Project description:The intestinal epithelium is an immunologically active barrier adapted for a low oxygen environment. Its role is implicated in the pathophysiology of various diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Patient-derived intestinal epithelial organoids (IEOs) mimic the architecture and cell type composition of the intestine and can be used for disease modeling and personalized drug screening. IEOs are usually cultured in atmospheric oxygen concentrations (20% oxygen) without accounting for the physiological hypoxia in the colonic epithelium, where the oxygen concentration range from 3% to <1%. Previously, we showed that human colon derived IEOs (colonoids) are viable and respond to the pro-inflammatory cytokines TNF and IL17 after short-term (40 hours) cultivation in low (2%) oxygen. We suggested that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of intestinal organoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia for the entire in vitro culture period and compare growth, differentiation, and immunological responses in colonoids at 2% and 20% oxygen. Growth from single cells to differentiated organoids was monitored by brightfield images captured throughout cultivation and evaluated with a linear mixed model. Cell composition in undifferentiated vs. differentiated colonoids was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing. Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. There were significantly more cells with proliferation potential (KI67 positive) in undifferentiated than in differentiated colonoids; and significantly higher expression of cell markers for goblet cells (MUC2) and absorptive cells (MUC2 negative, CK20 positive) in differentiated colonoids. Enteroendocrine cells (CGA positive) were only present in differentiated colonoids. No differences in expression of any of the cell marker proteins were found between colonoids cultured in 2% and 20% oxygen, but the single-cell RNA-sequencing analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CX3CL1, CXCL2, CXCL5, CXCL6, CXCL10, CXCL12, CCL20, CCL25, and NGAL upon TNF + poly(I:C) treatment. However, there appeared to be a lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

Project description:To investigate the detailed molecular mechanisms for the regulatory role of HIF-2α in experimental colitis, microarray gene expression analysis was performed on colon RNA isolated from 6- to 8-week-old Hif-2αF/F, Hif-2αlΔIE mice treated with 3%DSS for 3 days. Background & Aims: Hypoxic inflammation is characterized by decreased oxygen tension in inflammatory foci, and is a notable feature in inflammatory bowel disease (IBD). Hypoxic response is mediated by transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α, both of which are highly induced in IBD. HIF-1α is a protective factor that limits intestinal barrier dysfunction during inflammation. However, the role of HIF-2α has not been assessed in hypoxic inflammation and IBD. Methods: A hypoxia reporter mouse model was used to test the presence of hypoxia and HIF-2α in dextran sulfate sodium (DSS) and Citrobacter rodentium (C.rod)-induced colitis. The role of HIF-2α in these mouse models of colitis was further assessed in mice with an intestinal epithelium-specific gain- and loss-of-function of HIF-2α. Results: Induction of hypoxia and HIF-2α was confirmed in both murine experimental colitis models and human IBD samples. Disruption of HIF-2α attenuated colonic inflammation whereas stabilization of HIF-2α potentiated inflammation in mouse models of colitis. Interestingly, intestine specific overexpression of HIF-2α but not HIF-1α leads to spontaneous colitis and premature death in mice. Further mechanistic analysis showed that HIF-2α is a driver for pro-inflammatory response and is critical regulator of intestinal epithelial-derived tumor necrosis factor (TNF)-α. Blocking TNF-α completely ameliorated HIF-2α potentiated intestinal inflammation. Conclusions: These data demonstrate that HIF-2α is a critical transcription factor essential in intestinal epithelium elicited inflammatory response. Global gene expression profiling in colon RNAs isolated from 7-week-old Hif-2αF/F (n=6, Shah 007) and Hif-2αΔIE (n=5, Shah 008).

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ADH1C downregulation is a key hypoxia response in colon epithelium

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