of the host immune response. For instance, the bacterial endotoxin lipopolysaccharide (LPS) acts via TLR4 to synergize with the heat-sensitive ion channel TRPV1 in nociceptors, establishing a feed-forward sensitization loop that exacerbates chronic pain perception [12].

This review summarizes recent advances regarding the molecular mechanisms underlying the interaction between TRP channels and TLRs. We outline key structural features of relevant TRP channel subfamilies and analyze the three principal modes of direct physical coupling, indirect functional coupling via second messengers, and spatial co-organization within membrane microdomains. We further explore the implications of this interplay in regulating microscale cellular activity, host defense, and macroscale pathophysiological processes, including neuropathic pain sensitization, central neuroinflammation, and barrier organ dysfunction. Finally, we propose a conceptual model that frames TRP-TLR crosstalk as a convergence point for immune and sensory signaling. Decoding this interaction not only bridges traditional disciplinary boundaries but also establishes a solid theoretical foundation for developing precision-targeted therapeutics against infectious, inflammatory, and neuropathic diseases.

Structural and functional hallmarks of TRP channels in the immune-sensory axis

Based on sequence homology, the TRP superfamily is categorized into seven subfamilies: TRPV (vanilloid), TRPM (melastatin), TRPC (canonical), TRPA (ankyrin), TRPML (mucolipin), TRPP (polycystin), and TRPN (NO-mechanopotential) [13]. The TRPN subfamily is predominantly found in invertebrates such as Drosophila melanogaster and Caenorhabditis elegans, and is evolutionarily lost in the lineage leading to mammals [14]. While exhaustive overviews of the broad biological functions of TRP channels are available elsewhere [15], this section specifically distills the key structural and functional features of TRP channels that are most pertinent to their interaction with TLR signaling pathways and their roles in the immune-sensory axis.

Across all mammalian subfamilies, TRP channels are tetrameric ion channels built upon a conserved architectural scaffold: each subunit possesses six transmembrane helices (S1-S6) and a pore-forming loop between S5 and S6 [5]. This six-transmembrane (6TM) core, homologous to that of voltage-gated ion channels, forms the fundamental cation-permeable pathway [5]. However, the true functional identity of each TRP subfamily is defined by the large, modular, and often structurally unique domains that extend into the cytoplasm or extracellular space [16]. These domains serve as sophisticated sensory modules and allosteric control centers, coupling specific stimuli to the channel gate [16]. Ultimately, while they share this conserved structural framework, TRP channels exhibit remarkable functional diversity. Their specific activation profiles, distinct neuro-immune distribution, and overall relevance to inflammatory signaling are summarized in Table 1.

Table 1.

Overview of key TRP channel subfamilies and their relevance to TLR crosstalk

Subfamily	Members	Key Structural Features	Primary Functions & Activation	Relevance to TLR Crosstalk	Reference
TRPC	TRPC3, 6, 7	Lateral fenestrations in the pore domain for direct DAG binding (e.g., via S6 helix)	Non-selective cation channels; Activated directly by DAG downstream of PLC-coupled receptors	Indirect Coupling (PLC-DAG)	[26, 27]
	TRPC1, 4, 5	Conserved 6TM architecture	Activated by store depletion or PLC pathways (DAG-independent)	Indirect Coupling	[44, 45]
TRPV	TRPV1-4	N-terminal ARD; Extensive C-terminal region	Polymodal sensors (temperature, pH, osmolarity, mechanosensation); heat/capsaicin receptor (TRPV1)	Direct Coupling (TIR-cytoplasmic region) / Downstream Amplification	[12, 18]
	TRPV5-6	Conserved aspartate ring in the pore	Highly Ca²⁺-selective channels; Critical for epithelial Ca²⁺ reabsorption	Generally not linked to direct TLR crosstalk	[46, 47]
TRPA	TRPA1	Extensive N-terminal ARD (~ 14–16 repeats); C-terminal coiled-coil	Sensor for noxious chemicals, ROS, and inflammatory mediators	TLR4-independent sensing (via LPS-induced membrane perturbation)	[20, 25]
TRPM	TRPM2	Unique C-terminal Nudix box motif (Chanzyme)	Sensor for ADPR and oxidative stress (ROS)	Downstream Amplification	[48, 49]
	TRPM4, 5	N-terminal TRPM homology regions (MHRs)	Ca²⁺-activated monovalent cation-selective channels	Downstream Amplification	[33, 50]
	TRPM6, 7	C-terminal α-kinase domain (Chanzymes)	Regulate cellular Mg2+ and Ca2+ homeostasis	Indirect Coupling (TLR4 endocytosis)	[51, 52]
	TRPM8	N-terminal TRPM homology regions (MHRs)	Sensor for cool temperatures and cooling agents	(Not primary in TLR axis)	[35, 53]
TRPML	TRPML1-3	Large luminal/extracellular loop between S1 and S2	Regulate organellar ion homeostasis; Critical for vesicular trafficking across endolysosomes and ER	Spatial co-organization (endosome)	[39, 40]
TRPP	TRPP2, 3, 5	Large extracellular polycystin domain	Ciliary mechanosensors; form complexes with Polycystin-1 (PKD1)	Potential for indirect modulation via Ca2+ /mechanosensitive pathways	[41, 42]

TRPC2 is excluded from this table because it is a pseudogene in humans and does not encode a functional channel

N-Terminal ARDs as scaffolds for nociception and inflammation

The TRPV, TRPA, and TRPC subfamilies are characterized by N-terminal Ankyrin Repeat Domains (ARDs), which are versatile protein-protein interaction motifs that act as scaffolds for channel assembly and signal detection [15]. In the canonical pain and heat sensor TRPV1, the integration of diverse stimuli relies on the coordinated function of the entire channel architecture. For instance, the activation of TRPV1 by capsaicin and protons requires specific sites located within the transmembrane segments and extracellular loops, respectively [17], while the precise structural requirements for heat activation remain incompletely understood. Rather than serving as the primary binding interface for these direct agonists, the N-terminal ARD functions as an essential intracellular signaling scaffold. Specifically, it serves as a dynamic, competitive binding hub for calmodulin and ATP to tightly regulate channel desensitization (tachyphylaxis) during repeated stimulation [18]. In parallel, interactions with protein kinases are primarily scaffolded by the C-terminal domain (e.g., via AKAP79/150), highlighting a spatially segregated regulatory network [19].

This structural feature is even more pronounced in TRPA1, which possesses one of the most extensive ARDs in the TRP family, comprising approximately 16 ankyrin repeats. Cryo-electron microscopy (Cryo-EM) structures indicate that these repeats, together with a C-terminal coiled-coil assembly hub stabilized by inositol hexakisphosphate (IP₆), form an allosteric hub [20]. This architecture strategically positions key cysteine and lysine residues for covalent modification by endogenous and exogenous electrophiles generated during oxidative stress and inflammation, including 4-hydroxynonenal (4-HNE) and reactive aldehydes [21, 22]. While earlier hypotheses proposing TRPA1 as a primary sensor for noxious cold remain highly contested [23, 24], there is now broad consensus regarding its canonical function as a broadly tuned chemo-sensor. Critically, this extensive cytosolic scaffold allows TRPA1 to integrate both direct covalent damage and indirect downstream signals from PLC-coupled inflammatory receptors, translating the complex inflammatory milieu into unified nociceptive outputs [25].

Direct gating by lipid messengers: the TRPC subfamily

Within the canonical TRPC subfamily, members TRPC3, TRPC6, and TRPC7 serve as direct effectors for lipid second messengers. Unlike other calcium-permeable channels, these specific members are uniquely gated by diacylglycerol (DAG) in a tightly membrane-delimited, protein kinase C (PKC)-independent manner [26, 27]. While classic electrophysiological models primarily characterized this DAG-dependent gating at the plasma membrane, recent advances have expanded this sensory mechanism into the organellar interior. Crucially, during TLR4-driven macrophage activation, TRPC3 is directly gated by highly localized, intracellular DAG pools generated by the phosphatidic acid phosphatase lipin-1 at the endoplasmic reticulum (ER) [28]. This intracellular lipid-sensing capability positions TRPC3 as a pivotal component for translating innate immune signals into rapid organellar Ca²⁺ mobilization.

In stark contrast, the structurally divergent TRPC1, TRPC4, and TRPC5 subunits are entirely unresponsive to DAG. Instead, these members function predominantly as store-operated channels (SOCs), activating in response to the depletion of intracellular calcium stores downstream of IP₃-mediated Ca²⁺ release [5, 15, 26].

Chanzymes and organellar channels: TRPM and TRPML subfamilies

The TRPM and TRPML subfamilies demonstrate further functional diversification, with members acting as combined channel-enzymes (chanzymes) or as gatekeepers of intracellular organelles [15]. The TRPM family is functionally diverse, with TRPM2 being of particular relevance to immunity [29]. As a sensor for oxidative stress, its structure is unique among ion channels, featuring a C-terminal Nudix box motif homologous to ADP-ribose (ADPR) pyrophosphatases [30]. TRPM2 is gated by intracellular ADPR and reactive oxygen species (ROS), allowing this chanzyme to directly link the metabolic and oxidative state of an immune cell to Ca²⁺ influx, which in turn amplifies inflammatory responses like the production of the chemoattractant CXCL2 [31]. Beyond TRPM2, the TRPM subfamily exhibits remarkable structural and functional diversity. TRPM6 and TRPM7 also function as unique chanzymes, possessing an atypical C-terminal α-kinase domain critical for cellular Mg²⁺ and Ca²⁺ homeostasis [15, 32]. TRPM4 and TRPM5 are highly unique within the entire TRP superfamily as they are monovalent cation-selective channels that are impermeable to Ca²⁺, functioning primarily to regulate membrane potential following intracellular Ca²⁺ elevation [33, 34]. Finally, TRPM8 is the classic cold sensor and menthol receptor [35, 36]. In contrast, TRPML channels function as gatekeepers primarily localized to the membranes of late endosomes and lysosomes [37]. Their activation is tightly controlled by the organelle-specific lipid, phosphatidylinositol-3,5-bisphosphate (PI(3,5)P₂) [38]. Cryo-EM structures revealed a unique cytosolic Mucolipin Domain (MLD) that functions as a gating pulley mechanism, wherein lipid binding is mechanically transmitted to pull the S6 gate open [39]. This precise, localized gating mechanism is critical for regulating organellar ion homeostasis, and may provide opportunities for regulatory crosstalk with immune receptors such as endosomal TLRs that reside in the same compartment [40].

Group 2 channels with large extracellular domains: the TRPP subfamily

TRPP channels, along with TRPMLs, belong to a distinct TRP channel group characterized by a large extracellular/luminal loop between transmembrane helices S1 and S2 [41]. The cryo-EM structure of TRPP2 (also known as PKD2) revealed this region to be a large, structured polycystin domain. This domain is a hotspot for pathogenic mutations causing Autosomal Dominant Polycystic Kidney Disease (ADPKD) and is critical for proper channel assembly through extensive inter-subunit interactions [42]. Positioned above the transmembrane core, it makes strategic contacts with both the voltage-sensor-like domain (VSD) and the outer pore, suggesting it functions as a receptor domain for extracellular stimuli. Unlike most TRP channels, the VSD of TRPP2 contains two positively charged residues in its S4 helix, hinting at a potential role for voltage in its regulation [43].

