Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22PHOX, and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn’s disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.

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  1. 1.

    De Souza, H. S. & Fiocchi, C. Immunopathogenesis of IBD: current state of the art. Nat. Rev. Gastroenterol. Hepatol. 13, 13–27 (2016).

  2. 2.

    Neurath, M. F. Cytokines inInflammatoty bowel disease. Nat. Rev. Immunol. 14, 329–342 (2014).

  3. 3.

    Keshavarzian, A. et al. Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe. Gastroenterology 103, 177–185 (1992).

  4. 4.

    Simmonds, N. J. et al. Chemiluminescence assay of mucosal reactive oxygen metabolites in inflammatory bowel disease. Gastroenterology 103, 186–196 (1992).

  5. 5.

    McKenzie, S. J., Baker, M. S., Buffinton, G. D. & Doe, W. F. Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease. J. Clin. Invest. 98, 136–141 (1996).

  6. 6.

    Targan, S. R. et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s disease. Crohn’s Disease cA2 Study Group. N. Engl. J. Med. 337, 1029–1035 (1997).

  7. 7.

    Gálvez, J. Role of Th17 cells in the pathogenesis of human IBD. ISRN Inflamm. 25, 928461 (2014).

  8. 8.

    Abraham, C. & Cho, J. Interleukin-23/Th17 pathways and inflammatory bowel disease. Inflamm. Bowel Dis. 15, 1090–1100 (2009).

  9. 9.

    El-Benna, J. et al. Priming of the neutrophil respiratory burst: role in host defense and inflammation. Immunol. Rev. 273, 180–193 (2016).

  10. 10.

    Suh, Y. A. et al. Cell transformation by the superoxide-generating oxidase Mox1. Nature 401, 79–82 (1999).

  11. 11.

    El Hassani, R. A. et al. Dual oxidase2 is expressed all along the digestive tract. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G933–G942 (2005).

  12. 12.

    Bedard, Karen & Krause, Karl-Heinz The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007).

  13. 13.

    Corcionivoschi, N. et al. Mucosal reactive oxygen species decrease virulence by disrupting Campylobacter jejuni phosphotyrosine signaling. Cell Host Microbe 12, 47–59 (2012).

  14. 14.

    Leoni, G. et al. Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair. J. Clin. Invest. 123, 443–454 (2013).

  15. 15.

    Jones, R. M. et al. Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species. EMBO J. 32, 3017–3028 (2013).

  16. 16.

    Esworthy, R. S. et al. Nox1 causes ileocolitis in mice deficient in glutathione peroxidase-1 and -2. Free Radic. Biol. Med. 68, 315–325 (2014).

  17. 17.

    MacFie, T. S. et al. DUOX2 and DUOXA2 form the predominant enzyme system capable of producing the reactive oxygen species H2O2 in active ulcerative colitis and are modulated by 5-aminosalicylic acid. Inflamm. Bowel Dis. 20, 514–524 (2014).

  18. 18.

    Muniz, L. R., Knosp, C. & Yeretssian, G. Intestinal antimicrobial peptides during homeostasis, infection, and disease. Front. Immunol. 3, 310 (2012).

  19. 19.

    Yoo do, Y. et al. Bacteroides fragilis enterotoxin upregulates lipocalin-2 expression in intestinal epithelial cells. Lab. Invest. 93, 384–396 (2013).

  20. 20.

    Kjeldsen, L., Johnsen, A. H., Sengeløv, H. & Borregaard, N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J. Biol. Chem. 15, 10425–10432 (1993).

  21. 21.

    Flo, T. H. et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 16, 917–921 (2004).

  22. 22.

    Goetz, D. H. et al. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol. Cell 10, 1033–1043 (2002).

  23. 23.

    Roudkenar, M. H. 1st et al. Oxidative stress induced lipocalin 2 gene expression: addressing its expression under the harmful conditions. J. Radiat. Res. 48, 39–44 (2007).

  24. 24.

    Nielsen, B. S. et al. Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases. Gut 38, 414–420 (1996).

