A developmentally informed narrative review of Toll-like receptors in adult and pediatric acute respiratory distress syndrome

Abstract

Background and Objective

Acute respiratory distress syndrome (ARDS) is a severe inflammatory lung syndrome associated with high mortality and morbidity. Toll-like receptors (TLRs), key pattern-recognition receptors of innate immunity, mediate inflammatory responses to both infectious and sterile lung injury. Although TLR signaling has been increasingly implicated in ARDS pathogenesis, significant developmental and translational gaps remain, particularly in pediatric ARDS (PARDS). This review summarizes current evidence on TLR-mediated mechanisms in ARDS and PARDS, with emphasis on developmental regulation and translational relevance.

Methods

A narrative review was conducted using English-language studies identified from PubMed/MEDLINE and Google Scholar databases. Experimental, translational, and clinical studies investigating TLR signaling in ARDS and PARDS were reviewed, including studies involving cell-surface and endosomal TLRs, developmental immunology, animal models, genetic and transcriptomic analyses, and therapeutic modulation of TLR pathways or their key effector cytokines.

Key Content and Findings

TLR4 is an extensively studied receptor in ARDS, mediating inflammatory responses to endotoxins, sterile injury, sepsis, and ventilator-induced lung injury (VILI) through MyD88- and TRIF-dependent pathways. Endosomal TLRs, including TLR3, TLR7, and TLR9, contribute predominantly to viral-induced lung injury and antiviral immunity. Experimental studies demonstrate that TLR activation drives cytokine amplification, neutrophil recruitment, endothelial dysfunction, and alveolar-capillary barrier disruption. Human studies identify TLR-associated polymorphisms and transcriptomic alterations linked to ARDS susceptibility and outcomes. Importantly, pediatric immune responses exhibit developmental regulation of TLR signaling, with age-dependent variation in receptor expression and cytokine production, suggesting PARDS pathogenesis involving TLR activation cannot be directly inferred from adult disease models. However, direct TLR-focused studies in PARDS remain limited.

Conclusions

TLR signaling pathways are central to ARDS and PARDS pathogenesis and represent potential therapeutic targets. However, direct evidence in PARDS remains limited. Future research should prioritize age-stratified pediatric studies to define developmental differences in TLR signaling and guide therapeutic development.

Introduction

Acute respiratory distress syndrome (ARDS) in adults, and pediatric ARDS (PARDS) in children, are severe, life-threatening forms of acute hypoxemic respiratory failure caused by non-cardiogenic pulmonary edema (1). Mortality rates are high, varying with illness severity, geographic location, and ventilation strategy—ranging from 35–52% in adults (2) and 15–40% in children (3,4). Overall, the risk of death increases with age (5), though there are some reported mortality peaks at both ends of the age spectrum (6). Among survivors, they often accrue physical, cognitive, and psychological impairments that contribute substantially to long-term morbidity and healthcare burden (5,7-9).

ARDS and PARDS diagnostic criteria differ to address age-related factors. The Berlin definition [2012] outlines adult ARDS diagnosis, emphasizing PaO₂/FiO₂ ratios, bilateral infiltrates, and ruling out cardiac causes of hypoxemia (10). The Pediatric Acute Lung Injury Consensus Conference (PALICC) provided pediatric-specific criteria in 2015 (11). PALICC uses radiographic evidence of parenchymal lung disease, not requiring bilateral infiltrates, and prefer the oxygenation index over PaO₂/FiO₂, allowing for varied ventilation modes and oxygen saturation-based indices given limited arterial blood gas availability in children (11,12). Epidemiological studies like LUNG SAFE (2) in adults and PARDIE in pediatrics (3) help detail global ARDS incidence and management variability. There is currently no targeted drug therapy proven to consistently lower mortality in ARDS. Supportive, non-pharmacological therapies such as low tidal volume ventilation (13), prone positioning (14), and conservative fluid strategies (15) have reduced mortality. Knowledge gaps remain in PARDS, as most therapies are adapted from adult studies despite differences in disease progression and immune response by age (5,16).

The central tenet to ARDS pathogenesis involves the breakdown of the alveolar-capillary barrier, resulting in increased vascular permeability, alveolar flooding, hypoxemia, and respiratory failure (Figure 1) (15,17). This disruption is driven by injuries to both pulmonary endothelium and alveolar epithelium, alongside leukocyte infiltration, platelet aggregation, microvascular thrombosis, and fibrin deposition (18,19). Endothelial dysfunction—including inflammation and disruption of cell junctions—further increases vascular leakage and cell death, fueling lung injury through a self-amplifying immune response (18,20). ARDS often develops from a range of direct and indirect triggers such as sepsis, pneumonia, trauma, or transfusion (2,3), which activate innate immunity via pattern recognition receptors (PRRs) like Toll-like receptors (TLRs) (21-24). TLRs recognize both pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS), viral RNA, and fungal components, and damage-associated molecular patterns (DAMPs), including high mobility group box 1 protein (HMGB1), heat shock proteins (HSPs), mitochondrial DNA, and extracellular ATP (25-34). By sensing both infection and cellular injury, TLRs play an important role in regulating the immune activation in the lungs by bridging external insults to internal inflammatory responses. Studies on innate immunity have targeted upstream inflammatory sensors such as TLRs, which are essential in mediating host responses to both infectious and sterile lung injury (24,35,36). However, despite growing evidence, TLR-directed therapies have yet transitioned into clinical practice, indicating the need for more translational research in this area (24,36-38). This review summarizes the current and emerging knowledge on TLR signaling in ARDS, highlighting both infectious and sterile mechanisms of injury, with a special emphasis placed on the developmental regulation and translational relevance to PARDS. We aim to highlight limitations in current research models, developmental distinctions, key mechanistic insights, and knowledge gaps in PARDS. We present this article in accordance with the Narrative Review reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-2026-0138/rc).

Figure 1.

Pathophysiology of ARDS. In healthy lungs, the alveolar interface consists of intact epithelial and endothelial barriers, surfactant-producing AEC II, and quiescent AMs. When PAMPs—derived from viral, bacterial or fungal products—enter the alveolar space, they are recognized by TLRs. This recognition triggers downstream inflammatory cascades, including cytokines such as IL-1β, IL-6, TNF-α, CCL2, and IFN-β, many of which recruit neutrophils and monocytes. These cytokines disrupt the alveolar-endothelial barrier by injuring epithelial cells and pulmonary capillary endothelial cells. Barrier disruption increases vascular permeability, allowing protein-rich fluid and immune cells to enter the alveolar space. Proteinaceous fluid inactivates surfactant, while recruited neutrophils release ROS, promoting apoptosis of AEC II cells and further reducing surfactant production. Together, these processes exacerbate alveolar collapse and impair gas exchange. Damaged AEC II cells and other host cells also release DAMPs, such as HMGB1 and S100A8/A9, into the alveolar space, where they can re-stimulate TLRs and amplify cytokine and ROS production. This self-perpetuating inflammatory loop contributes to hallmark features of ARDS pathophysiology, including diffuse alveolar damage, protein-rich alveolar edema, epithelial cell shedding, and hypoxemia. AEC II, type II alveolar epithelial cells; AM, alveolar macrophage; ARDS, acute respiratory distress syndrome; DAMP, danger-associated molecular pattern; IL, interleukin; PAMP, pathogen-associated molecular pattern; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, tumor necrosis factor.

Methods

This review evaluated the role of TLRs in ARDS and PARDS by searching PubMed/MEDLINE and Google Scholar databases for English-language studies up to May 2026. Search terms included “acute respiratory distress syndrome”, “pediatric acute respiratory distress syndrome”, “Toll-like receptor”, “TLR”, “innate immunity”, “lung injury”, “sepsis”, “viral infection”, “inflammation”. and “acute lung injury”, etc. The review covered experimental, translational, and clinical studies on TLR signaling in ARDS/PARDS, prioritizing work on TLR-mediated inflammation, development of innate immunity, animal models, human genetic/transcriptomic data, and therapeutic strategies. Study selection was performed by a single author (C.W.T.F.). Foundational research on TLR biology and ARDS mechanisms was also included. Due to its narrative approach, study selection focused on relevance rather than adhering to systematic review criteria. The search strategy summary appears in Table S1.

Results

Overview of TLRs in ARDS and PARDS

TLRs are germline-encoded receptors which detect conserved microbial motifs termed as PAMPs for innate immune recognition. Each TLR exhibits distinct ligand specificity, cellular localization, and downstream signaling architecture (18,24,27-32,39-41). Surface-expressed TLRs—such as TLR1, TLR2, TLR4, TLR5, and TLR6—primarily recognize bacterial-derived PAMPs, including lipoproteins, peptidoglycan, flagellin, and LPS (Figure 2) (29,32,42,43). Among these, TLR4, which senses LPS, has been strongly implicated in sepsis-associated ARDS and endotoxin-induced lung injury, with knockout models demonstrating profound hypo-responsiveness to LPS (44), resulting in attenuated cytokine release, reduced neutrophilic infiltration, and protection from lung injury. In contrast, endosomal TLRs—TLR3, TLR7, TLR8, and TLR9—recognize nucleic acids and mediate viral lung inflammation (29,32,42,43,45), including severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-induced ARDS (45). Sterile forms of ARDS [e.g., trauma, transfusion-related acute lung injury (ALI)] also engage TLRs via DAMPs, for example, HMGB1 and HSP activate TLR2 and TLR4 (27,29,31), while oxidized phospholipids (18) and S100A8/A9 (30) interact with TLR4 (46). A summary of TLR characteristics is presented in Table 1.

Figure 2.

Canonical MyD88-dependent and TRIF-dependent pathways of surface and endosomal TLR receptors. In resting cells, surface TLRs exist as monomers. Ligand binding induces homo- or heterodimerization, bringing intracellular Toll/IL-1R (TIR) domains together and recruiting TIRAP, MyD88, IRAK1, and IRAK4 to form the Myddosome. IRAK4 phosphorylates IRAK1, enabling TRAF6 interaction, K63-linked auto-ubiquitination, IRAK1 degradation, and IRAK4 recycling. Endosomal TLRs recognize viral nucleic acids after viral uptake and endosomal trafficking, undergo homodimerization, and activate a similar MyD88-dependent cascade. Ubiquitinated TRAF6 activates the TAK1-TAB1/2/3 complex, which drives NF-κB and MAPK signaling. In the NF-κB pathway, TAK1 activates IKKβ, leading to IκBα degradation, NF-κB nuclear translocation, and transcription of inflammatory cytokines including IL-6, TNF-α, and IL-1β. In the MAPK pathway, TAK1 activates MAPKs and downstream JNK, p38, and ERK; JNK and p38 are particularly relevant to inflammatory signaling in ARDS. TLR4 can also initiate TRIF-dependent signaling after endocytosis, activating NF-κB/MAPK through RIP1, TRAF6, and TAK1, and IRF3-mediated type I interferon transcription through TRAF3, NAP1, TBK1, and IKKε. TLR3 signals through TRIF without TRAM. ARDS, acute respiratory distress syndrome; IL, interleukin; TLR, Toll-like receptor; TNF, tumor necrosis factor.

Table 1. A Summary of Toll-like receptors, their PAMP/DAMP recognition and signaling pathways.

