CTLA-4-expressing ILC3s restrain interleukin-23-mediated inflammation.

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms that shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.

Identification of anoctamin 1 (ANO1) as a key driver of esophageal epithelial proliferation in eosinophilic esophagitis.

BACKGROUND: The pathology of eosinophilic esophagitis (EoE) is characterized by eosinophil-rich inflammation, basal zone hyperplasia (BZH), and dilated intercellular spaces, and the underlying processes that drive the pathologic manifestations of the disease remain largely unexplored. OBJECTIVE: We sought to investigate the involvement of the calcium-activated chloride channel anoctamin 1 (ANO1) in esophageal proliferation and the histopathologic features of EoE. METHODS: We examined mRNA and protein expression of ANO1 in esophageal biopsy samples from patients with EoE and in mice with EoE. We performed molecular and cellular analyses and ion transport assays on an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI) and murine models of EoE to define the relationship between expression and function of ANO1 and esophageal epithelial proliferation in patients with EoE. RESULTS: We observed increased ANO1 expression in esophageal biopsy samples from patients with EoE and in mice with EoE. ANO1 was expressed within the esophageal basal zone, and expression correlated positively with disease severity (eosinophils/high-power field) and BZH. Using an in vitro esophageal epithelial 3-dimensional model system revealed that ANO1 undergoes chromatin modification and rapid upregulation of expression after IL-13 stimulation, that ANO1 is the primary apical IL-13-induced Cl- transport mechanism within the esophageal epithelium, and that loss of ANO1-dependent Cl- transport abrogated esophageal epithelial proliferation. Mechanistically, ANO1-dependent regulation of basal cell proliferation was associated with modulation of TP63 expression and phosphorylated cyclin-dependent kinase 2 levels. CONCLUSIONS: These data identify a functional role for ANO1 in esophageal cell proliferation and BZH in patients with EoE and provide a rationale for pharmacologic intervention of ANO1 function in patients with EoE.

17ß-Estradiol protects the esophageal epithelium from IL-13-induced barrier dysfunction and remodeling.

BACKGROUND: The incidence of eosinophilic esophagitis (EoE) is greater in male than female subjects, and the underlying molecular basis for this sex bias remains unclear. OBJECTIVE: We sought to delineate the contribution of the sex hormone estrogen to the EoE phenotype and esophageal epithelial barrier function and remodeling. METHODS: We performed demographic and incidence analyses of EoE in male and female subjects from a single-center pediatric cohort. Estrogen-responsive gene expression analyses and estrogen receptor (ESR) immunofluorescence staining of esophageal biopsy specimens from patients with EoE and control subjects were performed. The effect of 17ß-estradiol (E2) on IL-13-induced signaling pathways, gene expression, and esophageal epithelial architecture and barrier function in a primary human esophageal keratinocyte cell (EPC2) culture system (EPC2-air-liquid interface) was examined. RESULTS: We observed a male predominance in patients with EoE. Analyses of RNA sequencing data sets revealed a significant dysregulation of the estrogen-responsive gene network and expression of ESR1 and ESR2 in esophageal biopsy specimens from patients with EoE compared with control subjects. IL-13 stimulation of EPC2-air-liquid interface cells led to altered cellular architecture with induced dilation of intercellular spaces and barrier dysfunction. Pretreatment of EPC2s with E2 prior to IL-13 exposure abrogated IL-13-induced architectural changes and esophageal barrier dysfunction. Mechanistically, E2-protective effects were dependent on ESR2 and associated with diminishing of IL-13-induced tyrosine kinase 2 and signal transducer and activator of transcription 6 phosphorylation and EoE-dysregulated gene expression. CONCLUSIONS: Estrogen-responsive genes are modified in patients with EoE compared with control subjects. E2 attenuated IL-13-induced architectural changes and esophageal epithelial barrier dysfunction through inhibition of the IL-13/tyrosine kinase 2/signal transducer and activator of transcription 6 pathway via ESR2-dependent process. Estrogen hormone signaling may protect against development of EoE in female subjects.

Solute carrier family 9, subfamily A, member 3 (SLC9A3)/sodium-hydrogen exchanger member 3 (NHE3) dysregulation and dilated intercellular spaces in patients with eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by histopathologic modifications of esophageal tissue, including eosinophil-rich inflammation, basal zone hyperplasia, and dilated intercellular spaces (DIS). The underlying molecular processes that drive the histopathologic features of EoE remain largely unexplored. OBJECTIVE: We sought to investigate the involvement of solute carrier family 9, subfamily A, member 3 (SLC9A3) in esophageal epithelial intracellular pH (pHi) and DIS formation and the histopathologic features of EoE. METHODS: We examined expression of esophageal epithelial gene networks associated with regulation of pHi in the EoE transcriptome of primary esophageal epithelial cells and an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI). Molecular and cellular analyses and ion transport assays were used to evaluate the expression and function of SLC9A3. RESULTS: We identified altered expression of gene networks associated with regulation of pHi and acid-protective mechanisms in esophageal biopsy specimens from pediatric patients with EoE (healthy subjects, n = 6; patients with EoE, n = 10). The most dysregulated gene central to regulating pHi was SLC9A3. SLC9A3 expression was increased within the basal layer of esophageal biopsy specimens from patients with EoE, and expression positively correlated with disease severity (eosinophils/high-power field) and DIS (healthy subjects, n = 10; patients with EoE, n = 10). Analyses of esophageal epithelial cells revealed IL-13-induced, signal transducer and activator of transcription 6-dependent SLC9A3 expression and Na+-dependent proton secretion and that SLC9A3 activity correlated positively with DIS formation. Finally, we showed that IL-13-mediated, Na+-dependent proton secretion was the primary intracellular acid-protective mechanism within the esophageal epithelium and that blockade of SLC9A3 transport abrogated IL-13-induced DIS formation. CONCLUSIONS: SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in patients with EoE.

Dietary fiber is a critical determinant of pathologic ILC2 responses and intestinal inflammation.

Innate lymphoid cells (ILCs) can promote host defense, chronic inflammation, or tissue protection and are regulated by cytokines and neuropeptides. However, their regulation by diet and microbiota-derived signals remains unclear. We show that an inulin fiber diet promotes Tph1-expressing inflammatory ILC2s (ILC2INFLAM) in the colon, which produce IL-5 but not tissue-protective amphiregulin (AREG), resulting in the accumulation of eosinophils. This exacerbates inflammation in a murine model of intestinal damage and inflammation in an ILC2- and eosinophil-dependent manner. Mechanistically, the inulin fiber diet elevated microbiota-derived bile acids, including cholic acid (CA) that induced expression of ILC2-activating IL-33. In IBD patients, bile acids, their receptor farnesoid X receptor (FXR), IL-33, and eosinophils were all upregulated compared with controls, implicating this diet-microbiota-ILC2 axis in human IBD pathogenesis. Together, these data reveal that dietary fiber-induced changes in microbial metabolites operate as a rheostat that governs protective versus pathologic ILC2 responses with relevance to precision nutrition for inflammatory diseases.

PIR-B Regulates CD4+ IL17a+ T-Cell Survival and Restricts T-Cell-Dependent Intestinal Inflammatory Responses.

BACKGROUND & AIMS: CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. METHODS: We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non-inflammatory bowel disease patients and sorted human memory CD4+ T cells. RESULTS: We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B- Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1-dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. CONCLUSIONS: Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses.