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Original research
NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures
  1. Nai-Yun Hsu1,
  2. Shikha Nayar2,
  3. Kyle Gettler1,
  4. Sayali Talware3,4,
  5. Mamta Giri2,
  6. Isaac Alter2,
  7. Carmen Argmann5,
  8. Ksenija Sabic1,
  9. Tin Htwe Thin1,
  10. Huai-Bin Mabel Ko6,
  11. Robert Werner1,
  12. Christopher Tastad1,
  13. Thaddeus Stappenbeck7,
  14. Aline Azabdaftari8,
  15. Holm H Uhlig8,
  16. Ling-Shiang Chuang1,2,
  17. Judy H Cho1,2
  1. 1Department of Pathology, Molecular and Cell Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  2. 2The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  3. 3Division of Gastroenterology, Icahn School of Medicine at Mount Sinai Department of Medicine, New York, New York, USA
  4. 4The Icahn Genomic Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  5. 5Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  6. 6Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, USA
  7. 7Department of Inflammation and Immunity, Lerner Research Institute, Cleveland, Ohio, USA
  8. 8Translational Gastroenterology Unit, University of Oxford, Oxford, UK
  1. Correspondence to Dr Judy H Cho, Department of Pathology, Molecular and Cell Based Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA; judy.cho{at}mssm.edu

Abstract

Objective 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.

Design ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human 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.

Results TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα 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. Subclustering unexpectedly showed marked TNFα-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.

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

  • IBD - genetics
  • epithelial cells
  • ulcerative colitis
  • intestinal stem cell

Data availability statement

Data are available in a public, open access repository. We have submitted the data to GEO(#GSE202778). The data will release and be available to the public upon manuscript publication.

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Data availability statement

Data are available in a public, open access repository. We have submitted the data to GEO(#GSE202778). The data will release and be available to the public upon manuscript publication.

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Footnotes

  • Twitter @mountsinaiibd?lang=en, @shikha_nayar, @Felix_Chuang240, @JudyCho7

  • Contributors NYH, LSC and JHC: Experimental design and implementation. Data analyses and interpretation, and writing of manuscript. JHC: Guarantor. NYH, LSC, KS, IA, THT, HBMK and AA:Experimental studies and quality control. MG, SN, KG, ST and CT:Data analyses and interpretation. TS, HU and CA: Study design and interpretation.

  • Funding This study was supported by U01 DK062422, U24 DK052429, R01 DK123758 (JC), R01 DK106593 (JC) and the Sanford J. Grossman Charitable Trust. AA is supported by Deutsche Forschungsgemeinschaft (AZ 167/1-1).

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.