Collectively, the TRP superfamily achieves its functional diversity through remarkable structural innovations built upon a common channel scaffold. From the intricate intracellular allosteric hubs of TRPA1 to the organelle-specific lipid-sensing pulleys of TRPMLs and the large extracellular polycystin domains of TRPPs, these architectural adaptations equip different TRP channels to serve as highly specialized detectors of the diverse signals that characterize the immune microenvironment. It is this structural versatility that positions them as ideal partners for the TLR system in integrating pathogen-derived threat signals with physicochemical environmental context (Table 1).

Core mechanisms of TRP-TLR crosstalk

Modes of interaction

The interplay between TRP channels and TLRs is a coordinated process, far more intricate than random protein encounters. The mechanisms governing this crosstalk can be categorized into three principal modes of interaction: direct physical coupling, indirect functional coupling via shared signaling pathways, and spatial co-organization within specialized membrane microdomains [12, 54] (Fig. 1). For the sake of conceptual clarity, the mechanisms of interaction are delineated into three principal pillars; however, in physiological settings they are frequently interdependent and function synergistically.

Fig. 1.

Three core molecular modes of TRP-TLR crosstalk. A Direct physical coupling. In sensory neurons, the intracellular TIR domain of TLR4 physically interacts with the cytoplasmic region of TRPV1. This direct structural association anchors TRPV1 at the plasma membrane, effectively inhibiting agonist-induced channel endocytosis and subsequent lysosomal degradation. B Indirect functional coupling. Macrophages coordinate TLR4 downstream cascades via distinct TRP channel-mediated Ca²⁺ fluxes. At the endoplasmic reticulum (ER), lipin-1-derived DAG activates intracellular TRPC3 to release ER Ca²⁺ stores, driving the nuclear translocation of nuclear factor-kappa B (NF-κB) and the transcription of pro-inflammatory cytokines. In a parallel plasma membrane pathway, TRPM7-mediated extracellular Ca²⁺ influx controls the dynamin-dependent endocytosis of the TLR4-CD14 complex. This internalization shuttles TLR4 into early endosomes, initiating the TRIF-dependent signaling branch to trigger IRF3 activation. C Spatial co-organization. Cholesterol-rich lipid rafts function as dynamic signaling scaffolds that compartmentalize TLR4 and TRP channels (e.g., TRPV1). This membrane microdomain sequestration enforces the spatial co-localization of both receptor systems, maximizing the biochemical probability and fidelity of their physical interaction and downstream signal integration. Created in BioRender. You, X. (2026) https://BioRender.com/b1ambfx

Direct physical coupling

The most direct form of crosstalk involves physical binding between proteins, as exemplified by the interaction between TRPV1 and TLR4. Evidence from co-immunoprecipitation assays in heterologous expression systems and confocal microscopy has confirmed that these two receptors physically associate and colocalize within cells [12]. This interaction is mediated by the direct binding of the Toll/interleukin-1 receptor (TIR) domain of TLR4 to TRPV1 [12]. However, the specific structural motifs within the cytoplasmic region of TRPV1 responsible for this physical coupling remain to be fully elucidated. The involvement of the TIR domain is particularly notable, given that it conventionally serves as the core signaling hub for all TLRs to recruit downstream adaptors and initiate inflammatory cascades. Such direct physical association yields marked functional consequences. Specifically, this structural coupling prevents the agonist-induced endocytosis of TRPV1 and its subsequent lysosomal degradation, thereby abolishing a key mechanism of receptor desensitization [55]. In sensory neurons lacking TLR4, TRPV1 is cleared more rapidly from the cell membrane following activation. By anchoring TRPV1 at the membrane, TLR4 effectively blocks this desensitization process, leading to a potentiated and prolonged response to agonists [12]. The macroscopic pathophysiological consequences of this receptor sensitization, particularly how it translates into sustained nociceptive signaling at the systemic level, will be systematically detailed in Sect. 4.1.

Indirect functional coupling via shared signaling pathways

Beyond direct physical binding, TRP channels frequently function as downstream effectors for second messenger cascades initiated by TLRs. This indirect functional coupling relies on precise, compartmentalized signaling relays. A prominent example is the functional coupling between TLR4 and TRPC3, which operates via highly localized lipid metabolism at the organellar level [28]. Upon activation by LPS, the plasma membrane TLR4 complex recruits adaptor proteins that sequentially activate phospholipase D (PLD), leading to the generation of phosphatidic acid (PA) at the local ER membrane. Subsequently, the ER-resident enzyme lipin-1 dephosphorylates this PA to produce a highly concentrated, localized pool of DAG. This specific ER-anchored DAG pool serves as a direct gating ligand that opens TRPC3 channels, which are colocalized on the ER membrane. Activated TRPC3 then mediates the rapid release of Ca²⁺ from the ER lumen into the cytosol. Ultimately, this TRPC3-driven ER Ca²⁺ mobilization triggers the nuclear translocation of NF-κB and the transcription of pro-inflammatory cytokines [28].

In parallel to these ER events, full TLR4 signal transduction necessitates receptor endocytosis, a process tightly regulated by plasma membrane ion fluxes. Specifically, LPS-triggered Ca²⁺ influx mediated by the plasma membrane TRPM7 channel is a prerequisite for the internalization of the TLR4-CD14 signalosome. This TRPM7-dependent endocytosis physically shuttles the TLR4 complex from the plasma membrane into early endosomes, thereby initiating the TRIF-dependent signaling branch and the subsequent activation of the transcription factor IRF3 [52].

Beyond acute ion influx, the compartmentalized gating of organellar TRP channels dictates the baseline operational threshold of PRRs. A classic example is established by TRPP2 (Polycystin-2), which localizes to the primary cilia and ER membranes where it functions as a critical calcium-leak channel to maintain tonic intracellular Ca²⁺ balance. Under homeostatic conditions, this constitutive TRPP2-mediated Ca²⁺ signaling acts as a biochemical brake that constrains pro-inflammatory cascades. The genetic or functional loss of TRPP2 disrupts this tonic Ca²⁺ signaling and depletes organellar Ca²⁺ stores [56]. In the polycystin-deficient renal epithelium, this loss of Ca²⁺ homeostasis is accompanied by cell-autonomous pro-inflammatory transcriptional activation and upregulation of epithelial TLR4, licensing a hyper-inflammatory state that sensitizes the barrier to subsequent innate immune challenges [57, 58].

Spatial co-organization in membrane microdomains

The spatial co-organization of TRP channels and TLRs within specialized membrane microdomains further optimizes the efficiency and fidelity of their crosstalk. Lipid rafts—dynamic, nanoscale assemblies enriched in cholesterol, sphingolipids, and saturated phospholipids—serve as liquid-ordered platforms that laterally segregate and concentrate specific signaling molecules within the plane of the plasma membrane [41]. The macromolecular assembly of the TLR4 signaling complex is highly dependent on these domains; ligand molecules are initially presented to the TLR4/MD-2 complex by the glycosylphosphatidylinositol (GPI)-anchored protein CD14, which preferentially partitions into cholesterol-rich rafts. Consequently, these microdomains function as the primary organizing hubs for initial signalosome assembly [59, 60]. In addition, numerous TRP channels, including TRPV1 and TRPA1, partition into lipid rafts due to specific lipid-protein interactions [61, 62]. By segregating into the same restricted physical landscape, TLRs and TRP channels undergo a dramatic increase in their local concentration, which substantially enhances the probability of their reciprocal molecular encounters and functional integration without requiring constitutive physical coupling [60]. Biophysical perturbations that expand raft domains promote the lateral clustering of TLR4 and TRP channels, whereas the targeted biochemical disruption of lipid raft integrity uncouples these two sensory networks [63].

To translate this lateral lipid segregation into stable signaling microdomains, structural scaffolding proteins actively shape the three-dimensional geometry of the plasma membrane (Table 2). A prominent example of this structural enforcement is provided by Caveolin-1 (Cav-1), the primary architectural component that invaginates lipid rafts to form flask-shaped caveolae. Through its conserved caveolin scaffolding domain (CSD), Cav-1 physically tethers the TLR4 complex along with multiple TRP channels, including TRPC1 and TRPV4, within these membrane pockets [64–66]. This precise spatial sequestration restricts the lateral diffusion of intermediate signaling components away from the receptor complex. By positioning upstream PRRs and downstream calcium-permeable channels in immediate nanometer-scale proximity, the Cav-1 scaffold effectively minimizes signaling latency, establishing a highly efficient nanodomain that lowers the overall activation threshold for the innate immune response.

Table 2.

Molecular scaffolds organizing TRP and TLR components within distinct subcellular compartments

Scaffolding Protein	TRP Channel Interaction	TLR Pathway Connection	Physiological Context	Functional Outcome	Reference
Caveolin-1	Binds TRPC1, TRPV4 via CSD	Essential for TLR4 endocytosis; binds MyD88 pathway components	Endothelial permeability; Sepsis	Clusters signaling components; regulates signal duration	[64, 65]
PACSIN3	Binds TRPV4 N-terminus (PRD)	Modulated by PKC/PLD downstream of TLR4	Renal tubule function; Macrophage phagocytosis	Regulates subcellular trafficking and mechanosensitivity	[67, 68]
AKAP79/150	Tethers PKA/PKC to TRPV1	Recruits kinases activated by TLR signaling	Nociceptor sensitization	Facilitates rapid phosphorylation and channel sensitization	[19, 69]
Homer	Binds TRPC channels	Links to IP3R and Ca2+ stores	Neuronal Ca2+ signaling	Couples membrane channels to ER Ca2+ stores	[44, 70]

In addition to caveolar platforms, a distinct repertoire of intracellular adaptors and multi-protein scaffolds coordinate the spatial arrangement of the TRP-TLR signalosome across different subcellular compartments. For instance, the adaptor protein PACSIN3 binds to the N-terminal proline-rich domain of TRPV4, specifically dictating its mechanosensitivity and subcellular trafficking in response to distinct physiological and environmental stimuli [67, 68]. Similarly, A-kinase anchoring protein 79/150 (AKAP79/150) functions as a crucial molecular scaffold that physically tethers protein kinase A (PKA) and PKC directly to the C-terminal domain of TRPV1. This structural clustering ensures that kinases activated downstream of TLR cascades are positioned adjacent to their channel substrates, facilitating rapid post-translational phosphorylation and subsequent channel sensitization [19, 69]. In addition, at the interface between the plasma membrane and intracellular organelles, scaffolding proteins such as Homer couple membrane-resident TRPC channels to ER-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs). This structural alignment facilitates immediate store-operated calcium entry in response to IP₃ generation downstream of TLR pathways [44, 70]. Collectively, these molecular scaffolds transition TRP-TLR crosstalk from stochastic membrane encounters into tightly regulated, three-dimensional architectural networks that tightly control the magnitude and kinetics of cellular responses.

Parallel sensing via membrane biophysics (TLR-independent mechanism)

Complementing protein-mediated crosstalk, TRPA1 detects bacterial LPS through a parallel, TLR4-independent biophysical route. Rather than recognizing LPS via a stereoselective TLR4/MD-2 interface, TRPA1 senses the mechanical perturbation that lipid A imposes on the bilayer by inserting asymmetrically into the outer leaflet, thereby altering local membrane curvature and lateral tension [7, 71]. Under the force-from-lipids model, this perturbation is thought to be transduced by the channel’s exceptionally large N-terminal ARD, which may act as an elastic anchor that converts changes in membrane order into gating [25, 72].

