Inducible pluripotent stem cells to study human mast cell trajectories.

Mast cells (MCs) are derived from CD34+ hematopoietic progenitors, consist of different subtypes, and are involved in several inflammatory conditions. However, our understanding of human MC developmental trajectories and subtypes has been limited by a scarcity of suitable cellular model systems. Herein, we developed an in vitro model of human MC differentiation from induced pluripotent stem cells (iPSC) to study human MC differentiation trajectories. Flow cytometry characterization of hemopoietic cells derived from the myeloid cells-forming complex (MCFC) revealed an initial increase in Lin- CD34+ hematopoietic progenitors within Weeks 1-3, followed by an increase in CD34- CD45RA- SSClow and SSChigh hematopoietic cells. The Lin- CD34+ hematopoietic progenitors consisted of SSClow CD45RA- CD123± c-Kit+ FcεRI+ populations that were ß7-integrinhigh CD203c+ and ß7-integrinhigh CD203c- cells consistent with CMPFcεRI+ cells. Flow cytometry and cytologic analyses of the CD34- Lin- (SSClow) population revealed hypogranular cell populations, predominantly characterized by CD45RA- CD123± c-Kit+ FcεRI- ß7-integrinlow and CD45RA- CD123± c-Kit- FcεRI+ ß7-integrinMid cells. Analyses of hypergranular SSChigh cells identified Lin- CD34- CD45RA- c-Kit+ FcεRI- and Lin- CD34- CD45RA- c-Kit+ FcεRI+ cells. scRNA-seq analysis of the cells harvested at week 4 of the MCFC culture revealed the presence of monocyte and granulocyte progenitors (n = 547 cells, 26.7 %), Erythrocyte / unknown (n = 85, 4.1 %), neutrophils / myelocytes (n = 211 cells, 10.2 %), mast cell progenitor 1 (n = 599, 29.1 %), mast cell progenitor 2 (n = 152, 7.4 %), committed mast cell precursor (n = 113, 5.5 %), and MCs (n = 353, 17.1 %). In silico analyses of the MC precursor and mature MC populations revealed transcriptionally distinct MC precursor subtype and mature MC states (CMA1+ and CMA1- subtypes). Culturing MC precursor populations in MC maturation media (mast cell media II) led to homogenous mature MC populations as evidenced by high expression of high-affinity IgE receptor, metachromatic granules, presence of MC granule proteins (Tryptase and Chymase) and activation following substance P stimulation and FcεRI crosslinking. This human iPSC-based approach generates MC precursors and phenotypically mature and functional MC populations. This system will be a useful model to generate human MC populations and broaden our understanding of MC biology and transcriptional regulation of MC differentiation trajectories.

IL-13-induced STAT3-dependent signaling networks regulate esophageal epithelial proliferation in eosinophilic esophagitis.

BACKGROUND: Basal zone hyperplasia (BZH) and dilated intercellular spaces (DISs) are thought to contribute to the clinical manifestations of eosinophilic esophagitis (EoE); however, the molecular pathways that drive BZH remain largely unexplored. OBJECTIVE: We sought to define the role of IL-13-induced transcriptional programs in esophageal epithelial proliferation in EoE. METHODS: We performed RNA sequencing, bioinformatics, Western blot, reverse transcriptase quantitative PCR, and histologic analyses on esophageal biopsies from healthy control and patients with EoE, primary esophageal cells derived from patients with EoE, and IL-13-stimulated esophageal epithelial keratinocytes grown at the air-liquid interface (EPC2-ALI). Genetic (shRNA) and pharmacologic (proteolysis-targeting chimera degrader) approaches and in vivo model of IL-13-induced esophageal epithelial remodeling (Krt5-rtTA x tetO-IL-13Tg) were used to define the role of signal transducer and activator of transcription 3 (STAT3) and STAT6 and secreted frizzled-related protein 1 (SFRP1) in esophageal epithelial proliferation. RESULTS: RNA-sequencing analysis of esophageal biopsies (healthy control vs EoE) and EPC2-ALI revealed 82 common differentially expressed genes that were enriched for putative STAT3 target genes. In vitro and in vivo analyses revealed a link between IL-13-induced STAT3 and STAT6 phosphorylation, SFRP1 mRNA expression, and esophageal epithelial proliferation. In vitro studies showed that IL-13-induced esophageal epithelial proliferation was STAT3-dependent and regulated by the STAT3 target SFRP1. SFRP1 mRNA is increased in esophageal biopsies from patients with active EoE compared with healthy controls or patients in remission and identifies an esophageal suprabasal epithelial cell subpopulation that uniquely expressed the core EoE proinflammatory transcriptome genes (CCL26, ALOX15, CAPN14, ANO1, and TNFAIP6). CONCLUSIONS: These studies identify SFRP1 as a key regulator of IL-13-induced and STAT3-dependent esophageal proliferation and BZH in EoE and link SFRP1+ esophageal epithelial cells with the proinflammatory and epithelial remodeling response in EoE.

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.

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.