TLR	Location	PAMP (ligand)	DAMP (ligand)	Signaling pathways	Key outcomes
TLR1	Cell surface	Triacyl lipopeptide	–	NF-κB, MAPK via MyD88-dependent pathway	TNF-α, IL-1β, IL-6, IL-12
TLR2	Cell surface	Lipoteichoic acid; arabinomannan; peptidoglycan; zymosan; lipoprotein; pore protein	HMGB1; HSP60/70; hyaluronic fragments; biglycan	NF-κB, MAPK via MyD88-dependent pathway	TNF-α, IL-1β, IL-6, IL-10
TLR3	Endosomal	dsRNA	Self-dsRNA	NF-κB, IRF3 via TRIF-dependent pathway	IFN-α, IFN-β, IFN-λ, TNF-α, IL-6, IL-12
TLR4	Cell surface and endosome	Lipopolysaccharides; RSV fusion protein; Ebola glycoprotein	HMGB1; HSP60/70/90; S100A8/A9; biglycan; hyaluronic fragments; oxidized phospholipids	NF-κB, MAPK, IRF3 via MyD88- and TRIF-dependent pathway	TNF-α, IL-6, IL-1β, MIP-2
TLR5	Cell surface	Flagellin	–	NF-κB, MAPK via MyD88-dependent pathway	TNF-α, IL-8, IL-6, IL-1β
TLR6	Cell surface	Lipoteichoic acid; Diacylated lipopeptides	HMGB1; HSP60/70; hyaluronic fragments; biglycan	NF-κB, MAPK via MyD88-dependent pathway	TNF-α, IL-10, IL-6, IL-1β
TLR7	Endosome	ssRNA; imidazoquinoline	–	NF-κB, MAPK, IRF7 via MyD88-dependent pathway	IL-1β, IL-6, IL-12, TNF-α, IFN-α, IFN-β
TLR8	Endosome	ssRNA	–	NF-κB, MAPK, IRF7 via MyD88-dependent pathway	IL-1β, IL-6, IL-12, TNF-α, IFN-l
TLR9	Endosome	Unmethylated CpG DNA	Mitochondrial DNA; self-DNA	NF-κB, IRF7 via MyD88-dependent pathway	IL-1β, IL-6, TNF-α, IFN−α, IFN−β
TLR10†	Cell surface	dsRNA	–	NF-κB via MyD88-dependent pathway	Unknown

^†, TLR10 remains the least understood human TLR. While some studies suggest anti-inflammatory roles and dsRNA recognition, its function is still under investigation. CpG, cytosine-phosphate-guanine; DAMP, damage-associated molecular pattern; IL, interleukin; PAMP, pathogen-associated molecular pattern; RSV, respiratory syncytial virus; TLR, Toll-like receptor; TNF, tumor necrosis factor.

TLR10 is the most recently identified member of the TLR family and remains one of the least understood, with evidence suggesting both anti- and pro-inflammatory functions depending on cellular context (32,47,48). Unlike most TLRs, TLR10 appears to predominantly exert immunomodulatory effects, including suppression of NF-κB, MAPK, and type I interferon signaling pathways in response to selected bacterial and viral stimuli (47,49). Studies have also demonstrated a role for TLR10 in regulating responses to viral dsRNA (49), suggesting potential relevance in respiratory viral infections. However, the absence of a murine ortholog limits preclinical investigation (47,49-51), and the translational relevance of existing TLR10-transgenic mouse models remains uncertain. To date, TLR10 has not been directly studied in either adult ARDS or PARDS. Given its reported anti-inflammatory properties and involvement in viral innate immune responses, future studies should evaluate whether TLR10 contributes to the regulation of hyperinflammation in ARDS, particularly in virally mediated PARDS.

Understanding TLR structure, classification, and signaling is essential to discern their functions in immunity and disease. TLRs are type I transmembrane proteins with three domains: an extracellular leucine-rich repeat (LRR) region for ligand recognition, a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain that mediates downstream signaling (52). Synthesized in the endoplasmic reticulum, TLRs are trafficked by chaperone proteins to the cell surface or endosome. They remain as monomers until ligand binding triggers dimerization—either as homodimers (e.g., TLR4/TLR4) or as heterodimers (e.g., TLR2/TLR1)—to initiate downstream cascades (52).

TLR signaling pathways

The two primary TLR downstream signaling pathways are the MyD88-dependent and TRIF-dependent pathways (Figure 2). The MyD88-dependent pathway is utilized by all TLRs excluding TLR3, where the activation of NF-κB and MAPKs signaling results in the up-regulation and transcription of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) (21,32,52). On the other hand, the TRIF-dependent pathway is only used by TLR3 and partly by TLR4. The TRIF-dependent cascade activates interferon regulatory factors IRF3 and IRF7, inducing the production of type 1 interferons crucial for antiviral defense (32,43,52). TLR4 is unique because it employs both pathways: MyD88 signaling occurs at the plasma membrane, while TRIF signaling occurs post-endosomal internalization. This spatial segregation of MyD88 and TRIF signaling allows for a coordinated inflammatory and antiviral response (29,43,52). Spatial signaling dynamics influence the outcome—TRIF-dependent signaling is often localized to endosomal compartments, contributing to specificity.

To maintain homeostasis and prevent aberrant TLR signaling, several negative regulatory mechanisms are involved, including suppressor regulators (e.g., A20, IRAK-M, SOCS1), ubiquitin-mediated degradation, and decoy receptors (e.g., soluble TLRs) (29,43,52). Recent transcriptomic data from ARDS patients show alternative splicing of key TLR pathway genes, which suggests a context-specific, post-transcriptional fine-tuning of signaling responses (53). Additionally, TLR4 activation has been shown to upregulate TLR3 expression, indicating receptor crosstalk (23,54), and models propose that synergistic TLR/NOD-like receptor (NLR) co-activation may be the cause of the cytokine storm phenotype in ARDS and coronavirus disease 2019 (COVID-19) (46,55). These signaling pathways coincide with the pathophysiological processes central to ARDS described in adult patients: excessive cytokine release, leukocyte recruitment, and breakdown of the alveolar-capillary barrier. Though the question of whether these mechanisms are observed uniformly across all age groups remains unclear.

