TLRs are expressed by a range of immune cells including dendritic cells, macrophages and B cells, but also by non-immune cells including epithelial cells 16. All TLRs, except for TLR3, recruit myeloid-differentiation primary response protein 88 (MyD88) as an adaptor protein, crucial for the activation of NF-κB, which controls the expression of proinflammatory mediators 16. In TLR4 and TLR3 signaling, a second downstream pathway is involved. This pathway is mediated by Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF) protein and results in delayed NF-κB activation and activation of type I interferons 16.

Hyaluronan (HA) is part of the extracellular matrix and its high molecular weight (HMW) form is an important molecule in tissue architecture. Low molecular weight (LMW) HA is synthesized under inflammatory conditions by hyaluronan synthase-3 (HAS3) or is formed by the breakdown of HMW HA. LMW HA accumulates at sites of inflammation and stimulates macrophages to produce chemokines via TLR2 and TLR4 activation 17. Biglycan is a small leucin-rich proteoglycan, which is another constituent of the extracellular matrix. During tissue injury or by excretion from activated macrophages, biglycan becomes available in its soluble form and increases TNFα and macrophage inflammatory protein-2 (MIP2) levels via TLR2 and TLR4 18. Other extracellular matrix components such as versican, heparan sulfate, fibronectin, and tenascin-C can also activate TLR2 and/or TLR4 19.

The heat shock proteins (HSPs) are molecular chaperones abundantly present in the cytosol. Increased expression of HSPs is induced in response to a wide variety of insults, including heat stress. Intracellular HSPs have antiapoptotic properties, and extracellular HSPs interact with the immune system via TLR2 and TLR4 20. Caution is warranted when interpreting HSP data: LPS contamination of the HSP preparations used in studies is a well-known problem.

High-mobility box group-1 (HMGB1) is a nuclear chromatin-associated protein and an important mediator in sterile inflammation when released in the extracellular space 21. It is passively released from necrotic cells, but HMGB1 can also be actively secreted by several immune cells 8 to 12 hours after TLR ligation 21. TLR4-HMGB1 interactions activate the MyD88 pathway and are important for macrophage activation and cytokine release. Although there is debate whether highly purified HMGB1 can directly activate cells, it is now accepted that other molecules bound by HMGB1, at least in part, are responsible for the detrimental effects of extracellular HMGB1.

The S100 family of proteins, or calgranulins, consists of more than 20 intracellular calcium-binding proteins implicated in cell homeostasis and regulation of the cytoskeleton 22. S100A8 and S100A9 form heterodimers, their physiologically relevant form, and are secreted by activated phagocytes during inflammation. Extracellular S100A8/A9 activates endothelial cells, and murine S100A8 has been shown to induce chemotaxis in vitro 22. S100A8/A9 proteins are ligands for TLR4 23. β-defensins are host-derived antimicrobial peptides and signal via TLR4 and TLR1/2 2425.

In cellular injury, nucleic acids are released. Mammalian mRNA can activate TLR3, leading to IFNα secretion 26. Single-stranded RNA is sensed by TLR7 and TLR8 27. Mitochondria might carry bacterial molecular patterns, being evolutionary endosymbionts derived from bacteria. Mitochondrial DNA can activate TLR9 and stimulate neutrophil migration and degranulation 28.

Fatty acids and lipoproteins such as serum amyloid A, oxidized low-density lipoprotein and saturated fatty acids can activate TLR2 and TLR4 19. A more detailed list of all endogenous TLR activators has been previously published 19, and a schematic overview of DAMPs and TLR signaling pathways can be found in Figure 1.

Intracellular sensors of infection and tissue injury have been recently identified and termed NLRs 29. The NLR family consists of more than 20 members, but in the present review we focus on the function of NLRP3 (or NALP3 or cryoporin) in DAMP signaling.

