Latest Articles Include:
- The costs of being placed on hold
- Nat Immunol 12(5):365 (2011)
Nature Immunology | Editorial The costs of being placed on hold Journal name:Nature ImmunologyVolume: 12,Page:365Year published:(2011)DOI:doi:10.1038/ni0511-365Published online19 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Budget battles in the US Congress are precipitating higher costs at government facilities, including federal funding of biomedical research. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Jürg Tschopp 1951–2011
- Nat Immunol 12(5):367 (2011)
Nature Immunology | Obituary Jürg Tschopp 1951–2011 * Ralph C Budd1 * Pascal Schneider2 * Fabienne Mackay3 * Andreas Strasser4Journal name:Nature ImmunologyVolume: 12,Page:367Year published:(2011)DOI:doi:10.1038/ni0511-367Published online19 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The scientific community mourns the loss of Jürg Tschopp, who died recently while doing what he loved most, practicing sports in his beloved Swiss Alps. Jürg loved to escape to the rarefied air of the high peaks of the Valais to be with his family after busy periods of work or travel. Jürg was from Basel, Switzerland. An outstanding athlete in his youth, ranked nationally in the decathlon, he never lost his athleticism and competitive spirit, as anyone who ever went skiing, running or hiking with him soon noticed. After obtaining his PhD in biophysics with Professor Jürgen Engel at the Biocentre of the University of Basel, Jürg moved to the Scripps Research Institute as a postdoctoral fellow with Hans Mueller-Eberhard, where he discovered that complement pores are formed by C9 multimers. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Affiliations * Ralph C. Budd is at the Vermont Center for Immunology & Infectious Diseases, The University of Vermont College of Medicine, Burlington, Vermont, USA. * Pascal Schneider is in the Department of Biochemistry, University of Lausanne, Epalinges, Switzerland. * Fabienne Mackay is in the Department of Immunology, Faculty of Medicine, Nursing and Health Sciences, Central Clinical School, Alfred Hospital, Melbourne, Victoria, Australia. * Andreas Strasser is at the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. Corresponding author Correspondence to: * Andreas Strasser Author Details * Ralph C Budd Search for this author in: * NPG journals * PubMed * Google Scholar * Pascal Schneider Search for this author in: * NPG journals * PubMed * Google Scholar * Fabienne Mackay Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Strasser Contact Andreas Strasser Search for this author in: * NPG journals * PubMed * Google Scholar - The human condition: an immunological perspective
- Nat Immunol 12(5):369-372 (2011)
Nature Immunology | Commentary The human condition: an immunological perspective * Ronald N Germain1 * Pamela L Schwartzberg2 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:369–372Year published:(2011)DOI:doi:10.1038/ni0511-369Published online19 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Despite enormous progress in basic research, there are many gaps in understanding human immunity. Here we describe how new investigational tools and computational methods promise to improve the diagnosis, prognosis and therapy of the many diseases with components from the immune system. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Ronald N. Germain is with the Lymphocyte Biology Section, Laboratory of Immunology, the Program in Systems Immunology and Infectious Disease Modeling, National Institute of Allergy and Infectious Diseases, and the Trans-NIH Center for Human Immunology, Autoimmunity and Inflammation, National Institutes of Health, Bethesda, Maryland, USA. * Pamela L. Schwartzberg is with the Trans-NIH Center for Human Immunology, Autoimmunity and Inflammation and the Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ronald N Germain Author Details * Ronald N Germain Contact Ronald N Germain Search for this author in: * NPG journals * PubMed * Google Scholar * Pamela L Schwartzberg Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Flippin' lipids
- Nat Immunol 12(5):373-375 (2011)
Nature Immunology | News and Views Flippin' lipids * Marcus R Clark1Journal name:Nature ImmunologyVolume: 12,Pages:373–375Year published:(2011)DOI:doi:10.1038/ni.2024Published online19 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. B cell generation is disturbed in four newly identified mouse mutants bearing X-linked mutations in the gene encoding the ATPase ATP11C. These findings suggest that the distribution of membrane phospholipids confers a yet-to-be delineated developmental signal. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Marcus R. Clark is in the Department of Medicine, Section of Rheumatology and Gwen Knapp Center for Lupus Research, University of Chicago, Chicago, Illinois, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Marcus R Clark Author Details * Marcus R Clark Contact Marcus R Clark Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - An unexpected role for MHC class II
- Nat Immunol 12(5):375-376 (2011)
Nature Immunology | News and Views An unexpected role for MHC class II * Ghada S Hassan1 * Walid Mourad1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:375–376Year published:(2011)DOI:doi:10.1038/ni.2023Published online19 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. In addition to their classical function in antigen presentation and their lesser known ability to act as signal transducers, major histocompatibility complex class II molecules are now shown to promote Toll-like receptor signaling. This intriguing role requires intracellular association with the kinase Btk and the costimulatory molecule CD40. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Ghada S. Hassan and Walid Mourad are with the Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Département de Médecine, Hôpital Saint Luc, Montréal, Canada. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Walid Mourad Author Details * Ghada S Hassan Search for this author in: * NPG journals * PubMed * Google Scholar * Walid Mourad Contact Walid Mourad Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Bridging the information gap
- Nat Immunol 12(5):377-379 (2011)
Nature Immunology | News and Views Bridging the information gap * Michael D Milsom1, 2 * Andreas Trumpp1, 2 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:377–379Year published:(2011)DOI:doi:10.1038/ni.2026Published online19 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The interaction between hematopoietic stem cells and their niche is critical for the lifelong maintenance of the blood system. New research shows that crosstalk between stromal components of the niche mediates secretion of the chemokine CXCL12. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Michael D. Milson and Andreas Trumpp are with the Heidelberg Institute for Stem Cell Technology and Experimental Medicine, Heidelberg, Germany * Michael D Milsom * Division of Stem Cells and Cancer, German Cancer Research Center, Heidelberg, Germany. * Michael D Milsom & * Andreas Trumpp Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Andreas Trumpp Author Details * Michael D Milsom Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Trumpp Contact Andreas Trumpp Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Dampening insulin signaling by an NLRP3 'meta-flammasome'
- Nat Immunol 12(5):379-380 (2011)
Nature Immunology | News and Views Dampening insulin signaling by an NLRP3 'meta-flammasome' * Augustine M K Choi1 * Kiichi Nakahira1 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:379–380Year published:(2011)DOI:doi:10.1038/ni.2028Published online19 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The inflammasome has been linked to metabolic disorders such as obesity and type 2 diabetes. Data now suggest that the crosstalk between the inflammasome and autophagy critically mediates cytoplasmic receptor NLRP3–dependent activation of the inflammasome by the saturated fatty acids contained in a high-fat diet. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Augustine M.K. Choi and Kiichi Nakahira are in the Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Augustine M K Choi Author Details * Augustine M K Choi Contact Augustine M K Choi Search for this author in: * NPG journals * PubMed * Google Scholar * Kiichi Nakahira Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Research Highlights
- Nat Immunol 12(5):381 (2011)
Nature Immunology | Research Highlights Research Highlights Journal name:Nature ImmunologyVolume: 12,Page:381Year published:(2011)DOI:doi:10.1038/ni0511-381Published online19 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Double-stranded RNA is detected by the intracellular receptors TLR3 and Mda5, but how nucleic acids are captured and gain access to the intracellular compartments that contain these receptors is unclear. In the Journal of Biological Chemistry, Matsumoto and colleagues demonstrate that raftlin, a major lipid raft protein, is crucial for the uptake and segregation of the double-stranded RNA analog poly(I:C). Raftlin is normally expressed by a wide variety of cell types, including B cells, T cells, dendritic cells and epithelial cells. In the resting state, raftlin is present diffusely in the cytosol, but after the addition of poly(I:C), it is rapidly recruited to membrane rafts, where it associates with poly(I:C). Raftlin-poly(I:C) is then cycled to the endosomes, where it segregates with TLR3. Notably, knockdown of raftlin results in much lower expression of interferon-β after the addition of poly(I:C), which confirms its role in TLR3 activation. ZF J. Biol. Chem.286, 10702–10711 (2011) View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Immunology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22