Consistent with a mechanical mechanism, amphipaths that alter curvature (e.g., trinitrophenol) mimic or potentiate the response, whereas polymyxin B, which sequesters lipid A, abolishes it [7]. Notably, this modality operates in invertebrates lacking TLR4 (e.g., Drosophila), suggesting an ancestral, biophysically driven arm of innate defense [73, 74].

Critical insights and methodological caveats in receptor crosstalk

While the delineation of direct, indirect, and spatial crosstalk modalities provides a rigorous conceptual framework, several critical mechanistic ambiguities and methodological caveats must be addressed to define the current boundaries of the field.

First, a primary methodological limitation is that much of the definitive evidence supporting direct physical coupling (Sect. 3.1.1), such as the putative physical interaction between the TLR4 TIR domain and the cytoplasmic region of TRPV1, relies predominantly on heterologous over-expression systems and in vitro biochemical assays like co-immunoprecipitation [12]. These static, endpoint-based techniques inherently disrupt the native cellular architecture. Consequently, the real-time assembly, stability, and dissociation kinetics of the endogenous TRP-TLR signalosome under native pathophysiological conditions remain largely uncharacterized. Whether these direct physical complexes exist as constitutive macromolecular entities or function as transient, stimulus-evoked structures remains a fundamental unresolved question.

Second, the precise biophysical boundary separating lipid-enforced lateral co-localization (Sect. 3.1.3) from absolute macromolecular physical coupling remains poorly defined. Within the restricted nanoscale geometry of caveolae or lipid rafts, proteins are constrained in such extreme proximity that distinguishing between stochastic, lipid-driven collision and true allosteric structural coupling poses a formidable experimental challenge [60]. It remains to be determined whether membrane microdomains merely concentrate these sensory proteins to kinetically optimize encounter probability, or actively serve as structural templates that induce mandatory conformational changes necessary for channel gating.

To resolve these fundamental knowledge gaps, the field must complement bulk biochemical analyses with live-cell, native-state single-molecule investigations. The deployment of advanced biophysical modalities—such as single-molecule Fluorescence Resonance Energy Transfer (smFRET) and ultra-high-resolution imaging (e.g., photoactivated localization microscopy, PALM)—will be vital to definitively map the nanoscale topology and real-time operational dynamics of the TRP-TLR signaling axis [75, 76].

Regulation of TRP channel function by TLRs

The functional interaction between TRP channels and TLRs is tightly bidirectional. While TRP channels modulate downstream TLR signaling cascades, the activation of TLRs by PAMPs or DAMPs reciprocally controls TRP channel expression, subcellular trafficking, and gating biophysics. To achieve this precise regulatory control, the innate immune system employs a dual strategy consisting of long-term, sustained adaptations via genomic transcriptional remodeling, and rapid, short-term adjustments through post-translational modifications [12, 77].

Transcriptional remodeling of the TRP channel repertoire

One of the most notable modalities by which TLR signaling reorganizes cellular sensory capacity is through the transcriptional reshaping of the TRP channel expression landscape. Such genomic remodeling allows immune cells to dynamically transition from a baseline, resting state into a highly specialized effector phenotype tailored to environmental demands. Such transcriptional control exhibits high gene-specific selectivity, targeting distinct TRP channel subfamilies to coordinate discrete steps of the innate immune response.

Selective upregulation of TRPML2 in stimulated effector cells

The endolysosomal mucolipin channel TRPML2 represents a prominent example of a TLR-inducible ion channel that functions as a crucial component of the macrophage effector program. In various resting innate immune cell populations, including RAW 264.7 cells, primary bone marrow-derived macrophages (BMDMs), alveolar macrophages, and microglia—baseline Trpml2 mRNA and protein expressions are virtually undetectable [78]. However, upon encountering a diverse spectrum of classical TLR ligands, its expression is rapidly induced. Activation of TLR4 via LPS, TLR7/8 via R848, or TLR2 via Zymosan A all trigger marked increases in Trpml2 transcription [78]. The temporal kinetics of this transcriptional induction are cell-type specific, with expression levels peaking at approximately 6 h post-stimulation in primary BMDMs and alveolar macrophages, compared to 24 h in the RAW 264.7 cell line [78].

Notably, this transcriptional response is highly restricted to the TRPML2 subtype. Under identical TLR stimulation profiles, the mRNA levels of the other two mucolipin members, TRPML1 and TRPML3, remain entirely unaltered [78]. This divergence is governed by distinct upstream transcriptional regulatory mechanisms; for instance, TRPML1 expression is decoupled from acute inflammatory signals and is instead governed by metabolic stress-responsive transcription factors, such as transcription factor EB (TFEB) and TFE3, during homeostatic lysosomal biogenesis and autophagy [78, 79]. This differential regulation underscores the functional distinction between homeostatic organellar maintenance and acute inflammatory responsiveness.

The newly synthesized TRPML2 protein specifically localizes to perinuclear recycling endosomes, where it tightly colocalizes with the transferrin receptor (TfR) [78]. From this specialized subcellular compartment, TRPML2-mediated local calcium signals are indispensable for the intracellular trafficking and subsequent exocytosis of the critical chemokine CCL2. In the absence of functional TRPML2, newly synthesized CCL2 accumulates intracellularly within the endosomal trafficking pathway and its vesicular secretion is markedly diminished [80]. The macroscopic downstream consequences of this cellular trafficking defect on systemic immune cell recruitment and tissue-level infiltration in vivo are further explored in Sect. 4.3.

TLR-mediated regulation of TRPM2 expression

The melastatin-related channel TRPM2 is another essential sensory element whose genomic expression is selectively upregulated downstream of TLR cascades, providing an additional layer of regulatory control over global cytosolic calcium homeostasis. Upon TLR4 activation, downstream transcriptional networks upregulate TRPM2 abundance [81], which in turn shapes intracellular calcium mobilization, NADPH oxidase dynamics, and ROS production [82, 83].

Such synchronous yet compartmentalized co-regulation of TRPML2 and TRPM2 by TLR pathways reveals a highly integrated, multi-layered calcium signaling architecture. Rather than functioning redundantly, these two channels provide the activated cell with a multi-compartmental calcium signaling apparatus optimized for distinct timelines. Transcriptionally induced TRPML2 provides highly restricted, localized calcium pulses from endosomal lumen stores, which are spatially positioned to power the intracellular trafficking and secretion of chemokines such as CCL2 [80]. In parallel, the upregulation of TRPM2 contributes to broader, macroscale cytosolic calcium signals that are integrated with the cell’s oxidative state via its unique enzymatic sensitivity to ADPR, thereby fueling metabolic adaptations and global cytokine transcription [48, 84]. Ultimately, such dual-channel genomic remodeling illustrates that TLR activation does not merely upregulate isolated genes, but drives the de novo synthesis of an integrated physical signaling platform.

Constitutive and inducible TRP channels in macrophages

Unlike the dynamic, stimulus-dependent inducibility of TRPML2 and TRPM2, other TRP channels vital for cellular homeostasis are constitutively expressed, reflecting an alternative regulatory strategy. For instance, TRPV4, which participates in essential cellular functions such as phagocytosis and mechanosensitive matrix surveillance, shows invariant total protein expression profiles following LPS stimulation. Immunoblot analysis confirms that baseline TRPV4 protein levels remain completely unaltered post-TLR engagement across multiple macrophage populations [85].

This mechanistic divergence highlights two complementary strategies within the innate immune network. For channels like TRPV4, which must continuously survey physical parameters such as extracellular matrix compliance, constitutive expression ensures that the cell remains in a basal sensory-ready state to respond instantly to mechanical shifts in its surroundings [86]. Conversely, for channels like TRPML2 and TRPM2, which execute highly specialized, stimulus-evoked immunological effector programs, an inducible expression strategy is employed to conserve metabolic resources [80, 81]. This stimulus-dependent synthesis ensures that the components for escalating and amplifying systemic inflammatory cascades are deployed only after a pathogenic threat has been definitively recognized by upstream TLRs.

Dynamic post-translational control of channel activity and trafficking

Beyond altering gene expression profiles, TLR signaling pathways dynamically modulate the functional state of pre-existing TRP channels through sophisticated post-translational mechanisms. These rapid, real-time adjustments, encompassing direct physical associations, altered subcellular trafficking, and second-messenger-mediated gating modifications, allow for immediate and precise control over channel activity. Notably, such post-translational architecture enables upstream PRRs to drive opposing regulatory programs, ranging from signal amplification to signal termination, depending entirely on the specific channel partner and cellular context.

Membrane stabilization via direct TLR4-TRPV1 interaction

As detailed in Sect. 3.1.1, direct TLR4-TRPV1 coupling acts post-translationally to anchor TRPV1 at the plasma membrane and prevent desensitization, establishing a feed-forward sensitization loop whose systemic consequences are discussed in Sect. 4.1 [12, 55].

Signal termination via the indirect TLR4-PAR2-TRPV4 axis

In stark contrast to the sensitizing feed-forward loop observed with TRPV1, TLR4 engagement can also initiate an indirect, multi-step signaling cascade that suppresses TRP channel activity, serving as a critical negative feedback loop required for signal termination. This suppressive axis is initiated upon TLR4 activation, which triggers the localized release of the protease thrombin. Thrombin subsequently engages and cleaves the protease-activated receptor 2 (PAR2), mobilizing a Gαs-coupled signaling cascade that strongly activates adenylate cyclase to elevate intracellular cyclic AMP (cAMP) concentrations [87]. This localized rise in cAMP acts as a powerful inhibitory signal that blocks Ca²⁺ influx through the constitutively expressed TRPV4 channels [87]. Mechanistically, baseline TRPV4-mediated Ca²⁺ entry is vital for the sustained activation of the calcium/calcineurin-dependent transcription factor, Nuclear Factor of Activated T-cells (NFAT) [87]. In turn, activated NFAT is required to cooperate synergistically with NF-κB to drive long-lasting inflammatory gene expression [88]. By arresting TRPV4-mediated Ca²⁺ influx, the cAMP-dependent signal prevents NFAT activation and impairs its transcriptional cooperativity with NF-κB at specific target promoters, thereby effectively terminating the production of downstream pro-inflammatory cytokines [89]. The tissue-level and organ-system significance of this suppressive feedback loop in promoting the resolution of acute inflammation is further explored in Sect. 4.3.

TRP channels as critical modulators of TLR signaling

TRP channels function as intracellular signal integration hubs rather than passive ionic conduits. These channels interpret the physical and biochemical microenvironment by sensing danger-associated signals, such as intracellular oxidative stress or localized changes in membrane mechanics, and subsequently integrating this contextual information with the pathogen-derived signals received by TLRs [10]. Such molecular integration allows the individual cell to mount a context-dependent transcriptional and secretory response, rather than a simple binary reaction to a pathogen. Consequently, their modulatory influence spans the entire intracellular cascade of the innate immune response, from tightly governing initial ligand accessibility to amplifying downstream kinase signaling and precisely shaping the ultimate secretory and trafficking functions of the cell.

Regulation of ligand recognition and sensitivity

TRP channels are pivotal in setting the activation threshold of pattern recognition cascades. While certain TRP channels act as primary sensors, others exert regulatory control by precisely managing the spatial accessibility of internalized ligands to their cognate TLRs, particularly those sequestered within intracellular compartments [41, 90]. Consequently, TRP channels function as structural gatekeepers, providing stringent spatial checkpoints to ensure that TLR activation is confined to the appropriate subcellular organelles.