Surface TLR activation in experimental ARDS models

The TLR4 model is commonly used to study endotoxin-induced ARDS, as intratracheal or intravenous LPS delivery reliably triggers a dose-dependent inflammatory response that mimics key features of human ARDS. These include diffuse alveolar damage (56,57), neutrophilic infiltration (18,57,58), along with disruption of alveolar-capillary barrier integrity and the formation of hyaline membrane due to proteinaceous exudation into the alveolar space (56,57) and elevated pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β) in bronchoalveolar lavage fluid (59). TLR4 signaling initiates cytokine amplification, endothelial leak, and oxidative stress, closely reflecting the exudative phase seen in human ARDS.

TLR4-deficient mice demonstrated resistance to LPS, reduced pulmonary inflammation, and features resembling ARDS rescue, indicating the importance of TLR4 activation in endotoxin-induced injury (44,60). TLR4 deficiency reduced cytokine release, neutrophil infiltration, and lung injury (44,60), but in live polymicrobial sepsis, complete TLR4 deficiency impaired host defense and increased mortality due to poor immune response and bacterial spread (61,62). Endothelial-expressed TLR4 is critical for LPS-induced neutrophil sequestration to the lungs, highlighting the vascular compartment’s role in lung injury (63). TLR4 also detects certain viral components, such as respiratory syncytial virus (RSV) fusion protein and Ebola virus glycoproteins (64). Since viral infections are a primary cause of PARDS, further investigations into this TLR’s role in PARDS development and progression is warranted.

TLR4 activation has been demonstrated in many non-infectious models of ARDS. In a murine model of ventilator-induced lung injury (VILI), MyD88-dependent TLR4 signaling pathway is demonstrated to induce the release of IL-6 and macrophage inflammatory protein 2 (MIP-2), amplifying sterile inflammation via the TLR4-MyD88 axis (56). In acid aspiration-induced ALI, oxidized phosphatidylcholine—generated through reactive oxygen species-mediated oxidation of membrane polyunsaturated fatty acids—activates TLR4 independently of MD2 and CD14, requires the TRIF-TRAF6 pathway, and releases IL-6 (18). S100A8/A9 and HMGB1 molecules act as DAMPs exacerbating VILI via TLR4, associating intracellular damage to innate immune activation (30,33).

Lastly, TLR4 expression and responsiveness are developmentally regulated (65-67), with neonatal immune cells exhibiting attenuated TLR4 signaling compared to adults, influencing susceptibility and inflammatory responses in PARDS. Cord blood mononuclear cells from neonates produce significantly lower levels of pro-inflammatory cytokines such as TNF-α and IL-6 after TLR4 and TLR9 stimulation, with responses gradually increasing over the first year of life. By 12 months, cytokine production approached 80–90% of adult levels, suggesting progressive maturation (65). Similarly, monocyte expression of TLR4 in pre-term infants is reduced at birth but increases significantly over the first three months postnatally, indicating an age-dependent acquisition of innate immune capacity (66). Both findings support the concept that TLR signaling is dynamically programmed during early life, modulated by intrinsic developmental cues and environmental exposures (67).

While TLR4 remains the most extensively studied receptor in ARDS, the roles of other cell-surface TLRs—TLR1, TLR2, TLR5 and TLR6—have been implicated in pulmonary inflammation to a lesser degree. TLR2 recognizes bacterial lipoproteins and has been demonstrated in a few lung injury models. In a cecal ligation and puncture (CLP)-induced murine model of sepsis-associated ARDS, TLR2 knockdown attenuated histologic lung injury and suppressed pro-inflammatory cytokine expression, including TNF-α and IL-6 (19). Similarly, TLR2-/- mice subjected to pulmonary contusion exhibited reduced neutrophilic infiltration and alveolar damage, supporting its contributory role in sterile lung injury models (68). Evidence for TLR5 has been studied in bacterial infection-induced airway inflammation rather than in ARDS, as it did not demonstrate histopathologic or physiologic ARDS/ALI phenotypes (69). Overall, studies of TLR1, TLR5 and TLR6 in ARDS remain limited, suggesting TLR4 to be the dominant surface receptor driving lung injury pathogenesis.

Endosomal TLR activation in experimental ARDS models

Endosomal TLRs—TLR3, TLR7, TLR8, and TLR9—mediate responses to viral nucleic acids, triggering inflammation. In the human lung, these receptors are found on both non-immune structural cells and immune cells in the airway epithelium and alveolar space, including alveolar epithelial cells and macrophages (70-72): TLR3 is predominantly expressed in airway epithelial cells (70,71); TLR7/8 are found in the plasmacytoid, myeloid dendritic cells (73) and monocytes (74), and TLR9 in B cells and plasmacytoid dendritic cells (75,76). This is in contrast to TLR4, which is ubiquitously expressed across both structural and immune cells (77).

In viral infection models like influenza and SARS-CoV-2 (78), endosomal TLRs recognize nucleic acids: TLR3 detects dsRNA, TLR7/8 detects ssRNA, and TLR9 senses unmethylated cytosine-phosphate-guanine (CpG) DNA motifs (Table 1). Experimental models of endosomal TLR activation have utilized ligands such as Poly I:C (TLR3 agonist) (42), R848/Imiquimod (TLR7/8 agonists) (79,80), and CpG-oligonucleotides (TLR9 agonist) (29). Unlike TLR4 models, which trigger rapid cytokine release and endothelial dysfunction (58), endosomal TLR models focus on epithelial-centric responses with delayed inflammation and viral clearance (81). Epithelial TLRs coordinate neutrophil recruitment and cytokine responses, indicating their critical role in viral ARDS (82,83).

