IL-33 Induces Cellular and Exosomal miR-146a Expression as a Feedback Inhibitor of Mast Cell Function.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow-derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell-deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

Expression Efficiency of Multiple Il9 Reporter Alleles Is Determined by Cell Lineage.

Generation of allelic gene reporter mice has provided a powerful tool to study gene function in vivo. In conjunction with imaging technologies, reporter mouse models facilitate studies of cell lineage tracing, live cell imaging, and gene expression in the context of diseases. Although there are several advantages to using reporter mice, caution is important to ensure the fidelity of the reporter protein representing the gene of interest. In this study, we compared the efficiency of two Il9 reporter strains Il9citrine and Il9GFP in representing IL-9-producing CD4+ TH9 cells. Although both alleles show high specificity in IL-9-expressing populations, we observed that the Il9GFP allele visualized a much larger proportion of the IL-9-producing cells in culture than the Il9citrine reporter allele. In defining the mechanistic basis for these differences, chromatin immunoprecipitation and chromatin accessibility assay showed that the Il9citrine allele was transcriptionally less active in TH9 cells compared with the wild-type allele. The Il9citrine allele also only captured a fraction of IL-9-expressing bone marrow-derived mast cells. In contrast, the Il9 citrine reporter detected Il9 expression in type 2 innate lymphoid cells at a greater percentage than could be identified by IL-9 intracellular cytokine staining. Taken together, our findings demonstrate that the accuracy of IL-9 reporter mouse models may vary with the cell type being examined. These studies demonstrate the importance of choosing appropriate reporter mouse models that are optimal for detecting the cell type of interest as well as the accuracy of conclusions.

The Il9 CNS-25 Regulatory Element Controls Mast Cell and Basophil IL-9 Production.

IL-9 is an important mediator of allergic disease that is critical for mast cell-driven diseases. IL-9 is produced by many cell types, including T cells, basophils, and mast cells. Yet, how IL-9 is regulated in mast cells or basophils is not well characterized. In this report, we tested the effects of deficiency of a mouse Il9 gene regulatory element (Il9 CNS-25) in these cells in vivo and in vitro. In mast cells stimulated with IL-3 and IL-33, the Il9 CNS-25 enhancer is a potent regulator of mast cell Il9 gene transcription and epigenetic modification at the Il9 locus. Our data show preferential binding of STAT5 and GATA1 to CNS-25 over the Il9 promoter in mast cells and that T cells and mast cells have differing requirements for the induction of IL-9 production. Il9 CNS-25 is required for IL-9 production from T cells, basophils, and mast cells in a food allergy model, and deficiency in IL-9 expression results in decreased mast cell expansion. In a Nippostrongylus brasiliensis infection model, we observed a similar decrease in mast cell accumulation. Although decreased mast cells correlated with higher parasite egg burden and delayed clearance in vivo, T cell deficiency in IL-9 also likely contributes to the phenotype. Thus, our data demonstrate IL-9 production in mast cells and basophils in vivo requires Il9 CNS-25, and that Il9 CNS-25-dependent IL-9 production is required for mast cell expansion during allergic intestinal inflammation.

Lactic Acid Inhibits Lipopolysaccharide-Induced Mast Cell Function by Limiting Glycolysis and ATP Availability.

Sepsis has a well-studied inflammatory phase, with a less-understood secondary immunosuppressive phase. Elevated blood lactate and slow lactate clearance are associated with mortality; however, regulatory roles are unknown. We hypothesized that lactic acid (LA) contributes to the late phase and is not solely a consequence of bacterial infection. No studies have examined LA effects in sepsis models in vivo or a mechanism by which it suppresses LPS-induced activation in vitro. Because mast cells can be activated systemically and contribute to sepsis, we examined LA effects on the mast cell response to LPS. LA significantly suppressed LPS-induced cytokine production and NF-κB transcriptional activity in mouse bone marrow-derived mast cells and cytokine production in peritoneal mast cells. Suppression was MCT-1 dependent and reproducible with sodium lactate or formic acid. Further, LA significantly suppressed cytokine induction following LPS-induced endotoxemia in mice. Because glycolysis is linked to inflammation and LA is a byproduct of this process, we examined changes in glucose metabolism. LA treatment reduced glucose uptake and lactate export during LPS stimulation. LA effects were mimicked by glycolytic inhibitors and reversed by increasing ATP availability. These results indicate that glycolytic suppression and ATP production are necessary and sufficient for LA effects. Our work suggests that enhancing glycolysis and ATP production could improve immune function, counteracting LA suppressive effects in the immunosuppressive phase of sepsis.

Designer covalent heterobivalent inhibitors prevent IgE-dependent responses to peanut allergen.

Allergies are a result of allergen proteins cross-linking allergen-specific IgE (sIgE) on the surface of mast cells and basophils. The diversity and complexity of allergen epitopes, and high-affinity of the sIgE-allergen interaction have impaired the development of allergen-specific inhibitors of allergic responses. This study presents a design of food allergen-specific sIgE inhibitors named covalent heterobivalent inhibitors (cHBIs) that selectively form covalent bonds to only sIgEs, thereby permanently inhibiting them. Using screening reagents termed nanoallergens, we identified two immunodominant epitopes in peanuts that were common in a population of 16 allergic patients. Two cHBIs designed to inhibit only these two epitopes completely abrogated the allergic response in 14 of the 16 patients in an in vitro assay and inhibited basophil activation in an allergic patient ex vivo analysis. The efficacy of the cHBI design has valuable clinical implications for many allergen-specific responses and more broadly for any antibody-based disease.

A conserved enhancer regulates Il9 expression in multiple lineages.

Cytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production.