A prominent manifestation of this spatial control is the role of the endolysosomal channel TRPML1 in facilitating the trafficking of nucleic acids to endosomal TLRs. The ligand-induced activation of TLR7 and TLR9 is restricted to the endolysosomal lumen, an evolutionary strategy that prevents the aberrant recognition of self-derived nucleic acids within the extracellular space or the cytosol [91]. Such strict spatial segregation necessitates high-fidelity vesicular transport mechanisms to deliver internalized microbial nucleic acids into signaling-competent organelles. TRPML1 operates as a critical regulator of this vesicular transport apparatus. Specifically, the process is initiated by the lipid kinase PIKfyve, which synthesizes the phosphoinositide PtdIns(3,5)P₂ on the lysosomal membrane. PtdIns(3,5)P₂ directly binds and activates TRPML1, triggering localized, organelle-derived Ca²⁺ release from the lysosomal lumen into the surrounding cytosol [38]. Ultimately, such compartmentalized Ca²⁺ signal is an indispensable prerequisite for the efficient transport and delivery of single-stranded RNA (ssRNA) into TLR7-positive endolysosomal compartments [92].

In primary dendritic cells (DCs) lacking functional TRPML1 (Mcoln1⁻/⁻), the vesicular delivery of ssRNA to endosomal TLR7 is disrupted, resulting in impaired TLR7 transactivation and a reduced downstream cytokine secretory response [92]. Pharmacological modulation further validates the precise operation of this intracellular axis; the synthetic TRPML1 small-molecule agonist ML-SA1 specifically accelerates this vesicular delivery pathway, strongly enhancing TLR7-mediated cellular transcriptional outputs [92, 93]. Such regulatory checkpoint is not restricted to TLR7 pathways, as the PIKfyve-TRPML1 axis constitutes a broader control system for nucleic acid routing. Indeed, the pharmacological inhibition of PIKfyve similarly impairs the endolysosomal transport of specific TLR9 ligands (such as CpG-A DNA) to their respective signaling compartments [92, 94]. Collectively, these findings position the TRPML1 channel as a key regulator of ligand spatial accessibility for endosomal TLR signaling. This mechanism represents a crucial layer of homeostatic oversight that transcends simple signal amplification, functioning as a macromolecular checkpoint that links the cell’s metabolic state (reflected by PIKfyve lipid kinase activity) to its transcriptional readiness to respond to nucleic acid PAMPs [79]. Thus, the TRPML1-dependent relay redefines the lysosome as an active signaling hub where ion channels, lipid kinases, and endosomal immune receptors converge to gate the fidelity of intracellular pattern recognition.

Amplification and fine-tuning of downstream cascades

TRP channels tightly regulate the intensity and duration of TLR intracellular signaling through precise positive feedback loops and biphasic Ca²⁺ fluxes. This spatiotemporal regulation translates distinct ionic signals into specific biochemical outcomes, ranging from substantial signal amplification to the precise temporal modulation of cytokine transcription.

In endothelial cells, the TRPC6 channel drives a positive feedback loop that amplifies TLR4-initiated signaling [95]. The localized Ca²⁺ influx mediated by TRPC6 activates nonmuscle myosin light chain kinase (MYLK). In addition to phosphorylating myosin to alter intracellular cytoskeletal tension, this activated kinase simultaneously acts as a crucial molecular scaffold to facilitate the physical assembly of MyD88 and IRAK4 adapter proteins [95]. Such dual biochemical function enhances the stability of the TLR4 signalosome, effectively converting transient receptor engagement into a sustained downstream transcriptional response.

As exemplified by the DAG-dependent TRPC3 gating in macrophages described in Sect. 3.1.2, organelle-derived calcium signals supply the localized biochemical threshold required to drive NF-κB dissociation and nuclear translocation [28]. This compartmentalized signal amplifies the transcriptional output of TLR4 signaling without requiring sustained global calcium influx across the plasma membrane [28].

TRP channels also enable the intricate temporal fine-tuning of intracellular cascades, as exemplified by TRPV2. This channel mediates a biphasic Ca²⁺ signal that regulates specific cytokine transcription in two distinct temporal waves [96]. The initial, rapid release of Ca²⁺ from the ER triggers the degradation of IκBα, activating the canonical NF-κB pathway to induce primary Tnf and Il6 gene expression [96]. Subsequently, the LPS-induced translocation of TRPV2 to the plasma membrane facilitates a sustained influx of extracellular Ca²⁺. This secondary, prolonged calcium signal selectively sustains IL-6 production via an alternative signaling pathway that functions entirely independently of the initial NF-κB activation event [96]. This precise temporal separation allows the cell to dynamically modulate its cytokine secretory profile over time, preventing an indiscriminate burst of transcriptional mediators.

Shaping of immune cell effector functions

The intracellular modulation of TLR signaling by TRP channels translates directly into the shaping of specific cellular effector programs. By integrating physical microenvironmental cues with biochemical pathogen-derived signals, TRP channels act as critical autonomous determinants that dictate the nature and magnitude of cellular responses, including phagocytosis, directed motility, vesicular secretion, and inflammatory cell death pathways.

Regulation of autonomous phagocytosis and transcriptional polarization

The cross-system integration of physical and biochemical cues is prominently illustrated by the regulation of cellular phagocytosis and polarization via TRPV4. As a fundamental innate immune function, phagocytosis requires highly coordinated cytoskeletal rearrangements and membrane remodeling. While extracellular LPS acts through TLR4 to biochemically prime the cell, optimal phagocytic activity emerges only when cells simultaneously encounter a mechanical substrate compliance within a specific pathophysiological range (≈ 8–25 kPa) [85, 97, 98]. Under these dual conditions, the mechanical matrix stiffness enhances TRPV4 channel activity, which cooperates synergistically with TLR4-evoked signaling to mediate the sustained Ca²⁺ influx required for effective actin polymerization and particle internalization. Genetic deletion or pharmacological blockade of TRPV4 abolishes this stiffness- and stimulus-dependent facilitation of phagocytic capacity, effectively preventing the engulfment of both non-opsonized (e.g., bacteria) and opsonized targets [85].

Beyond physical internalization, the TRPV4-dependent axis actively modulates the qualitative profile of transcriptional outputs, shifting the cellular state toward a distinct pro-resolution phenotype. Upon TLR4 engagement, cells lacking functional TRPV4 display an elevated synthesis of the pro-inflammatory mediator IL-1β alongside a diminished expression of the anti-inflammatory cytokine IL-10 [85]. Such cytokine profiling demonstrates that TRPV4 acts as a finely tuned regulator of cellular polarization after threat clearance, rather than a generic signal amplifier [99, 100].

Control of vesicular logistics and directional cell migration

TRP channels are equally instrumental in controlling cellular deformation and directed migration by regulating vesicular trafficking. As established in Sect. 3.2.1, the TLR-induced expression of TRPML2 is required for the directed chemotaxis of innate immune effector cells [78, 80]. A parallel requirement operates in dendritic cells, where the related endolysosomal channel TRPML1 mediates localized lysosomal Ca²⁺ release that is required to activate the motor protein myosin IIA [101]. This localized calcium-dependent motor activation provides the structural driving force for fast, directional amoeboid motility and cell shape deformation [101]. The macroscopic, in vivo consequences of these localized trafficking and motility defects on systemic cell recruitment and regional tissue migration are further explored in Sect. 4.3.

Modulation of NLRP3 inflammasome assembly

TRP channels also serve as critical autonomous checkpoints governing the assembly of the NLRP3 inflammasome, a multi-protein platform responsible for proteolytic cytokine processing and the initiation of pyroptotic cell death cascades. While upstream TLR signaling provides the essential priming signal (Signal 1) by upregulating the transcription of Nlrp3 and Il1b, the subsequent physical assembly and activation of the inflammasome complex (Signal 2) heavily rely on intracellular ionic shifts and metabolic stress indicators (Fig. 2B) [102]. The Ca²⁺-permeable chanzyme TRPM2 tightly integrates these dual requirements. Functioning as an intrinsic sensor of oxidative stress, TRPM2 is activated by ROS generated downstream of TLR engagement, acting in concert with intracellular ADPR to gate a sustained influx of extracellular Ca²⁺ [103]. Ultimately, such prolonged Ca²⁺ flux acts as an essential signaling trigger for NLRP3 inflammasome assembly and subsequent caspase-1 activation, driving the proteolytic cleavage of pro-IL-1β into its mature form [48].

Fig. 2.

Pathological integration of TRP-TLR signaling in hyperalgesia and vascular hyperpermeability. A Hyperalgesia / Neuropathic Pain Sensitization. In peripheral sensory neurons, chemotherapeutic agents (e.g., paclitaxel) drive neurotoxicity by altering plasma membrane lipid raft dynamics. Paclitaxel increases lipid raft content and promotes the nanoscale co-organization of TLR4 and TRPV1. This structural proximity facilitates direct physical coupling that anchors TRPV1 at the cell surface, inhibiting agonist-induced endocytosis and lysosomal degradation. The resulting sustained TRPV1 sensitization lowers its activation threshold, driving pathological ectopic discharges and severe tactile allodynia. Targeted disruption of TLR4-lipid rafts by the apolipoprotein A-I binding protein (AIBP) intercepts this feed-forward sensitization. B Central Neuroinflammation and Paracrine Sensitization. During neuroinflammation, glial-neuronal paracrine signaling amplifies nociceptive transmission. Resident microglia detect danger signals via TLR4, triggering NADPH oxidase (NOX)-mediated ROS generation. This oxidative burst activates the redox-sensitive TRPM2 channel, driving sustained Ca²⁺ influx and the subsequent release of pro-inflammatory cytokines (e.g., IL-6, TNF- α, IL-1β). These glial-derived mediators diffuse across the synaptic cleft and act on adjacent sensory neurons, where cytokine receptor signaling sensitizes TRPV1, ultimately culminating in neuronal hyperexcitability and central sensitization. C Vascular Hyperpermeability and ARDS. Within the alveolo-capillary microenvironment, giant vascular endothelial cells function as signal integrators for both chemical and physical pathological cues. The TLR4-TRPC6-MYLK axis couples LPS recognition to TRPC6-mediated Ca²⁺ influx, subsequently activating MYLK. At the same time, the TRPV4 mechanosensory axis detects the increased stiffness of the underlying basement membrane/extracellular matrix (BM/ECM). The convergence of these calcium-driven pathways provokes substantial actomyosin cytoskeletal contraction. This mechanical retraction forcefully disrupts endothelial junctions, generating paracellular gaps that allow uncontrolled fluid extravasation from the vascular lumen into the alveolar space, clinically manifesting as ARDS. Created in BioRender. You, X. (2026) https://BioRender.com/3szy672

Tonic control of cytotoxic granule exocytosis

The regulatory influence of TRP channels also extends to the secretory dynamics of cytotoxic lymphocytes. In natural killer (NK) cells, the endolysosomal channel TRPML1 functions as a critical negative regulator of cell-autonomous degranulation. TRPML1 tightly modulates the homeostatic volume and content of secretory lysosomes, which sequester cytotoxic effectors such as granzyme B [104]. Pharmacological inhibition or genetic silencing of TRPML1 in these cells accelerates the exocytosis of these lytic granules and enhances IFN-γ production upon target cell contact [104]. Such autonomous negative feedback indicates that tonic TRPML1-mediated calcium flux sets the precise physiological threshold for cytotoxic granule exocytosis, thereby finely tuning the basal activation state of the cell [104, 105].