- Nat Immunol 12(5):383-390 (2011)
Nature Immunology | Review Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22 * Gregory F Sonnenberg1, 2 * Lynette A Fouser3 * David Artis1, 2 * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:383–390Year published:(2011)DOI:doi:10.1038/ni.2025Published online19 April 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The maintenance of barrier function at exposed surfaces of the mammalian body is essential for limiting exposure to environmental stimuli, preventing systemic dissemination of commensal and pathogenic microbes and retaining normal homeostasis of the entire body. Indeed, dysregulated barrier function is associated with many infectious and inflammatory diseases, including psoriasis, influenza, inflammatory bowel disease and human immunodeficiency virus, which collectively afflict millions of people worldwide. Studies have shown that interleukin 22 (IL-22) is expressed at barrier surfaces and that its expression is dysregulated in certain human diseases, which suggests a critical role in the maintenance of normal barrier homeostasis. Consistent with that, studies of mouse model systems have identified a critical role for signaling by IL-22 through its receptor (IL-22R) in the promotion of antimicrobial immunity, inflammation and tissue repair at barrier surfaces. In this review! we will discuss how the expression of IL-22 and IL-22R is regulated, the functions of the IL-22–IL-22R pathway in regulating immunity, inflammation and tissue homeostasis, and the therapeutic potential of targeting this pathway in human disease. View full text Figures at a glance * Figure 1: Functional consequences of IL-22–IL-22R signaling at barrier surfaces. (–) IL-22 is dynamically regulated at barrier surfaces, including the skin (), lung () and intestine (). Depending on the cytokine milieu and tissue in which it is expressed, IL-22 can regulate the expression of genes encoding molecules associated with inflammation, repair or chemotaxis or the expression of antimicrobial peptides, which can orchestrate host-protective immunity, tissue inflammation, repair or homeostasis. MMP, matrix metalloproteinase. * Figure 2: Regulation, function and lineage relationships of IL-22-producing ILCs. ILCs have been identified as a critical source of IL-22. (–) Mouse () and human () IL-22-producing ILCs can be distinguished by phenotypic surface-marker expression, transcriptional profile, effector cytokine expression or functional ability. The origins, lineage relationships, plasticity, regulation and functions of IL-22-producing ILCs are not fully understood and are now an area of intense research. LN, lymph node; BAFF, B cell–activating factor. ILC, innate lymphoid cell; LTi, lymphoid tissue inducer cell. * Figure 3: Differentiation, regulation and function of IL-22-producing T cell populations. IL-22 expression can be induced in a broad range of T cell populations, including the innate-like γδ T cells and NKT cells, as well as TCRαβ-expressing CD4+ TH1, TH17 and TH22 cells and CD8+ TC17 and TC22 cells. The generation of these cell populations depends on particular cognate interactions and cytokine milieus. After lineage commitment, these cells are transcriptionally regulated by many factors and coexpress a range of effector cytokines. TN, naive T cell; TLR2, Toll-like receptor 2. Author information * Abstract * Author information Affiliations * Department of Microbiology and Institute for Immunology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Gregory F Sonnenberg & * David Artis * Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Gregory F Sonnenberg & * David Artis * Inflammation and Immunology Research Unit, Biotherapeutics Research and Development, Pfizer Worldwide Research & Development, Cambridge, Massachusetts, USA. * Lynette A Fouser Competing financial interests L.A.F. is employed by Pfizer. Corresponding author Correspondence to: * David Artis Author Details * Gregory F Sonnenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Lynette A Fouser Search for this author in: * NPG journals * PubMed * Google Scholar * David Artis Contact David Artis Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions
- Nat Immunol 12(5):391-398 (2011)
Nature Immunology | Article CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions * Amir Schajnovitz1 * Tomer Itkin1 * Gabriele D'Uva1, 2 * Alexander Kalinkovich1 * Karin Golan1 * Aya Ludin1 * Dror Cohen3 * Ziv Shulman1 * Abraham Avigdor1, 4 * Arnon Nagler4 * Orit Kollet1 * Rony Seger5 * Tsvee Lapidot1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:391–398Year published:(2011)DOI:doi:10.1038/ni.2017Received12 October 2010Accepted04 March 2011Published online27 March 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact–dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34+ progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP–protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells. View full text Figures at a glance * Figure 1: Secretion of functional CXCL12 in the bone marrow correlates with expression of Cx43 and Cx45 and gap-junction activity. () Real-time quantitative PCR analysis of Cxcl12, Cx43 and Cx45 mRNA among total mRNA extracted from tibia of mice (n = 4 per group) injected subcutaneously for 5 consecutive days with G-CSF (300 μg/kg), presented relative to that of mice injected with PBS (control (Ctrl)), set as 100%. *P < 0.05 and **P < 0.005 (t-test). () Immunoblot analysis of Cx43, Cx45 and the kinases Erk1 and Erk2 (Erk) in lysates of femoral bone marrow cells obtained as in ; each lane represents an individual mouse. () Enzyme-linked immunosorbent assay (ELISA) of CXCL12 in bone marrow (BM) supernatants of mice (n = 8 per group) injected intraperitoneally for 2 consecutive days with CBX (5 mg per kg body weight), presented relative to that of mice injected with PBS, set as 100%. *P < 0.001 (t-test). () Femoral homing of bone marrow mononuclear cells labeled with the cytosolic dye CFSE and transplanted into mice (n = 4 per group) treated with CBX, assessed 3 h after transplantation; results are presen! ted relative to that of mice treated with PBS, set as 100%. *P < 0.01 (t-test). Data are representative of four experiments (mean and s.e.m. in ,,). * Figure 2: CXCL12 secretion is cell contact dependent, but CXCL12 production is not. () ELISA of CXCL12 and HGF in the conditioned media (CM) of contacting (C) or noncontacting (N) primary human BMSCs. *P < 0.05 (t-test). () Migration of G2 cells toward conditioned media collected from contacting or noncontacting primary human BMSCs. *P < 0.005 (t-test). () Real-time quantitative PCR analysis of CXCL12 mRNA in contacting or noncontacting primary human BMSCs after 1 d or 4 d of culture. () Immunocytochemistry of CXCL12 (green) in contacting or noncontacting primary human BMSCs after 1 d or 4 d culture (blue, nuclei). Original magnification, ×40. Data are from three independent experiments (–; mean and s.e.m.) or are representative of three independent experiments (). * Figure 3: Cx43 and Cx45 gap junctions directly regulate CXCL12 secretion. () ELISA of CXCL12 and HGF in the conditioned media of primary human BMSCs treated for 1 h with 100 μM CBX or PBS. *P < 0.05, **P < 0.0001 (t-test). () Migration of G2 cells toward unconditioned media (UCM) with (+CBX) or without (–CBX) supplementation with 100 μM CBX, and toward the conditioned media of primary human BMSCs treated for 1 h with 100 μM CBX (+CBX) or PBS (–CBX). *P < 0.005 (t-test). () ELISA of CXCL12 in the conditioned media of primary human BMSCs treated for 12 h with medium (Med), control mimetic peptide (Ctrl), Cx43 mimetic peptide (Cx43), Cx45 mimetic peptide (Cx45) or a mixture of Cx43 and Cx45 mimetic peptides (Cx43+Cx45). *P < 0.0001 and **P < 0.05 (t-test). () Real-time quantitative PCR analysis of CXCL12 mRNA in primary human BMSCs treated for 12 h as in . Data are pooled from nine experiments () or are from four () or three (,) independent experiments (mean and s.e.m.). * Figure 4: Cx43 and Cx45 regulate Cxcl12 transcription by localizing Sp1 to the nucleus. () Real-time quantitative PCR analysis of Cxcl12 mRNA in Cx45-FL and Cx45-KO primary mouse BMSCs transfected with control siRNA (Ctrl siRNA) or Cx43-specific siRNA (Cx43 siRNA). *P < 0.05 (t-test). () ELISA of CXCL12 in conditioned media from Cx45-FL and Cx45-KO primary mouse BMSCs transfected as in . *P < 0.00005 and **P < 0.05 versus Cx45-KO and control siRNA (t-test). () Real-time quantitative PCR analysis of Sp1 mRNA in Cx45-FL and Cx45-KO primary mouse BMSCs