Herein we describe a mechanism whereby serine proteases promote the development of allergen-induced AHR through activation of PAR-2. Confirming our previous work , and that of others  we again demonstrate that mucosal sensitization to GC frass is reduced in mice lacking PAR-2, resulting in reduced AHR and airway inflammation. Previous studies have focused on the role for PAR-2 in direct activation of bronchial epithelial cells [20, 30–32] triggering the development of innate immunity by causing the release of chemotactic factors specific for the growth and recruitment of pulmonary mDCs (i.e. CCL20, GM-CSF). We demonstrate here that in vivo PAR-2-deficient mice display reduced production of both Th2 and Th17-associated cytokines, suggesting that PAR-2 differentially influences multiple T cell responses. As mucosal exposure to GC frass induces upregulation of PAR-2 on pulmonary mDCs, but not pDCs, we sensitized naïve mice with GC-frass pulsed wild type BMDCs, or BMDCs from PAR-2-deficient mice. As previously observed , the adoptive transfer of allergen-pulsed mDCs yields robust production of IFNγ and IL-17A and airway neutrophilia along with the typical Th2 cytokines and eosinophilia, a response which more closely resembles the mixed Th1/Th2/Th17 response observed in severe asthmatics. However, in this context, the lack of PAR-2 on sensitizing DCs markedly impacts Th2 cytokine production, providing evidence that PAR-2 on mDCs is involved in promoting Th2 immune responses. In contrast, the Th17 response is only slightly diminished in mice sensitized with PAR-2-deficient DCs suggesting that PAR-2 expression on other cell types controls Th17 cytokine production. Thus, the present study suggests that specifically targeting PAR-2 activation of pulmonary mDCs may allow one to limit the development of Th2 responses at mucosal sites.
The observation that the induction of Th2 responses by GC frass is reduced in both PAR-2 deficient mice and mice sensitized with PAR-2 deficient DCs strongly implicates PAR-2 expression on mDCs on the ability of GC frass to induce a Th2-polarized immune response at mucosal surfaces. However, the mechanism whereby PAR-2 promotes the development of Th2 responses is unclear. While we observed that PAR-2 deficient BMDCs produce significantly reduced levels of all cytokines examined (IL-6, IL-23, TNFα) we have previously reported that GC frass-induced production of both IL-6 and IL-23 is completely abrogated in MyD88 -/- BMDC , suggesting that PAR-2 expression may amplify TLR-triggered cytokine production. However, as LPS-depleted serine proteases still demonstrated marked Th2-skewing capacity in vivo, it seems unlikely that the reduced Th2-skewing capacity of PAR-2 DCs is the direct result of reduced cytokine production. In contrast, PAR-2 activation has been shown to enhance maturation of BMDCs (as evidenced by increased MHC Class II and CD86 expression) , suggesting that reduced co-stimulatory molecule expression may be involved. In support of this possibility, we observe decreased expression of the co-stimulatory molecule CD80 and CD86 on pulmonary mDCs from PAR-2 -/- mice. Collectively, these data suggest that PAR-2 expression on pulmonary mDCs is required for optimal induction of Th2 immunity following exposure to GC frass.
While our data suggests that DCs lacking PAR-2 demonstrate reduced Th2 skewing capacity we cannot completely rule out a role for the epithelium in the ability of the GC frass to induce a Th2 response. Epithelial cells treated with allergens with active proteases (Aspergillus extract, Derp1) induce IL-25, a strong inducer of Th2 immunity  in an ERK/p38 dependent manner . As we have shown that GC protease mediated induction of IL-8 from epithelial cells is dependent upon ERK activation , it is likely that IL-25 may also be produced. Moreover, Alternaria mediated PAR-2 cleavage has also been shown to induce TSLP production from bronchial epithelial cells . TSLP in turn directly enhances the ability of DCs to induce a Th2 response [36, 37], suggesting an additional mechanism whereby PAR-2 may amplify Th2 immunity. However, additional studies making use of mice lacking PAR-2 specifically in DC populations or pulmonary epithelial cells will be required to conclusively determine the relative contributions of PAR-2 on DCs and epithelial cells.
It is interesting to note that while there is a substantial (~75%) decrease in the number of neutrophils in the BAL in PAR-2 -/- mice, a dramatic impact on neutrophil recruitment was not observed in mice sensitized with GC frass-pulsed PAR-2 -/- BMDCs (~20% decrease). This is especially striking given that in both PAR-2 -/- and mice sensitized with GC frass-pulsed PAR-2 -/- BMDCs levels of IL-17A are only partially affected. As IL-17A is a strong promoter of neutrophilia, this is somewhat surprising. However, a recent report by Fei et al suggests that elevated IL-17A is not sufficient to drive neutrophilia in a model of allergic bronchopulmonary aspergilliosis . Rather, a combination of both TNFα and IL-17A are required to get maximal neutrophil recruitment, whereas the production of IL-17A alone was associated with a more pronounced eosinophilia . In this report we also demonstrate that PAR-2 expression on mDCs is required for maximal GC frass-induced production of TNFα, suggesting that differential TNFα production may explain differences in neutrophilia observed PAR-2 -/- versus mice sensitized with GC frass-pulsed PAR-2 -/- BMDCs. Indeed, in PAR-2 -/- mice, GC frass sensitization results in limited IL-5, but robust IL-17A production. Moreover, at challenge, PAR-2 -/- mice also lack DC-derived, GC frass-stimulated TNFα production resulting in a milieu with low IL-5 and TNFα, but high IL-17A levels that is not conducive to strong recruitment of eosinophils or neutrophils. In contrast, while sensitization of mice with GC frass-pulsed BMDCs also results in a low IL-5 and high IL-17A levels, GC frass challenge of these mice induces high levels of TNFα as endogenous DCs can respond to serine protease in the GC frass. This resulted in a low IL-5, high TNFα/IL-17A milieu which permits neutrophil recruitment, but resulted in limited recruitment of eosinophils.
The data presented here suggest that the initial response to inhaled GC frass is complex, and that synergy between different components is likely required for maximal allergenic capacity. However, these studies do highlight the importance of protease activity and activation of PAR-2 receptors on pulmonary mDCs in inducing the development of Th2 responses at mucosal surfaces. Taken together with a number of other studies showing the ability of proteases to induce the development of Th2 responses [30, 32, 39], these studies suggest that activity of proteases at mucosal surfaces may be a key factor regulating the development of Th2 immune responses. It is important to note that while there is good evidence to support that cockroach proteases and PAR-2 are involved in the Th2 responses in the airways, this study does not demonstrate the development of antigen-specific T cell responses by cockroach proteases and PAR-2. A greater understanding of the processes that lead to the development of Th2 responses may prove invaluable in therapeutic regulation of Th2 responses that are both undesirable (i.e. allergy, asthma), or desirable (parasitic clearance).