Cell-type specific dichotomy in TRP channel outputs

It is worth noting that the ultimate immunomodulatory output of TRP channels is highly contingent upon the specific cellular lineage and microenvironmental context. For instance, while TRPC3 clearly functions as a pro-inflammatory signal amplifier downstream of TLR4 in peripheral macrophages, brain-derived neurotrophic factor (BDNF)-driven TRPC3 activation conversely suppresses pro-inflammatory mediator release, dampening nitric oxide and TNF-α production in central nervous system (CNS)-resident microglia [106]. Ultimately, such lineage-specific divergence underscores that TRP channels function as context-dependent regulators rather than fixed binary switches, with their output tuned to the distinct homeostatic demands of peripheral and central innate immune compartments.

Collectively, the precise molecular and subcellular mechanisms detailed throughout this section not only shape the autonomous effector functions of innate immune cells, but also dictate the overarching pathophysiological outcomes observed at the systemic level (Table 3).

Table 3.

Molecular and pathophysiological mechanisms of TRP-TLR crosstalk

TRP-TLR Axis	Core Cell Type & Subcellular Localization	Primary Crosstalk Mechanism & Signaling Cascade	Key Second Messenger(s) & Downstream Effector(s)	Pathophysiological Outcome & Clinical Significance	Reference
TRPV1-TLR4	Nociceptors; plasma membrane lipid rafts	Direct physical coupling (TIR to C-terminus); inhibition of channel endocytosis	Extracellular Ca²⁺ influx; neuropeptide release (CGRP, SP)	Feed-forward nociceptor sensitization; CIPN, chronic pain, and pruritus	[12, 55, 107]
TRPC3-TLR4	Macrophages; ER	TLR4-activated PLD–lipin-1 cascade; ER-localized DAG-dependent TRPC3 gating	ER Ca²⁺ release; NF-κB nuclear translocation	Sustained pro-inflammatory cytokine transcription; systemic inflammation	[28]
TRPV4-TLR4	Alveolar macrophages; plasma membrane	TLR4-induced thrombin–PAR2–Gαs cascade; cAMP-dependent inhibition of TRPV4 gating	Decreased Ca²⁺ influx; blockade of calcineurin/NFAT and NF-κB cooperativity	Termination of acute pro-inflammatory signaling; resolution of acute lung injury (ALI)	[87, 89]
TRPML1-TLR7/9	Dendritic cells, macrophages; endolysosomes	PIKfyve-dependent PI(3,5)P₂ generation; TRPML1-mediated lysosomal Ca²⁺ release	Activation of vesicle fusion machinery; viral nucleic acid delivery to TLR7/9	Enhanced endosomal TLR7/9 activation; antiviral immunity	[79, 91–93]
TRPM7-TLR4	Macrophages; plasma membrane	LPS-induced TRPM7-mediated Ca²⁺ influx; dynamin-dependent endocytosis of TLR4–CD14	Endosomal translocation of TLR4; IRF3 and NF-κB activation	Activation of TRIF-dependent pathways; type I IFN production	[52]
TRPML2-TLR2/4/7	Alveolar macrophages, BMDMs; Recycling endosomes	TLR-induced selective Trpml2 transcriptional upregulation	Localized vesicular Ca²⁺ release; trans-Golgi/endosomal CCL2 trafficking	Effector phenotype transition; directional macrophage chemotaxis	[78, 80]
TRPM2-TLR4	Macrophages, Microglia; Plasma membrane & organelles	TLR4-triggered ROS and intracellular ADPR-dependent TRPM2 gating	Intracellular Ca²⁺ influx; NLRP3 inflammasome assembly and caspase-1 cleavage	Mature IL-1β release; pyroptosis and neuroinflammation-induced neuronal damage	[48], 81–84, 103]
TRPA1 (TLR4-independent)	Sensory neurons, epithelial barriers; plasma membrane	Asymmetric membrane insertion of LPS conical lipid A moiety	Increased membrane lateral tension; proposed ARD-mediated mechanotransduction (force-from-lipids model)	Immediate nociceptive defensive reflexes; hyper-acute neurogenic inflammation	[7, 25, 61, 71, 72]
TRPV2-TLR4	Macrophages; ER & plasma membrane	Stimulus-evoked targeted TRPV2 vesicular surface translocation	Biphasic Ca²⁺ signal (early ER leak, late-stage extracellular influx)	Early-phase NF-κB priming; late-phase independent stabilization of sustained Il6 transcripts	[96]
TRPC6-TLR4	Vascular endothelial cells; Plasma membrane	TLR4-dependent TRPC6 gating	Intracellular Ca²⁺ surge; MYLK activation and MyD88/IRAK4 signalosome scaffolding	Actin stress fiber contraction; endothelial junction tearing and vascular barrier breakdown in ARDS	[95]
TRPP2-TLR4	Renal tubular epithelial cells; primary cilia and ER	Loss of TRPP2-mediated homeostatic ER Ca²⁺ leak	Cytosolic Ca²⁺ depletion; cell-autonomous NF-κB/STAT3 hyperactivation and Tlr4 transactivation	Breach of epithelial immune tolerance to DAMPs; sterile inflammation and cystogenesis in ADPKD	[56–58]

Core pathophysiological mechanisms of the TRP-TLR axis

While the subcellular biophysics and organellar trafficking of the TRP-TLR axis ensure cell-autonomous signaling fidelity, the true pathological impact of this crosstalk becomes apparent only at the tissue level. Within the complex architecture of metazoan organs, individual receptor-channel interactions coalesce into integrated networks that drive chronic inflammation, somatic and visceral hypersensitivity, and barrier disruption. Shifting the focus from single-cell biochemistry towards multicellular paracrine networks, microenvironmental deterioration, and the temporal evolution of disease, this section illustrates how identical molecular crosstalk modules are translated by local microenvironments into diverse clinical syndromes.

Peripheral nociceptive sensitization and visceral hypersensitivity

At the organismal level, the bidirectional crosstalk between TRP channels and TLRs transitions from localized subcellular events into substantial alterations in tissue-level sensory processing. Mechanistically, as established in Sect. 3.1.1, the direct TLR4-TRPV1 coupling anchors the channel at the cell surface and prevents desensitization [12]. This sustained membrane retention amplifies nociceptive signaling, lowering the mechanical and thermal activation thresholds of peripheral sensory neurons and driving pathological hypersensitivity across diverse visceral and somatic disease models [55].

Somatic hyperalgesia in chemotherapy-induced peripheral neuropathy and peripheral infections

Such persistent non-desensitized state of the surface-anchored TRP-TLR complex constitutes a fundamental driver of somatic hyperalgesia, as prominently manifested in chemotherapy-induced peripheral neuropathy (CIPN). During clinical oncological regimens involving paclitaxel, this chemotherapeutic agent acts as a potent exogenous ligand that continuously targets and activates the surface-localized TLR4 complex on dorsal root ganglion (DRG) nociceptors [108]. Instead of triggering the standard homeostatic internalization of TRPV1, the chronic paclitaxel-TLR4 engagement maintains adjacent TRPV1 channels in a sensitized, hyper-responsive state [63, 108]. In vivo, this membrane stabilization translates minor, innocuous thermal or mechanical stimuli into high-frequency pathological bursts of action potentials, clinically manifesting as excruciating tactile allodynia and thermal hyperalgesia (Fig. 2A) [108].

Similarly, in acute localized bacterial infections, such as dental pulpitis, this uncoupled desensitization program drives severe orofacial pain cascades. Infiltrating bacterial LPS triggers a coordinated, two-stage nociceptive response: an immediate, direct activation of TRPA1 channels that evokes acute nociceptive signaling [7], followed by a prolonged, TLR4-dependent trafficking phase that mobilizes intracellular TRPA1-containing vesicles to the plasma membrane [109]. At the same time, the activation of adjacent TLR4 persistently sensitizes the colocalized TRPV1 pool, maintaining the nociceptor in a state of chronic hyperexcitability [107]. Such coordinated neuronal activation drives the sustained exocytosis of pro-nociceptive neuropeptides, such as calcitonin gene-related peptide (CGRP) and Substance P (SP), from the peripheral nerve terminals into the local dental microenvironment, generating intense neurogenic inflammation and intractable localized pain [7, 107].

Amplification and polarization of pruritic signaling

Beyond pain transduction, the persistent operational dynamics of the TRP-TLR axis position TLR4 as a critical regulator that amplifies and sustains acute and chronic pruritus (itch) within the dermal microenvironment. In histamine-dependent pruritic cascades, the sensory transmission relies on the pre-formed, constitutive TLR4-TRPV1 macromolecular complex to maintain sustained, non-desensitizing calcium influx, thereby preventing the normal sensory adaptation of itch-sensing fibers [110].

Conversely, in non-histaminergic pruritic pathways, the axis transitions into long-term transcriptional control, directly governing the expression and membrane abundance of the upstream MrgprA3 receptor, which subsequently engages the TRPA1 pruritic signaling cascade [110]. TLR4-dependent intracellular signaling cascades (e.g., PI3K/AKT) sensitize adjacent TRPV4 channels within the neuronal membrane, substantially exacerbating chronic, repetitive scratching behaviors in vivo [111]. Ultimately, such multi-channel post-translational and transcriptional modifications render the peripheral nervous system structurally hyper-responsive to ambient inflammatory or pathogen-derived pruritic challenges [110].

Visceral hypersensitivity and epithelial barrier breakdown in inflammatory bowel disease

The translation of cellular non-desensitization into full-scale organ system pathology is prominently manifested in visceral hypersensitivity models, particularly ulcerative colitis and other manifestations of inflammatory bowel disease (IBD). Such pathological progression operates as a distinct two-stage cascade involving initial tissue priming followed by sustained microenvironmental exposure [112]. During the acute inflammatory priming phase, localized mucosal tissue disruptions upregulate the baseline genomic expression of both TLR4 and TRPV1 within colonic sensory neurons [113].

As the disease progresses into the chronic sensitization phase, the extensive breakdown of the intestinal epithelial barrier permits the continuous translocation of luminal bacterial LPS into the underlying lamina propria microenvironment [112]. Consequently, such sustained permeation subjects the overexpressed and poorly desensitized neuronal TLR4-TRPV1 complexes to chronic activation. The subsequent failure of TRPV1 to undergo agonist-induced internalization establishes a self-amplifying pathological loop of chronic visceral pain, where innocuous mechanical or chemical stimuli from the gut are misinterpreted by the central nervous system as intense noxious events [12]. The causal validation of this axis is reinforced by genetic and pharmacological interventions; targeted in vivo disruption of the upstream TLR4 signaling component successfully interrupts this persistent hyper-sensitizing relay, substantially attenuating visceral mechanical hyperalgesia and restoring baseline visceral sensory thresholds [113].

Translational perspectives: acute-to-chronic phase transitions

From a translational medicine perspective, the persistent operational dynamics of the peripheral TRP-TLR axis introduce a major clinical barrier in managing the acute-to-chronic pain transition. In the hyperacute phase of somatic or visceral injury, the activation of the receptor-channel complex relies primarily on exogenous initiating insults, such as paclitaxel infusions or luminal bacterial LPS translocation [7, 108]. However, as the pathology transitions into a chronic state, the sustained electrical discharge from non-desensitized nociceptors drives local neurogenic inflammation, which chronically remodels the extracellular matrix [114]. Such tissue damage triggers the continuous enzymatic shedding of endogenous DAMPs from damaged host cells [10]. Consequently, these endogenous ligands replace the original exogenous stimuli, binding back onto the overexpressed TLR4-TRP complex to fuel a self-sustaining pathological amplification loop [10, 115]. Ultimately, such mechanistic divergence explains the marked translational gap in clinical trial outcomes: downstream antagonists fail to achieve long-term disease modification in chronic cohorts because the axis has already transitioned into an autonomous, self-perpetuating tissue program that is entirely decoupled from the initial triggering event [116, 117]. Therefore, therapeutic timing must be strategically aligned with these distinct chronological windows.

