Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages Jane Atesoh Awuha,b, Markus Hauga,b, Jennifer Mildenbergera,c, Anne Marstada,b, Chau Phuc Ngoc Doa,b, Claire Loueta,b, Jørgen Stenvika,b, Magnus Steigedala,b, Jan Kristian Damåsa,b,d, Øyvind Halaasb, and Trude Helen Floa,b,1 aCentre of Molecular Inflammation Research, Norwegian University of Science and Technology, 7491 Trondheim, Norway; bDepartment of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway; cDepartment of Technology, University College of Sør-Trøndelag, 7004 Trondheim, Norway; and dDepartment of Infectious Diseases, St. Olavs Hospital, 7006 Trondheim, Norway Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved June 29, 2015 (received for review December 11, 2014) Several mechanisms are involved in controlling intracellular sur- duced by Candida albicans in DCs is largely dependent on the CLR vival of pathogenic mycobacteria in host macrophages, but how Dectin-1 and signaling through the Syk kinase and IRF5 (15). these mechanisms are regulated remains poorly understood. We Listeria monocytogenes and Mtb may disrupt the phagosomal report a role for Kelch-like ECH-associated protein 1 (Keap1), an membrane of macrophages and trigger type I IFN responses oxidative stress sensor, in regulating inflammation induced by through cytosolic Nod1/Nod2/RIP2 and IRF3 (L. monocytogenes) infection with Mycobacterium avium in human primary macro- or IRF5 (Mtb) responding to bacterial cell-wall peptidoglycan, or phages. By using confocal microscopy, we found that Keap1 asso- STING/TBK1/IRF3 responding to bacterial DNA (6, 8). There is ciated with mycobacterial phagosomes in a time-dependent manner, more evidence that TBK1 and IKKe are also activated by TLR whereas siRNA-mediated knockdown of Keap1 increased M. avium- ligands that signal via MyD88, leading to IFN-β production without induced expression of inflammatory cytokines and type I interferons the activation of IRF3 (16), and we recently showed that IRF5 (IFNs). We show evidence of a mechanism whereby Keap1, as part of activation in human monocytes by Staphylococcus aureus ssRNA β an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiq- was dependent on IKK and not TBK1 (17). uitination and degradation of IκB kinase (IKK)-β thus terminating IKK Inflammatory mediators shape the immune response and the activity. Keap1 knockdown led to increased nuclear translocation of outcome of infection (reviewed in refs. 2, 3, 5, 7, 18). Mice deficient transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 in the adaptor MyD88 that conveys signals from TLRs and IL-1 driving the expression of inflammatory cytokines and IFN-β.Further- and IL-18 receptors are extremely susceptible to infections with more, knockdown of other members of the Cul3 ubiquitin ligase Mtb and M. avium. Mice deficient in TNF or IL-1 receptor die complex also led to increased cytokine expression, further impli- rapidly after Mtb infection, and people receiving anti-TNF therapy cating this ligase complex in the regulation of the IKK family. have increased risk for reactivation of latent tuberculosis (3). On the contrary, mice deficient in typeIIFNsactuallydobetterthan Finally, increased inflammatory responses in Keap1-silenced cells WT mice, arguing for a role of IFN-α/β in supporting Mtb survival contributed to decreased intracellular growth of M. avium in pri- (19). PRR activation induces the production of reactive oxygen mary human macrophages that was reconstituted with inhibitors species (ROS) via phagosomal NADPH oxidase (NOX) (20). of IKKβ or TANK-binding kinase 1 (TBK1). Taken together, we However, excessive oxidative signaling may be damaging to propose that Keap1 acts as a negative regulator for the control cells; hence, an efficient mechanism for modulation is imperative. of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained Significance or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages. Inflammatory signaling is a central mechanism controlling host defenses to pathogens. Members of Mycobacterium avium Keap1 | human primary macrophages | infection | Mycobacterium avium | complex cause disease in immunocompromised patients and in inflammation individuals with predisposing lung abnormalities. We provide evidence of a mechanism in human primary macrophages whereby the oxidative stress sensor Kelch-like ECH-associated he Mycobacterium avium complex consists of widely distrib- protein 1 (Keap1) negatively regulates inflammatory responses Tuted opportunistic mycobacteria that cause nontuberculous and thus facilitates intracellular growth of M. avium. Our find- pulmonary disease in immunocompromised individuals (1, 2). Like ings are of high biological and clinical significance, as opportu- the more virulent Mycobacterium tuberculosis (Mtb), M. avium nistic infections with nontuberculous mycobacteria are receiving invades host macrophages and may escape killing by prevent- renewed attention