TLR3-deficient mice exhibited attenuated cytokine production, reduced pulmonary inflammation, and improved survival following lethal influenza A infection, despite higher viral loads, indicating TLR3-driven inflammation mainly causes disease severity (84). In contrast, TLR3/TRIF signaling reduced viral load and disease severity in SARS-CoV-infected mice, suggesting weak TLR3 responses may worsen ARDS by impairing antiviral immunity (78). Non-infectious stimuli like mechanical ventilation can also activate TLR3-driven inflammation through a MyD88-dependent, TLR4-independent mechanism (85).

TLR7, like TLR3, contributes to viral-induced lung injury. In mice, TLR7 deficiency increased upper airway inflammation but impaired lower respiratory immunity following influenza A infection, indicating its compartmentalized immune functions (86). TLR7 contributes to both acute and chronic viral-induced lung injury. Experimental studies demonstrate that TLR7 activation promotes cytokine release, barrier dysfunction, and sustained airway inflammation (87-89). Recent translational profiling in severe viral PARDS further demonstrated interferon-driven immune dysregulation with impaired TLR7-induced IL-1β responses, suggesting that excessive antiviral interferon signaling may paradoxically contribute to dysfunctional innate immunity in pediatric lung injury (74).

Experimental lung injury models on TLR8 and TLR9 are limited. Due to limited models and lack of functional mouse TLR8 homologs, TLR8’s role in ARDS remains unclear. Transgenic mice expressing TLR8 display lung expression and cytokine response during Mycobacterium tuberculosis infection (90), but their relevance for viral studies remains undetermined. TLR9 activates pulmonary inflammation in murine models, causing neutrophilic infiltration and cytokine release (91). In a lung contusion model, TLR9-/- mice exhibited reduced cytokines and lung injury, implicating TLR9 activation in sterile trauma-induced lung inflammation (92).

Models of viral lung injury provide some insight into nucleic acid sensing and cytokine production but do not capture important aspects of human ARDS like endothelial injury, multi-phase progression, and immune dysregulation (93,94). It is possible that ARDS animal models are limited by simplified triggers and difficulties replicating human physiology (95).

Multi-TLR activation in experimental ARDS models

Individual TLR activation models are limited in replicating the complex and varied nature of clinical ARDS. Animal models often fail to capture factors like immune senescence, co-morbidities, and polymicrobial triggers, leading to a disconnect between preclinical mechanistic success and clinical outcomes (96-98). The omission of these important variables contributes to the translational disconnect between mechanistic success in animal models and their failure at bedside, highlighting the complexity of replicating human ARDS in simplified systems (99). While these models may provide mechanistic insights, they do not replicate the entirety of the pathophysiology of ARDS per se (96-98). Sepsis-induced ARDS features broad TLR activation, with increased TLR2 and TLR4 expression and cytokine production observed in septic mice; however, murine sepsis models yield inconsistent ARDS phenotypes and mortality rates (77,100,101).

A CLP mouse model was used to study the activation of multiple TLRs in sepsis-induced ARDS. The release of fecal material into the peritoneal cavity triggers an immune response induced by polymicrobial sepsis, characterized by increased pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), neutrophil infiltration and alveolar structural damage (19). Knockdown of TLR2, TLR4, and TLR9 attenuated histopathological lung injury and pro-inflammatory cytokines, namely TNF-α and IL-6 (19), while TLR3 knockdown showed no effect, implying that it is not a pivotal contributor to sepsis-induced lung injury (19). However, variability in microbial content due to CLP technique limits reproducibility and complicates pathogen-TLR interaction analysis, affecting the translation of findings to human cases.

Sterile lung injury models show varying roles for TLRs compared to microbial models. The bleomycin-induced lung injury model is extensively utilized to investigate sterile inflammation and fibrosis, as intratracheal administration of bleomycin induces epithelial injury, alveolar inflammation, and progressive pulmonary fibrosis, which closely resemble human ARDS characteristics (102). Inhibiting TLR9 in this model reduces lung inflammation, cell death, and fibrosis, suggesting endogenous CpG-rich DNA may drive ARDS via the TLR9 pathway (102). Conversely, mice lacking both TLR2 and TLR4 suffer worse lung injury and survival after bleomycin exposure due to impaired repair mechanisms, highlighting the complex and context-dependent functions of multiple TLRs in sterile ARDS (39). Additionally, trauma-hemorrhagic shock models indicate TLR4 activation by DAMPs or gut bacteria links systemic and lung inflammation (103).

Collectively, these studies suggest that multiple TLRs contribute to lung injury in ARDS of infective or sterile etiologies, although their roles may vary depending on the context. Consequently, a comprehensive understanding of stimulus-specific and receptor-specific roles in ARDS is crucial for the development of targeted interventions and models.

Large-animal models and translational relevance

Large-animal ARDS models, particularly swine models, have been used less frequently to investigate specific TLR pathways and are more commonly employed to study the mechanical aspects of VILI due to their close resemblance to human pulmonary anatomy and physiology. In VILI models, where lung size influences mechanical stretch and injury, injurious mechanical ventilation post polytrauma has been shown to induce alveolar damage and inflammation (102), potentially involving HMGB1-induced TLR4 activation (33). Other models combine acid aspiration or sepsis with injurious mechanical ventilation and reproduce key clinical features of ARDS, including hypoxemia, elevated inflammatory cytokines, and multi-organ dysfunction (103-105). Although direct investigation of TLR signaling in large-animal ARDS models remains limited, these systems provide an important translational platform for studying lung injury mechanisms and validating findings generated in small-animal and in vitro models.