transfected as in . () Immunoblot analysis of Sp1 in nuclear extracts of Cx45-FL and Cx45-KO primary mouse BMSCs transfected as in ; histone H2B serves as a loading control. () Immunocytochemistry of Sp1 (green) in Cx45-FL and Cx45-KO primary mouse BMSCs transfected as in (blue, nuclei); arrows indicate cytoplasmic Sp1. Original magnification, ×60 (left and left middle) or ×20 (right and right middle). Data are from three experiments (–; mean and s.e.m.) or are representative of three independent experiments (,). * Figure 5: Calcium internalization controls the secretion of CXCL12 from BMSCs. () Real-time calcium-conduction imaging before and 20 min after treatment of primary human BMSCs with CBX. () Kinetics of the calcium-conduction experiments in , presented as the change in fluorescence normalized to baseline fluorescence (dF/F) before CBX (black lines) and after CBX (red lines). *P < 0.05 (t-test). () ELISA of mouse CXCL12 in the conditioned media of MS-5 cells treated for 1 h or 12 h with BAPTA-AM, presented relative to that of control cells treated with dimethyl sulfoxide (DMSO), set as 100%. *P < 0.05 and **P < 0.005 (t-test). () ELISA of mouse CXCL12 in the conditioned media of MS-5 cells treated for 1 h or 12 h with ionomycin, presented as in . *P < 0.005 (t-test). Data are representative of six independent experiments () or are from six experiments (; mean ± s.e.m.) or three experiments (,; mean and s.e.m.). * Figure 6: Calcium internalization results in more cAMP and PKA activation, which in turn induces the secretion of CXCL12 from BMSCs via RalA. () Real-time quantitative PCR analysis of ADCY isoforms in human BMSCs, presented relative to the expression of HPRT1 (encoding hypoxanthine guanine phosphoribosyl transferase). () Flow cytometry analysis of the phosphorylation of CREB in MS-5 cells pretreated for 20 min with H89 or PBS, then treated for 30 min with BAPTA-AM or ionomycin, presented relative to that of control cells treated with dimethyl sulfoxide (DMSO), set as 100%. *P < 0.005 (t-test). () ELISA of CXCL12 in the conditioned media of MS-5 cells pretreated for 20 min with H89 or PBS, then treated for 1 h with DMSO or ionomycin. *P < 0.01 (t-test). () ELISA of CXCL12 in the conditioned media of MS-5 cells treated for 12 h with MDL. *P < 0.005 (t-test). () ELISA of CXCL12 in the conditioned media of MS-5 cells pretreated for 20 min with H89 or PBS, then treated for 1 h with DMSO or forskolin. *P < 0.005 (t-test). () ELISA of CXCL12 in the conditioned media of Cx45-FL mouse BMSCs transfected with control siRNA (! Cx45-FL+ctrl siRNA) and Cx45-KO mouse BMSCs transfected with Cx43-specific siRNA (Cx45-KO+Cx43 siRNA) and treated for 2 h with DMSO, ionomycin or forskolin. *P < 0.005 (t-test). () ELISA of CXCL12 in the conditioned media of human BMSCs transfected with empty plasmid (Ctrl) or with plasmid encoding dominant negative RalA (RalA-DN) after treatment for 1 h with DMSO or ionomycin. *P < 0.005 (t-test). Data are from three experiments with three donors (; mean and s.e.m.) or are from three experiments (–; mean and s.e.m.). * Figure 7: Maintenance of CD34+CD38− primitive properties by stromal networks. (,) Light microscopy of purified human CD34+ cells cultured for 4 d together with contacting primary human BMSCs () or noncontacting primary human BMSCs (). Black arrows indicate hematopoietic cells captured in the same plane as BMSCs; white arrows indicate BMSCs. Insets present a wider field of the micrograph. Original magnification, ×20 (insets), with 5.4× enlargement for main images. () Immunocytochemistry of the expression of membrane-bound CXCL12 (green) on the surface of contacting versus noncontacting primary human BMSCs (blue, nuclei). Original magnification, ×20. () Flow cytometry analysis of CD34 expression in purified CD34+ cells after 4 d of culture with contacting or noncontacting primary human BMSCs, presented relative to that in cells immediately after purification (0), set as 100%. *P < 0.005 (t-test). () Flow cytometry of CD34+CD38− primitive cells after 4 d of culture together with contacting or noncontacting primary human BMSCs, presented as in . *P - The E3 ligase Itch is a negative regulator of the homeostasis and function of hematopoietic stem cells
- Nat Immunol 12(5):399-407 (2011)
Nature Immunology | Article The E3 ligase Itch is a negative regulator of the homeostasis and function of hematopoietic stem cells * Chozhavendan Rathinam1, 4 * Lydia E Matesic2 * Richard A Flavell1, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:399–407Year published:(2011)DOI:doi:10.1038/ni.2021Received06 January 2011Accepted14 March 2011Published online10 April 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Although hematopoietic stem cells (HSCs) are the most thoroughly characterized type of adult stem cell, the intricate molecular machinery that regulates their self-renewal properties remains elusive. Here we showed that the E3 ubiquitin ligase Itch negatively regulated the development and function of HSCs. Itch−/− mice had HSCs with enhanced frequency, competence and long-term repopulating activity. Itch-deficient HSCs showed accelerated proliferation rates and sustained progenitor properties, as well as more signaling by the transcription factor Notch1, due to more accumulation of activated Notch1. Knockdown of Notch1 in Itch-mutant HSCs resulted in reversion of the phenotype. Thus, we identify Itch as a previously unknown negative regulator of HSC homeostasis and function. View full text Figures at a glance * Figure 1: Itch deficiency results in a greater frequency of HSCs in the bone marrow. () Real-time PCR analysis of Itch mRNA expression in sorted LT-HSCs (LSK CD150+CD48−), ST-HSCs (LSK CD150+CD48+) and MPPs (LSK CD150−CD48+), presented relative to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase). () Distribution of Lin− cells (top), LSK cells (middle) and various LSK subsets (bottom) among total Itch+/+ and Itch−/− bone marrow cells: cells in gate G1 (top) were further discriminated on the basis of their expression of Sca-1 and c-Kit (middle), then gated c-Kit+Sca-1+ cells (G2) were defined further on the basis of the expression of CD34 and Flt3 (bottom). FSC, forward scatter. Numbers adjacent to outlined areas indicate percent cells in each throughout. () Absolute number of cells in HSC subsets in 4-week-old Itch+/+ and Itch−/− mice (n = 5 per group) based on the gates in . () Distribution (top) and absolute number (bottom) of cells in LSK subsets in 4-week-old Itch+/+ and Itch−/− mice (n = 5 per group), a! ssessed on the basis of expression of CD150 and CD48. () Expression of Flt3 and CD34 on cells of Itch+/+ and Itch−/− bone marrow LSK subsets identified as in . () Absolute number of cells in LSK subsets in 4-week-old Itch+/+ and Itch−/− mice (n = 5 per group), stained as in . *P < 0.05 (Student's t-test). Data are representative of two independent experiments (; mean and s.e.m. of duplicates) or ten (–) or three () independent experiments (mean ± s.e.m. in ,,). * Figure 2: Cell-intrinsic defects and greater competence of Itch-mutant HSCs. () Distribution Lin− cells (top) and LSK cells (bottom) in Itch+/+ and Itch−/− fetal livers (n = 5 mice per genotype). (,) Distribution (top) and absolute number (bottom) of cells in LSK subsets in Itch+/+ and Itch−/− fetal livers (n = 5 mice per genotype), assessed on the basis of the expression of CD34 and Flt3 () or of CD150 and CD48 (). () Distribution of donor-derived sorted LSK cells (top) or total bone marrow cells (bottom) from Itch+/+ and Itch−/− (CD45.2+) mice in the total bone marrow of wild-type (CD45.1+) congenic recipient mice 18 weeks after transplantation. (,) Hematopoiesis of donor-derived (CD45.2+) cells in the peripheral blood () and absolute number of LSK cells in the bone marrow () of wild-type (CD45.1+) recipients (n = 10 per group) of donor Itch+/+ or Itch−/− (CD45.2+) LSK cells mixed at various ratios (horizontal axes) with wild-type competitor (CD45.1+) cells. () Hematopoiesis of donor-derived (CD45.2+) cells in the peripheral blood! of wild-type (CD45.1+) recipients (n = 10 per group) of Itch+/+ or Itch−/− (CD45.2+) bone marrow cells mixed at various ratios (horizontal axis) with wild-type competitor (CD45.1+) cells. () Absolute number of cells of various LSK subsets in 4- and 20-week-old Itch+/+ and Itch−/− mice (n = 5 per genotype). (,) Hematopoiesis of Itch+/+ or Itch−/− donor-derived (CD45.2+) cells in primary and secondary wild-type recipients (; n = 10 per group) or number of Itch+/+ or Itch−/− donor-derived LSK cells in secondary wild-type recipients (). *P < 0.05 (Student's t-test). Data are representative of three (–,) or two (–,,) independent experiments (mean ± s.e.m. in ,,,–). * Figure 3: Accelerated proliferation of Itch-mutant HSCs. () In vivo proliferative potential of Itch+/+ and Itch−/− LSK subsets, as assessed by BrdU incorporation. Numbers below bracketed lines indicate percent proliferating (BrdU+) cells. () Ex vivo cell cycle status of Itch+/+ and Itch−/− LSK cells, as assessed by staining with pyronin Y and Hoechst. () In vitro proliferative potential of Itch+/+ (shaded histograms) and Itch−/− (black lines) CD150+CD48− LSK cells, as