IL-9, a pleiotropic cytokine, has been implicated in the pathogenesis of many atopic diseases; including allergic rhinitis, asthma, atopic dermatitis, contact dermatitis, and food allergy; through a variety of activities on mast cells, epithelial cells and smooth muscle cells18–20. Previous studies have addressed the role of IL-9 in inducing changes associated with human asthma by investigating the airways of animals by blockade or genetic knock-down of IL-9 designed to induce allergic inflammation or over expression of IL-96,9,21. The present study further explored the role of IL-9 by comparing the differences in airway inflammation and percentages and numbers of target cells (ILC2 cells, Th2 cells and mast cells) in murine asthma models of WT and Il9−/−mice challenged with HDM. It has been well documented that type 2 cytokines IL-5 and IL-13 are implicated in the pathogenesis of the eosinophil-rich airway inflammation, which typically characterizes asthma22,23. In the present study we demonstrated that IL-9 deficiency clearly attenuated the production of type 2 cytokines. This finding is in accordance with a previous study, which showed that IL-9 blockade reduced the concentrations of these mediators in the BALF of OVA-sensitized and challenged mice8. Using HDM-challenged murine models of asthma, our data further indicated that IL-9 deficiency reduced proliferation of target cells (ILC2 cells, Th2 cells and mast cells) and infiltration of eosinophils. Furthermore, IL-9 deficiency may also partly account for reduced goblet cell hyperplasis and hypertrophy observed in the present study, particularly as type 2 cytokines and eosinophils have been implicated as contributory factors to these phenomena24–26.
The epithelium-derived cytokines IL-33, TSLP, and IL-25 have been implicated in pathogenesis of asthma because they promote type 2 cytokine synthesis27,28. Correspondingly, our data showed that the expression of these cytokines increased in the HDM-sensitized and challenged mice. Interestingly, IL-9 deficiency did not further reduce the expression of IL-33, IL-25 and TSLP, although the airway infiltration of inflammatory cells, goblet cell hyperplasia, and type 2 cytokines production were attenuated. These observations suggest that IL-9 may be a critical downstream component of IL-33, IL-25 and TSLP signaling pathways in asthma pathogenesis. Moreover, these data are consistent with previous studies which have shown that IL-9 was required for allergic airway inflammation mediated by TSLP and IL-9 deficiency attenuated airway inflammation in the IL-33-induced asthma model17,29. These observations might also, partially explain why deletion of the IL-9 gene could not completely abolish HDM-induced airways inflammation.
It has been reported that ILC2 cells are able to produce IL-9, which is crucial for survival of the ILC2 cells30. Whilst the numbers of ILC2s in helminth-induced lung inflammation have been shown to be reduced in IL-9 receptor deficient mice30, neutralization of IL-9 has not been found to affect the ILC2 numbers in lung tissues in a papain-induced murine model of lung inflammation21. However, our data have shown that IL-9 deficiency markedly attenuated ILC2 cell accumulation and production of IL-5 and IL-13, which are consistent with the findings from other studies. For example, IL-9 deficiency in ILC2 cells led to a decrease in IL-5 and IL-13 production in cells cultured in the presence of TSLP and IL-33, despite the number of ILC2 cells not being affected21,31,32. Taken together, these results suggest that IL-9 may promote allergic inflammation by amplifying the function of ILC2.
With reference to the different Th subsets, it has been shown that IL-9 acts as a growth factor for especially Th2 and Th17 cells13,20. Previous studies have shown that anti-IL-9 antibody treatment was able to inhibit airway inflammation by reducing the number of Th17 cells and IL-17 levels9. Additionally, it has been also shown that IL-9 mediates Th17 differentiation in vitro33,34. Furthermore, pulmonary overexpression of IL-9 appears to induce Th2 differentiation leading to pathologic changes in the lungs35. Our findings for Th2 cells are also in accordance with these studies, and have further demonstrated that IL-9 deficiency significantly decreased Th2 cells infiltration and production of IL-4, IL-5, IL-13 and IL-17A; suggest that IL-9 can act on Th2 cells and Th17 cells, which are major contributors to allergic inflammation, either directly or indirectly.
Mast cells are a main target of IL-9, which serves as a growth factor for these cells and also influences their function36,37. The present study demonstrated that mast cell accumulation in the lung tissue of HDM-challenged mice was clearly attenuated by IL-9 deficiency, and this was supported by the reduction of mMCP-1 in the lung tissue homogenate of the experimental animals. These findings are in accordance with previous studies, which indicated that accumulation of mast cells in a model of acute allergic inflammation was dependent on IL-9, because neutralizing IL-9 dramatically reduced the number of mast cells infiltrating the lungs10,38. These data suggest that IL-9 inhibition might be required to diminish pulmonary mast cell numbers, possibly providing some beneficial effects in asthmatics.
Our studies have shown that IL-9 deficiency markedly reduced the numbers of ILC2 cells, Th2 cells and mast cells, in BALF and the lung tissues of HDM-challenged mice. Thus, it is reasonable to speculate that IL-9 deficiency might inhibit ILC2 cells, Th2 cells and mast cells accumulation in the lung by leading to decreased proliferation of these cells. Indeed, assessment of the expression of Ki67 in these cells, as a marker for cells that were in active phases of the cell cycle, demonstrated that the number of Ki67+ILC2 cells, Ki67+Th2 cells and Ki67+mast cells in the Il9−/−mice was clearly reduced compared to that in WT mice following HDM challenge, thus supporting the hypothesisthat IL-9 deficiency may partly affect proliferation of ILC2, Th2 and mast cells.
In summary, our study has demonstrated a critical role for IL-9 in regulating numbers of ILC2 cells, Th2 cells, mast cells and expression of cytokines produced by these cells in the airways of HDM sensitized mice, following allergen challenge. Furthermore, IL-9 deficiency results in a profound decrease in inflammatory cellular infiltration and goblet cell hyperplasia. It is possible that IL-9 might promote proliferation of ILC2 cells, Th2 cells and mast cells. These findings should improve the understanding of the pathogenesis of asthma and facilitate the development of novel therapies for treatment of this disease.