A growing body of evidence implicates TH
17 cells and their associated cytokines in allergic asthma, especially in severe cases where inflammation is predominantly driven by neutrophils3,4
. The data from this study indicate a novel mechanism for TH
17 cells in asthma pathogenesis. We show for the first time that IL-17A enhances the contractile response of airway smooth muscle to MCh and KCl. Furthermore, we show that activation of latent TGF-β by integrin αvβ8 on dendritic cells is required for TH
17 cell development in the lung in response to sensitization and challenge with OVA and Alum. Interestingly, mice lackingαvβ8 on dendritic cells were protected from AHR without changes in inflammatory cell numbers, goblet cell differentiation, or lymphocyte recall responses, and were also protected from AHR in the house dust mite asthma model. Overall, these findings suggest that IL-17A can contribute to AHR through direct effects on airway smooth muscle, without grossly modulating airway inflammation or mucus metaplasia.
We performed a number of experiments to test if enhanced contraction provoked by IL-17A was due direct or indirect interactions between IL-17A and IL-17 receptors on airway smooth muscle. We depleted epithelium from tracheal rings by mechanical brushing to distinguish effects of IL-17A on epithelium and airway smooth muscle. Increased contraction was still observed in epithelium-denuded tracheal rings, which indicates that the effects of IL-17A on airway contractility do not depend on its known effects on airway epithelial cells. In line with previous reports, we saw that epithelial removal in and of itself was sufficient to enhance contractile responses of tracheal rings to MCh and KCl34,35
, and we show here that this effect is potentiated further by pretreatment with IL-17A. We considered the possibility that a contaminant (e.g., LPS) in our IL-17A preparations might inadvertently stimulate contractile responses. However, enhanced contractile responses were completely prevented by adding IL-17A blocking antibodies, indicating that this result was due to the cytokine and not to contaminating factors. We also tested the specificity of IL-17A for its cognate receptor on airway smooth muscle. The IL-17 cytokine family signals through heterodimeric complexes of the IL-17 Receptor A (IL-17RA)-IL-17RE receptors, and IL-17A and F are known to bind the IL-17RA/RC heterodimer in other tissues28
. We found that both of the IL-17RA/RC isoforms are expressed in airway smooth muscle and that enhanced contraction triggered by IL-17A was completely prevented in tracheal rings isolated from IL-17RC-deficient mice, and that these mice were protected from AHR induced by OVA sensitization and challenge. Collectively, these data indicate that IL-17A acts through IL-17RA/RC receptors expressed by airway smooth muscle to increase contractile responses. It was somewhat surprising that IL-17F, which like IL-17A can ligate IL-17RA/RC heterodimers, did not affect airway smooth muscle contractility over a range of concentrations that were sufficient to see the effects of IL-17A. We suspect that this result reflects the lower affinity of IL-17F for these receptors 43
The NF-κB transcription factor is a major target of pro-inflammatory cytokines and we found in this study that IL-17A causes a robust activation of NF-κB upon MCh stimulation of airway smooth muscle. Since our primary phenotype after IL-17A treatment was enhanced contraction, we looked for NF-κB target genes that would modulate airway smooth muscle contraction. IL-17A treatment increased RhoA and ROCK2 expression, which are two prominent regulators of myosin light chain (MLC) phosphorylation and subsequent smooth muscle contraction36
. Several groups have reported in studies of smooth muscle that activation of RhoA stimulates ROCK2 to phosphorylate myosin light chain phosphatase (MYPT)44–46
. Phosphorylation deactivates MYPT and results in a net increase in MLC phosphorylation. Accordingly, our data show that IL-17A-induced expression of RhoA and ROCK2 correlated with enhanced phosphorylation of MLC and MYPT1. Phosphorylation of these proteins and expression of RhoA and ROCK2 was blocked with BAY 11-7082, a small molecule inhibitor of NF-κB, indicating that these proteins are target genes of NF-κB. This pathway was also stimulated in airway smooth muscle of OVA sensitized and challenged mice, and was impaired in mice lacking integrin αvβ8 on dendritic cells. Furthermore, reduced tracheal ring contraction in OVA sensitized and challenged mice lacking integrin αvβ8 on dendritic cells could be completely restored by ex vivo
IL-17A treatment. Together these data suggest that protection from AHR in mice lacking integrin αvβ8 on dendritic cells results from impaired IL-17A signaling. Since these mice had a near-absence of pulmonary IL-17A producing αβ T cells (TH
17), but minimal changes in IL-17A producing γδ T cells, our data suggest a model whereby IL-17A produced by TH
17 cells acts on airway smooth muscle to induce AHR.