Human ARDS studies: genetic and transcriptomic variation in TLR pathways

Animal studies have clarified important TLR mechanisms in ARDS, but human ARDS is complicated by immune diversity, co-morbidities, and multi-organ dysfunction. Animal models rarely capture this complexity, especially for PARDS, where juvenile animal models are rarely used. Age-specific immune responses and lung development are often overlooked, which challenge the adequacy of adult-centric models to represent pediatric lung pathophysiology (106). Conversely, age-specific TLR development, immune composition, and lung architecture remains largely unexamined in preclinical studies, limiting their translational application in pediatric disease. To bridge these gaps, human research has focused on blood transcriptomics, immune signaling, and genetics to better understand TLR pathways in ARDS. These studies can clarify the specific roles of TLRs across different ages, causes, and clinical outcomes.

Genetic variations in TLR pathways may influence ARDS susceptibility and outcomes. In esophageal cancer surgery patients, TLR4 single-nucleotide polymorphisms (SNPs) increased the risk and severity of developing postoperative ARDS (107). SNPs in the TLR4/NF-κB signaling axis, TNF-α (rs1800629), IL-6 (rs1800769), and MyD88 (rs7744) were associated with increased gene expression, greater ARDS incidence, and worse prognosis, indicating their potential as prognostic biomarkers (108). Conversely, the TLR4 Asp299Gly (rs4986790) variant was protective against severe COVID-19, highlighting the role of TLR4 genotypes in modulating viral-induced lung injury (109).

Children with inherited TLR3 deficiency in PARDS developed severe influenza pneumonitis, linked to TLR3-loss-of-function mutations (e.g., P554S, P680L) (110). These mutations impaired type I & III interferon responses and viral clearance, highlighting TLR3’s antiviral role in the respiratory epithelium (110). Additionally, TLR3 rs5743313 CT genotype (one C allele, one T allele) is associated with severe pandemic H1N1 influenza cases (111).

Polymorphisms in the promoter of TLR1 (rs5743551, -7202A>G) were found to increase TLR1 surface expression upon LPS exposure, leading to excessive TNF-α and IL-6 levels (112). Carriers of this G allele were more likely to develop ALI and had worse outcomes in sepsis-related ARDS, demonstrating that hypermorphic TLR1 signaling may amplify harmful inflammation. This suggests broader TLR variants beyond TLR4 justify investigation into ARDS susceptibility and prognosis.

Beyond DNA-level polymorphisms, post-transcriptional regulation of TLR pathway genes may affect individual ARDS outcomes. Peripheral blood mononuclear cells (PBMCs) from ARDS patients revealed alternative splicing in key components like MyD88 and IRAK1 (53). Lower levels of the inhibitory MyD88 short isoform correlated with greater disease severity, while increased expression of an IRAK1 variant lacking exon 11 was observed (53). These splice variants likely impact inflammation and may explain variability in ARDS severity and treatment responses.

Pediatric perspectives: developmental regulation of TLR signaling

Infants and young children show age-dependent differences in TLR expression and function that may influence susceptibility, disease severity, and therapeutic responses in PARDS. Compared with adults, neonates and infants display attenuated TLR-mediated cytokine activity and a relative Th2-skewed immune profile, suggesting developmental modulation of innate immune responses (66,80,113,114). Furthermore, TLR2 regulation appears to be developmentally variable whereas TLR4-mediated endotoxin sensing remains relatively conserved during early life, indicating receptor-specific maturation patterns (115). Despite increasing recognition that TLR signaling is developmentally regulated (116-118), few studies have directly examined how these age-related differences influence PARDS pathobiology. Understanding these developmental trajectories is essential for interpreting pediatric lung injury and for designing age-appropriate immunomodulatory therapies.

In early life, TLR signaling is tightly regulated, with neonates and children displaying distinct receptor profiles and cytokine responses compared to adults. Research indicates that innate immune responses, especially those mediated by TLRs, mature at different rates: for example, TNF-α and IL-6 responses approach adult levels by 12 months (65,67), while TLR3-driven IFN-β production continues increasing through 24 months (67,119). These findings show that innate immunity develops along unique timelines for each TLR, suggesting infants and toddlers may have different lung-inflammatory profiles and thus varying risks and treatment responses in PARDS.

At the cellular level, pre-term and term neonates exhibit attenuated TLR-mediated responses, including reduced IL-6 and TNF-α production following TLR4 (LPS) and TLR9 (CpG) stimulation, which may contribute to impaired early-life infection control (120). Developmental differences extend beyond receptor expression, with age-related changes in transcriptional regulation influencing cytokine responses following TLR activation (121,122). Neonatal immune cells also preferentially adopt regulatory and Th2-skewed cytokine programs, including increased IL-10 production, potentially limiting excessive inflammation at the expense of efficient pathogen clearance (118). Consistent with these findings, longitudinal studies have demonstrated age-dependent increases in TLR-induced cytokine responses throughout infancy and early childhood, confirming that TLR function continues to mature beyond the neonatal period (116,123). Together, these observations support the concept that pediatric innate immune responses are qualitatively distinct from adults, consistent with pulmonary immune profiling studies in severe viral PARDS demonstrating interferon-dominant immune signatures and altered TLR7-associated cytokine responses (74).

Recent experimental work extends this concept: co-stimulation of TLR7/8 and the C-type lectin Mincle in newborn dendritic cells uniquely enabled Th-1 polarization—a pathway underrepresented in early life (124). Furthermore, transcriptomic signatures in pediatric septic shock patients differ with increasing age, implying that innate programs keep evolving well into childhood (125). Fundamental disparities in lung injury responses between childhood and adults are noted too (126,127), emphasizing the importance of integrating age-specific differences in immunologic responses in PARDS models. Therefore, these studies paint a unified picture: TLR pathways are not simply “dampened” in children but mature in a ligand- and receptor-specific manner, implying that the severity of pediatric lung injury will vary with both age and nature of the PAMP or DAMP stimulus.