NLRP3, expressed by immune cells, epithelial cells, and osteoblasts, assembles with adaptor protein ASC (apoptosis-associated speck-like protein) and caspase-1 to form a multiprotein complex termed the NLRP3 inflammasome 29. NLRP3 inflammasome-dependent caspase-1 activation leads to maturation and release of IL-1β and IL-18. A two-step process is required for NLRP3 inflammasome signaling: a priming step (signal 1) and a NLRP3 activation step (signal 2). Signal 1 induces NF-κB-dependent production of pro-IL-1β and pro-IL-18 via, for example, activation of TLRs. Signal 2 can be provided by the presence of PAMPs, inhaled large particles, and DAMPs. How these various stimuli are recognized by NLRP3 and trigger NLRP3 activation is still not completely elucidated. Potassium efflux, production of reactive oxygen species, and lysosomal damage with cathepsin B release are intracellular features associated with NLRP3 activation 30.

Intracellular ATP is a nucleotide with a primary function in energy metabolism. Extracellular ATP has a function in cell communication and its release from healthy cells is tightly regulated 31. Cell damage and/or cellular stress leads to an uncontrolled release of ATP. The ATP-induced NLRP3 inflammasome activation requires activation of the P2X₇ receptor in combination with the pannexin-1 channel and decreases intracellular potassium levels 2932.

Another NLRP3-activating DAMP is uric acid, the main metabolite of the cellular catabolism of purines that at high local concentrations precipitates and forms crystals 33. Phagocytosis of uric acid crystals can induce lysosomal damage and the release of cathepsin B 33. Uric acid crystals may also trigger potassium efflux and/or induce reactive oxygen species production.

Extracellular matrix components HA and biglycan have also been shown to induce NLRP3 activation 3435. The association of HA with CD44 and the mechanism of HA catabolism, such as endocytosis and lysosmal hyaluronidase activity, are needed to trigger intracellular NLRP3 34. How these small intracellular saccharides activate NLRP3 is still unclear. Biglycan can interact with multiple receptors, including the P2X₇ receptor, in order to form multireceptor complexes 35. Furthermore, biglycan induces the formation of reactive oxygen species.

A schematic overview of NLRP3 inflammasome signaling can be found in Figure 2.

RAGE is a multiligand receptor that is highly expressed in lung tissue, primarily by type I alveolar cells 3637. The RAGE receptor contains one V-domain and two C-domains extracellularly. The receptor has a transmembrane part and a cytoplasmic tail, which is important for intracellular signaling. Although the exact downstream signaling after RAGE-ligand interaction is largely unknown, it ultimately leads to the activation of the NF-κB and mitogen-activated protein kinase signaling pathways 36. Soluble RAGE, a RAGE isoform lacking the transmembrane domain, can be used as a marker of alveolar epithelial type I cell injury 37. Furthermore, it is thought that soluble RAGE can act as a decoy receptor by competing with full-length RAGE for ligand binding 3637.

RAGE interacts with various ligands. S100A12, also a member of the S100 family of proteins, is mainly found in granulocytes, and contributes to endothelial and leukocyte activation via binding with RAGE 22. HMGB1- RAGE interactions stimulate chemotaxis, immune cell differentiation and migration, cell growth, and the up-regulation of receptors such as TLR4 and RAGE 21. Other known RAGE ligands are amyloid, β-sheet fibrils, S100B, and S100P 36.

Previously it was shown that HA levels in bronchoalveolar lavage fluid (BALF) were six times higher in ARDS patients compared with control patients 38. The role of HA in VILI was first studied in vitro 39. Cyclic stretch of human fibroblasts triggered the release of soluble LMW HA. Furthermore, stretch-induced LMW HA was able to increase the production of IL-8 in both static and stretched epithelial cells 39. Other investigators observed that LMW HA can induce epithelial-to-mesenchymal transition in alveolar type II cells via the MyD88 pathway 40. When injured, alveolar type II cells are capable of self-renewal but can also undergo epithelial-to-mesenchymal transition, which may contribute to the development of lung fibrosis and subsequently respiratory failure.

Mice ventilated for 5 hours with high V_T (30 ml/kg) were found to have increased LMW HA and HAS3 mRNA levels in lung tissue 41. Ventilated HAS3 knockout (KO) mice had significantly reduced LMW HA levels, neutrophil infiltration, and MIP2 (rodent equivalent for human IL-8) production compared with ventilated wild-type mice 41. In search for a treatment strategy that would reduce LMW HA levels produced by HAS3, phosphodiesterase-3 inhibitors were studied 42. Treatment of septic ventilated rats resulted in decreased HAS3 expression levels in lung tissue. Moreover, alveolar protein levels, neutrophil influx, and lung injury scores were reduced. Whether these anti-inflammatory properties are indeed all mediated by inhibition of HAS3 and a reduction of LWM HA levels remains to be elucidated.