assessed by CFSE dilution. () Ex vivo population expansion of Itch+/+ and Itch−/− CD150+CD48− LSK cells in response to a cytokine 'cocktail'. Data are representative of two (,) or five (,) independent experiments (mean ± s.e.m. of duplicates in ). * Figure 4: Augmented repopulation activity of Itch−/− HSCs after myeloablation. () Distribution of LSK cells in Itch+/+ and Itch−/− bone marrow (n = 5 mice per group) after injection of 5-FU. () Differential blood counts and hematocrit (HCT) of Itch+/+ and Itch−/− mice (n = 5 per group) after injection of 5-FU. K/μl, 1 × 103 platelets or red blood cells (RBC) per microliter. *P < 0.05 (Student's t-test). () Survival of wild-type recipients (n = 10 per group) of Itch+/+ or Itch−/− bone marrow cells, assessed after sequential 5-FU treatment (log-rank nonparametric test) and presented as a Kaplan-Meier survival curve. P = 0.0082 (Student's t-test). Data are representative of two independent experiments (mean ± s.e.m. in ). * Figure 5: Less spontaneous differentiation by Itch-deficient HSCs in vitro. () Expression of lineage markers in Itch+/+ and Itch−/− CD150+CD48− LSK cells after in vitro culture in the presence of a cytokine 'cocktail'. Numbers below bracketed lines indicate percent Lin+ cells. () Expression of Sca-1 and c-Kit in Itch+/+ and Itch−/− CD150+CD48− LSK cells after in vitro culture in the presence of a cytokine 'cocktail'; Lin− cells were pre-gated. () Colony-forming unit (CFU) assay of Itch+/+ and Itch−/− CD150+CD48− LSK cells. () Hematopoiesis of donor-derived (CD45.2+) cells in the peripheral blood of wild-type (CD45.1+) recipients (n = 8 mice per group) of LSK cells cultured in vitro (top) or freshly isolated LSK cells (Naive; bottom). Numbers below bracketed lines indicate percent CD45.2+ cells. () Multilineage reconstitution by donor-derived (CD45.2+) cells (myeloid, CD45.2+CD11b+; B lineage, CD45.2+CD19+; T lineage, CD45.2+CD3ε+) in recipients (n = 8 mice per group) of cells as in . *P < 0.05 (Student's t-test). Data are repres! entative of five (,) or two (–) independent experiments (mean ± s.e.m. in ,). * Figure 6: Itch deficiency results in more Notch1 protein and signaling in HPCs. () Full-length Notch1 and cleaved Notch1 (transmembrane and intracellular domains (TM+ICD)) in Itch+/+ and Itch−/− Lin− bone marrow cells. kDa, kilodaltons. Actin serves as a loading control throughout. () Intracellular Notch1 expression in Itch+/+ and Itch−/− CD150+CD48− LSK cells; Isotype (left), cells stained with isotype-matched control antibody (control). () Geometric mean fluorescence intensity (GMFI) of the results in . () Expression of green fluorescent protein (GFP) in CD150+CD48− LSK cells from Itch+/+ and Itch−/− transgenic Notch reporter (TNR+) mice. () Geometric mean fluorescence intensity of the results in . () Cleaved Notch1 (ICD) in Itch+/+ and Itch−/− Lin− bone marrow cells. () Cleaved Notch1 in the nucleus (left) and cytoplasm (right) of Itch+/+ and Itch−/− Lin− bone marrow cells. HDAC1 (histone deacetylase) and GAPDH (glyceraldehyde phosphate dehydrogenase) serve as controls. (,) Real-time PCR analysis of Hes1 and Myc in Itch+! /+ and Itch−/− CD150+CD48− LSK cells () and of Notch1 in Itch+/+ and Itch−/− CD150+CD48− LSK cells (LT-HSC) or Lin− cells (). () Immunoprecipitation analysis of the interaction between Itch and Notch1 in Lin− cells. α-ICD-Notch1, antibody to the intracellular domain of Notch1; α-Notch1, anti-Notch1. () Ubiquitination assay of the involvement of Itch in the ubiquitination of Notch1 protein. *P < 0.05 (Student's t-test). Data are representative of five (,), three (–) or two (–) independent experiments (average ± s.e.m. in ,; mean ± s.e.m. in ,). * Figure 7: Knockdown of Notch1 in Itch-deficient HSCs results in reversion of their phenotype. () Distribution of donor-derived (CD45.2+) LSK cells in total bone marrow from wild-type (CD45.1+) congenic recipients 8 weeks after transplantation of Itch+/+ or Itch−/− (CD45.2+) CD150+CD48− LSK cells transduced with control or Notch1-specific shRNA. () Incorporation of BrdU in vivo by donor-derived (CD45.2+) LSK cells from wild-type (CD45.1+) congenic recipients 8 weeks after transplantation as described in . () Ex vivo population expansion of LSK cells obtained from wild-type (CD45.1+) congenic recipients 8 weeks after transplantation as described in , then cultured in vitro in the presence of the HSC cytokine 'cocktail'. (,) Real-time PCR analysis of Hes1 () and Myc () in Itch+/+ and Itch−/− CD150+CD48− LSK cells obtained from wild-type (CD45.1+) congenic recipients 8 weeks after transplantation as described in . *P < 0.05 (Student's t-test). Data are representative of two independent experiments (mean ± s.e.m. of duplicate samples in ,). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA. * Chozhavendan Rathinam & * Richard A Flavell * Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, USA. * Lydia E Matesic * Howard Hughes Medical Institute, New Haven, Connecticut, USA. * Richard A Flavell * Present address: National Institutes of Health, Center for Biomedical Research Excellence in Stem Cell Biology, Roger Williams Medical Center, Boston University School of Medicine, Providence, Rhode Island, USA. * Chozhavendan Rathinam Contributions C.R. conceived of, designed and did the study, analyzed and interpreted all data, and wrote the manuscript; L.E.M. provided the Itch−/− mice and corrected the manuscript; and R.A.F. provided advice and corrected the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Richard A Flavell Author Details * Chozhavendan Rathinam Search for this author in: * NPG journals * PubMed * Google Scholar * Lydia E Matesic Search for this author in: * NPG journals * PubMed * Google Scholar * Richard A Flavell Contact Richard A Flavell Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–11 Additional data - Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling
- Nat Immunol 12(5):408-415 (2011)
Nature Immunology | Article Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling * Haitao Wen1, 2, 6 * Denis Gris1, 2, 6 * Yu Lei1, 3 * Sushmita Jha1 * Lu Zhang1, 3 * Max Tze-Han Huang1, 3 * Willie June Brickey1 * Jenny P-Y Ting1, 2, 4, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:408–415Year published:(2011)DOI:doi:10.1038/ni.2022Received17 February 2011Accepted15 March 2011Published online10 April 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg High-fat diet (HFD) and inflammation are key contributors to insulin resistance and type 2 diabetes (T2D). Interleukin (IL)-1β plays a role in insulin resistance, yet how IL-1β is induced by the fatty acids in an HFD, and how this alters insulin signaling, is unclear. We show that the saturated fatty acid palmitate, but not unsaturated oleate, induces the activation of the NLRP3-ASC inflammasome, causing caspase-1, IL-1β and IL-18 production. This pathway involves mitochondrial reactive oxygen species and the AMP-activated protein kinase and unc-51–like kinase-1 (ULK1) autophagy signaling cascade. Inflammasome activation in hematopoietic cells impairs insulin signaling in several target tissues to reduce glucose tolerance and insulin sensitivity. Furthermore, IL-1β affects insulin sensitivity through tumor necrosis factor–independent and dependent pathways. These findings provide insights into the association of inflammation, diet and T2D. View full text Figures at a glance * Figure 1: Palmitate activates NLRP3-ASC inflammasome. (–) ELISA for IL-1β (–), IL-18 () and IL-6 () in supernatants of resting or LPS-primed bone marrow-derived macrophages (BMMs) stimulated with palmitate conjugated to BSA (PA-BSA) or BSA control. (,) ELISA for IL-1β () and IL-18 () in supernatants. Resting or LPS-primed BMMs generated from wild-type (WT), Nlrp3−/−, Pycard−/− or Nlrc4−/− mice were stimulated with PA-BSA. () IL-1β ELISA of supernatants from LPS-primed BMMs stimulated with PA-BSA in the absence or presence of the pan-caspase inhibitor zVAD or caspase-1 inhibitor zYVAD. () ELISA for IL-1β in supernatants. Resting or LPS-primed BMMs generated from WT or Casp1−/− mice were stimulated with PA-BSA, BSA control, ATP or nigericin. () ELISA for TNF in supernatants. Resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− or Casp1−/− mice were stimulated with PA-BSA as indicated. Values are expressed as mean ± s.d., and the results are representative of three independent expe! riments. * Figure 2: Palmitate induces IL-1β and caspase-1 processing, which is dependent on NLRP3 and ASC. Immunoblotting for procaspase-1 and activated caspase-1 (p10), pro-IL-1β and cleaved IL-1β (p17) in supernatants (Sup) and cell lysates (Lys) from resting or LPS-primed BMMs generated from WT, Nlrp3−/−, Pycard−/− () or Nlrc4−/− () mice stimulated with PA-BSA (0.2 or 0.5 mM) as indicated at top. Data are representative of two independent experiments. * Figure 3: Palmitate-induced inflammasome activation requires ROS. () ROS production determined by flow cytometry using the fluoroprobe dihydrorhodamine 123 (DHR) in LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of ROS inhibitor APDC (50 μM). MFI, mean fluorescence intensity. (,) ELISA for IL-1β () and immunoblotting for caspase-1 p10 and IL-1β p17 () on