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Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2

2020; Springer Nature; Volume: 39; Issue: 21 Linguagem: Inglês

10.15252/embj.2020105139

1460-2075

Kiramage Chathuranga, Tae Hwan Kim, Hyun‐Cheol Lee, Jun‐Seol Park, Jaehoon Kim, W. A. Gayan Chathuranga, Pathum Ekanayaka, Youn Jung Choi, Chul‐Ho Lee, Chul‐Joong Kim, Jae U. Jung, Jong‐Soo Lee,

RNA modifications and cancer

Article16 September 2020Open Access Source DataTransparent process Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2 Kiramage Chathuranga orcid.org/0000-0002-0279-2895 College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Tae-Hwan Kim College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Search for more papers by this author Hyuncheol Lee College of Veterinary Medicine, Chungnam National University, Daejeon, Korea California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA Search for more papers by this author Jun-Seol Park College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Jae-Hoon Kim Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology (UST), Daejeon, Korea Search for more papers by this author Wijesinghe A Gayan Chathuranga orcid.org/0000-0001-8348-727X College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Pathum Ekanayaka orcid.org/0000-0002-2388-3905 College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Youn Jung Choi Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Chul-Ho Lee Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology (UST), Daejeon, Korea Search for more papers by this author Chul-Joong Kim College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Jae U Jung Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Jong-Soo Lee Corresponding Author [email protected] orcid.org/0000-0001-5119-0711 College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Kiramage Chathuranga orcid.org/0000-0002-0279-2895 College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Tae-Hwan Kim College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Search for more papers by this author Hyuncheol Lee College of Veterinary Medicine, Chungnam National University, Daejeon, Korea California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA Search for more papers by this author Jun-Seol Park College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Jae-Hoon Kim Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology (UST), Daejeon, Korea Search for more papers by this author Wijesinghe A Gayan Chathuranga orcid.org/0000-0001-8348-727X College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Pathum Ekanayaka orcid.org/0000-0002-2388-3905 College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Youn Jung Choi Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Chul-Ho Lee Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology (UST), Daejeon, Korea Search for more papers by this author Chul-Joong Kim College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Jae U Jung Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Jong-Soo Lee Corresponding Author [email protected] orcid.org/0000-0001-5119-0711 College of Veterinary Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Author Information Kiramage Chathuranga1,‡, Tae-Hwan Kim1,2,‡, Hyuncheol Lee1,3,‡, Jun-Seol Park1,‡, Jae-Hoon Kim4, Wijesinghe A Gayan Chathuranga1, Pathum Ekanayaka1, Youn Jung Choi5, Chul-Ho Lee4, Chul-Joong Kim1, Jae U Jung5 and Jong-Soo Lee *,1 1College of Veterinary Medicine, Chungnam National University, Daejeon, Korea 2Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea 3California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA 4Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology (UST), Daejeon, Korea 5Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +82 42 821 6753; Fax: +82 42 821 8903; E-mail: [email protected] EMBO J (2020)39:e105139https://doi.org/10.15252/embj.2020105139 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract NF-κB essential modulator (NEMO) is a key regulatory protein that functions during NF-κB- and interferon-mediated signaling in response to extracellular stimuli and pathogen infections. Tight regulation of NEMO is essential for host innate immune responses and for maintenance of homeostasis. Here, we report that the E3 ligase MARCH2 is a novel negative regulator of NEMO-mediated signaling upon bacterial or viral infection. MARCH2 interacted directly with NEMO during the late phase of infection and catalyzed K-48-linked ubiquitination of Lys326 on NEMO, which resulted in its degradation. Deletion of MARCH2 resulted in marked resistance to bacterial/viral infection, along with increased innate immune responses both in vitro and in vivo. In addition, MARCH2−/− mice were more susceptible to LPS challenge due to massive production of cytokines. Taken together, these findings provide new insight into the molecular regulation of NEMO and suggest an important role for MARCH2 in homeostatic control of innate immune responses. Synopsis NEMO/IKKγ plays a key role in regulating both the NF-κB and type I IFN signaling pathways. Here, E3 ubiquitin ligase MARCH2 is shown to catalyze Lys48-linked polyubiqutination and degradation of NEMO/IKKγ, thereby negatively regulates the innate immune response to viral and bacterial infection. MARCH2−/− mice are more resistant to viral and bacterial infection. MARCH2 specifically interact with NEMO/IKKγ upon viral and bacterial infection. MARCH2 catalyzes the K48-linked polyubiquitination of Lys326 on NEMO/IKKγ. Introduction The innate immune system is the first line of host defense against viral and bacterial infections and functions by recognizing pathogen-associated molecular patterns (PAMPs), which are usually components of viruses and bacteria (Akira et al, 2006). PAMPs are detected by host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) or RIG-I-like receptors (RLRs) (Creagh & O'Neill, 2006; Eyster et al, 2011; Lee et al, 2019). After sensing PRRs, a series of downstream signaling events facilitated by adaptor molecules is triggered, leading to production of type I interferons (IFNs) and pro-inflammatory cytokines, and synthesis of antiviral interferon-stimulated genes; this inhibits the spread of viruses and bacteria and activates adaptive immune responses (Sadler & Williams, 2008; Liu et al, 2011). Upon viral or bacterial infection, the cellular signaling cascades that generate IFNs and pro-inflammatory cytokines are triggered by molecules that activate transcriptional factors, including NF-κB and IRFs (Heaton et al, 2016). Activation of NF-κB is regulated by the IκBα kinase (IKK) complex (which is composed of the catalytic subunits IKKα and IKKβ) and the regulatory subunit IKKγ/NF-κB essential modulator (NEMO). Upon activation of the IKK complex via K63-linked polyubiquitination of NEMO, IκB is phosphorylated by IKKα and IKKβ, followed by its K48-linked polyubiquitination and proteasomal degradation, thereby releasing NF-κB, which then translocates to the cell nucleus to activate transcription of pro-inflammatory cytokines and related genes (Heaton et al, 2016). Activation and nuclear translocation of IRF3 and IRF7 via the IKK-related kinases TANK-binding kinase (TBK1) and IKKε (Fitzgerald et al, 2003; Sharma et al, 2003) also occur in parallel; this leads ultimately to induction of type I interferons and other antiviral genes (McWhirter & Maniatis, 2005). NEMO/IKKγ plays a key role in regulating both the NF-κB and type I IFN signaling pathways (Xing et al, 2016). NEMO/IKKγ is the integral regulatory scaffolding protein of the canonical IKK complex (Häcker & Karin, 2006) and a key protein for cell survival and immune responses (Schmidt-Supprian et al, 2000). For this reason, interrupting NEMO/IKKγ has severe consequences for tissue homeostasis, and mutation of NEMO/IKKγ can lead to hereditary human diseases such as incontinentia pigmenti, anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), and immunodeficiency 33 (Filipe-Santos et al, 2006; Fusco et al, 2015). By contrast, continuous or excessive activation of NEMO/IKKγ induces autoimmune disease, tumor development, and chronic inflammation (Ben-Neriah & Karin, 2011; Ruland, 2011). Therefore, control of NEMO/IKKγ must be tightly regulated to maintain immune homeostasis. Recent studies identified both positive (mainly) and negative regulators of NEMO/IKKγ in the NF-κB signaling pathway (Chariot et al, 2002; Field et al, 2003; Stilmann et al, 2009; Ashida et al, 2010; Xing et al, 2016; Zhang et al, 2017). However, other negative regulators of NEMO/IKKγ remain to be identified. Membrane-associated RING-CH 2 (MARCH2), an E3 ubiquitin ligase, is a member of the MARCH family; this family comprises 11 members and localizes mainly to the endoplasmic reticulum (ER), Golgi, endosome, and plasma membrane (Nathan & Lehner, 2009). MARCH2 participates in vesicular trafficking between the trans-Golgi network and endosomes, as well