Conversely, HMW HA exerts anti-inflammatory and anti-apoptotic effects 1743. Transgenic mice that overexpress HMW HA are protected from bleomycin-induced lung injury 17. Furthermore, HMW HA pretreatment inhibited inflammatory cell infiltration, cytokine production and the extent of lung injury in a rat model of sepsis combined with mechanical ventilation 43. It is thought that the beneficial effects of HMW HA are secondary to an altering in the balance of HA in favor of the HMW form, thereby maintaining the integrity of the extracellular matrix.

Injurious MV (2 hours, V_T = 30 ml/kg) resulted in elevated lung levels of versican, heparan sulfate proteoglycan and biglycan compared with non-injurious ventilated rats (2 hours, V_T = 8 ml/kg) 44. The dynamic alterations of extracellular matrix components during VILI can influence viscoelastic properties of the lung but may also contribute to the progression of pulmonary inflammation. Further studies are warranted.

Induction of HSPs mediates cellular protection against harmful stimuli. Kira and colleagues documented the upregulation of pulmonary HSP70 expression due to injurious MV (30 minutes, positive inspiratory pressure with 30 cmH₂O) in rats 45. Induction of HSP70 sup-pressed cytokine-induced IL-8 and TNFα expression in respiratory epithelial cells 46. HSP induction was shown to stabilize inhibitory κB factor, indicating attenuated NF-κB activation 46. In line with this, Ribeiro and colleagues observed lower levels of proinflammatory cytokines and a better lung compliance in ex vivo rat lungs pretreated with heat stress 18 hours before the start of injurious MV (2 hours, V_T = 40 ml/kg) 47. However, timing is essential for the cytoprotective effects of the heat shock response. Induction of HSPs in cells already primed by inflammation might lead to cell death by apoptosis 48.

More recently, the role of extracellular HSPs has become an area of interest. Presence of extracellular HSP72 was reported in BALF and plasma of ALI patients 49. In vitro, extracellular HSP72 induced a dose-dependent increase in IL-8 expression and activation of NF-κB in bronchial epithelial cells 50. Moreover, intratracheal installation of HSP72 in mice induced upregulation of keratinocyte-derived chemokine and TNFα levels, and increased neutrophil influx in BALF 50. TLR4 mutant mice did not develop a similar inflammatory response to intratracheal HSP72, stressing the important role of TLR4 in HSP-mediated inflammation. Further research is needed to understand the divergent response to HSPs. Strategies that activate HSP genes may be beneficial in the pathogenesis of VILI, and antibodies that block extracellular HSP could reduce pulmonary inflammation.

A rise in HMGB1 levels in response to MV has been observed 5152. HMGB1 concentrations in the BALF of patients subjected to long-term ventilator therapy (V_T <8 ml/kg and optimal positive end-expiratory pressure) were increased when compared with a control group of healthy volunteers 51. In an in vivo VILI model, levels of HMGB1 in BALF were fivefold higher in the high V_T group (4 hours, V_T = 30 ml/kg) when compared with the lower V_T group (V_T = 8 ml/kg) 52. Blocking HMGB1 led to a significant reduction in neutrophil influx and TNFα concentration in BALF, improved oxygenation, and limited microvascular permeability 52. These results indicate that the presence of enhanced HMGB1 levels exerts detrimental effects in the lung. In line with this hypothesis, Abraham and colleagues observed pulmonary neutrophil accumulation, lung edema, and increased production of IL-1β, TNFα, and MIP2 due to intratracheal administration of HMGB1 in mice 53. Numerous experimental disease models (including sepsis, ischemia-reperfusion injury and arthritis) responded to therapy targeting HMGB1, which might indicate that removing or neutralizing HMGB1 attenuates innate immune system activation and reduces tissue damage 21. HMGB1 is a promising target for future therapy. Careful patient selection will be necessary, however, since antibodies may also interfere with beneficial qualities of HMGB1 such as potential antitumor activities 21.