Central neuroinflammatory cascades and glial-neuronal communication

Within the immune-privileged environment of the CNS, the TRP-TLR signaling axis transitions from cell-autonomous events into a pathological, multicellular network that drives progressive neurodegeneration. Mechanistically, as established via the lipid-metabolic relays in Sect. 3.1.2 and the oxidative stress-sensing mechanisms detailed for TRPM2 in Sect. 3.2.1 and 3.3.3, TLR4 engagement on central myeloid lineages promotes direct TLR4-TRPV1 physical coupling and intracellular ROS bursts [118]. Rather than terminating within the myeloid compartment, these TRPV1- and TRPM2-mediated calcium fluxes drive the hyperactivation of canonical NF-κB and NLRP3 inflammasome complexes [48, 119]. At the tissue level, this localized transcriptional transactivation polarizes microglia into a chronic, neurotoxic M1 effector phenotype, establishing a self-sustaining paracrine amplification cascade that drives synaptic dysfunction, neural network hyperexcitability, and severe brain tissue damage [120–122].

Glial phenotypic locking and cross-synaptic paracrine amplification

This cell-autonomous channel hyperactivation operates as a critical driver of glial phenotypic locking during major CNS insults, most notably in the acute and chronic phases of ischemic stroke and epileptogenesis. Following focal cerebral ischemia or during paroxysmal seizure activity, the injured brain parenchyma releases abundant intracellular DAMPs into the surrounding microenvironment [123]. These endogenous ligands bind to microglial TLR4, which initiates the genomic upregulation and functional assembly of the TRPM2 and TRPV1 channel platforms [124]. Once synthesized and mobilized, these channels drive sustained calcium influx, persistently locking the resident microglia into a highly reactive, neurotoxic M1 polarization state [119, 122].

The structural and physiological consequence of this phenotypic locking is the sustained synthesis and release of pro-inflammatory mediators, such as IL-1β, TNF-α, and IL-6, into the local parenchymal microenvironment [125]. Crucially, these glia-derived soluble mediators do not remain isolated; they diffuse rapidly across the synaptic cleft to target adjacent nociceptive and excitatory neurons.

Upon binding to their respective cognate receptors on the neuronal membrane, these paracrine mediators mobilize downstream intracellular kinase cascades (such as PKA and PKC) that rapidly phosphorylate the intracellular domains of neuronal-resident TRPA1 and TRPV1 channels [19]. This post-translational phosphorylation markedly lowers the activation threshold of neuronal channels, rendering them hyper-responsive to ambient mechanical, thermal, or chemical stimuli, thereby driving an immune-initiated, neuron-amplified paracrine positive feedback loop that overcomes physiological sensory desensitization. This cascade transforms localized microglial stress into a generalized state of chronic neural network hyperexcitability that drives progressive brain tissue destruction, cognitive decline, and seizure propagation in vivo (Fig. 2B) [122, 126].

Translational perspectives: glial-neuronal target specificity

From a neuropharmacological perspective, the cross-synaptic paracrine amplification loop driven by the central TRP-TLR axis imposes two major hurdles for clinical translation: the spatial constraints of blood-brain barrier (BBB) permeability and the cellular specificity of target interventions. Systemic administration of generic TRP or TLR antagonists is often suboptimal for central neurodegenerative disease. High-molecular-weight, hydrophilic, or biologic agents, including antibody-based TLR antagonists, are largely excluded by the intact blood-brain barrier, whereas the small lipophilic channel blockers that do penetrate the CNS tend to produce on-target central toxicity [127–129].

Furthermore, even with successful central delivery, global non-selective channel blockade risks disrupting the vital homeostatic operations of resident microglia. Under physiological conditions, basal TRP-dependent calcium signaling is essential for microglial surveillance, synaptic pruning, and the clearance of metabolic debris [130–132]. Completely silencing these channels to halt M1 polarization inadvertently impairs the cell’s neuroprotective, anti-inflammatory functions, potentially exacerbating secondary neurodegeneration [130–132]. Consequently, future therapeutic strategies must transition away from generic systemic channel inhibition. Innovation must focus on either developing BBB-permeable nanocarriers targeted to reactive microglial sub-populations [97, 133], or biochemically decoupling the downstream paracrine interfaces, such as the specific kinase-mediated phosphorylation sites on neuronal TRPA1/TRPV1 [19, 134], thereby silencing pathological hyperexcitability while preserving the physiological surveillance capacity of the central innate immune apparatus.

Barrier organ dysfunction and tissue-level functional impairment

At the interface of epithelial and endothelial boundaries, the subcellular mechanisms of the TRP-TLR axis translate into complex, tissue-wide responses to simultaneous biochemical and biophysical threats. Within barrier organs, most notably the respiratory tract, this molecular crosstalk manifests in two opposing directions. On the one hand, resident immune populations synergistically integrate chemical pattern recognition with local mechanical strain to maximize host defense and pathogen clearance [85]. On the other hand, the pathological hyperactivation of these shared receptor-channel complexes within the vascular lining generates excessive cytoskeletal contraction, disrupting inter-endothelial junctions and contributing to organ failure [95, 135]. Such functional bifurcation is prominently illustrated during the evolution of acute lung injury (ALI) and sepsis-induced acute respiratory distress syndrome (ARDS) [97].

Synergistic chemical and mechanical defense in alveolar macrophages

During the hyperacute phase of ARDS, resident alveolar macrophages constitute the primary cellular shield against pathogen dissemination within the alveolar space [136]. At this early stage, isolated biochemical stimulation by PAMPs, such as bacterial LPS, merely induces a baseline priming state. As established in Sect. 3.3.3, the dual integration of TLR4-mediated biochemical priming and TRPV4-dependent mechanosensory surveillance allows alveolar macrophages to dynamically adapt to pathological shifts in septal compliance without requiring a secondary independent stimulus [85].

Ultimately, such synergistic integration at the single-cell level broadens exponentially, dictating the overall host defense capacity within the alveolar space. The molecular convergence of pattern recognition and mechanical strain detection drives the efficient engulfment, internalization, and lysosomal clearing of invading microbial pathogens across the blood-air interface [85]. In addition, such coordinated protective program actively limits collateral parenchymal destruction by steering macrophage activation toward a pro-resolution transcriptional state, thereby preserving tissue structural integrity when the barrier organ is under maximum biophysical stress [85, 98].

Endothelial barrier disruption and pulmonary edema

In contrast to the protective, defense-enforcing program executed within the alveolar space, a parallel endothelial TRPC6-TLR4 crosstalk module drives a highly destructive pathological cascade within the pulmonary vasculature, serving as the primary driver of endothelial barrier breakdown [95]. When systemic sepsis or severe localized pneumonia triggers the large-scale accumulation of circulating LPS and endogenous DAMPs within the microcirculation, endothelial TLR4 receptors are subjected to sustained activation.

Under these conditions, sustained TLR4 signaling becomes coupled to the opening of plasma membrane TRPC6 channels, and the resulting Ca²⁺ influx activates MYLK [95, 137]. As depicted in Fig. 2C, MYLK-driven actomyosin contraction pulls adjacent endothelial cells apart, destabilizing claudin- and cadherin-based junctions and producing the microvascular permeability that drives fluid exudation, pulmonary edema, and the ventilation-perfusion mismatch of ARDS (Fig. 2C) [95, 135, 137].

Aberrant vesicular trafficking and disrupted regional immune shuttling

Beyond immediate barrier disruption, the organellar ion channels of the TRP-TLR axis coordinate long-term tissue-level pathology by governing aberrant vesicular trafficking. During the progression phase of barrier organ injury, sustained TLR activation drives the de novo synthesis of TRPML2 within the tissue parenchyma [78]. As detailed in Sect. 3.2.1, TRPML2 then sustains the trafficking and secretion of chemokines such as CCL2 [80]. Under chronic stimulation, this persistent chemokine release establishes an uncontrolled trans-endothelial gradient that drives the excessive influx of neutrophils and monocytes into the lung parenchyma, dismantling the blood-air barrier and worsening ARDS [78, 80].

In parallel, this inflammatory microenvironmental deterioration fundamentally impairs adaptive immune priming by targeting the related endolysosomal channel TRPML1. As established in Sect. 3.3.3, the TRPML1-driven myosin IIA motor powers the directed migration of dendritic cells [101]. Within the pathologically altered matrix of the injured barrier organ, the disruption of this TRPML1-dependent motor apparatus severely impairs the capacity of antigen-presenting DCs to undergo directed migration. Consequently, despite full phenotypic maturation triggered by direct TLR signaling, these Mcoln1^⁻/⁻ DCs exhibit severe mechanical trafficking stagnation, failing to egress from the inflamed peripheral parenchyma and traverse regional lymphatic conduits [92, 101]. This localized motility defect translates macroscopically into a marked delay in DC shuttling to regional draining lymph nodes, limiting clonal T-cell expansion and disrupting the fidelity of host adaptive immune responses in vivo [37, 101].

Translational perspectives: compartmentalized microenvironmental regulators

From a clinical and pharmacological perspective, the diametrically opposed actions of the TRP-TLR axis across the alveolar-capillary membrane highlight the major limitations of conventional, non-selective systemic interventions and mandate a shift toward compartmentalized therapeutics. In clinical trials for sepsis-induced ARDS, broad-spectrum TLR4 inhibitors or systemic calcium channel blockers have largely failed to improve survival [138]. Such translational failure stems directly from the disruption of the spatial heterogeneity inherent to the axis: globally silencing TLR4 or TRPV4 impairs the mechanosensitive phagocytic function of alveolar macrophages, compromising host pathogen clearance and predisposing patients to secondary bacterial colonization and infection escalation [85]. Conversely, failing to arrest the endothelial TLR4-TRPC6-MYLK axis allows severe endothelial barrier disruption and edema to proceed unchecked [95].

Consequently, future translational strategies must transition away from generic systemic channel blockade in favor of engineered, tissue- or compartment-selective nanocarriers. For example, endothelial-targeted immunoliposomes can be designed to selectively deliver TRPC6 inhibitors or MYLK-decoupling peptides to the vascular lining, attenuating the endothelial contractile program while entirely preserving the force-dependent pathogen-clearing capacity of alveolar macrophages within the airspace. Only through such spatially segregated, cell-specific molecular tuning can the TRP-TLR axis be transitioned from a pathological amplifier into a highly refined, localized immunoregulatory control point capable of modulating inflammation without compromising host defense.

Epithelial immune tolerance breach and cystogenesis

At the interface of specialized epithelial barriers, the chronic disruption of organellar ion homeostasis triggers a substantial breakdown of tissue-level immune tolerance, converting a structural epithelial layer into an active driver of progressive fibroinflammatory disease. Mechanistically, as established in Sect. 3.1.2, the loss of functional TRPP2 depletes tonic intracellular Ca²⁺ [56] and relieves the constraint on pro-inflammatory cascades, driving cell-autonomous pro-inflammatory transcriptional activation and epithelial TLR4 upregulation [57]. At the organ level, such intracellular signaling shift translates into an aberrant microenvironmental dialogue, where renal epithelial cells become hypersensitive to endogenous danger signals [57], culminating in sustained macrophage recruitment and progressive cyst expansion in ADPKD [57, 58].

