Protease allergen-induced HMGB1 contributes to NLRC4 inflammasome-mediated inflammation in experimental asthma.

Abstract

NLRC4 inflammasomes, an essential component of innate immunity, respond to stimuli during bacterial infections, specifically cytosolic flagellin and T3SS components, thereby inducing immune responses.1 Although inflammasomes are critical to host defense by activating caspase-1 to cleave pro-cytokines of IL-1β and IL-18 into their bioactive forms, overt inflammasome activation results in collateral damage to the host.1 With reference to asthma, the role of the NLRC4 inflammasomes remains unclear. In this study, we aimed to explore the role of NLRC4 inflammasomes in Aspergillus protease (AP) allergen-induced asthma and the mechanisms underlying NLRC4 inflammasome regulation via the damage-associated molecule pattern (DAMP) high-mobility group box 1 (HMGB1). To explore the role of the NLRC4 inflammasomes, we stimulated air–liquid interface (ALI)-cultured normal human bronchial epithelial cells (NHBEs; Figure 1A,B) with Aspergillus protease (AP) after measuring the proteolytic activity and endotoxin level of AP (Figure S1A,B; Appendix S1). Compared with control cells, AP-treated ALI-cultured NHBEs exhibited higher NLRC4, caspase-1, and IL-1β expression; no change in NLRP3 expression; and increased IL-1β release (Figure 1C–E). We then compared asthma symptoms in a mouse model. NLRC4-deficient and wild-type (WT) mice were challenged intranasally with a mixture of chicken egg ovalbumin (OVA) and AP (designated APO) (Figure 1F). Compared to WT mice, NLRC4-deficient mice showed reduced asthma with significantly decreased airway hyper-responsiveness (AHR) (Figure 1G); decreased serum immunoglobulin (IgE) (Figure 1H); decreased IL-4, IL-5, IL-13, TNF-α, and IL-17A expression; and fewer neutrophils and eosinophils in the bronchoalveolar lavage fluid (BALF) (Figure 1I–O). The lung and tracheal epithelium of APO-challenged, NLRC4-deficient mice showed decreased expression of inflammatory cell infiltration, mucus secretion (Figure S1C,D), caspase-1 and IL-1β expression (Figure 1R,S; Figure S1E), and decreased IL-1β secretion in the BALF (Figure 1P,Q). HMGB1 has various immunological activities, affecting chemotaxis and cytokine production and HMGB1 level is elevated in asthma.2 In addition, HMGB1 promotes inflammasome activation through Toll-like receptor (TLR) and NF-κB pathway.3, 4 Glycyrrhizic acid (GA), a direct inhibitor of HMGB1, binds directly to each of the two HMG boxes of HMGB15 and controls IL-1β levels by inhibiting the HMGB1-mediated TLR4/NF-κB pathway.6 Thus, we hypothesized that HMGB1 could be involved in IL-1β secretion and inflammasome assembly in AP-induced asthma. We found that AP treatment increased the HMGB1 level in ALI-cultured NHBEs (Figure S2A,B), and HMGB1 treatment (50 ng/mL) enhanced NLRC4, IL-1β, and caspase-1 activation and IL-1β release in a time-dependent manner (Figure S2C,D). Furthermore, we found that NLRC4 expression was reduced upon siRNA-mediated HMGB1 silencing (Figure S2F,G). Glycyrrhizic acid suppressed NLRC4, caspase-1, and pro-IL-1β expression in ALI-cultured NHBE lysates (Figure 2A). IL-1β release, which was increased after 1 h of AP stimulation, was decreased by GA treatment (Figure 2B,C). Next, we used an asthma model to determine whether the observed effects of AP-induced HMGB1 in vitro also occurred in vivo. The increased levels of AHR; serum IgE; and HMGB1, IL-4, IL-5, IL-13, TNF-α, IL-17A, IL-1 β, eosinophils, and neutrophils in BALF upon APO challenge were decreased by GA treatment (Figure 2D–O). In addition, GA treatment partially reversed the increased expression of NLRC4, caspase-1, and IL-1β in the lung of APO-challenged mice (Figure 2P–R). These results demonstrate that GA inhibits asthma symptoms and IL-1β release, likely through a decrease in the HMGB1/NLRC4-dependent caspase-1 level. To the best of our knowledge, this study is the first to provide experimental evidence that NLRC4 inflammasomes are involved in allergic asthma. Furthermore, we showed that DAMP HMGB1 is involved in increased NLRC4 inflammasome-dependent IL-1β release during the allergic response to AP. However, in this study, we used only fungal protease to induce asthma. Thus, further studies using other types of allergen-induced asthma models are needed to elucidate the role of NLRC4 inflammasomes in asthma, as specimens from patients with asthma have hypersensitivity to protease allergens. YHK conceived and designed the study. YHK and J-OL drafted the manuscript for important intellectual content. YHK, J-OL, JSK, B-YK, B-JP, S-JL, DEK, S-HL, I-SS, and TK performed the experiments, analysis, and interpretation of the manuscript. I-SS and TK supervised all processes. The authors thank Professor Jong-Hwan Park at Chonnam National University (CNU, Gwangju, Korea) for providing Nlrc4−/− mice and helpful discussion. This study was supported by a grant from the Korea Institute of Oriental Medicine (Grant No. KSN2013330), funded by the Korean Government (MSIP). The authors declare that there are no conflicts of interest. Figures S1–S2 Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.