Distinct dampening of IL‐33 following inhalation of interferon‐lambda in the respiratory epithelium of in vivo asthma

The allergen-mediated inflammation begins with increased secretion of epithelial cell-derived cytokines, which provide critical signals to enhance T-helper (Th) 2 immune responses and interleukin (IL)-33, which is dominantly produced by the airway epithelium, is a key epithelial-derived cytokine involved in the pathogenesis of allergic asthma.1 Interferon (IFN)-regulated innate immune system serves as the first line of defense against respiratory viruses in the respiratory epithelium and inverse links between low expression of IFNs and the severity of allergic response have been described in the airways of asthma patients.2 Our previous study demonstrated that inhalation of IFN-λs might improve the asthma phenotypes in an in vivo lung and had therapeutic potential for allergic asthma.3 However, the exact mechanism by which IFN-λ suppresses Th2 immune response remain largely elusive. The cellular receptors of IFN-λs are mainly distributed in the respiratory epithelium and we presumed that the suppression of Th2 immune responses via inhaled IFN-λs might be associated with the reduction of IL-33 in the lung of asthma. For induction of allergic asthma, BALB/C mice were sensitized with house dust mites (HDM) extract (50 μg), and were administered recombinant IFN-λ (IFN-λ2 [2 μg] and IFN-λ3 [2 μg]) with HDM (50 μg) challenge (Figure 1A) (Supplementary material and methods). We found that IFN-stimulated genes were induced after inhalation delivery of IFN-λs, confirming that IFN-λ was well delivered to the lungs of asthmatic mice, and IFN-λ itself did not cause any pathologic findings in the lungs (Figure S1). Our data revealed that methacholine-increased total lung resistance was reduced in IFN-λs-treated asthmatic mice and goblet cell metaplasia were also improved after inhalation of IFN-λs (Figure 1B,C). Transcriptions of Il-4, Il-5, Il-13, and Il-17A were significantly downregulated in the lung of asthmatic mice following inhalation of IFN-λs (Figure 1D). We sought to determine whether inhalation of IFN-λ inhibited the function of innate lymphoid cells (ILCs) and CD4+ T cells using flow cytometry (Figure S2). Inhalation of IFN-λs significantly attenuated the number of HDM-induced Il-5, Il-13, and Il-17A-producing ILCs (Figure 1E) and CD4+ T cells in the lung of asthma (Figure 1F). However, no change in IFN-γ-producing ILCs and CD4+ T was observed in asthmatic mice exhibited through IFN-λ inhalation. Based on previous findings, lung epithelial cells might be the main cellular sources of IL-33 production in allergic airway diseases and full-length IL-33 could be cleaved during allergic inflammation, which generates mature forms of IL-33 with higher bioactivity.4-6 Our data revealed that mRNA and secreted protein of IL-33 were significantly reduced in the lung of asthmatic mice after IFN-λ inhalation (Figure 2A,B). In particular, both full-length and cleaved IL-33 protein in the total lung of asthmatic mice following inhalation of IFN-λ. The results showed that both full-length and cleaved IL-33 were induced in the lung of asthmatic mice, and inhalation of IFN-λ significantly decreased both forms of IL-33 (Figure 2C). To determine the effects of IFN-λ on respiratory epithelium, epithelial cells were isolated from mice lungs using EpCAM microbead and MACS (Figure 2D). We analyzed expression levels of IL-33 in the epithelial cell from the lung of HDM-challenged mice depending on inhalation of IFN-λ. Western blot showed that expression levels of full-length IL-33 were higher in the lung of asthmatic mice, and was significantly reduced in the lung with inhalation of IFN-λ (Figure 2E). The findings suggest that significant of full-length IL-33 by IFN-λ occurs in respiratory epithelial cells to which IFN-λ was directly delivered. These data provide compelling evidence that inhalation of IFN-λ led to reduced IL-33 generation in the respiratory epithelial cells resulting in the decrease of Th2 immune responses at the level of ILCs and CD4+ T cells. We estimate that a strategy to deliver IFN-λ to the respiratory epithelium will be an effective therapeutic model for inhalation therapy of allergic asthma. Jina Won: Designed and performed the research, analyzed the data, and wrote the paper. Ara Jo: Performed research. Haeun Shin: Performed research. Sujin Kim: Analyzed data. Hyun Jik Kim: Drafted the article and revised it critically for important intellectual content; provided approval of the version to be submitted. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education (2022R1A2C2011867) awarded to H.J.K and by the Ministry of Science and ICT (RS-2023-00222762), Korea awarded to H.J.K. This research was also supported by a grant from the Korean Health Technology R&D Project through the Korean Health Industry Development Institute, funded by the Ministry of Health and Welfare of the Republic of Korea (HI23C0795 awarded to H.J.K). The authors declare no conflicts of interest. The data that support the findings of this study are available in the supplementary material of this article. 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