Microenvironmental Sensitization and the Overwhelming of Non-Coding RNA Brakes.

This loss of tonic biophysical restraint directly primes the renal epithelial tissue microenvironment for sustained inflammation and structural remodeling. In Pkd2-deficient renal epithelial cells, the chronic activation of intrinsic transcriptional networks induces a pronounced upregulation of baseline TLR4 expression [57]. This transcriptional upregulation creates a state of persistent hypersensitivity to endogenous DAMPs that continuously accumulate within the expanding cystic fluid and renal interstitial spaces [57].

During the progression of ADPKD, the sustained engagement of this upregulated epithelial TLR4 complex by interstitial danger signals activates a sustained paracrine TLR4/IFN-γ/STAT1 signaling axis. Under homeostatic conditions, the activation of this axis transactivates interstitial non-coding RNAs, specifically microRNA-214 (miR-214), which functions as a critical post-transcriptional negative feedback regulator [57]. In healthy tissues, miR-214 directly targets the 3’ untranslated region (UTR) of Tlr4 mRNA, executing a precise gene-silencing program to resolve transient inflammatory responses and maintain epithelial immune tolerance [57]. However, within the pathologically altered microenvironment of the ADPKD kidney, this protective non-coding RNA brake is overcome by the continuous influx of endogenous DAMPs and chronic organellar calcium depletion. The failure of this miR-214-dependent silencing mechanism leads to persistent, unmitigated epithelial TLR4 activation. This sustained molecular drive triggers the continuous synthesis and baseline secretion of pro-inflammatory cytokines and chemokines into the renal interstitium, breaking the innate immune privilege of the tubular epithelium [57, 58].

Macrophage reciprocal recruitment and interstitial cyst expansion

The long-term organ-system consequence of this epithelial tolerance breach is the establishment of a self-sustaining pathological amplification loop driven by intense interstitial macrophage recruitment. The chemokines continuously secreted by the sensitized Pkd2-deficient epithelial cells establish a strong, localized chemotactic gradient that drives the substantial infiltration of circulating monocyte-derived macrophages into the renal parenchyma [58, 139].

Once inside the interstitial space, these recruited macrophages are polarized by the DAMP-rich environment into a highly active M2-like phenotype [57, 140]. Rather than clearing debris to promote tissue resolution, these infiltrating immune cells engage in a reciprocal pathogenic dialogue with the adjacent epithelial cells [12]. The macrophages secrete a variety of mitogenic and pro-inflammatory factors that act on the epithelial cells, further driving their uncoordinated proliferation, fluid secretion, and fluid-filled cyst expansion [58, 141]. This reciprocal cellular communication permanently remodels the surrounding extracellular matrix, physically compressing adjacent healthy nephrons and driving progressive interstitial fibrosis, which ultimately leads to terminal renal failure in vivo. Thus, the TRPP2-TLR4 axis represents a pivotal checkpoint where ion channel dysfunction translates into a breach of epithelial immune tolerance, highlighting that TRP-TLR crosstalk is fundamental to the maintenance of tissue architecture beyond the nervous system.

Translational perspectives: non-coding RNA restoration and disease-modifying strategies

From a therapeutic and clinical translation perspective, the elucidation of the TRPP2-TLR4-miR-214 axis shifts the drug discovery strategy for ADPKD from traditional symptomatic fluid management toward mechanistic disease-modifying interventions. Current clinical regimens rely heavily on vasopressin V2 receptor antagonists, such as tolvaptan, to delay cyst progression by non-selectively slowing fluid accumulation. However, these fluid-targeted interventions fail to address the underlying interstitial inflammation and epithelial tolerance breach, frequently causing severe systemic side effects like substantial aquaresis and hepatic toxicity [142].

The molecular characterization of the TRPP2-TLR4 crosstalk opens an alternative therapeutic window focused on restoring endogenous post-transcriptional regulators. Future innovative strategies may involve the deployment of synthetic miR-214 mimics or target-specific oligonucleotides delivered via renal epithelium-targeted nanocarriers [57]. By artificially restoring the post-transcriptional silencing of Tlr4 mRNA, these interventions can selectively desensitize the Pkd2-deficient epithelium to interstitial DAMPs, thereby interrupting the macrophage recruitment cascade and attenuating cyst proliferation at its mechanistic source [57]. Crucially, because this approach targets a localized tissue-level tolerance breach rather than globally silencing systemic pattern recognition, it preserves the broader host innate immune defense infrastructure, offering a promising targeted strategy for long-term clinical management.

Therapeutic targeting of the TRP-TLR axis

Pharmacological strategies from broad modulation to precision targeting

Existing therapies for chronic pain, refractory neuroinflammation, and autoimmune diseases often have limited efficacy or significant side effects [143]. In this context, the interaction between TLRs, the core molecules of immune sensing, and TRP channels, key mediators of environmental and nociceptive stimuli, has emerged as a crucial therapeutic frontier [82]. During disease progression, this dynamic interplay within the TLR-TRP signaling axis constitutes a critical convergent pathway in the pathological cycle of immune activation and pain sensitization. Consequently, innovative pharmacological strategies targeting this interaction open a promising new avenue for addressing these clinical challenges.

The upstream/downstream targeting dichotomy in CIPN

Paclitaxel is a first-line chemotherapeutic agent for common solid malignancies, but the associated CIPN impairs the rehabilitation, functional capacity, and quality of life of cancer survivors. Studies show that both paclitaxel and LPS can directly excite peripheral sensory and spinal neurons and enhance TRPV1-mediated capsaicin responses through the TLR4 signaling pathway [108]. Enhanced TRPV1 activity in cutaneous afferents is associated with a cutaneous burning sensation, while heightened TRPV1 signaling in deep somatic afferents leads to musculoskeletal aches and cramps [144]. Intervention strategies targeting this axis show therapeutic potential, but key differences in the mechanisms of action of different drugs provide an informative framework to elucidate two distinct therapeutic philosophies.

The TRPV1 blocker AMG9810 represents a downstream intervention strategy for treating established hyperalgesia (Fig. 3). It acts directly on the ion channel function of upregulated and sensitized TRPV1 channels, thereby interrupting the output of pain signals and reversing established hyperalgesia [108]. This approach aims to manage the symptoms of a system that has already entered a pathological state.

Fig. 3.

Pharmacological targeting of the TRP-TLR signaling axis. The schematic illustrates multi-level pharmacological strategies targeting specific TRP channels and TLRs across distinct subcellular compartments (plasma membrane, ER, and endolysosomes). At the plasma membrane, these agents present contrasting strategies for pain management: LPS-RS antagonizes TLR4 to intercept the upstream sensitization signal, acting as a disease-modifying strategy to prevent hyperalgesia. Conversely, AMG9810 directly blocks the TRPV1 pore, providing a downstream symptomatic blockade of nociceptive output. Cannabidiol (CBD) exerts analgesic and anti-inflammatory effects by inducing TRPV1 desensitization. At the ER membrane, Pyr10 selectively inhibits intracellular TRPC3. This targeted inhibition intercepts DAG-dependent localized Ca²⁺ release from the ER, thereby suppressing systemic inflammation without compromising normal plasma membrane Ca²⁺ entry mechanisms. At the endolysosome, the TRPML1 synthetic agonist ML-SA1 promotes lysosomal Ca²⁺ release, which drives the fusion of ssRNA-containing vesicles with lysosomes. This localized calcium signaling facilitates the transport of viral ligands to endosomal TLR7, ultimately enhancing host antiviral immunity. Created in BioRender. You, X. (2026) https://BioRender.com/axqx680

In contrast, the TLR4 antagonist LPS-RS exemplifies an upstream preventative strategy (Fig. 3). Inhibiting TLR4 signaling blocks the initial signaling events that lead to both the increased expression and functional sensitization of the TRPV1 protein, thus preventing the development of hyperalgesia [108].

The distinction between AMG9810 and LPS-RS reveals a strategic insight, suggesting that targeting the axis offers different therapeutic windows for the prophylaxis of a sensitized state or the therapeutic intervention for established pain. Upstream inhibition of TLR4 is a disease-modifying approach, as it prevents the molecular and cellular changes (i.e., TRPV1 upregulation and sensitization) that constitute the pathological state [108]. Mechanistically, blocking TLR4 prevents the neuron from entering a pathological state, maintaining it at a homeostatic level. Downstream blockade of TRPV1, however, is a symptomatic treatment that acts on a neuron that has already undergone pathological changes, silencing the final output of the pain signal without reversing the underlying sensitized state. This insight shifts the therapeutic choice from drug selection to a time-dependent decision of whether the goal is prevention or management of an existing condition.

Multi-target modulation by cannabidiol

Cannabidiol (CBD), a phytocannabinoid isolated from Cannabis sativa, modulates a multi-target synergistic mechanism to modulate TLR4 and TRPV1 functions for treating chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) (Fig. 3) [145]. At the level of inflammatory regulation, CBD activates CB2 receptors to inhibit the TLR4/NF-κB signaling pathway, markedly reducing the expression of pro-inflammatory factors IL-6, TNF-α, and cyclooxygenase-2 (COX-2) [146]. In addition, it indirectly enhances endocannabinoid activity to amplify CB2-mediated anti-inflammatory signals [147]. In a rat model of CP/CPPS, this effect is manifested by the dynamic regulation of CB2 expression, in which elevated CB2 levels during inflammation return to physiological baseline after CBD intervention, signifying restoration of inflammatory homeostasis [145]. At the analgesic level, CBD first acts as an agonist to briefly activate the TRPV1 channel, which subsequently induces a lasting desensitization of the channel [148]. This mechanism, similar to that of capsaicin, effectively blunts the responsiveness of TRPV1 to painful stimuli. It also cooperatively reduces COX-2 release in the prostatic microenvironment, thereby simultaneously alleviating pain and local inflammation [145]. This dual modulation of the CB2-TLR4/NF-κB anti-inflammatory axis and TRPV1-COX-2 analgesic axis has been validated in both human prostate epithelial cells (RWPE-1) and animal models, highlighting the potential of CBD as a multi-target therapeutic strategy for CP/CPPS [145].

Intracellular and organellar targeting strategies

The scope of pharmacological intervention has expanded from ion channels on the plasma membrane to intracellular organelles, and therapeutic goals have shifted from simple inhibition to bidirectional modulation.

Targeting the ER demonstrates the potential to shift therapeutic focus from the plasma membrane to intracellular organelles [28]. The TRPC3 blocker Pyr10 exemplifies this approach. By intercepting the DAG-dependent TRPC3-mediated Ca²⁺ release from the ER, Pyr10 effectively attenuates systemic inflammation without compromising plasma membrane Ca²⁺ entry mechanisms (Fig. 3) [28]. This indicates that targeting signaling branches in specific subcellular compartments allows for fine-grained intervention in inflammatory pathways [28].

Targeting the lysosome reveals that the therapeutic goal is not restricted to immunosuppression but also includes immune enhancement. The TRPML1 agonist ML-SA1 illustrates this shift (Fig. 3) [149]. By potentiating the lysosomal trafficking of single-stranded RNA to TLR7, this agonist boosts the delivery of ssRNA to TLR7, thereby amplifying antiviral interferon responses [92]. Consequently, the TRP-TLR axis can be targeted to suppress pathological inflammation, as with Pyr10, or harnessed to enhance protective immunity, as seen with ML-SA1 enhancing TLR7-dependent antiviral responses [92]. This capacity for bidirectional modulation characterizes the axis as a central immunoregulatory node whose set point can be adjusted according to the clinical context, moving beyond the view of this pathway as solely a pro-inflammatory target.

