NLRC4 Regulates Th2 Differentiation in Mice With Allergic Airway Inflammation Induced by House Dust Mite

Abstract

Inflammasomes, as components of the innate immune system, help defend the host and maintain cellular homeostasis by sensing invading pathogens or danger signals, which leads to the proteolytic maturation of IL-1β and IL-18 [1]. NLRC4 inflammasomes are well known for protecting mucosal barriers, including the lung and intestine, from bacterial pathogens by sensing flagellin or type 3 secretion system [2]. However, the excessive activation of NLRC4 inflammasomes can induce pathophysiological alterations, leading to various inflammatory and tumor-like diseases [3, 4]. Several studies have suggested that NLRC4 regulates eosinophilic inflammatory responses, and our previous research revealed its role in a fungal protease-induced allergic airway inflammation (AAI) model [5, 6]. However, the potential function of NLRC4 inflammasomes in the regulation of Th cell differentiation in AAI remains unclear.

In this study, we extended our investigation to house dust mite (HDM)-induced AAI, focusing on elucidating the underlying mechanism of NLRC4 in Th2 differentiation in the lung. We observed a notable elevation of NLRC4 expression in lungs from AAI mice (Figure S1A,B). We then explored the role of NLRC4 in the pathogenesis of AAI triggered by HDM using NLRC4 knock-out (KO) mice and adeno-associated virus (AAV)-mediated NLRC4 overexpression mice. To investigate the effects of NLRC4 deficiency in AAI, NLRC4 KO and wild-type (WT) mice were intranasally challenged with HDM extracts (Figure 1A). Compared to WT HDM mice, NLRC4 KO HDM mice showed reduced AAI symptoms with decreased airway hyperresponsiveness (AHR) (Figure 1B); lower eosinophils and neutrophils in the bronchoalveolar lavage fluid (BALF) (Figure 1C,D); decreased IL-4, IL-5, IL-13, IL-6, IL-1β, TNF-α, CCL22, and CCL17 levels in the BALF (Figure 1E–L); and decreased serum immunoglobulin E (IgE) (Figure 1M). The lungs of NLRC4 KO HDM mice exhibited decreased inflammatory cell infiltration, reduced mucus secretion (Figure S2A–C), lower caspase-1 and IL-1β expression (Figure 1N–R), and decreased populations of activated T cells (CD4⁺ CD69⁺) and Th2 cells (CD4⁺ GATA3⁺) (Figure 1S).

In addition, we performed in vitro experiments by incubating the splenic naïve CD4⁺ T cells from WT and NLRC4 KO mice under Th2 polarization conditions. Compared to the WT Th2 group, the NLRC4 KO Th2 group showed a decrease in the proportion of activated T cells and Th2 cells (Figure 1T) and in the level of IL-5 and IL-13 in the supernatant (Figure S2D,E).

To determine the effects of NLRC4 overexpression in the lungs during AAI development, AAV-NLRC4 or AAV-GFP (control) were intratracheally injected 7 days before the start of the HDM challenges (Figure 2A). Compared to AAV-GFP HDM mice, AAV-NLRC4 HDM mice showed exacerbated AAI symptoms with increased AHR (Figure 2B); higher eosinophils and unchanged neutrophils in the BALF (Figure 2C,D); increased IL-4, IL-5, IL-13, IL-6, IL-1β, TNF-α, CCL22, and CCL17 levels in the BALF (Figure 2E–L); and elevated serum IgE (Figure 2M). The lungs of AAV-NLRC4 HDM mice showed increased expression of inflammatory cell infiltration (Figure S3A,B), elevated caspase-1 and IL-1β expression (Figure 2N,O), and higher Th2 cell subset (Figure 2P). However, mucus production (Figure S3A,C) and the activated T cell populations (Figure 2P) were not affected.

To the best of our knowledge, this study is the first to suggest an unconventional role for NLRC4 in the regulation of Th2 cell responses to HDM allergens in the lung using NLRC4 KO and AAV-mediated NLRC4 overexpression mice. Our results provide a valuable insight into the role of NLRC4 in AAI.

Y.H.K. and I.-S.S. designed the experiments. S.-W.P. performed most of the experiments and drafted the manuscript. W.-I.K., S.-J.L., J.L., M.-J.P., J.-H.P., and J.-C.K. provided experimental support. T.K., J.-S.K., Y.H.K., and I.-S.S. discussed the results and reviewed the manuscript. T.K., J.-S.K., and I.-S.S. received grant support. I.-S.S. supervised the project.

The authors declare no conflicts of interest.