LPS-primed BMMs stimulated with PA-BSA in the absence of or presence of increasing concentrations of APDC. () ELISA for IL-1β. Resting or LPS-primed BMMs were stimulated with PA-BSA (0.5 mM) in the absence or presence of the ROS inhibitors APDC (10 or 50 μM) and N-acetyl-cysteine (NAC, 5 or 25 mM) or the NAPDH oxidase inhibitor diphenyleneiodonium (DPI, 5 or 25 μM). Values are expressed as mean ± s.d. In , one of two independent experiments is shown. In each of –, the results are representative of three independent experiments. * Figure 4: Palmitate-induced inflammasome activation involves AMPK. () Thr172-phosphorylated (p-) and total AMPK-α subunit determined by immunoblotting. Resting or LPS-primed BMMs were untreated or stimulated with PA-BSA (0.5 mM) for 24 h. (–) Analysis of LPS-primed BMMs stimulated with PA-BSA (0.5 mM) in the absence or presence of AMPK agonist AICAR. () ROS production determined using the fluoroprobe DHR. (,) IL-1β () and IL-6 () in supernatants, measured by ELISA. () Immunoblotting for caspase-1, IL-1β and AMPK signaling molecules. () Transfection of BMMs with empty vector (EV) or constitutively active (CA) AMPK-α1 subunit. N, non-transfected. Efficiency was determined 16 h later by flow cytometric analysis of GFP expression in macrophages cotransfected with CA AMPK-α1 and pmaxGFP. Values are percentages of GFP-positive cells in the ranges indicated by the bars. () ELISA for IL-1β in supernatants of cells transfected as in and stimulated with PA-BSA (0.5 mM) for 24 h. In each of , and , one of two independent experiments is shown. ! In each of – and , the results are representative of three independent experiments. Values are expressed as mean ± s.d. *P < 0.05 versus controls. * Figure 5: Palmitate-induced AMPK inactivation leads to defective autophagy and the generation of mitochondrial ROS. () Immunoblotting for AMPK activation (Thr172 p-AMPK-α) and LC3B (top), and densitometric analysis to quantify ratio of LC3B-II to actin (bottom). BMMs were pretreated with LPS (200 ng/ml) for 3 h, followed by PA-BSA (0.5 mM) treatment for 24 h in the absence or presence of chloroquine (50 μM). Med, medium control. (,) LC3B and autophagosomes in BMMs pretreated with LPS for 3 h, followed by PA-BSA treatment for 24 h in the absence or presence of AICAR. Cells were fixed and stained for LC3B (, left). Quantitation of autophagosomes was performed by counting LC3B puncta in 100 cells (, right). Cells were fixed and examined by transmission electron microscopy for autophagosomes (). Quantitation (, right) is based on counting autophagosomes in ten cells per treatment. () Immunoblotting (left) and densitometric analysis to quantify ratio of LC3B-II to actin and ULK1 species (right) in treated BMMs. () Mitochondrial ROS production determined using the MitoSOX fluorescence indicat! or. In each of and , one of three independent experiments is shown. In each of and , one of two independent experiments is shown. Values are expressed as mean ± s.d. *P < 0.05 versus controls. * Figure 6: Inflammasome-generated IL-1β inhibits insulin signaling in vitro. (,) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) () or TNF (2 ng/ml) () for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (). p-Akt (Ser473) was determined by immunoblotting (). () p-IRS1 (Ser307), determined by immunoblotting, in FL83B mouse liver cells treated with either IL-1β or TNF for 24 h. (–) Akt Ser473 phosphorylation measured by flow cytometry () or immunoblotting (). FL83B cells (,) or WT primary hepatocytes () were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (), Nlrp3−/− or Casp1−/− () macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation. () Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 recep! tor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin. () p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d. * Figure 7: IL-1β and TNF cooperatively mediate insulin resistance in vivo. () ITT performed 2 h after IL-1β injection. Eight-week old male C57BL/6 mice were intraperitoneally injected with saline or mouse recombinant IL-1β (1 μg/kg). () ITT in WT mice on regular diet (RD) or HFD for 12 weeks, or Il1b−/− mice on HFD for 12 weeks. (,) ITT () and serum IL-1β (, left) and TNF (, right) determined by ELISA, 2 h after IL-1β injection. Recombinant mouse IL-1β was administered to WT and Tnfa−/− mice (n = 5 for each group). One of two independent experiments is shown (mean ± s.d.). *P < 0.05 versus controls. * Figure 8: The NLRP3-ASC inflammasome promotes insulin resistance in vivo. () Blood glucose and insulin levels measured in WT (n = 6), Nlrp3−/− (n = 5) or Pycard−/− (n = 6) mice under fasting or refed conditions after 12 weeks of HFD. (–) Glucose tolerance test (GTT) (,) and insulin tolerance test (ITT) (,) performed for WT, Nlrp3−/− (,) and Pycard−/− (,) mice on HFD for 12 weeks. () GTT conducted on the indicated bone marrow chimeric mice on HFD for 12 weeks. (,) Insulin-stimulated phosphorylation of IRβ, IRS1 and Akt (Ser473) in liver tissues of individual WT and Pycard−/− mice (), and p-Akt (Ser473) in liver, white adipose (WAT) and muscle tissues of individual WT and Nlrp3−/− mice () on HFD for 12 weeks, after insulin (2 IU/kg) infusion. Graphs at right of blots show the quantitation of each molecule. pY, phosphotyrosine. (,) Expression of Tnfa and Mcp1 mRNA relative to Actb in liver tissues of Nlrp3−/− mice () and Pycard−/− mice () on regular diet (RD) or HFD for 12 weeks. One of two independent experiments i! s shown (mean ± s.d.). *P < 0.05 versus controls. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Haitao Wen & * Denis Gris Affiliations * Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. * Haitao Wen, * Denis Gris, * Yu Lei, * Sushmita Jha, * Lu Zhang, * Max Tze-Han Huang, * Willie June Brickey & * Jenny P-Y Ting * Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. * Haitao Wen, * Denis Gris & * Jenny P-Y Ting * Department of Oral Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. * Yu Lei, * Lu Zhang & * Max Tze-Han Huang * Center for Translational Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. * Jenny P-Y Ting * Inflammatory Diseases Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. * Jenny P-Y Ting Contributions H.W., D.G. and J.P.-Y.T. designed the experiments; H.W., D.G., Y.L., S.J., L.Z., M.T.-H.H. and W.J.B. performed experiments and provided intellectual input; J.P.-Y.T. supervised the study. H.W., D.G. and J.P.-Y.T. interpreted the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jenny P-Y Ting Author Details * Haitao Wen Search for this author in: * NPG journals * PubMed * Google Scholar * Denis Gris Search for this author in: * NPG journals * PubMed * Google Scholar * Yu Lei Search for this author in: * NPG journals * PubMed * Google Scholar * Sushmita Jha Search for this author in: * NPG journals * PubMed * Google Scholar * Lu Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Max Tze-Han Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Willie June Brickey Search for this author in: * NPG journals * PubMed * Google Scholar * Jenny P-Y Ting Contact Jenny P-Y Ting Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (602K) Supplementary Figures 1–5 and Supplementary Table 1 Additional data - Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk
- Nat Immunol 12(5):416-424 (2011)
Nature Immunology | Article Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk * Xingguang Liu1 * Zhenzhen Zhan1 * Dong Li2 * Li Xu1 * Feng Ma2 * Peng Zhang1 * Hangping Yao2 * Xuetao Cao1, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:416–424Year published:(2011)DOI:doi:10.1038/ni.2015Received25 October 2010Accepted28 February 2011Published online27 March 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The molecular mechanisms involved in the full activation of innate immunity achieved through Toll-like receptors (TLRs) remain to be fully elucidated. In addition to their classical antigen-presenting function, major histocompatibility complex (MHC) class II molecules might mediate reverse signaling. Here we report that deficiency in MHC class II attenuated the TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and dendritic cells, which protected mice from endotoxin shock. Intracellular MHC class II molecules interacted with the tyrosine kinase Btk via the costimulatory molecule CD40 and maintained Btk activation, but cell surface MHC class II molecules did not. Then, Btk interacted with the adaptor molecules MyD88 and TRIF and thereby promoted TLR signaling. Therefore, intracellular MHC class II molecules can act as adaptors, promoting full activation of TLR-triggered innate immune responses. View full text Figures at a glance * Figure 1: Deficiency in MHC class II protects mice from challenge with TLR ligands. (–) Enzyme-linked immunosorbent assay (ELISA) of TNF (), IL-6 () and IFN-β () in the serum of H2−/− or H2+/+ mice (n = 5 per genotype) 2 h after intraperitoneal administration of PBS or LPS, CpG-ODN (CpG) or poly(I:C) (at a dose of 15, 20 or 20 mg per kg body weight, respectively). *P < 0.01 (Student's t-test). () Survival of H2−/− mice and H2+/+ mice (n = 10 per genotype) given intraperitoneal injection of LPS (15 mg