To better summarize this section, the following table (Table 4) outlines the key pharmacological agents that target the TRP-TLR interaction axis.

Table 4.

Pharmacological Agents Targeting the TRP-TLR Axis

Agent	Target(s)	Mechanism of Action	Therapeutic Effect	Reference
AMG9810	TRPV1	Direct TRPV1 antagonist; blocks nociceptive output.	Reverses established CIPN hyperalgesia.	[108]
LPS-RS	TLR4 / TRPV1	TLR4 antagonist; prevents TLR4-mediated sensitization and subsequent upregulation of TRPV1.	Prevents the development of paclitaxel-induced hyperalgesia.	[108]
CBD	TRPV1 / TLR4	Induces TRPV1 desensitization; inhibits TLR4/NF-κB signaling via CB2 receptor activation.	Alleviates chronic pain and inflammation in CP/CPPS.	[145]
Pyr10	TRPC3	Selective TRPC3 blocker; prevents ER Ca2+ release required for NF-κB activation.	Ameliorates systemic inflammation by suppressing LPS-induced pro-inflammatory gene expression.	[28]
ML-SA1	TRPML1 / TLR7	TRPML1 agonist; enhances transport of ssRNA ligands to endolysosomal TLR7.	Boosts TLR7-driven antiviral immunity.	[92]

Advanced therapeutic modalities

Precision gene editing

Translating molecular insights into targeted therapies is frequently hindered by the limitations of broad-spectrum pharmacology, as systemic TRP channel antagonists often disrupt essential physiological homeostasis [150]. Consequently, non-viral CRISPR-Cas9 technologies have emerged as a promising tool for precision intervention. Recent advances utilizing cationic lipid-based transfection have successfully introduced functional gene-editing systems into primary human DRG neurons. By specifically targeting pain-associated loci such as the TRPV1 gene, this non-viral modality reduced neuronal hypersensitivity to agonists like capsaicin [151].

Within the TRP-TLR immune-sensory axis, the value of precision editing lies in targeting specific intracellular interaction domains rather than ablating the entire channel. Because the TLR4-TRPV1 coupling is mediated by the TIR domain interface detailed in Sect. 3.1.1 [12], CRISPR-based interventions could in principle be engineered to edit only these interaction motifs. By selectively altering the binding interface, it may become possible to uncouple the pathological TLR4-mediated sensitization loop while preserving the independent, homeostatic sensory capabilities of the TRP channel, thereby overcoming the limitations of conventional pharmacological antagonists.

Wireless, transgene-free neuromodulation

To circumvent the off-target effects and spatial limitations associated with systemic drug delivery, advanced physical interventions are being developed. For example, a recent advance in wireless, transgene-free neuromodulation utilizes near-infrared (NIR) light-responsive nanoparticles to achieve precise spatiotemporal control over channel activity [133]. These engineered nanoparticles function as specialized nanoantennae, converting externally applied NIR light into highly localized thermal energy to specifically gate endogenous heat-sensitive TRPV1 channels.

Within the specific context of the TRP-TLR immune-sensory axis, this targeted approach offers a promising but still conceptual therapeutic strategy. The bidirectional crosstalk between TLRs and TRP channels is inherently localized, often restricted to specific anatomical sites of acute infection or discrete membrane microdomains, such as lipid rafts [152]. However, systemic pharmacological blockade of these channels frequently disrupts physiological homeostasis, best exemplified by the clinical failure of early TRPV1 antagonists due to severe systemic hyperthermia [150]. By deploying NIR-activated nanoparticles selectively to sites of TLR-driven inflammation, such as a localized neuropathic lesion, it becomes possible to achieve on-demand, spatially restricted activation or desensitization of TRP channels. This modality allows for the precise modulation of local immune effector functions and nociceptive signaling while preserving systemic thermoregulation.

Challenges and considerations for clinical translation

While targeting the TRP-TLR axis holds substantial therapeutic promise, the path to clinical translation is associated with challenges rooted in the complexity of the TRP channels themselves. The development of TRPV1 antagonists serves as an instructive example, providing a framework for understanding the core obstacles that must be overcome.

The promise and peril of TRPV1 antagonists

TRPV1 antagonists were once highly anticipated for pain medicine, showing potent analgesic effects in preclinical models and attracting extensive investment across numerous clinical trials. Most of these trials failed because of on-target hyperthermia [153, 154]. The reason is that TRPV1 is both a pain sensor and a central regulator of core body temperature, so systemic blockade disrupts thermoregulation even as it suppresses pathological pain [155]. This tension between physiological and pathological roles is common among TRP channels, which are frequently embedded in homeostatic processes; TRPML channels, for example, contribute to both viral infection and antiviral immunity, again requiring selective intervention [105]. A viable strategy must therefore target pathological channel activity without compromising basal physiological function.

The translational gap and species-specific discrepancies

A major challenge in pharmacological development is accurately predicting the severity and clinical tolerability of target-mediated adverse effects. While early preclinical studies documented transient hyperthermic responses in rodent and non-human primate models following the administration of TRPV1 antagonists, these animal models underestimated the magnitude, duration, and clinical severity of the hyperthermia subsequently observed in human cohorts [150]. This translational gap stems primarily from divergent species-specific pharmacological adaptations. In preclinical species, the hyperthermic response rapidly attenuated following repeated daily dosing, leading to a premature assumption of long-term clinical safety [156]. However, in human phase I trials (e.g., AMG 517), the hyperthermic response was severe (reaching up to 40.2 °C), persistent, and did not attenuate upon repeated administration [13, 150]. In addition, intrinsic species-specific differences in thermoregulatory physiology, such as the human reliance on TRPV1-coupled cutaneous vasodilation and active sweating versus rodent tail vasodilation, likely exacerbated this clinical disparity. This failure of preclinical models to predict the sustained nature of human hyperthermia led to the termination of numerous clinical trials, underscoring the need for human-centric validation platforms in early drug discovery.

Achieving spatiotemporal selectivity in therapeutic targeting

The failure of TRPV1 antagonists also underscores the substantial challenge of selectivity. This requirement extends beyond simply targeting one TRP channel subtype over another, which is already difficult due to structural homology. Therapeutic precision requires state-dependent or context-dependent targeting. An optimal intervention would preferentially block TRPV1 channels that are in a pathologically sensitized state in nociceptors, while leaving resting channels involved in thermoregulation untouched. The functional context dependency of TRP-TLR interactions, in which the TRPV4-TLR4 axis promotes itch in neurons but inflammation resolution in macrophages, implies that a systemic drug can have opposing effects in different tissues [41, 85]. This functional mosaicism demands therapeutic strategies that can achieve a high degree of spatial, cellular, and state-specific precision.

In summary, the clinical development of TRPV1 antagonists provides a valuable framework for anticipating future challenges. Future success in targeting the TRP-TLR axis will depend not just on developing more potent molecules, but on designing more refined therapeutic strategies. These must be guided by a comprehensive understanding of channel function in both physiological and pathological contexts, validated in human-relevant model systems, and aimed at achieving a level of selectivity that can distinguish a pathologically-active channel from a homeostatic one.

Conclusion and future perspectives

The interaction between TRP channels and TLRs connects environmental sensing with host innate immune defense. This review has systematically elucidated the bidirectional regulatory mechanisms of this immune-sensory signaling axis. TRP channels not only modulate TLR ligand recognition and signal transduction, but their own expression and function are also tightly controlled by TLR signaling. The functional outcomes of this interplay are highly dependent on cell type and subcellular compartmentalization, playing a pivotal role in a multitude of pathophysiological processes, including pain, neuroinflammation, infectious diseases, and metabolic disorders. Although our understanding of the TRP-TLR signaling axis has advanced substantially, numerous major unanswered questions remain, pointing the way for future research.

Future research directions

Elucidating the structural basis and regulatory specificity

Future investigations must transition from phenomenological observations to mechanistic characterizations. To date, direct physical interaction has only been preliminarily characterized for a few TRP-TLR pairs (e.g., TRPV1-TLR4), while the structural basis for most interactions remains unknown. Future research should use high-resolution techniques like Cryo-EM to resolve the three-dimensional structures of TRP-TLR complexes within their native membrane environments (e.g., lipid nanodiscs), thereby revealing their specific binding interfaces and activation conformations to inform rational drug design. Furthermore, the regulatory specificity that TLR signaling exhibits toward TRP channel subtypes remains largely uncharacterized. For instance, why do TLR ligands selectively induce the transcription of TRPML2, yet have no significant effect on the structurally similar TRPML1 and TRPML3? Answering such questions requires comprehensive profiling of transcription factor binding sites within the promoter regions of different TRP genes, coupled with proteomics approaches to systematically map the protein interactome that constitutes the TRP-TLR signalosome in various cellular contexts.

Expanding the scope of known interactions

Current research has predominantly focused on the TRPV subfamily, while the potential roles of other subfamilies, such as TRPC, TRPM, TRPA, and TRPML, in modulating TLR signaling pathways are only beginning to be elucidated. While the interaction between TRPP2 and TLR4 in renal inflammation has recently been characterized, the potential interplay between other TRPP members and PRRs in different tissue contexts warrants further investigation.

Decoding spatiotemporal dynamics

The function of a TRP-TLR signaling pair is dictated by its subcellular localization (e.g., plasma membrane vs. endolysosome). Future studies should employ advanced live-cell imaging and organelle-specific probes to track the trafficking, assembly, activation, and dissociation of these complexes in real-time. Elucidating how TRP-TLR interactions are initiated, sustained, and terminated in different compartments to produce distinct functional outputs is essential for determining their specific roles in physiology and pathology.

Bridging the translational gap

Overcoming the limitations of preclinical animal models is a key objective. Future research must increasingly leverage human-centric model systems. This strategy includes the use of patient-derived cells, induced pluripotent stem cell (iPSC)-derived neurons and immune cells, and organoid/organ-on-a-chip platforms. These models are essential for validating findings in a human genetic context and for understanding the specific pathophysiological contributions of TRP-TLR crosstalk in complex human diseases, such as chronic pain, inflammatory disorders, and neurodegeneration. This shift toward human-centric models is a necessary strategic response to past clinical failures (e.g., TRPV1 antagonists). By providing human-relevant efficacy and safety data before clinical trials are initiated, these models can substantially de-risk the drug development pipeline.

Concluding remarks

By pursuing these directions, the field can transition from cataloging isolated interaction events to constructing an integrated regulatory logic for the TRP-TLR signaling network. This integrated framework will not only advance our fundamental understanding of immune-sensory interplay, but will also facilitate the development of more refined immunomodulatory and sensory-intervention therapies.

Authors’ contributions

Xiaoxing You: Conceptualization, Visualization, Funding acquisition. Chi Zhang: Visualization, Formal analysis. Qianrui Zeng: Writing-original draft, Formal analysis. Chang Liu: Writing-original draft, Formal analysis. Na Xie: Writing-original draft, Formal analysis. Shutong Chen: Writing-review & editing. Haodang Luo: Writing-review & editing, Funding acquisition.

Funding

This work was supported by National Natural Science Foundation of China (82302567), and the Science and Technology Innovation Project of Hengyang Science and Technology Bureau (202330046180).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.