17 cells and IL-17A have been implicated in several mouse models of asthma, although some of these data are conflicting. Adoptive transfer of antigen-specific TCR transgenic T cells polarized in vitro
to the Th17 phenotype into antigen challenged mice enhances AHR, as does pulmonary administration of an IL-17A overexpressing adenovirus47
. Furthermore, as we observed in this study with IL-17RC-deficient mice, IL-17RA-deficient mice have been reported to show dramatic protection from AHR in an OVA sensitization and challenge model of allergic asthma48
. However, this same study found that neutralization of IL-17A at the time of OVA challenge exacerbated AHR and local administration of IL-17A reduced eosinophilia and AHR. Another study using a similar model showed impaired induction of AHR in IL-17A-deficient mice crossed to OVA-specific TCR transgenic mice49
. Several other studies that either neutralized IL-17A with antibodies or administered exogenous IL-17A have shown mixed effects on allergen-induced AHR, but the preponderance of evidence supports an important role for IL-17A in allergic airway inflammation50–53
. It should be noted that the relative contributions of various cells and cytokines to models of allergic asthma can differ among inbred strains of mice, which might be one explanation for the apparently conflicting reports about the contributions of IL-17 to allergic asthma. Since all of the studies described in the current manuscript were performed in C57BL/6 mice, we cannot be certain that the same effects would be seen in other strains (e.g. Balb/c) in which TH
2 cytokines have more potent effects. IL-17A was recently shown to act synergistically with IL-13 to enhance AHR in the A/J mouse strain, whereas the C3H/HeJ strain is naturally protected from AHR through decreased production of IL-17A5
. Indeed, we and others have previously reported that inhibition of IL-13 can potently inhibit AHR in models of allergic asthma and that over-expression or administration of IL-13 is sufficient to induce AHR, in part through direct effects of IL-13 on airway epithelial cells. Based on the results of this study, we suspect transgenic or intranasal administration of IL-13 results in higher concentrations of IL-13 than those released in the context of allergen challenge, and that under those circumstances IL-17 is not required for the development of AHR. Although inhibitor studies confirm that released IL-13 is important in the development of allergen-induced AHR, our results suggest that the small amounts of IL-13 released in response to allergen challenge are not in themselves sufficient to induce AHR, but require the additional effect of IL-17.
The results of this study provide several novel insights into how TH17 cells and TH17 cytokines contribute to the development of allergic asthma, and thus identify a number of new potential therapeutic targets. Our results identify the integrin, αvβ8, as a critical upstream activator that is essential for TH17 cell generation in allergic airway disease. We also show that loss of this integrin on dendritic cells dramatically inhibits TH17 cell generation without effects on the IL-17A producing γδ T cells, suggesting it might be possible to inhibit this TH17 induction without eliminating the key role played by IL-17A producing γδ T cells in pulmonary defense against bacterial and fungal pathogens. Furthermore, our findings are the first demonstration that IL-17A, but not IL-17F or IL-22, can contribute to allergic asthma by directly enhancing the contractile responses of airway smooth muscle cells, through a pathway that involves activation of NF-κB and induction of RhoA and ROCK2 expression. The αvβ8 integrin, IL-17A and NF-κB and its downstream effectors could thus all be attractive targets for improved treatment of allergic asthma.