per kg body weight). P < 0.01 (Wilcoxon test). () ELISA of TNF, IL-6 and IFN-β in serum from wild-type mice lethally irradiated and given intravenous transplantation of 1 × 107 bone marrow cells from H2−/− or H2+/+ mice 3 weeks before challenge with PBS or LPS, assessed 2 h after challenge. *P < 0.01 (Student's t-test). Data are from three independent experiments (mean ± s.e.m.). * Figure 2: Deficiency in MHC class II protects mice from sepsis induced by live E. coli. (,) ELISA of TNF () and IL-6 () in serum from H2−/− or H2+/+ mice (n = 3 per genotype) 4 h after intraperitoneal infection with E. coli 0111:B4 (1 × 107 colony-forming units per mouse). *P < 0.01 (Student's t-test). () Survival of mice (n = 10 per genotype) treated as described in ,. P < 0.01 (Wilcoxon test). Data are from three independent experiments (mean ± s.e.m.). * Figure 3: Deficiency in MHC class II attenuates TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and DCs. () ELISA of cytokines in supernatants of H2−/− or H2+/+ macrophages (top row) or DCs (bottom row) left unstimulated (Med) or stimulated for 6 h with LPS (100 ng/ml), CpG ODN (CpG; 0.3 μM) or poly(I:C) (10 μg/ml). (,) ELISA of cytokines in supernatants of H2−/− or H2+/+ macrophages given mock transfection (Mock) or transfected with vectors for the expression of MHC class II α-chain and β-chain (α+β-chain) or MHC class II α-chain alone (α-chain) or β-chain alone (β-chain) and, 36 h later, stimulated for 6 h with LPS (), or CpG ODN or poly(I:C) (). () Immunoblot analysis of the expression of MHC class II β-chain (MHCIIβ) and β-actin in lysates (left) and flow cytometry analysis of the surface expression of MHC class II (MHCII; right) of macrophages 48 h after transfection with control siRNA (Ctrl (left) or solid line with no shading (right)) or siRNA specific for MHC class II β-chain (siRNA (left) or solid line with gray shading (right)). Dotted line (righ! t), isotype-matched control antibody. () ELISA of cytokines in supernatants of macrophages transfected as in and, 48 h later, left unstimulated or stimulated for 6 h with LPS, CpG ODN or poly(I:C). () ELISA of cytokines in serum from wild-type mice first depleted of endogenous macrophages and then transplanted with 1 × 107H2−/− or H2+/+ bone marrow–derived cells 6 h before challenge with LPS, CpG ODN or poly(I:C), measured 2 h after challenge. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from three independent experiments (–,–; mean ± s.e.m.) or are representative of three independent experiments with similar results (). * Figure 4: Deficiency in MHC class II impairs the MyD88-dependent and TRIF-dependent activation of mitogen-activated protein kinases, NF-κB, IRF3 and IRF7 in TLR-triggered macrophages. () Immunoblot analysis of phosphorylated (p-) or total protein in lysates of H2−/− or H2+/+ macrophages stimulated for 0–60 min (above lanes) with LPS (100 ng/ml). () Immunoblot analysis of IRF3 among nuclear proteins from macrophages stimulated with LPS; lamin A serves as a loading control. () Luciferase activity in lysates of H2−/− or H2+/+ macrophages transfected with luciferase reporter plasmids for NF-κB, AP-1, IRF3 or IRF7 (vertical axes) and, 36 h later, left unstimulated or stimulated for 4 h with LPS (100 ng/ml), CpG ODN (0.3 μM) or poly(I:C) (10 μg/ml); results are presented relative to the activity in unstimulated H2+/+ macrophages, set as 1. (,) Immunoblot analysis (IB) of IRAK1 and MyD88 () or TBK1 and TRIF () immunoprecipitated (IP) with anti-MyD88 () or anti-TRIF () from lysates of H2−/− or H2+/+ macrophages stimulated for 0–90 min (above lanes) with LPS; immunoglobulin G (IgG) serves as an immunoprecipitation control. () In vitro kinase ass! ay of IRAK1, TAK1 and TBK1 in lysates of H2−/− or H2+/+ macrophages left stimulated (Med) or stimulated for 30 min with LPS, CpG ODN or poly(I:C), assayed with the substrates MBP (for IRAK1), MKK4 (for TAK1) or recombinant IRF3 (for TBK1). *P < 0.01 (Student's t-test). Data are from one experiment representative of three independent experiments with similar results (,,,; mean ± s.d. of four samples in ) or are from three independent experiments (; mean ± s.e.m.). * Figure 5: MHC class II molecules promote TLR-triggered inflammatory innate responses by maintaining Btk activation. (,) Immunoblot analysis of Btk phosphorylated at Tyr550 (p-Btk(Y550)) or Tyr222 (p-Btk(Y222)) or total Btk in lysates of H2−/− or H2+/+ peritoneal macrophages left unstimulated or stimulated for 0–60 min with LPS (100 ng/ml; ) or for 30 min with CpG ODN (0.3 μM) or poly(I:C) (10 μg/ml; ). () ELISA of TNF, IL-6 and IFN-β in supernatants of Btk+/+ or Btk−/− peritoneal macrophages left unstimulated or stimulated for 6 h with LPS, CpG ODN or poly(I:C). () ELISA of TNF, IL-6 and IFN-β in supernatants of H2−/− or H2+/+ peritoneal macrophages mock-transfected or transfected with constitutively active Btk(E41K) and, 48 h later, stimulated for 6 h with LPS, CpG ODN or poly(I:C). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three independent experiments with similar results (,) or are from three independent experiments (,; mean ± s.e.m.). * Figure 6: Intracellular MHC class II molecules interact with CD40 and Btk. (,) Immunoblot analysis of Btk (), CD40 () or MHC class II (,) immunoprecipitated with antibody to MHC class II from cytoplasmic and plasma membrane proteins in lysates of peritoneal macrophages stimulated for 0–15 min with LPS. Immunoglobulin G serves as an immunoprecipitation control. (–) Confocal microscopy of macrophages left unstimulated (0 min) or stimulated for 15 min with LPS (100 ng/ml), then labeled with antibodies to the appropriate molecules (above images). Original magnification, ×630. () ELISA of TNF, IL-6 and IFN-β in supernatants of Cd40+/+ or Cd40−/− peritoneal macrophages left unstimulated or stimulated for 6 h with LPS (100 ng/ml), CpG ODN (0.3 μM) or poly(I:C) (10 μg/ml). *P < 0.01 (Student's t-test). Data are representative of three independent experiments with similar results (–) or are from three independent experiments (; mean ± s.e.m.). * Figure 7: Activated Btk interacts with MyD88 and TRIF, promoting the activation of MyD88-dependent and TRIF-dependent pathways. () Immunoblot analysis of MyD88, TRIF or Btk immunoprecipitated with anti-Btk from lysates of H2−/− or H2+/+ macrophages stimulated for 0–90 min with LPS. () Immunoblot analysis of HEK293 cells 48 h after cotransfection of Flag-tagged MyD88 or Flag-tagged TRIF plus hemagglutinin (HA)-tagged Btk, followed by immunoprecipitation with anti-hemagglutinin. TCL, immunoblot analysis of total cell lysates with anti-Flag. () Luciferase assay of the activation of NF-κB or IRF3 in lysates of HEK293 cells 24 h after transfection of luciferase reporter plasmid for NF-κB or IRF3, plus empty vector control (Ctrl) or plasmid expressing MyD88 or TRIF either alone (MyD88 or TRIF) or together with plasmid expressing Btk(E41K) (MyD88+E41K or TRIF+E41K; dose, horizontal axis); results were normalized to renilla luciferase activity and are presented relative to the activity in cells transfected with empty vector control, set as 1. *P < 0.05 and **P < 0.01 (Student's t-test). Data are from! one experiment representative of three independent experiments with similar results (mean ± s.d. of six samples in ). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Entrez Nucleotide * NM_010378.2 * NM_207105.2 * BC094338 * NM_010851 * NM_013482.2 Mouse Genome Informatics * 003584 * 002536 * 002928 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China. * Xingguang Liu, * Zhenzhen Zhan, * Li Xu, * Peng Zhang & * Xuetao Cao * Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, China. * Dong Li, * Feng Ma & * Hangping Yao * Chinese Academy of Medical Sciences, Beijing, China. * Xuetao Cao Contributions X.C. and X.L. designed the experiments; X.L., Z.Z., D.L., L.X., F.M., P.Z. and H.Y. did the experiments; X.C. and X.L. analyzed data and wrote the paper; and X.C. was responsible for research supervision, coordination and strategy. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Xuetao Cao Author Details * Xingguang Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Zhenzhen Zhan Search for this author in: * NPG journals * PubMed * Google Scholar * Dong Li Search for this author in: * NPG journals * PubMed * Google Scholar * Li Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Ma Search for this author in: * NPG journals * PubMed * Google Scholar * Peng Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Hangping Yao Search for this author in: * NPG journals * PubMed * Google Scholar * Xuetao Cao Contact Xuetao Cao Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–16 Additional data - STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development
- Nat Immunol 12(5):425-433 (2011)
