title: Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages.
abstract: Several mechanisms are involved in controlling intracellular survival of pathogenic mycobacteria in host macrophages, but how these mechanisms are regulated remains poorly understood. We report a role for Kelch-like ECH-associated protein 1 (Keap1), an oxidative stress sensor, in regulating inflammation induced by infection with Mycobacterium avium in human primary macrophages. By using confocal microscopy, we found that Keap1 associated with mycobacterial phagosomes in a time-dependent manner, whereas siRNA-mediated knockdown of Keap1 increased M. avium-induced expression of inflammatory cytokines and type I interferons (IFNs). We show evidence of a mechanism whereby Keap1, as part of an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiquitination and degradation of IκB kinase (IKK)-β thus terminating IKK activity. Keap1 knockdown led to increased nuclear translocation of transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 driving the expression of inflammatory cytokines and IFN-β. Furthermore, knockdown of other members of the Cul3 ubiquitin ligase complex also led to increased cytokine expression, further implicating this ligase complex in the regulation of the IKK family. Finally, increased inflammatory responses in Keap1-silenced cells contributed to decreased intracellular growth of M. avium in primary human macrophages that was reconstituted with inhibitors of IKKβ or TANK-binding kinase 1 (TBK1). Taken together, we propose that Keap1 acts as a negative regulator for the control of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Jane Atesoh
authors.initials: JA
authors.lastname: Awuh
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Markus
authors.initials: M
authors.lastname: Haug
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Technology, University College of Sør-Trøndelag, 7004 Trondheim, Norway;
authors.forename: Jennifer
authors.initials: J
authors.lastname: Mildenberger
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Anne
authors.initials: A
authors.lastname: Marstad
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Chau Phuc Ngoc
authors.initials: CP
authors.lastname: Do
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Claire
authors.initials: C
authors.lastname: Louet
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Jørgen
authors.identifier: http://orcid.org/0000-0002-1051-9258
authors.initials: J
authors.lastname: Stenvik
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Magnus
authors.initials: M
authors.lastname: Steigedal
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Infectious Diseases, St. Olavs Hospital, 7006 Trondheim, Norway.
authors.forename: Jan Kristian
authors.initials: JK
authors.lastname: Damås
authors.affiliation: Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway;
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Halaas
authors.affiliation: Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway; trude.flo@ntnu.no.
authors.forename: Trude Helen
authors.initials: TH
authors.lastname: Flo
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D055785:Gene Knockdown Techniques; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007249:Inflammation / Q000473:pathology*; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism; D050835:Interferon Regulatory Factors / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology*; D009162:Mycobacterium avium / Q000254:growth & development / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D055550:Protein Stability; D021381:Protein Transport; D059748:Proteolysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D014376:Tuberculosis / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D054875:Ubiquitination; D015854:Up-Regulation
chemical_list: D002352:Carrier Proteins; D016207:Cytokines; D050856:Interferon Regulatory Factor-1; D050835:Interferon Regulatory Factors; D047908:Intracellular Signaling Peptides and Proteins; C494258:Irf1 protein, mouse; C498178:Irf5 protein, mouse; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D016328:NF-kappa B; C479529:RBX1 protein, human; D017382:Reactive Oxygen Species; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human; D051550:I-kappa B Kinase
keywords: Keap1; Mycobacterium avium; human primary macrophages; infection; inflammation