We demonstrate that the Th17 response to EC sensitization is exaggerated in DKO mice and drives airway inflammation and AHR in response to airway challenge with antigen independently of Th2 cytokines.
EC sensitization with OVA elicits a systemic and local Th17 immune response that is partially decreased in MyD88-/-
, suggesting that it is partially dependent on endotoxin. In the absence of IL-4 and IL-13, we observed an exaggerated systemic Th17 response and more importantly, an exaggerated IL-17 response in antigen challenged airways that drove airway inflammation and AHR. IL-4 was identified as the major Th2 cytokines that downregulates the Th17 response to EC sensitization, because IL-4 KO mice, but not IL-13 KO mice, had an increased systemic Th17 response and increased IL-17 expression in antigen challenged lungs (). This is consistent with the fact that T cells express IL-4R, by not IL-13R32
. Absence of IL-4 and IL-13 had no demonstrable effect on the infiltration of sensitized skin by CD4+
cells (), but was associated with lack of infiltration by eosinophils and with higher, albeit not significant, expression of IL-17A and IL-17F mRNA (). DKO mice had markedly exaggerated expression of IL-17A and IL-17F mRNA in lungs and increased secretion of IL-17A by OVA stimulated lung cells following OVA airway challenge (). The observation that the local Th17 response in lungs was markedly more exaggerated than in the skin, suggests that tissue specific factors regulate IL-17 expression by locally infiltrating T cells or that IL-17-secreting cells may home less efficiently to skin than to lung.
We confirmed the importance of IL-4 and IL-13 in driving Th2 dependent responses in our model. DKO mice had undetectable serum levels of OVA-specific IgE, and severely diminished eosinophil infiltration in OVA sensitized skin and in BALF and parenchyma of OVA challenged lungs. In addition, they exhibited decreased mucus production evidenced by decreased PAS staining, consistent with IL-13 being a key mediator of mucus production in the airway 5
. EC sensitized DKO mice had a comparable increase in circulating eosinophils as WT controls and their splenocytes secreted normal amounts of IL-5 in response to OVA stimulation (Supplementary Fig 1B and C). The latter finding is in agreement with the previously reported normal IL-5 production in DKO mice infected with Schistosoma mansoni 24
. A likely explanation of the absent tissue eosinophilia at sites of antigen challenge in DKO mice is the failure to upregulate the expression of the eosinophil attracting chemokine CCL11, which is induced by IL-4 and IL-13, and is essential for eosinophil recruitment to skin and lungs in our model 28
IL-4 downregulates the expression of IFN-γ. DKO mice infected with Schistosoma mansoni
have increased production of IFN-γ and IgG2a antibodies 24
immunized, airway challenged DKO mice have increased expression of IFN-γ in bronchial DLN 25
. We were unable to detect a change in the amount of IFN-γ secreted by OVA stimulated splenocytes or in the expression of mRNA in sensitized skin or challenged lungs in DKO mice. The increased IgG2a response of these mice may be due to absence of the inhibitory effect of IL-4 on IgG2a producing B cells 33
Airway challenge of EC sensitized DKO mice resulted in a dramatic influx of neutrophils in BALF with increased expression of CXCL2, dense peribronchial mononuclear cell infiltrate and increased response to methacholine. All these responses were inhibited by neutralizing anti-IL-17A mAb. Since this rat mAb is specific to IL-17A and does not cross-react with IL-17F, which was elevated in the lungs of DKO mice. This result suggests that exaggerated IL-17A expression was responsible for the airway inflammation and AHR in EC sensitized DKO mice. In contrast, in a previous study with EC sensitized WT mice, IL-17 contributed only partially to airway inflammation and AHR18
. A role for IL-17 in human asthma is suggested by the observation that severe asthma is associated with accumulation of IL-17 and neutrophils in the airways12, 13, 34, 35
, and that IL-17 sputum level correlates with airway hyper-reactivity to methacholine36
Although BALF neutrophilia was present in EC sensitized DKO mice, their peribronchial infiltrate contained little, if any, neutrophils. This was unexpected given the fact that IL-17 mRNA expression was >20 fold higher in the challenged lungs of EC sensitized DKO mice than WT mice (). It was previously reported that transgenic mice that selectively overexpress IL-17A in the lung also develop a mononuclear peribronchial infiltrate with virtually no neutrophils 37
. These observations suggest that high expression levels of IL-17A in the lungs may interfere with peribronchial accumulation of neutrophils. The reason for this is at present unknown. Although modest, the accumulation of PAS positive material in the airway of challenged DKO mice is consistent with the observation that PAS positive material accumulates in the airway of mice that selectively overexpress IL-17A in the lung 37
The development of airway inflammation and AHR in EC sensitized DKO mice was rather unexpected because it has been reported that airway inflammation and AHR are absent in i.p.
immunized DKO mice25
. We demonstrated that i.p.
immunization, which does not induce a detectable Th17 response in WT mice18
, induced only a modest Th17 response in DKO mice (). The weak Th17 response of i.p
immunized DKO mice may explain their failure to develop airway inflammation and AHR following airway challenge.
Skin is an important portal for sensitization to environmental allergens in AD 38
and it is believed that asthma can be precipitated in patients with AD upon inhalation of allergens that have been first introduced via the skin39
. IL-17 is expressed in acute AD skin lesions 17
and we recently found that serum IL-17 levels are elevated in 6 of 20 patients with AD, but undetectable in 20 healthy controls (p<0.01). These observations suggest that EC sensitization in humans with AD may elicit a Th17 response. Although mouse models have limitations as to whether they replicate the pathogenesis of human disease, should a similar mechanism apply in humans, blockade of IL-4 and IL-13 in patients with AD could result in a heightened IL-17 response and IL-17 driven airway inflammation and AHR. This may explain the failure of therapies targeting IL-4Rα in several clinical trials for asthma 40