The results of this study show that IL-22 and its receptor are highly expressed in the airways of severe asthmatics, and that bronchial epithelial cells from severe asthmatics are more sensitive to the effects of IL-22 stimulation in the context of TGF-β1 exposure, thus supporting a role for this cytokine in more severe, steroid refractory phenotypes of this disease.
It has become clear in recent years that different phenotypes of asthma are differentially regulated by cytokines. While Th2 cytokines are involved in milder forms of allergic asthma, Th17 cytokines (IL-17A, IL-17 F and IL-22) are more strongly associated with severe, difficult to treat asthma [3–6]. However, there is currently limited information on the role of Th17 associated cytokines, including IL-22, in human asthma. Zhao et al. demonstrated that the percentage of Th17 cells and plasma concentrations of IL-17 and IL-22 are increased in proportion to the severity of allergic airway disease . In vitro, it has been shown that IL-22 promotes the proliferation and migration of airway smooth muscle cells [28, 29]. It has also been shown that ovalbumin (OVA)-sensitized and challenged Balb/C mice express IL-22 in the lung, whereas this cytokine is undetectable in control animals . Thus, it is likely that the co-expression of IL-22 along with other cytokines, for example IL-17A or TGF-β1, may have different effects than if IL-22 is expressed alone. In severe asthma, there is significantly higher expression of TGF-β1 compared to milder forms of asthma , suggesting the possibility that, in severe asthma, IL-22 may have different effects than in acute or mild disease because of the associated expression of TGF-β1. TGF-β1 is a potent promoter of EMT in airway epithelial cells . Recently, it has been shown that TGF-β1-induced EMT in human bronchial epithelial cells is enhanced by IL-1β  and TNF-α , but the role of other cytokines such as IL-22 in the induction of EMT has not been explored.
The results from this study corroborate the findings of Zhao et al.  as IL-22 expression was predominantly detected in the subepithelial region of inflamed airways in severe asthma patients. As further support for the increased activity of IL-22 in severe asthma, primary bronchial epithelial cells obtained from severe asthmatics expressed significantly higher levels of the IL-22 receptor. Taken together, these results suggest that IL-22 expression and signaling is associated with severe allergic airway disease rather than milder forms of asthma. However, as some studies have demonstrated a tissue-protective role of IL-22 in terms of reducing the expression of proinflammatory cytokines such as IFN-γ  and enhancing barrier function , it was important to evaluate the impact of IL-22 stimulation on airway epithelial cells, both alone and in the context of stimulation with TGF-β1, a cytokine that is closely associated with severe asthma and tissue remodeling due to its role in the induction of EMT.
Previous studies have demonstrated that well-differentiated airway epithelial cell cultures from asthmatics undergo EMT more readily compared to control subjects, suggesting that epithelial repair in asthmatic airways is dysregulated , a finding which is supported by the results of the current study. Based on cellular morphology following 5 days of stimulation with TGF-β1, either with or without concomitant IL-22 stimulation, primary epithelial cells derived from patients with severe asthma underwent a more complete transition to a mesenchymal phenotype compared to cells from mild asthmatics and normal control subjects. This change from a typical epithelial cobblestone-like morphology to spindle-shaped mesenchymal cells driven by TGF-β1 is well-described in the literature, not only regarding airway epithelial cells in the context of asthma [17, 32], but also in the context of tumor cell metastasis . The results of this study show that the morphological change induced by TGF-β1 in airway epithelial cells is a factor of disease severity in the patients from whom the cells were derived, supporting previous studies , but covering a broader range of disease severity.
The switch from an epithelial to a mesenchymal phenotype was assessed by evaluating changes in the expression of epithelial E-cadherin and mesenchymal N-cadherin by qPCR, along with the expression of MUC5AC, an airway epithelial marker, and vimentin, a mesenchymal marker which is frequently investigated in studies on EMT . TGF-β1 robustly decreased the expression of MUC5AC (by 80-90%) in primary bronchial epithelial cells from all subjects, demonstrating the loss of a characteristic airway epithelial cell marker under these conditions, although no further reduction in MUC5AC levels was observed when IL-22 was given to these cells along with TGF-β1. Conversely, TGF-β1 stimulation induced a milder (~50%) reduction in E-cadherin mRNA expression, which was only significant in cells from healthy control and severe asthmatics, suggesting that E-cadherin is more robustly expressed and tightly regulated than mucin genes under EMT conditions. IL-22 stimulation in the context of TGF-β1 exposure led to a further reduction in the expression of E-cadherin mRNA, although these changes were only statistically significant in cells derived from severe asthmatics. qPCR analysis was also performed for N-cadherin and vimentin to evaluate the impact of IL-22 and TGF-β1 stimulation on the expression of mesenchymal genes in bronchial epithelial cells. As expected, a significant upregulation in N-cadherin and vimentin mRNA was seen in the cells from all three patient groups following 3 days of stimulation with TGF-β1, while no effects of IL-22 were observed on the expression of mesenchymal genes, either when given alone or in combination with TGF-β1. These results demonstrate that, unlike TGF-β1, IL-22 is not a bona fide EMT-inducing cytokine, as it does not appear to induce a global change in epithelial and mesenchymal gene expression as observed in cells treated with TGF-β1. However, the further decrease in E-cadherin mRNA expression in severe asthmatic cells when IL-22 was added with TGF-β1 suggests that IL-22 may facilitate EMT in severe disease by further depressing E-cadherin expression.
