We have previously shown that short fragments of HA mediate ozone-induced AHR (25
). In this study we demonstrate that TLR4 is necessary for both ozone-induced AHR and HA-induced AHR. Furthermore, we show that after both ozone exposure and HA challenge, TLR4 is necessary for the production of a number of proinflammatory cytokines implicated in the pathogenesis of AHR. Our data suggest a central role for macrophage-derived TLR4 in the release of proinflammatory cytokines and likely the development of AHR. Our observations also suggest that activation of airway epithelia could contribute to the biological response to both ozone and HA. Our report therefore links innate immunity to the development of AHR after a relevant environmental challenge and further supports that endogenous ligands of TLR4 can act as innate immune activators in sterile environmental lung injury.
The role of innate immunity in the response to noninfectious injury has recently received some attention. Toll-like receptors can mediate the immunologic response to noninfectious cell damage such as ischemia-reperfusion injury (38
), autoimmunity (39
), bleomycin lung injury (31
), and asbestos-related lung injury (35
) through recognition of endogenous ligands released during tissue injury (danger-associated molecular patterns). Danger-associated molecular patterns include extracellular matrix components including fibronectin, fibrinogen, and HA. Of these, HA has been most frequently implicated in innate immune activation. Previous work supports the conclusion that oxidant tissue injury can lead to release of short-fragment HA, which acts as a proinflammatory mediator and enhances cellular inflammation in sterile lung injury (40
). Although in this model we did observe clear differences in both cytokines and AHR in a manner dependent on TLR4, we did not observe notable differences in cellular inflammation. The role of TLR4 in HA-mediated cell recruitment remains unknown. However, our data support that TLR4 contributes to HA-induced activation of macrophages. Previous work suggests that TLR4 interaction with long-fragment HA can promote epithelial cell integrity and recovery from sterile lung injury (31
), whereas short-fragment HA elicits immuno-stimulatory responses by macrophages. It is therefore plausible that the response to HA is ultimately dependent on the intensity of lung injury, HA fragment size, and the cell types that encounter this ligand.
TLR4 and other HA receptors, such as CD44, are located on several lung cell types that can regulate TLR4-dependent response to inhaled endotoxin, including both airway epithelia (42
) and alveolar macrophages (43
). Previous work suggests that each of these cell types have the potential to bind HA and mediate HA-induced effects. We observed that ozone induced activation in both airway epithelia and macrophages, and direct challenge to HA led to enhanced activation of NF-κB primarily in both cell types. Additionally, after exposure to ozone, we observed colocalization of HA and TLR4 on lung macrophages but not on bronchial epithelia. Furthermore, direct challenge of cultured macrophages with HA reconstituted a similar cytokine profile as observed with in-vivo
ozone and HA challenge in the lung. Therefore, although we cannot rule out a direct effect of HA on airway epithelia in this model, we suspect that alveolar macrophages may play a major role in the development of ozone-induced and HA-induced AHR, whereas airway epithelia may be indirectly activated by downstream HA-induced factors (44
). Interestingly, we also observed localization of HA on the cell surface of subepithelial smooth muscle cells. It has been previously recognized that biological stress can induce production of HA in airway smooth muscle (45
). The role of both airway epithelia and airway smooth muscle in this context will be a focus of future investigation. In our model, HA alone only partially reconstituted the physiological response to ozone. We suspect that other factors, in addition to HA, contribute to the full response to ozone inhalation. However, our data do support that release of short-fragment HA contributes to TLR4-dependent activation of macrophages resulting in release of proinflammatory cytokines and the development of AHR.
We have previously shown that HA binding through inter-α trypsin inhibitor and CD44 is necessary for AHR after ozone exposure (25
). Taylor and colleagues recently demonstrated that HA released during sterile tissue injury binds to a receptor complex consisting of TLR4, CD44, and MD-2 and initiates the innate immune response (32
). We speculate, based on CD44-TLR4 coimmunoprecipitation in alveolar macrophages and whole lung lysates, that a similar receptor complex is operative in ozone-induced lung injury. However, the precise coreceptor mechanism between TLR4 and CD44 remains unknown. CD44 may act as a platform, stabilizing HA for TLR4 to bind. Alternatively, CD44 may facilitate dimerization of TLR4 and TLR4 signaling. Finally, the cytoplasmic tail of CD44 may bind to tyrosine kinases, which may in turn help activate the TLR4 signaling pathway. It is possible that other coreceptors (MD-2 and CD14) contribute to the full response to HA. A previous report from our group supports the observation that ozone exposure can prime innate immunity through trafficking of TLR4 to the cellular surface of lung macrophages, which can sensitize the organism to subsequent TLR4 activation (36
). The current report demonstrates that TLR4 activation is also important for the immediate response to inhaled ozone. The binding of TLR4 to HA appears partially necessary for the development of ozone-induced AHR. However, the mechanism by which macrophage-derived soluble factors leads to AHR remains unknown. We speculate that TLR4 interaction with HA results in the generation of inflammatory cytokines like TNF-α, which can promote airway smooth muscle constriction (47
). In this way, ozone exposure could lead to enhanced AHR. The increased availability of both cell receptor (TLR4) and ligand (HA) significantly improves conditions for their interaction, and may explain, in part, the clinical observation that AHR peaks at 24 to 48 hours after inhaled ozone exposure. However, it is notable that CD44 and inter-α-trypsin inhibitor deficiency leads to complete abolition of ozone-induced AHR (25
), whereas TLR4 deficiency only partially ameliorates inflammation and AHR. Therefore, it remains plausible that other HA-dependent but partially TLR4-independent pathways exist that lead to the development of ozone-induced AHR and inflammation.
In summary, we demonstrate that ozone-induced AHR is partially mediated by the endogenous innate immune ligand HA and the surface receptor TLR4. This report provides important insight into the pathogenesis of environmental airways disease and implicates innate immune activation through the endogenous ligand HA in the pathogenesis of AHR.