Scientists at Mount Sinai say they have published one of the first studies to demonstrate the importance of reactive oxygen species in maintaining stem cell function and preventing inflammation during wound repair. The team believes its findings could provide greater insights into the prevention and treatment of inflammatory bowel diseases (IBD). The researchers published their study (“NOX1 is essential for TNFαlpha-induced intestinal epithelial ROS secretion and inhibits M cell signatures”) in Gut.

Reactive oxygen species are highly reactive chemicals formed from oxygen. They serve as prime signals of cellular dysfunction that contribute to diseases. Secretion of reactive oxygen species in the intestine is necessary for maintaining stem cell function and important for wound repair. However, it can cause inflammatory effects as well.

The Mount Sinai team found the key transcription factors driving abnormal stem cell changes, suggesting a significant role of reactive oxygen species in maintaining healthy intestines.

“Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined,” the investigators wrote.

“ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFalpha stimulation. Clustering of epithelial cells from human ulcerative colitis (UC; inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (Ulex Europaeus Agglutin-1 lectin) and in vivo with DSS injury.

“TNFalpha induces ROS production more in NOX1-WT vs. NOX1-deficient murine colonoids under a range of Wnt- and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFalpha-stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT Enhancer Binding Protein Delta), DNMT1 (DNA methyltransferase), and HIF1A (hypoxia-inducible factor) baseline.

Marked TNFalpha-mediated induction of M cells

“Subclustering unexpectedly showed marked TNFalpha-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis, and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation.

“NOX1 deficiency plus TNFalpha stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritize ROS modulation for future therapies.”

“While it’s clear that regulation of oxygen and reactive oxygen species plays a critical role in chronic diseases generally, and IBD in particular, this study provides a major advance in defining the key role of oxygen species in maintaining a healthy epithelial barrier for IBD,” said senior author Judy H. Cho, MD, dean for translational genetics, the Ward-Coleman chair in translational genetics, and vice chair of pathology, molecular and cell-based medicine at the Icahn School of Medicine at Mount Sinai.

The researchers studied the role of reactive oxygen species and NOX1, the protein used to produce these chemicals, by examining single-cell gene expression in vitro and in vivo in mice models, as well as ex vivo in the form of human intestinal biopsies obtained following routine colonoscopies. They measured the amount of reactive oxygen species and analyzed the gene expression profile of intestine barrier cells from mice and human patients with a subtype of IBD known as ulcerative colitis.

Intestine barrier cells cover the intestine surface and help to digest food, absorb nutrients, and prevent the invasion of gut bacteria. The Mount Sinai researchers compared gene expression data in both inflamed and uninflamed colon tissues.

The researchers found that a combination of NOX1, loss of function (which results in decreased reactive oxygen species), and the presence of tumor necrosis factor (TNF) that causes inflammation leads to an abnormal increase of microfold cells.

Microfold cells, also known as M cells, are crucial to regulating gut immune response. The scientists found this abnormal increase in M cells, in stem cells in both the human and mice models, as a result of loss of reactive oxygen species. This increase in epithelial M cells drives increased recruitment of immune cells in mice. By treating intestine cells with reactive oxygen species, they were able to reverse the initial defect caused by losing reactive oxygen species during inflammation.

“Reactive oxygen specifies released by stem cells are critical in maintaining a healthy gut via maintaining proper balance of intestine barrier cell types,” said lead author Nai-Yun Hsu, PhD, associate scientist in the Judy Cho Laboratory. The researchers encourage further studies, which they said could include direct reactive oxygen species-stem cell modulation therapy to IBD patients in future treatments.

The University of Oxford contributed to the research.


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