S100A9 mRNA is upregulated in rat lungs undergoing injurious MV (30 minutes, V_T = 25 ml/kg) 54. In mice, 4 hours of MV with a conventional V_T of 10 to 12 ml/kg also enhanced the pulmonary transcription of S100A9 mRNA 14. Both studies used whole lung tissue as the source of mRNA, making the interpretation of which cells are responsible for the changes in gene expressions difficult. The expression of S100A12 in lung tissue and the concentration of S100A12 in BALF were both significantly higher in patients with ARDS when compared with healthy controls 5556. In addition, S100A8/A9 protein levels were elevated in the BALF of ARDS patients relative to healthy controls 56. Although studies revealed the presence of S100 proteins in the inflamed lung, the role of these proteins has not been studied extensively. Their shown potential to activate leukocytes and endothelial cells warrants further research.

Animal studies demonstrated increased ATP concentrations in BALF due to injurious MV (30 minutes, positive inspiratory pressure 40 cmH₂O and 1 hour, V_T = 40 ml/kg) 5758. To determine whether extracellular ATP contributed to VILI development, ATP was administered before the onset of low-pressure ventilation (30 minutes, positive inspiratory pressure 15 cmH₂O). Intratracheal ATP significantly increased the volume of epithelial lining fluid during low-pressure MV 57. In line with this, other investigators studied the effects of intratracheal ATP in nonventilated mice 58. ATP increased the pulmonary wet to dry ratio, the lung permeability index and mRNA expression of IL-6, TNF-α, and MIP2, and induced aggregation of inflammatory cells in alveolar tissue. ATP signals via P2 purinergic receptors and blockage with a specific P2 receptor antagonist, just before the onset of MV, partially mitigated the inflammatory response during high tidal ventilation 58. Recently, elevated pulmonary ATP concentrations were observed in patients with lung fibrosis and mice with bleomycin-induced lung injury 59. In mice, ATP levels were reduced using an ATP-degrading enzyme - this resulted in attenuated inflammatory cell recruitment, lung IL-1β levels and matrix remodeling protein tissue inhibitor of metalloproteinase-1 concentrations. ATP-induced inflammation was mediated by the ATP/P2X₇ axis. Whether this axis also contributes to VILI development remains an interesting topic for future studies.

Injurious MV (5 hours, V_T = 15 ml/kg) increased uric acid levels in BALF of mice 60. When mice were pretreated with allopurinol (uric acid synthesis inhibitor) or uricase (degrades uric acid), alveolar barrier dysfunction was attenuated 60. Gasse and colleagues demonstrated pulmonary uric acid accumulation in a bleomycin-induced lung injury model 61. Local administration of uric acid crystals induced dose-dependent cell recruitment in BALF, IL-1β production and tissue inhibitor of metalloproteinase-1 expression, establishing uric acid as a major endogenous danger signal. Uric-acid-induced lung inflammation was mediated by the NLRP3 inflammasome, MyD88, and the IL-1 receptor 1 pathway and the combined action of TLR2 and TLR4 for optimal inflammation 61.

After 4 hours of low tidal MV (V_T = 8 ml/kg), TLR4 KO mice displayed lower pulmonary cytokine and chemokine levels compared with ventilated wild-type animals 62. Moreover, MV caused release of endogenous TLR4 ligands in BALF 62. In line with this, lower pulmonary IL-1β and keratinocyte-derived cytokine levels and NF-κB activity were observed in similarly ventilated TRIF KO mice 63. The TLR4-TRIF pathway was therefore suggested as a dominant signaling cascade involved in MV-induced inflammation 62. However, a role for TLR3 signaling cannot be excluded.