as in recycling of endosomes via interaction with syntaxin 6 (Nakamura et al, 2005). Deficiency of MARCH2 suppresses the growth of colon cancer cells by activating ER stress mechanisms (Xia et al, 2017). As an E3 ubiquitin ligase, MARCH2 transports ubiquitin to substrates such as DLG1, β2AR, CFTR, and ERGIC3, as well as to the envelope protein of HIV-1 (Cao et al, 2008; Han et al, 2012; Cheng & Guggino, 2013; Zhang et al, 2018; Yoo et al, 2019). To date, however, the specific immunomodulatory function of MARCH2 upon viral or bacterial infection remains unknown. In this study, we used MARCH2 knockout mice to demonstrate the physiological role of MARCH2 and showed that MARCH2 is a negative regulator of the NF-κB and type I IFN signaling pathways upon viral or bacterial infection. MARCH2 interacts specifically with NEMO/IKKγ and mediates K48-linked polyubiquitination and degradation of NEMO in response to infection. These findings suggest that interaction between MARCH2 and NEMO is essential for maintenance of homeostasis in the innate immune system. Results MARCH2 plays a critical role in host defense in vivo The MARCH family of E3 ligases plays diverse roles in cell metabolism. These roles include membrane protein trafficking, antigen presentation, and regulation of innate immunity (Liu et al, 2019). However, its functions during innate immune responses are unclear, although we know that MARCH5 regulates MAVS (Yoo et al, 2015). Based on our preliminary MARCH family screening study, we identified MARCH2 as a potential negative regulator of the IFN-β pathway. To evaluate the potential roles of MARCH2 in innate immune responses, we generated MARCH2−/− mice on a C57BL/6 background using the CRISPR-Cas9 system and verified genetic knockout of the MARCH2 gene (Fig EV1A–G). MARCH2−/− mice did not show any external abnormalities. First, to investigate the important role of MARCH2 in viral infection in vivo, we challenged MARCH2+/+ and MARCH2−/− mice intravenously with VSV-GFP (Fig 1A). The serum of MARCH2−/− mice contained fewer replicating viruses than that of MARCH2+/+ mice. In addition, when we measured the amount of cytokines in the serum, we found that the concentrations of IFN-β, IL-6, IL-12, and TNF-α were higher in MARCH2−/− mice than in MARCH2+/+ mice (Fig 1B). Furthermore, we measured serum cytokine levels in mice injected with the viral RNA mimic ligand, poly(I:C) (Fig 1C). As observed for virus infection, serum cytokine levels in MARCH2−/− mice after ligand stimulation were higher than those in MARCH2+/+ mice. These results strongly suggest that MARCH2 is involved in antiviral innate immune responses in vivo. Click here to expand this figure. Figure EV1. MARCH2 knockout mice generation A. Schematic representation of the genomic target site in the MARCH2 gene and gRNA sequence. B. Sequencing alignment result of MARCH2 gene and mice genomic DNA. C–F. Genotyping of MARCH2 mice (F2 generation). Heterozygous MARCH2+/− mice (F1 generation) were mated, and genomic DNA was extracted from tail ends from the pups (F2 generation) (C, D) Agarose gel electrophoresis of nested PCR products. First PCR product size, 582 bp (C). Second PCR product size, 264 bp (D). (E) Agarose gel electrophoresis of melted, reannealed, and T7 endonuclease 1-treated DNA. (F) MARCH2+/+ or MARCH2−/− mice DNA was (Mouse number: 1, 3, 4, 7) individually mixed with equal amount of MARCH2+/+ DNA then, melted, and reannealed, and T7 endonuclease 1 was treated. G. BMDMs isolated from MARCH2+/+ and MARCH2−/− mice were infected with PR8-GFP (MOI = 3) virus. Whole-cell lysates were used for immunoblot with anti-MARCH2 antibody, which is normalized by β-actin. Data information: (+/+, wild-type; +/−, heterozygous; −/−, knockout), gRNA: guide RNA. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Deficiency of MARCH2 strengthens antimicrobial immunity in vivo A, B. MARCH2+/+ (n = 8) or MARCH2−/− (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 h post-infection (hpi). (A) Virus titer was analyzed in a plaque assay. (B) Secretion of IFN-β, IL-6, IL-12, and TNF-α was measured in specific ELISAs. C. MARCH2+/+ (n = 6) or MARCH2−/− (n = 6) mice were intravenously injected with poly(I:C) (200 μg/mouse). Serum samples were collected at 2 hpi, and secretion of IFN-β, IL-6, IL-12, and TNF-α was measured in specific ELISAs. D–J. MARCH2+/+ (n = 8) or MARCH2−/− (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (D) The percentage of surviving mice (D, log-rank