While these developmental studies are not PARDS specific, they provide important biological context for age-stratified pediatric lung injury. In a sepsis-related ARDS model, administration of IFN-β restored alveolar macrophage function, reduced TNF-α and IL-6 levels, improved bacterial clearance, and enhanced survival (128). Although direct studies in ARDS remain limited, IFN-β has also been proposed—based on its known epithelial-protective properties—to strengthen alveolar barrier integrity and promote surfactant release, suggesting additional mechanisms by which TRIF/TLR3-driven IFN-β might ameliorate lung injury. Given that TLR3-mediated IFN-β responses continue to mature throughout early childhood (119), developmental differences in this pathway may contribute to age-dependent variation in host responses to lung injury and infection. By contrast, TLR2/4 signaling reaches near-adult levels by approximately 12 months of age (119), suggesting that receptor-specific maturation may influence how PARDS manifests across different pediatric age groups. These developmental differences may partly contribute to the distinct clinical phenotypes and outcome patterns observed between pediatric and adult ARDS, although direct evidence remains limited.

Despite growing evidence that TLR signaling is developmentally regulated, direct investigation of TLR pathways in PARDS remains extremely limited. To date, only a small number of studies have identified polymorphisms in TLR2 (rs5743708) and TLR4 (rs4986790) associated with susceptibility to infectious ARDS in children (129). Most pediatric studies have instead focused on downstream inflammatory mediators, with elevated levels of IL-6, IL-8, MCP-1, IP-10, TNF-α, IL-12p70, IL-17A, GM-CSF, and IFN-α in severe PARDS patients (130). Several of these cytokines are canonical downstream products of TLR signaling pathways, suggesting that innate immune activation contributes to disease pathogenesis. Recent multi-omics pulmonary immune profiling further demonstrated interferon-driven immune dysregulation and suppression of TLR7-induced IL-1β responses in severe viral PARDS, highlighting pediatric-specific innate immune mechanisms distinct from those described in adult (74). Collectively, these findings suggest that while downstream signatures of TLR activation are detectable in PARDS, the upstream receptor-level mechanisms remain largely unexplored. Consequently, future studies should integrate age-stratified analyses with direct assessment of TLR expression, signaling, and function to better define the contribution of innate immune pathways to PARDS pathobiology.

Emerging TLR-targeted therapies in ARDS

Given the central role of TLR signaling in ARDS pathogenesis, several TLR-targeted therapies have been evaluated in preclinical models and early-phase clinical studies. However, their limited success in clinical trials illustrates a recurrent translational problem: inhibition of a single innate immune receptor rarely matches the biological complexity of ARDS. Eritoran, a TLR4 antagonist, improved survival in influenza-infected mice (131,132), but did not reduce mortality in patients with severe sepsis (133). This failure likely reflects several factors: trial populations were not enriched for patients with demonstrable TLR4- or endotoxin-driven biology; treatment may have been administered after downstream inflammatory cascades were already established; and sepsis-associated ARDS involves redundant PAMP- and DAMP-sensing pathways, including TLR2, TLR9, complement, inflammasomes, and endothelial injury pathways. Thus, blockade of TLR4 alone may be biologically insufficient once systemic inflammation, immunosuppression, coagulopathy, and multi-organ dysfunction have evolved. Direct viral lung injury may also differ fundamentally from indirect extrapulmonary ARDS, as pulmonary-compartmentalized antiviral inflammation may engage distinct TLR-adaptor programs compared with polymicrobial sepsis.

Other agents indirectly modulating TLR4 have shown benefit in preclinical studies. Propofol, a common anesthetic agent, was shown to suppress the TLR4-HMGB1 axis (134), attenuating cytokine release and lung injury in experimental models. TAK-242, a selective inhibitor of intracellular TLR4-TIR domain signaling (135), potently suppressed TLR4-mediated signaling in murine and human cell models, yet failed to significantly reduce IL-6 levels or improve clinical outcomes in a randomized placebo-controlled severe sepsis trial (136). These results suggest that target engagement in simplified experimental systems does not guarantee meaningful pharmacodynamic suppression in critically ill patients. Clinical ARDS encompasses variable timing of injury onset, mixed infectious and sterile triggers, differences in hyperinflammatory versus hypoinflammatory phenotypes, variable pathogen burden, and concurrent supportive therapies. Without biomarker-guided enrichment, a therapy that may benefit a narrow TLR-driven subgroup can appear ineffective when tested across an unselected syndrome-level population.

Endosomal TLR antagonists further highlight the difficulty of balancing immune suppression against host defense. Chloroquine (102), hydroxychloroquine (137), and ODN2088 (TLR7/9 inhibitor) (102,138) reduced inflammation in murine models by impairing ligand binding or endosomal acidification. However, the direction of effect is highly timing-dependent: early TLR7 antagonism can impair viral clearance, whereas later inhibition may reduce immune-mediated injury (88). This duality may explain why broadly dampening antiviral innate sensing has not consistently improved clinical outcomes. IL-1 blockade using anakinra in a murine COVID-19 ARDS model displayed efficacy in reducing classical hallmarks of inflammation (139), but did not shorten time to clinical improvement in adults (140). IL-6 blockade with tocilizumab showed encouraging observational signals (141), but larger randomized trials, including COV-AID (140), failed to demonstrate consistent survival benefits or improvements in primary clinical endpoints. In contrast, the JAK1/2 inhibitor baricitinib demonstrated a reduction in mortality in the phase III COV-BARRIER trial (142,143) despite not meeting all primary endpoints, supporting the possibility that broader modulation of convergent downstream inflammatory pathways may be more effective than receptor- or cytokine-specific blockade in heterogeneous ARDS populations.