Chun and colleagues studied TLR involvement in VILI using a model in which MV (6 hours, V_T = 10 ml/kg) was combined with TLR3-mediated lung inflammation 64. Interestingly, they demonstrated that MV-associated augmentation of TLR3-induced lung inflammation occurred via the MyD88 pathway. Since TLR4 signals via MyD88, their next step was to identify the role of TLR4 in this model. In contrast to Vaneker and colleagues' study 62, TLR4 KO mice displayed no significant differences in cytokine concentrations, neutrophil influx or lung permeability when compared with wild-type mice 64. These results suggest that other MyD88-dependent receptors are required for MV augmentation of TLR3-induced lung inflammation - the TLR4-MyD88 pathway, however, seems not to be involved. The use of a TLR3 agonist makes it difficult to separate out the effect of MV on TLR4-TRIF activation, since a part of this signaling cascade is already activated by the TLR3 agonist. In isolated perfused lungs of C3H/HeJ mice, which lack functional TLR4, injurious MV (3.5 hours, V_T = 15 ml/kg) still resulted in NF-κB activation 8. These results suggest that other PRRs may contribute to MV-induced inflammation.

The exact role of TLR2 in VILI is a matter of debate. Although TLR2 mRNA expression levels in lung tissue of healthy mice increased due to MV (4 hours, V_T = 8 ml/kg), lungs from TLR2 KO mice did not show attenuated cytokine levels 62. In contrast to the findings in the mouse model, a more recent study conducted in rats demonstrated that TLR4 mRNA, but not TLR2 mRNA, was upregulated by injurious MV (4 hours, V_T = 15 ml/kg) 65. TLR3 expression is enhanced in lung histological slides of ARDS patients 66. Although not studied in VILI, TLR3 KO mice were protected from hyperoxia-induced ALI 66. The negative regulator of MyD88-dependent TLR signaling, IL-1-receptor-associated kinase-M (IRAK-M), is a possible regulator of VILI 65. In the absence of infection, IRAK-M was downregulated by injurious MV (4 hours, V_T = 15 ml/kg).

TLR signaling pathways that are activated in response to pathogens are modulated by MV. In septic rats (induced by cecal ligation and puncture), injurious MV (4 hours, V_T = 20 ml/kg) influenced TLR4 and IRAK-M protein levels, pulmonary and systemic cytokines, and mortality 67. CD14, an important accessory protein for LPS-TLR4 ligand interaction, was upregulated due to 4 hours of injurious MV (V_T = 20 ml/kg) in rabbits 68. In addition, alveolar macrophages from these ventilated animals were stimulated with LPS: TNFα release in the injurious MV group was 20-fold higher than in the low V_T group (5 ml/kg). These results suggest that mechanical stress can sensitize lungs to endotoxin, partially via CD14 upregulation 68.

Up to now, many studies have been conducted to unravel the role of TLRs, TLR ligands, and their specific signaling pathways. However, there are still many questions to be answered. Whether TLR4 plays an important role in VILI is uncertain but is of potential interest as TLR4 antagonists are available for use in humans.

Preclinical and clinical studies previously demonstrated an important role for IL-1β, the main product of activated NLRP3 inflammasome, in the pathogenesis of VILI 6970. IL-1 receptor KO mice displayed less alveolar barrier dysfunction due to injurious MV (3 hours, V_T = 30 ml/kg) when compared with wild-type mice 70.

The role of NLRP3 inflammasome in VILI has not been studied. Recently, NLRP3 signaling in a bleomycin-induced lung injury model was established. NLRP3 and ASC KO mice, both unable to assemble the NLRP3 inflammasome, displayed less lung inflammation compared with wild-type mice 61. Given the presence of NLRP3 ligands ATP and uric acid in VILI and the proven role of IL-1β, we expect there is a role for NLRP3 in VILI.

Soluble RAGE levels are significantly higher in pulmonary edema fluid and plasma from ALI/ARDS patients compared with patients with hydrostatic edema 37. Soluble RAGE concentrations are also elevated in BALF from rats with LPS or hydrochloric acid-induced ALI 37. In mice with LPS-induced lung injury, soluble RAGE treatment attenuated neutrophil influx, lung permeability, pathological lung changes, and NF-κB activity 71.

RAGE is highly expressed in lung tissue and might have a role in cell physiology of healthy lungs. Spontaneous development of pulmonary fibrosis-like alterations in aged RAGE KO mice has been demonstrated 72. However, lack of RAGE in younger mice with bleomycin or hyperoxia-induced lung injury resulted in reduced lung inflammation 7374. We speculate that RAGE signaling also exerts detrimental effects in VILI, but future studies are mandatory to elucidate the exact role of RAGE.