test, **P < 0.01) and body weight changes for each group (E) are shown. (F, G) At 3 dpc, the bacterial load in the spleen (F) and liver (G) was examined. (H–J) Amount of IL-6, IL-12, CCL5, and CXCL10 in serum (H), spleen (I), and liver (J) of mice in each group at 24 hpc. Data information: *P < 0.05, **P < 0.01 (two-tailed Student's t-test). Data are expressed as the mean ± SEM. Source data are available online for this figure. Source Data for Figure 1 [embj2020105139-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Second, to investigate whether MARCH2 plays a role in response to bacterial infection, we challenged MARCH2+/+ and MARCH2−/− mice with a lethal dose of Listeria monocytogenes (Lm). As shown in Fig 1D and E, MARCH2−/− mice were more resistant to bacterial infection than MARCH2+/+ mice. To examine the effects on pathogen clearance, we examined the bacterial burden in the spleen and liver using colony-forming unit (CFU) titration assays (Fig 1F and G). The results correlated with those of the survival assay; the bacterial load in MARCH2−/− mice was significantly lower than that in MARCH2+/+ mice. Next, we investigated whether the low bacterial burden in MARCH2−/− mice was due to higher inflammatory responses in these animals. Serum, spleen, and liver samples from MARCH2−/− mice contained higher levels of IL-6, IL-12, CCL5, and CXCL10 than those from MARCH2+/+ mice (Fig 1H–J). These results also indicate that MARCH2 is involved in antibacterial innate immune responses in vivo. Third, to investigate whether MARCH2 is responsible for susceptibility to lipopolysaccharide (LPS)-induced septic shock in vivo, MARCH2+/+ and MARCH2−/− mice were injected intraperitoneally with LPS. All MARCH2+/+ mice survived, whereas over 60% of MARCH2−/− mice died (Fig 2A and B). These results suggest that MARCH2−/− mice are more susceptible to endotoxin shock than MARCH2+/+ mice. Furthermore, hematoxylin and eosin staining of spleen sections revealed greater inflammatory cell infiltration in MARCH2−/− mice than in MARCH2+/+ mice (Fig 2C). Therefore, we hypothesized that MARCH2 is involved in inflammatory responses during LPS-mediated septic shock. To test this, we used an ELISA to measure cytokine levels in serum (Fig 2D). As expected, MARCH2−/− mice showed markedly higher expression of inflammatory mediators than MARCH2+/+ mice. We also measured expression of mRNA encoding pro-inflammatory cytokine/chemokine genes in the spleen and liver at 6 hpt. As shown in Fig 2E and F, expression of mRNA encoding IL-6, TNF-α, CXCL10, and IL-1β was significantly higher in MARCH2−/− mice than in MARCH2+/+ mice. These data indicate that MARCH2 attenuates inflammatory responses under conditions of excessive inflammation. Collectively, the results provide in vivo evidence that MARCH2 is a critical regulator in innate immune responses against viral and bacterial infection. Figure 2. Deficiency of MARCH2 induces susceptibility to LPS-induced septic shock A, B. MARCH2+/+ (n = 8) or MARCH2−/− (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). (A, B) Percentage of surviving mice (A, log-rank test, **P < 0.01) and body weight changes (B) in each group. C. MARCH2+/+ (n = 4) or MARCH2−/− (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6 mm. D. MARCH2+/+ (n = 6) or MARCH2−/− (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, and CCL-5 in serum from mice in each group were measured at 12 hpc by ELISA. E, F. MARCH2+/+ (n = 5) or MARCH2−/− (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, and IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR. Data information: *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Data are expressed as the mean ± SEM. Source data are available online for this figure. Source Data for Figure 2 [embj2020105139-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint MARCH2 negatively regulates pathogen-mediated innate immune responses To further assess the critical role of MARCH2 in virus- or bacteria-mediated innate immune responses, we isolated bone marrow-derived macrophages (BMDMs), peripheral blood mononuclear cells (PBMCs), and peritoneal macrophages (PMs) from MARCH2+/+ and MARCH2−/− mice. First, we infected BMDMs with a RNA virus (influenza A virus [PR8-GFP] or vesicular stomatitis virus [VSV-GFP]) and a DNA virus (herpes simplex virus [HSV-GFP]) and found that virus titers in MARCH2−/− BMDMs were lower than those in MARCH2+/+ BMDMs (Fig 3A–C). Next, we used an ELISA to measure the amount of