This finding was supported by Western blot analysis of the cadherin switch in these cells, with significantly higher levels of N-cadherin and a virtual disappearance of E-cadherin seen in the cells from severe asthmatics following stimulation with TGF-β1. As seen on the mRNA level, a trend for a further decrease in E-cadherin expression was observed in severe asthmatic cells treated with both IL-22 and TGF-β1 compared to expression levels following TGF-β1 stimulation alone. This effect was more evident when the ratio of E-cadherin to N-cadherin was determined in these cells, as severe asthmatic cells demonstrated a more profound cadherin switch when IL-22 stimulation occurred in the context of TGF-β1 exposure. These results confirm that TGF-β1 potently suppresses the expression of epithelial adherens junction proteins in primary bronchial epithelial cells, and that concurrent stimulation with IL-22 contributes to this suppression, predominantly in cells taken from patients with severe asthma pathology. This finding is especially interesting given previous studies showing impaired intestinal epithelial barrier function in IL-22 deficient mice . In the present study, treatment with IL-22 led to a slight but not significant increase in the expression of E-cadherin protein levels in healthy control cells; however, an assessment of barrier function in cultured airway epithelial cells was not within the scope of the present investigation.
The effects of TGF-β1 on epithelial and mesenchymal gene expression in human airway epithelial cells have been explored in a number of studies [21, 32, 33, 38, 39]. The results obtained in this study, with decreased expression of E-cadherin as well as increased expression of vimentin and N-cadherin, agree with these previous reports. However, the role of IL-22 in EMT, either alone or in the context of TGF-β1 stimulation, has not yet been investigated. This study provides novel results in that the combined impact of IL-22 with TGF-β1 was associated with an additive effect on the suppression of E-cadherin in primary bronchial epithelial cells, thus promoting the loss of adherens junctions in these cells, which has been previously described as an early event in the process of EMT . It is important to highlight the fact that IL-22 mediated its most robust effects in the context of TGF-β1 stimulation in cells obtained from severe asthmatics. This result corroborates previous studies showing that asthmatic epithelial cells more readily progress through EMT , but provide novel insight into the mechanism by which this occurs. As IL-22 is highly expressed in severe asthmatics compared to mild asthmatics and normal control subjects, exposure to IL-22 in vivo may increase the sensitivity of these cells to EMT-promoting stimuli such as TGF-β1 in vitro. Further studies are certainly warranted to investigate the molecular mechanisms responsible for this, as well as the impact of other cytokines expressed in severe asthma, such as IL-17A, on the ability of bronchial epithelial cells to progress through EMT.
IL-22 mediates its signaling through a heterodimeric receptor composed of the IL-22R1 chain and the IL-10R2 chain ; downstream signaling is mediated predominantly via STAT3 . Conversely, TGF-β1 signals through the type II TGF-β receptor (TGF-βRII), which then phosphorylates and activates signaling Smads such as Smad2, Smad3 and Smad4. Once activated, these Smads translocate to the nucleus to mediate their effects on the transcription of target genes . To investigate the transcriptional regulation of EMT in primary bronchial epithelial cells stimulated with IL-22, TGF-β, and IL-22+TGF-β1, changes in the expression of EMT-associated transcription factors were investigated by qPCR. As expected, TGF-β1 stimulation alone potently upregulated the mRNA expression of all these transcription factors, most notably in cells derived from severe asthmatics. Costimulation with IL-22 and TGF-β1 had variable effects, with no change in the expression of Snail2 and Zeb2, a trend for a reduction in the expression of Twist1 and Twist2, and a significant increase in the expression of Snail1 and Zeb1 relative to expression levels following stimulation with TGF-β1 alone. Interestingly, the highest levels of Snail1 and Zeb1 were observed in cells obtained from severe asthmatics, with evidence of a synergistic effect of IL-22 and TGF-β1 on the mRNA expression of these key EMT-associated transcription factors in severe asthmatic bronchial epithelial cells, which may explain the profound cadherin switch observed in these cells. Previous studies have demonstrated that Snail1 forms a transcriptional repressor complex with Smad3 and Smad4 to promote EMT in epithelial cells; suppression of both Snail and Smad4 by siRNA potently suppressed the induction of EMT, supporting the key role played by these transcription factors in this process . In the present study, concurrent stimulation of severe asthmatic bronchial epithelial cells with IL-22 and TGF-β1 led to a robust upregulation in Snail1 expression. This result may explain the effect of combined IL-22/TGF-β1 stimulation on E-cadherin repression in severe asthmatic cells, as this gene is highly sensitive to repression by the Snail1/Smad complex , whereas Twist transcription factors have been found to affect E-cadherin expression only indirectly .
Taken together, the results of this study suggest that the process of EMT as a factor contributing to the development of airway remodeling may only be clinically meaningful in patients with severe asthma. However, a strategy to inhibit the expression or signaling of cytokines that play a role in this process in milder stages of the disease may have a beneficial impact on lung structure and function by impeding this process. Further in vivo investigations are required to establish the effect of IL-22 inhibition on the progression of airway remodeling in chronic allergic asthma.