  25. 25.

    Moschen, A. R., Gerner, R. R. & Wang, J. Lipocalin 2 protects from inflammation and tumorigenesis associated with gut microbiota alterations. Cell Host Microbe 19, 455–469 (2016).

  26. 26.

    Singh, V., Yeoh, B. S. & Chassaing, B. Microbiota-inducible innate immune, siderophore binding protein lipocalin 2 is critical for intestinal homeostasis. Cell. Mol. Gastroenterol. Hepatol. 2, 482–498 (2016).

  27. 27.

    Yan, L. & Borregaard, N. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J. Biol. Chem. 276, 37258–37265 (2001).

  28. 28.

    Debbabi, M. et al. NOXO1 phosphorylation on serine 154 is critical for optimal NADPH oxidase 1 assembly and activation. FASEB J. 27, 1733–1748 (2013).

  29. 29.

    Amatya, N., Garg, A. V. & Gaffen, S. L. IL-17 signaling: The Yin and the Yang. Trends Immunol. 38, 310–322 (2017).

  30. 30.

    Brenner, D., Blaser, H. & Mak, T. W. Regulation of tumour necrosis factor signalling: live or let die. Nat. Rev. Immunol. 15, 362–374 (2015).

  31. 31.

    Stallhofer, J. et al. Lipocalin-2 is a disease activity marker in inflammatory bowel disease regulated by IL-17A, IL-22, and TNF-α and modulated by IL23R genotype status. Inflamm. Bowel Dis. 21, 2327–2340 (2015).

  32. 32.

    Karlsen, J. R., Borregaard, N. & Cowland, J. B. Induction of neutrophil gelatinase-associated lipocalin expression by co-stimulation with interleukin-17 and tumor necrosis factor-alpha is controlled by IkappaB-zeta but neither by C/EBP-beta nor C/EBP-delta. J. Biol. Chem. 285, 14088–14100 (2010).

  33. 33.

    Motoyama, M., Yamazaki, S., Eto-Kimura, A., Takeshige, K. & Muta, T. Positive and negative regulation of nuclear factor-kappaB-mediated transcription by IkappaB-zeta, an inducible nuclear protein. J. Biol. Chem. 280, 7444–7451 (2005).

  34. 34.

    Alex, P. et al. Distinct cytokine patterns identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflamm. Bowel Dis. 15, 341–352 (2009).

  35. 35.

    Yokota, H. et al. NOX1/NADPH oxidase expressed in colonic macrophages contributes to the pathogenesis of colonic inflammation in trinitrobenzene sulfonic acid-induced murine colitis. J. Pharmacol. Exp. Ther. 360, 192–200 (2017).

  36. 36.

    Gomollón, F. et al., ECCO. 3rd European evidence-based consensus on the diagnosis and management of Crohn’s Disease 2016: Part 1: diagnosis and medical management. J. Crohns Colitis 11, 3–25 (2017).

  37. 37.

    Onishi, R. M. & Gaffen, S. L. Interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease. Immunology 129, 311–321 (2010).

  38. 38.

    Anderson, P. 1 Post-transcriptional control of cytokine production. Nat. Immunol. 9, 353–359 (2008).

  39. 39.

    De Sousa Abreu, R., Penalva, L. O., Marcotte, E. M. & Vogel, C. Global signatures of protein and mRNA expression level. Mol. Biosyst. 5, 1512–1526 (2009).

  40. 40.

    Grasberger, H. et al. Increased expression of DUOX2 is an epithelial response to mucosal dysbiosis required for immune homeostasis in mouse intestine. Gastroenterology 149, 1849–1859 (2015).

  41. 41.

    Pircalabioru, G. et al. Defensive mutualism rescues NADPH oxidase inactivation in gut infection. Cell Host Microbe 19, 651–663 (2016).

  42. 42.

    Rokutan, K. et al. NADPH oxidases in the gastrointestinal tract: a potential role of Nox1 in innate immune response and carcinogenesis. Antioxid. Redox Signal. 8, 1573–1582 (2006).