because of increased incidence and difficul- ing phagosomal maturation while retaining access to essential – ties in treatment. Altered Keap1 gene expression may also have nutrients (2 4). Initial infection is sensed by various pattern rec- vital clinical implications for other inflammation-associated con- ognition receptors (PRRs) from surface Toll-like receptors ditions, opening novel research venues for translational research, (TLRs) and C-type lectin receptors (CLRs), to endosomal TLRs, for instance in the expanding field of host-targeted therapy for cytosolic NOD-like receptors (NLRs), and nucleic acid sensors infectious diseases. (5–8). Engagement of these PRRs initiates production of inflam- matory mediators and type I IFNs through activation of the canonical Author contributions: J.A.A., M.H., and T.H.F. designed research; J.A.A., M.H., J.M., A.M., IκB kinase (IKK)-α/β kinases and the transcription factor NF-κBor C.P.N.D., C.L., J.S., Ø.H., and T.H.F. performed research; J.A.A., M.H., J.M., A.M., C.P.N.D., the IKK-related kinase TBK1 and IFN regulatory factors (IRFs) C.L., J.S., M.S., J.K.D., Ø.H., and T.H.F. analyzed data; and J.A.A., M.H., M.S., J.K.D., and T.H.F. (9, 10). Individual PRRs initiate overlapping and distinct signaling wrote the paper. pathways in various cell types. Endosomal TLRs act as sensors of The authors declare no conflict of interest. foreign nucleic acids and trigger the production of type I IFNs and This article is a PNAS Direct Submission. inflammatory cytokines. In plasmacytoid dendritic cells (DCs), this Freely available online through the PNAS open access option. results from RNA and DNA activation of NF-κB, IRF5, and IRF7, 1To whom correspondence should be addressed. Email: [email protected]. whereas IRF1, rather than IRF7, drives type I IFNs downstream of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. TLR7 and TLR9 in myeloid DCs (11–14). IFN-β production in- 1073/pnas.1423449112/-/DCSupplemental. E4272–E4280 | PNAS | Published online July 20, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1423449112 Downloaded by guest on October 1, 2021 Kelch-like ECH-associated protein 1 (Keap1) plays a well-established Untr TBHP M. avium PNAS PLUS A B 10 - NAC role as a sensor for ROS for the protection of cells against ox- *** +NAC * idative damage (21). Keap1 is characterized by several distinct -NAC 8 ** binding domains, allowing its self-interaction and interaction with other proteins such as cullin-ring 3 E3 ubiquitin ligase (Cul3), the 6 transcription factor nuclear factor (erythroid-derived 2)-like 2 4 (Nrf2), and p62 (22–25). Modification of Keap1 by electrophilic or +NAC Mean fluorescence intensity units/cell oxidative stress leads to stabilization of Nrf2. Nrf2 translocates to 2 the nucleus and activates the transcription of cytoprotective genes, 0 including p62, heme oxygenase, and NADPH dehydrogenase, Untr TBHP M. avium – quinone 1. The Keap1 p62 interaction may lead to Keap1 degra- CFP-M. avium a647-Keap1 CD40 E40 * F40 dation by autophagy, resulting in a noncanonical mechanism of * regulation of Nrf2 (24, 26, 27). There is some evidence suggesting a * role for the Keap1 E3-ligase complex in the regulation of NF-κB 30 30 30 signaling in the context of tumorigenesis (26–28). Although the role of Keap1 in ROS signaling has been well established, its role in merge DIC 20 20 20 regulation of inflammatory signaling has not been clearly eluci- dated. We show here that Keap1 down-regulates the inflammatory 10 10 10 response necessary for the control of M. avium infection through %Keap1-M. avium phagosome association 0 0 0 the canonical IKK complex and its homolog, TBK1. time p.i.: 4h 24h Untr NAC DPI siNTC sip62 Results M. avium Induces ROS and the Recruitment of Keap1 to Its Phagosomes in Human Primary Macrophages. ROScanbetriggeredbyinvading bacteria, and, despite the fact that pathogenic mycobacteria have Fig. 1. M. avium induced cellular ROS generation and recruitment of Keap1 to developed strategies to counter it, ROS modulates antimycobacterial its phagosome. (A) M. avium induction of cellular ROS production in human host defenses, including inflammation (29–31). We investigated primary macrophages detected by confocal microscopy. After a 1-h infection, whether M. avium induced ROS production in human primary ROS was analyzed by a fluorogenic marker for ROS in live cells, 5-(and-6)-car- ′ ′ monocyte-derived macrophages (MDMs). Cells were infected with boxy-2 ,7 -dichlorodihydrofluoresceindiacetate (carboxy-H2DCFDA), and TBHP M. avium, and, 1 h after infection, ROS was significantly increased was used as a positive control. NAC 5 mM was used to inhibit ROS production. as shown by confocal fluorescence microscopy (Fig. 1 A and B). (B) Quantification of ROS induction per cell by using Imaris Cell module from Small amounts of ROS were detected in untreated macrophages. three independent experiments counting at least 100 cells for each condi- tion.