IFN-β and IL-6 secreted by BMDMs either infected with viruses or treated with poly(I:C) and poly(dA:dT). Consistent with the results of in vivo experiments, we found that MARCH2−/− cells secreted more cytokines than MARCH2+/+ cells (Fig 3D and E), suggesting that MARCH2 deficiency increases production of type I IFNs and pro-inflammatory cytokines and suppresses RNA virus infection of primary immune cells. Antiviral innate immune responses are initiated by host sensors, which activate key signaling molecules such as TBK1, IRF3, and P65. Since phosphorylation on specific amino acids within these molecules acts as an activation signal, we investigated phosphorylation in response to infection by VSV-GFP. We found that the phosphorylation levels of these molecules in MARCH2−/− BMDMs were markedly higher than those in MARCH2+/+ BMDMs (Appendix Fig S1A). Additionally, real-time qPCR revealed that expression of mRNA encoding IFN-β, IFN-α, or other antiviral-related genes was higher in MARCH2−/− BMDMs (Appendix Fig S1B). Also, we found that PBMCs or PMs infected with VSV-GFP or Coxsackievirus B (CVB-GFP), respectively, showed similar phenotypes (Fig 3F–I). These results suggest that MARCH2 negatively regulates both the type I IFN signaling pathway and antiviral gene expression in primary immune cells in response to virus infection. Figure 3. Deficiency of MARCH2 leads to augmented innate immune responses upon microbial infection in immune cells A–C. BMDMs isolated from MARCH2+/+ or MARCH2−/− mice were infected with PR8-GFP (A, MOI = 3), VSV-GFP (B, MOI = 5), or HSV-GFP (C, MOI = 3), and virus titer was measured in a plaque assay. D, E. BMDMs isolated from MARCH2+/+ or MARCH2−/− mice were infected with viruses or treated with poly(I:C) (80 μg/ml) or poly (dA:dT) (1 μg/ml). The concentration of secreted IFN-β (D) and IL-6 (E) in the supernatants was measured in an ELISA. F, G. PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2−/− mice were infected with VSV-GFP (MOI = 1). (F) The virus titer was measured in a plaque assay. (G) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA. H, I. PMs isolated from MARCH2+/+ or MARCH2−/− mice infected with CVB-GFP (MOI = 3). (H) Virus titer was measured in a plaque assay. (I) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA. J, K. BMDMs (J) or PMs (K) isolated from MARCH2+/+ or MARCH2−/− mice were infected with Salmonella typhimurium or Listeria monocytogenes, or treated with LPS or zymosan. The concentration of IL-6 and IL-12 in supernatant was analyzed in an ELISA. L, M. RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with PR8-GFP (MOI = 1), VSV-GFP (MOI = 0.5), poly(I:C) (20 μg/ml) or HSV-GFP (MOI = 1) poly (dA:dT) (1 μg/ml), and IFN-β (L), and IL-6 (M) levels in the supernatant were measured in an ELISA. N. RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with S. typhimurium or L. monocytogenes, or treated with LPS (100 ng/ml) or zymosan (100 μg/ml), and IL-6 secretion into the cell supernatant was measured in an ELISA Data information: *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Data are representative of three independent experiments, each with similar results, and expressed as the mean ± SD of two biological replicates. Source data are available online for this figure. Source Data for Figure 3 [embj2020105139-sup-0005-SDataFig3.xlsx] Download figure Download PowerPoint Second, to determine whether MARCH2 is involved in antibacterial innate immune responses, BMDMs or PMs from MARCH2+/+ and MARCH2−/− mice were exposed to Lm, Salmonella typhimurium (Stm), Zymosan (Zym), or LPS, followed by measurement of IL-6 and IL-12 secretion into the culture supernatant at 12 h or 24 h. As shown in Fig 3J and K, stimulated MARCH2−/− cells secreted more cytokines than MARCH2+/+ cells, a finding consistent with those from the virus infection experiments. Next, to examine the effect of MARCH2 on NF-κB signaling, we treated MARCH2+/+ and MARCH2−/− BMDMs with LPS and then subjected the cells to immunoblotting with the indicated antibodies (Appendix Fig S1C). The results showed that MARCH2 deficiency triggered increased phosphorylation of IkBα and NF-κB. We also confirmed that stimulated MARCH2−/− BMDMs showed higher expression of IL-6 mRNA than stimulated MARCH2+/+ BMDMs (Appendix Fig S1D). Collectively, the data from primary cells suggest that MARCH2 negatively regulates virus- or bacteria-mediated innate immune responses. Third, to confirm the effects of MARCH2 in