Newer strategies include prophylactic TLR activation to prime antiviral immunity. Rather than suppressing inflammation, this approach seeks to induce a transient antiviral state prior to infection by enhancing innate immune readiness and promoting early interferon-mediated responses (144). Intranasal TLR2 agonism is being investigated as a preventative therapy capable of providing broad protection against respiratory viruses (144), an approach that may be particularly relevant to viral PARDS, where respiratory viral infections such as rhinovirus are important etiologic contributors (145). Nevertheless, this strategy may be risky in pediatric populations, particularly in tropical regions where respiratory virus circulation is less predictable, because unnecessary TLR activation could provoke inflammation in uninfected individuals or in children with developmentally distinct immune responses. A summary of potential TLR-associated therapies for ARDS is presented in Table 2.

Table 2. Proposed TLR pharmacologic therapies for ARDS or ARDS like conditions.

Pharmacologic agent	Mechanism of action/target	Clinical development
Eritoran	TLR4 antagonist	Phase III severe sepsis trial: ACCESS (133)—no difference in mortality or clinical outcomes
Tocilizumab/sarilumab	IL-6 receptor	Phase III COVID-19 trials:
		RECOVERY (146), REMAP-CAP (147), EMPACTA (148)—reduced mortality and improved outcomes
		COVACTA (149)—no difference in mortality
		WHO REACT (IL6 antagonists) metanalysis (150)—reduced mortality in hospitalized COVID-19 patients
Anakinra	IL-1 receptor antagonist	Phase III COVID-19 trials:
		SAVE-MORE (151)—reduced mortality in hospitalised COVID-19 patients
		CORIMUNO-ANA-1 (152)—no difference in mortality
		Phase III sepsis trials—no difference in mortality (153) but reanalysis of the subgroup with macrophage activation syndrome showed improved mortality (154)
Sivelestat	Neutrophil elastase inhibitor	Phase III acute lung injury trial: STRIVE (155)—terminated prematurely due to negative trend in long-term mortality
Baricitinib	JAK1/2 inhibitor	Phase III COVID-19 trials:
		COV-BARRIER (143), RECOVERY (156) trials—reduced mortality in hospitalized COVID-19 patients
		ACTT-2 (157), ACTT-4 trials (158)—no difference in mortality
Tofacitinib	JAK inhibitor downstream of TLR-mediated cytokine storm	Phase II/III STOP-COVID trial (159)—reduced risk of mortality in hospitalized COVID-19 patients
TAK-242 (Resatorvid)	Inhibits TLR4 signaling at TIR domain	Phase III severe sepsis trial (136)—no difference in mortality of cytokine levels
Enpatoran	TLR7/8 inhibitors	Phase II COVID-19 trial: ANEMONE (160) trial—no difference in recovery time in hospitalized COVID-19 patients
Propofol	Indirectly inhibits HMGB1-TLR4 axis	Preclinical studies only (134)
Albiflorin	TLR4/NF-kB inhibitor	Preclinical studies only (161)
IL-35	Anti-inflammatory cytokine; inhibits TLR4-NF-kB	Preclinical studies only (162)
TLR9 antagonist (ODN2088)	CpG inhibition	Preclinical studies only (138)

ARDS, acute respiratory distress syndrome; COVID-19, coronavirus disease 2019; CpG, cytosine-phosphate-guanine; IL, interleukin; TLR, Toll-like receptor.

Translational challenges

The translational challenge for TLR-targeted therapy in ARDS lies not in the absence of biological rationale, but in the difficulty of matching a specific immune pathway to the right patient, disease stage, and clinical endpoint. The failure of TLR-directed agents in clinical trials should therefore not be interpreted as evidence that TLRs are irrelevant to ARDS pathobiology. Rather, it highlights several recurring barriers: ARDS is a syndrome with heterogeneous infectious and sterile triggers, TLR activation may be protective early but pathogenic later, and downstream inflammatory cascades may already be established by the time treatment is initiated. In addition, single-receptor blockade may be insufficient in a disease state characterized by redundant PAMP- and DAMP-sensing pathways, receptor crosstalk, endothelial injury, immunosuppression, and multi-organ dysfunction. These issues are especially relevant to PARDS, where developmental differences in TLR expression and cytokine responses may alter both therapeutic efficacy and risk.

Several evidence gaps further limit translation. First, this review focuses specifically on TLR signaling and therefore does not comprehensively address the broader network of innate immune receptors involved in ARDS and PARDS. Other PRRs, including NLRs, RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs), also contribute to pathogen sensing, sterile inflammation, and immune regulation, often through extensive crosstalk with TLR pathways. Consequently, a TLR-centered framework provides only a partial view of the innate immune landscape underlying lung injury. Second, much of the mechanistic evidence is derived from in vitro systems and animal models that may not fully recapitulate the heterogeneity, temporal evolution, compartment-specific biology, and clinical complexity of human ARDS. Third, direct studies of TLR signaling in PARDS remain scarce, requiring substantial reliance on adult ARDS data and developmental immunology studies to infer pediatric mechanisms. Future translational studies should therefore incorporate PAMP- and DAMP-associated biomarkers, inflammatory subphenotyping, longitudinal and compartment-specific immune profiling, multi-omics approaches, and age-stratified enrollment across adult and pediatric cohorts. Such designs will be essential to determine whether TLR signaling is protective, pathogenic, or already bypassed at the time of intervention, and to define when TLR modulation is most likely to be safe and biologically effective.

Conclusions

TLRs play a mechanistic role in pathogenesis of ARDS, especially TLR4 in sepsis-induced lung injury and TLR3/TLR7 in viral lung injury. However, understanding of TLR involvement in PARDS remains limited and is largely extrapolated from adult and animal studies that do not adequately account for age-specific differences in TLR expression and signaling. These developmental differences may contribute to distinct immune responses and clinical phenotypes between ARDS and PARDS, emphasizing the need for pediatric-focused research. Future studies should prioritize age-stratified clinical and translational investigations that account for developmental stage, disease etiology, immune phenotype, and treatment timing to better define the role of TLR signaling in PARDS.

Supplementary

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