Munc13‐4 regulates asthma and obesity in mice by controlling functions of CD11c+ antigen‐presenting cells

Asthma and obesity are both increasing in prevalence globally. Although the causal or pathophysiological relationship between these two diseases is unclear, a significant genetic correlation between these two diseases was recently reported.1 Among the shared disease risk loci, the UNC13D locus is of interest, because this locus has been associated with multiple immune and metabolic traits.2 The risk allele of this locus for two diseases decreases expression levels of UNC13D in immune cells (Figure S1), suggesting that the Munc13-4 protein encoded by UNC13D, which are expressed in both hematopoietic and non-hematopoietic cells, and regulates secretion of cytotoxic granules,3 plays a common protective role in two diseases by regulating immune cell functions. To clarify this hypothesis, we investigated the phenotypes of Jinx mice, which harbor the Unc13d gene mutation and lack Munc13-4 protein,4 in mouse models of diseases. In a mouse model of asthma, enhanced eosinophilic inflammation (Figure 1A) and Th2 cytokine production (Figure 1B) in the lungs, as well as higher production of Th2 cytokines by splenocytes in response to ovalbumin (OVA) re-stimulation (Figure 1C), were observed in Jinx mice. In a high fat diet (HFD)-induced mouse model of obesity and diabetes, Jinx mice persistently showed more adiposity (Figure 1D) with mildly decreased glucose tolerance and insulin sensitivity (Figure 1E) compared with wild type (WT) mice. Further, we clarified that Munc13-4 deficiency in bone marrow (BM) cells phenocopied those observations in Jinx mice (Figure S2A–D). In addition to its well-accepted role in induction of insulin resistance, we recently uncovered that white adipose tissue (WAT) inflammation promotes adiposity through upregulation of GDF3 in adipose tissue macrophages (ATM) and subsequent inhibition of lipolysis in adipocytes.5 In accordance with this, expression levels of MCP-1, F4/80, and GDF3 (Figure 1F) and ATM numbers (Figure 1G) in WAT were upregulated in Jinx mice, suggesting that Munc13-4 deficiency promoted adiposity by enhancing WAT inflammation. As Munc13-4 deficiency enhanced the Th2 response induced by antigen sensitization (Figure 1C Figure S2B), we examined the role of Munc13-4 in CD11c+ antigen-presenting cells (APC) which are also known to play an essential role in the induction of obesity-related chronic inflammation in WAT. Munc13-4 were expressed in mouse splenic CD11c+ APC with being enriched in the CD11chigh MHC-classIIhigh dendritic cell population, and in BM-derived CD11c+ APC (Figure 2A). Although Munc13-4 deficiency did not affect either MHC-classII expression levels or the antigen-presenting capacity of CD11c+ APC (Figure 2B), it significantly suppressed secretion of immuno-regulatory cytokines, IL-10 (Figure 2C) and IL-12 (that also regulates chronic inflammation)6 (Figure 2D), by CD11c+ APC. Transfer of Jinx mice-derived OVA-pulsed BM-CD11c+ APC into OVA/alum-sensitized mice significantly increased eosinophilic lung inflammation, which was mainly medicated by MHC-classII+ fraction (Figure 2E). Transfer of Munc13-4-deficient splenic CD11c+ APC into HFD-fed mice also exacerbated WAT inflammation and insulin resistance (Figure 2F). These results suggest that Munc13-4 deficiency in CD11c+ APC plays an essential role in the induction of the phenotypes observed in Jinx mice. In the current study, we newly established that Munc13-4 plays an important regulatory role in inflammation by controlling CD11c+ APC functions. Furthermore, to our best knowledge, this is also the first study to determine the unknown pathophysiological roles in vivo of such a gene identified by genome-wide association studies as a common risk locus for both asthma and obesity. Our findings suggests that the risk allele of the UNC13D locus with reduced Munc13-4 expression exacerbates human disorders by modulating CD11c+ APC functions. We hope that these findings will inform the development of new therapeutic strategies that target and potentiate Munc13-4 functions in the simultaneous treatment of asthma and obesity. This work was supported by JSPS KAKENHI Grant Numbers 26,461,484, 17 K09994, 20 K08796, and 23H03302 to K.O. and 14F04104, 18 K19554, and 19H03449 to T.I. It was also supported by grants from Gunma University, Takeda Science Foundation, Senshin Medical Research Foundation, Naito Foundation, Astellas Foundation for Research on Metabolic Disorders, Ono Medical Research Foundation, and the Basic Research Support Program from Japanese Society of Allergology to K.O., Sanofi Scholarship Donation, Mitsubishi Tanabe Pharma Scholarship Donation to T.I., by the joint research program of the Institute for Molecular and Cellular Regulation, Gunma University, 21,012 to Y.K., and 19,012 to K.N., and by a grant from Ministry of Education, Culture, Sports, Science and Technology to Y.K. K.O. designed and performed experiments, wrote the manuscript, and supervised the whole project. Y.K. investigated the correlation between the UNC13D locus at 17q25.1 and diseases through GWAS catalog, and eQTL effect of the locus through data bases such as the GTEx project and the ImmuNexUT project, analyzed the data, and wrote genetic parts in the manuscript. M.Z. and H.W. performed experiments. K.N. analyzed data, proposed experimental plans, and gave fruitful comments during manuscript preparation. T.I. supervised the project, analyzed data, and helped K.O. write the manuscript by giving substantial comments. The authors thank the members of the Laboratories of Molecular Endocrinology and Metabolism and of Endocrine and Metabolic System Regulation, Gunma University, particularly K. Hoshino for performing the initial experiments, T. Nara, E. Kobayashi, Y. Izawa, and M. Suto for their technical assistance and for colony maintenance of mice, T. Ushigome for colony maintenance of mice, and S. Shigoka for general management. The authors also thank the staffs at Bioresource Center, Gunma University, for their help in breeding of mice. The authors have declared that no conflict of interest exists. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. 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