vitro, we prepared MARCH2 knockdown mouse macrophage cells (Raw264.7) by subjecting them to siRNA-mediated gene silencing (Appendix Fig S2A). Similar to MARCH2−/− BMDMs or PMs, virus replication in MARCH2 knockdown RAW264.7 cells was lower than that in control cells upon infection with PR8-GFP, VSV-GFP, or HSV-GFP (Appendix Fig S2B–D). Also, we measured the concentration of IFN-β and IL-6 in the supernatant of cells infected with virus and in cells exposed to poly(I:C) and poly(dA:dT) (Fig 3L and M). We found that knockdown of MARCH2 increased secretion of IFN-β and IL-6. In addition, we confirmed similar activation of signaling molecules and induction of mRNA encoding IFN-β and antiviral-related genes in response to PR8-GFP infection (Appendix Fig S2E and F). To evaluate antibacterial innate immune responses, we infected control and MARCH2 knockdown cells with Stm and Lm or treated them with LPS and Zym. As shown in Fig 3N, secretion of IL-6 by MARCH2 knockdown cells was higher than that by control cells; knockdown of MARCH2 increased expression of mRNA encoding inflammatory cytokines and antimicrobial genes in response to LPS stimulation (Appendix Fig S2G). Next, to confirm these results, we generated stable MARCH2-overexpressing RAW264.7 cells and confirmed overexpression by immunoblot analysis (Appendix Fig S3A). As in the previous experiments, control cells and MARCH2-overexpressing cells were infected with viruses, followed by measurement of GFP expression, virus titers, and IFN-β or IL-6 concentrations. Overexpression of MARCH2 increased virus replication and reduced secretion of IFN-β and IL-6 in response to infection by VSV-GFP, PR8-GFP, or HSV-GFP (Appendix Fig S3B–G). Activation of signaling molecules in MARCH2-overexpressing cells was lower than that in control cells (Appendix Fig S3H). We also examined the physiological role of MARCH2 in antibacterial innate immune responses by exposing MARCH2-overexpressing Raw264.7 cells to diverse bacteria and TLR ligands. To do this, cells were infected with Stm or Lm or treated with LPS, Zym, imiquimod, or CpG oligodeoxynucleotides (CpG-ODN). We found that secretion of IL-6 and IL-12 into the supernatant of MARCH2-overexpressing cells in response to all bacteria and ligands was lower than that into the supernatant of control cells (Appendix Fig S3I and J), confirming the function of MARCH2 in antibacterial innate immune responses. Taken together, the data strongly suggest that MARCH2 plays a negative regulatory role in virus- or bacteria-mediated innate immune responses. To exclude the possibility that negative regulation of pathogen-mediated innate immune signaling by MARCH2 is a cell type-specific phenomenon, we examined the function of MARCH2 in HEK293T cells. Upon infection with PR8-GFP virus, HEK293T cells transiently expressing MARCH2 showed higher susceptibility to virus infection (Appendix Fig S4A–C). Next, we generated a MARCH2−/− HEK293T cell line to test whether regulatory function was restored by reconstitution of MARCH2. MARCH2+/+, MARCH2−/−, and MARCH2−/− cells transiently transfected with MARCH2 were infected with VSV-GFP. Virus titration and cytokine secretion assays showed that reconstitution of MARCH2 restored its innate immune function (Fig EV2A and B). These results were confirmed in a luciferase assay and by immunoblot analysis of activated signaling molecules (Fig EV2C and D). Taken together, these results suggest that MARCH2 is a negative regulator of both antiviral and antibacterial innate immune responses. Click here to expand this figure. Figure EV2. Reconstitution of MARCH2 in MARCH2−/− HEK293T cells reduces the immune response leading to increased virus replication A, B. MARCH2+/+ HEK293T cells transfected with Flag-tagged empty vector or MARCH2−/− HEK293T cells transfected with Flag-tagged empty vector or MARCH2WT were infected with VSV-GFP (MOI = 0.5). (A) Viral replication was determined at 24 hpi by fluorescence microscopy, fluorescence absorbance, and plaque assay. (B) Concentration of IFN-β and IL-6 secreted in supernatants was determined at 12 and 24 hpi by ELISA, Scale bar, 50 μm. C. IFN-β luciferase reporter assay. MARCH2+/+ or MARCH2−/− HEK293T cells were transfected with firefly luciferase reporter plasmid encoding INF-β promoter, TK-Renilla plasmid, and expression plasmids of RIG-I 2CARD, MAVS, or TBK-1. MARCH2−/− HEK293T cells were reconstituted with increasing amount of Flag-tagged MARCH2 plasmid (100, 200 ng). D. MARCH2+/+, M