  43. 43.

    Hayes, P. et al. Defects in NADPH oxidase genes NOX1 and DUOX2 in very early onset inflammatory bowel disease. Cell. Mol. Gastroenterol. Hepatol. 1, 489–502 (2015).

  44. 44.

    Rada, B. & Leto, T. L. Oxidative innate immune defenses by Nox/Duox family NADPH oxidases. Contrib. Microbiol. 15, 164–187 (2008).

  45. 45.

    Chung, K. W. et al. Involvement of NF-κBIZ and related cytokines in age-associated renal fibrosis. Oncotarget 8, 7315–7327 (2017).

  46. 46.

    Katz, L. H. et al. Expression of IL-2, IL-17 and TNF-alpha in patients with Crohn’s disease treated with anti-TNF antibodies. Clin. Res. Hepatol. Gastroenterol. 38, 491–498 (2014).

  47. 47.

    Fujino, S. et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 52, 65–70 (2003).

  48. 48.

    Joo, J. H. et al. NADPH oxidase 1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteasomal degradation of NoxO1 in colon cancer cells. Cancer Res. 76, 855–865 (2016).

  49. 49.

    Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

  50. 50.

    Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA 76, 4350–4354 (1979).

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The authors wish to thank Dr. Martine Torres for her editorial help, Pr. Anne Couvelard for the histological images of the colon sections, and Olivier Thibaudeau for preparation of the paraffin sections. This work was supported by La Ligue Nationale Contre le Cancer, Comité De Paris, Grant no. RS17/75-46, and no. RS18/75-13, INSERM, CNRS and University Denis-Diderot Paris 7.

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Author notes

  1. These authors contributed equally: Nesrine Makhezer, Marwa Ben Khemis, Dan Liu


  1. INSERM-U1149, CNRS-ERL8252, Centre de Recherche sur l’Inflammation, Paris, France

    • Nesrine Makhezer
    • , Marwa Ben Khemis
    • , Dan Liu
    • , Yamina Khichane
    • , Viviana Marzaioli
    • , Asma Tlili
    • , Marjan Mojallali
    • , Coralie Pintard
    • , Philippe Letteron
    • , Margarita Hurtado-Nedelec
    • , Jamel El-Benna
    • , Jean-Claude Marie
    •  & Pham My-Chan Dang
  2. Université Paris Diderot, Sorbonne Paris Cité, Laboratoire d’Excellence Inflamex, DHU FIRE, Faculté de Médecine, Site Xavier Bichat, Paris, France

    • Nesrine Makhezer
    • , Marwa Ben Khemis
    • , Dan Liu
    • , Yamina Khichane
    • , Viviana Marzaioli
    • , Asma Tlili
    • , Marjan Mojallali
    • , Coralie Pintard
    • , Philippe Letteron
    • , Margarita Hurtado-Nedelec
    • , Jamel El-Benna
    • , Jean-Claude Marie
    •  & Pham My-Chan Dang
  3. Département d’Immunologie et d’Hématologie, UF Dysfonctionnements Immunitaires, HUPNVS, Hôpital Bichat, Paris, France

    • Margarita Hurtado-Nedelec
  4. Département de Pathologie, Hôpital Bichat – Claude Bernard, Paris, France

    • Aurélie Sannier
  5. Service d’Hépato-Gastroentérologie et Cancérologie Digestive, Hôpital Bichat – Claude Bernard, Paris, France

    • Anne-Laure Pelletier


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N.M., M.B.K., D.L., Y.K., A.T., M.M., C.P., P.L., and A.S. designed and performed the experiments and analyzed the data. V.M., M.H.N., J.C.M., and J.E.B. analyzed the data and contributed to the critical reading of the manuscript. A.L.P. recruited the patients, contributed to the discussion, and to the critical reading of the manuscript. P.M.C.D. supervised the project, designed the experiments, and wrote the manuscript. All the authors have read the manuscript and agreed with the data.

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The authors declare no competing interests.

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Correspondence to Pham My-Chan Dang.

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