Nature Immunology | Article STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development * Andre Limnander1 * Philippe Depeille2 * Tanya S Freedman1 * Jen Liou3 * Michael Leitges4, 5 * Tomohiro Kurosaki6, 7 * Jeroen P Roose2 * Arthur Weiss1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:425–433Year published:(2011)DOI:doi:10.1038/ni.2016Received27 February 2011Accepted02 March 2011Published online27 March 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca2+-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide–exchange factor RasGRP and depended on the concentration of the Ca2+ sensor STIM1, which controls the magnitude of Ca2+ entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca2+-dependent Erk signaling as a critical proapoptotic pathway ! that mediates the negative selection of B cells. View full text Figures at a glance * Figure 1: Sensitization of B cells to antigen-induced apoptosis correlates with Ca2+-dependent activation of Erk. () Intracellular Ca2+ in wild-type DT40 cells (DT40; left) and DT40 cells overexpressing STIM1 (DT40 + STIM1; right) stimulated with 1 μM thapsigargin (Thaps) or M4 antibody to the BCR (α-BCR), followed by lysis (maximum Ca2+-bound emissions) for termination of stimulation, and Ca2+ chelation with EGTA (maximum Ca2+-free emissions). () Annexin V (AnnV) staining on the surface of cells left unstimulated (−) or stimulated for 8 h as in . () Immunoblot analysis (IB) of lysates of cells left unstimulated or stimulated for 2 or 5 min (above lanes) with thapsigargin, probed with antibodies 4G10 and RC20 to phosphorylated tyrosine (p-Tyr). () Immunoprecipitation (with 4G10) of proteins containing phosphorylated tyrosine from lysates of unstimulated or thapsigargin-stimulated cells, followed by SDS-PAGE and immunoblot analysis (left) or staining with colloidal blue (CB; right); arrow indicates band excised and identified as Erk2 by mass spectrometry. () Flow cytometry of thapsig! argin-stimulated cells, assessing the intensity of phosphorylated Erk. () Mean fluorescence intensity (MFI) of the phosphorylated Erk in , over time. Data are representative of at least five experiments each (–,,; error bars (), s.e.m.) or one experiment (). * Figure 2: BCR stimulation activates Erk via two distinct pathways activated mainly by either Ca2+ or DAG. () Flow cytometry of cells left unstimulated (Unstim) or stimulated for 5, 15 or 30 min with anti-BCR alone or anti-BCR plus EGTA, assessing the intensity of phosphorylated Erk. () Mean fluorescence intensity of the phosphorylated Erk in , over time. () Flow cytometry analysis of the abundance of phosphorylated Erk in cells transiently transfected for 16–20 h with a construct encoding CD16 plus either vector alone or construct encoding DGK-ζ, then left unstimulated or stimulated for 5 min with 1 μM thapsigargin or anti-BCR alone or anti-BCR plus EGTA; staining of surface CD16 serves as a marker to distinguish transfected and untransfected populations in the same sample (gated populations in ). () The phosphorylated Erk response in each population in ; numbers above bracketed lines indicate percent cells positive for phosphorylated Erk in the corresponding CD16+ population. Data are representative of at least five (,) or three (,) independent experiments. * Figure 3: Ca2+-dependent Erk activation occurs selectively at stages of negative selection in primary bone marrow B cells. () Flow cytometry of wild-type C57BL/6 bone marrow cells stimulated with thapsigargin, then fixed and made permeable and stained for phosphorylated Erk along with cell surface markers; B220+ cells were gated (top) in four subsets defined as B220+IgM−IgD− (precursors (Prec)), B220+IgM+IgD− (immature (Imm)), B220+IgMhiIgDint (transitional (Trans)) and B220+IgMloIgD+ (mature recirculating (Mat)). Below, the phosphorylated Erk response of each subset. () Mean fluorescence intensity of the phosphorylated Erk response for each subset in over time. Data are representative of at least three experiments. * Figure 4: STIM1 overexpression confers a competitive disadvantage to developing B cells. Flow cytometry of bone marrow, splenic and lymph node (LN) cells from the lethally irradiated CD45.2+CD45.2+ recipients (n = 5) of CD45.1+CD45.1+ or CD45.1+CD45.2+ purified bone marrow hematopoietic stem cells infected with retrovirus encoding eYFP or eYFP-STIM1, respectively, mixed at a ratio of 1:1 and injected into hosts along with uninfected carrier bone marrow of the same genotype, analyzed 8–10 weeks after injection after staining for cell surface markers. Results are presented as the ratio of CD45.1+CD45.2+ cells to CD45.1+CD45.1+ cells for the infected (eYFP+) or uninfected (eYFP−) populations at various stages in B cell development (corresponding to the ratio of eYFP-STIM1+ to eYFP+ in the infected population): bone marrow B cell subsets as defined Figure 3; splenic B cell subsets defined as B220+CD21-CD23- (transitional 1 (T1)), B220+IgMhiIgDhi (transitional 2 (T2)), B220+IgMloIgDhi (follicular mature (FM)). Data are from one experiment representative of at lea! st three independent experiments (error bars, s.e.m.). * Figure 5: Activation of Erk downstream of Ca2+ requires RasGRP. () Intensity of phosphorylated Erk in the eYFP-STIM1+ population of wild-type (WT), Rasgrp1−/−Rasgrp3−/− or Sos1−/−Sos2−/− DT40 cells transiently transfected with the eYFP-STIM1 retrovirus, then stimulated with thapsigargin, fixed and made permeable and stained for phosphorylated Erk. () Annexin V on the surface of cells stably overexpressing STIM1, left unstimulated or stimulated for 8 h with thapsigargin or anti-BCR, presented relative to that on unstimulated cells. () Intensity of phosphorylated Erk in wild-type DT40 cells stably overexpressing STIM1, left untreated (No inhibitor) or treated for 5 min with the PKC inhibitor Gö6976 or GF-109203X, then left unstimulated or stimulated for 5 min with thapsigargin (top row) or anti-BCR alone or with EGTA (bottom row). Data are representative of two independent experiments (), five experiments (; error bars, s.e.m.) or at least two independent experiments (). * Figure 6: Phosphorylation of RasGRP1 at Ser332 is required for the restoration of Ca2+-dependent Erk activation by RasGRP1 in RasGRP-deficient cells. (,) Intensity of the phosphorylated Erk response over time in RasGRP-deficient cells overexpressing STIM1, transiently transfected with constructs encoding wild-type or S332A Myc-tagged human RasGRP1, then stimulated with thapsigargin () or with anti-BCR plus EGTA (). (,) Analysis of the phosphorylated Erk response as in , but in cells transfected with constructs encoding wild-type RasGRP1 or mutant RasGRP1 with substitution of aspartic acid for serine at position 332 (S332D), then stimulated with thapsigargin () or anti-BCR plus EGTA (). () Three-dimensional homology model of the structure of the CDC25 domain of mouse RasGRP1 made with the RasGRF1 CDC25 domain as a template, showing the potential phosphorylation site at Ser332 (blue sticks) in flap2 abutting the helical hairpin; stick-and-ball indicates residues within 3.2 Å of Ser332. Data are representative of at least four independent experiments (,) or two independent experiments (,). * Figure 7: PKC-δ is required for Ca2+-dependent activation of Erk in developing bone marrow cells. () Ratio of Prkdc−/− cells to wild-type (Prkdc+/+) cells throughout B cell development in lethally irradiated recipients (n = 8) of Prkdc−/− and wild-type bone marrow cells (bearing different CD45 congenic markers) mixed at a ratio of 1:1, analyzed 6–8 weeks after injection. P value, paired t-test. () The phosphorylated Erk response in Prkcd−/− and wild-type bone marrow cells left unstimulated or stimulated with thapsigargin, analyzed by flow cytometry. () Intensity of the phosphorylated Erk in , presented relative to that in resting cells. Data are from one experiment representative of two independent experiments (; error bars, s.e.m.) or at least four independent experiments (,). * Figure 8: PKC-δ is required for STIM1-mediated sensitization of bone marrow B cells to negative selection. Flow cytometry of cells from lethally irradiated CD45.1+CD45.2+ heterozygous recipients reconstituted with CD45.1+CD45.1+ hematopoietic stem cells infected with eYFP-expressing retrovirus and mixed at a ratio of 1:1 with wild-type (black bars; n = 8 recipients) or Prkdc−/− (gray bars; n = 11 recipients) CD45.2+CD45.2+ hematopoietic stem cells infected with the eYFP-STIM1 retrovirus, then injected into hosts along with uninfected carrier bone marrow of the same genotype, followed by analysis 8–10 weeks after injection (as in Fig. 4). P value, unpaired t-test. Data are from one experiment representative of three independent experiments (error bars, s.e.m.). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 2IJE * 2IJE Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Medicine, Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis, University of California at San Francisco, San Francisco, California, USA. * Andre Limnander, * Tanya S Freedman & * Arthur Weiss * Department of Anatomy, University of California at San Francisco, San Francisco, California, USA. * Philippe Depeille & * Jeroen P Roose * Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Jen Liou * Division of Nephrology, Department of Medicine, Hannover Medical School, Hannover, Germany. * Michael Leitges * The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway. * Michael Leitges * Laboratory for Lymphocyte Differentiation, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan. * Tomohiro Kurosaki * Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan. * Tomohiro Kurosaki Contributions A.L. designed and did experiments, analyzed the data and wrote the manuscript; P.D. collaborated with A.L. in determining expression of various proteins on sorted bone marrow B cell populations and in collecting reconstituted mice; T.S.F. did homology modeling of RasGRP1; J.L. and T.K. provided reagents; M.L. generated the Prkcd−/− mice; J.P.R. designed experiments and provided reagents; and A.W. designed experiments, supervised the research, revised the manuscript and provided support. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Arthur Weiss Author Details * Andre Limnander Search for this author in: * NPG journals * PubMed * Google Scholar * Philippe Depeille Search for this author in: * NPG journals * PubMed * Google Scholar * Tanya S Freedman Search for this author in: * NPG journals * PubMed * Google Scholar * Jen Liou Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Leitges Search for this author in: * NPG journals * PubMed * Google Scholar * Tomohiro Kurosaki Search for this author in: * NPG journals * PubMed * Google Scholar * Jeroen P Roose Search for this author in: * NPG journals * PubMed * Google Scholar * Arthur Weiss Contact Arthur Weiss Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (725K) Supplementary Figures 1–10 Additional data - The P4-type ATPase ATP11C is essential for B lymphopoiesis in adult bone marrow
- Nat Immunol 12(5):434-440 (2011)
Nature Immunology | Article The P4-type ATPase ATP11C is essential for B lymphopoiesis in adult bone marrow * Owen M Siggs1 * Carrie N Arnold1 * Christoph Huber2 * Elaine Pirie1 * Yu Xia1 * Pei Lin1 * David Nemazee2 * Bruce Beutler1 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:434–440Year published:(2011)DOI:doi:10.1038/ni.2012Received10 November 2010Accepted23 February 2011Published online20 March 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg B lymphopoiesis begins in the fetal liver, switching after birth to the bone marrow, where it persists for life. The unique developmental outcomes of each phase are well documented, yet their molecular requirements are not. Here we describe two allelic X-linked mutations in mice that caused cell-intrinsic arrest of adult B lymphopoiesis. Mutant fetal liver progenitors generated B cells in situ but not in irradiated adult bone marrow, which emphasizes a necessity for the affected pathway only in the context of adult bone marrow. The causative mutations were ascribed to Atp11c, which encodes a P4-type ATPase with no previously described function. Our data establish an essential, cell-autonomous and context-sensitive function for ATP11C, a putative aminophospholipid flippase, in B cell development. View full text Figures at a glance * Figure 1: A heritable B cell deficiency. () Frequency of CD19+ and Thy-1.2+ blood lymphocytes in wild-type (WT) and emptyhive mice. (–) Frequency (,,) and number () of B cell subsets in the bone marrow (), spleen () or peritoneal cavity () or all three (). Hardy fractions in bone marrow (A–F) were gated as follows: A, B220+CD43+BP-1−CD24−; B, B220+CD43+BP-1−CD24+; C, B220+CD43+BP-1+CD24+; D, B220+CD43−IgM−IgD−; E, B220+CD43−IgM−IgD+; and F, B220+CD43−IgM+IgD+; the C′ fraction (B220+CD43+BP-1+CD24hi) was not resolved. Fractions A and B–D may also be gated as CD19− and CD19+ populations among B220+IgM− cells, respectively (). IgM+ splenocytes were separated into the following subsets: T1, CD93+CD23−; T2, CD93+CD23+IgMint; T3, CD93+CD23+IgMhi; marginal zone (MZ), CD93−CD23−IgMhiCD21hi; and follicular (Fo), CD93−CD23+. Peritoneal lymphocytes were separated into the following subsets: B-2, CD19+B220hi; B-1, CD19+B220lo–int; B-1a, CD5+CD43+; and B-1b, CD5−CD43−. () IgM allotype! expression on CD19+ blood lymphocytes from wild-type and emptyhive mice on a (C57BL/6 × BALB/c)F2 background (heterozygous IgM allotype). Each symbol (,) represents an individual mouse; numbers adjacent to outlined areas or in quadrants (–,,) indicate percent cells in each. Data are representative of three independent experiments (–) or one experiment () with three mice per genotype (error bars (), s.e.m.). * Figure 2: Immunoglobulin secretion in emptyhive mice. () Total immunoglobulins in the serum of 12- to 24-week-old naive mice. (,) NP-specific antibodies 7, 14 or 28 d after immunization of 12-week-old mice with NP-Ficoll () or alum-precipitated NP-CGG (), presented as absorbance at 450 nm (A450). Different capture antigens were used to discriminate between total IgG1 (NP23-BSA) and high-affinity IgG1 (NP4-BSA) in response to NP-CGG. Each symbol represents an individual mouse. NS, not significant (P > 0.05); P values, wild-type versus emptyhive (unpaired t-test). Data are from one experiment (error bars, s.e.m.). * Figure 3: A cell-intrinsic failure of adult B cell development. () Lymphocyte repopulation in blood 8 weeks after reconstitution of irradiated RAG-1-mutant recipients with a 1:1 mixture of emptyhive (CD45.2+) and wild-type (C57BL/6 (B6); CD45.1+) bone marrow. (,) Reconstitution of bone marrow, spleen and peritoneal B cell subsets with donor-derived cells 8 weeks after reconstitution of irradiated wild-type recipients (CD45.2+) with unmixed bone marrow (,) or fetal liver cells at embryonic day 16.5 () from either mutant or wild-type donors (CD45.1+). () NP-specific immunoglobulin titers in bone marrow chimeras (key) immunized with NP-Ficoll at 8 weeks after reconstitution, assessed 7 and 14 d later. () Absolute number of follicular and marginal zone B cells as a function of age. () Expression of intracellular IgM (cμ) in B220+ bone marrow cells negative for mIgM. Rag1mal/mal (right), mouse homozygous for the maladaptive mutation of Rag1 (negative control). Each symbol (,,) represents an individual mouse; numbers adjacent to outlined area! s or quadrants (,,) indicate percent cells in each. Data are from one experiment (–) or two independent experiments () with six (), four to seven () or three () mice per genotype (error bars (,,), s.e.m.). * Figure 4: Sensitivity to IL-7 and a failure to sustain expression of Ebf1. () Frequency of 7-AAD− lymphoblasts (FSChiSSChi) among B220+ mIgM− bone marrow cells sorted by flow cytometry and cultured ex vivo in the presence or absence of IL-7 (100 ng/ml). SSC, side scatter; FSC, forward scatter. () Frequency (left) and number (right) of 7-AAD− mIgM+ cells among B220+ mIgM− bone marrow cells sorted from wild-type (CD45.1+) mice or emptyhive (CD45.2+) mice and cultured together for 4 d in the presence of various concentrations of IL-7. () RT-PCR analysis of cDNA from pre-pro-B cells (7-AAD−B220+IgM-IgD−CD19−NK1.1−Ly6C−); each lane represents an individual mouse. () Expression of λ5 (CD179b; solid lines) on the surface of B220+CD43+ bone marrow lymphocytes; shaded histograms, rat IgG2a isotype-matched control antibody. () Expression of IL-7Rα (CD127) on the surface of B220+IgM− bone marrow lymphocytes (left), or B220+IgM−λ5+ cells (right; presented as mean fluorescence intensity (MFI)). () Splenic B cell numbers in emptyhive mic! e also heterozygous (Flt3+/wmfl) or homozygous (Flt3wmfl/wmfl) for an additional mutation (warmflash) of Flt3. Numbers adjacent to outlined areas or in quadrants (,,,) indicate percent cells in each. Data are representative of two independent experiments, each with three mice per genotype (error bars, s.e.m.). * Figure 5: Partial correction of the emptyhive phenotype by BCR transgenes. (,) Frequency of CD19+ cells in the blood () and B cell subsets in the bone marrow () of mice with the emptyhive mutation either alone (MM4−) or combined with the MM4 transgene (MM4+), assessed by flow cytometry. (,) Frequency of CD19+ cells in the blood () and B cell subsets in the bone marrow () of mice with the emptyhive mutation either alone (−) or combined with the LC2 light chain transgene or the knock-in allele of heavy-chain variable region 10 (VH10) or both (SWHEL), assessed by flow cytometry. Hardy fractions (,) were gated as follows: A, B220+IgM−IgD−CD19−; B–D, B220+IgM−IgD−CD19+; E, B220+IgM+IgD−; F, B220+IgM+IgD+. Numbers adjacent to outlined areas indicate percent cells in each; each symbol represents an individual mouse. *P < 0.05 and **P < 0.001, versus nontransgenic emptyhive (unpaired t-test () or one-way analysis of variance followed by Bonferroni's post-test ()). Data are representative of two independent experiments (error bars, s.e.m.). * Figure 6: The emptyhive phenotype is caused by a recessive mutation of Atp11c. (,) Chromosomal mapping () and fine mapping () of the emptyhive phenotype. LOD, logarithm of odds score; Mb, megabases. () DNA sequence chromatograms of the mutated nucleotides in Atp11c (cytosine to thymine at position 2113, resulting in the substitution of a stop codon for glutamine at position 655; and thymine to adenine at position 1214, resulting in the substitution of lysine for isoleucine at position 355), as detected in male emptyhive and spelling mice. (,) Analysis of phenotypic complementation in the progeny of a mating between a male spelling-hemizygous (spl/Y) mouse and a female emptyhive-heterozygous (+/emp) mouse (to assess allelism of spelling and emptyhive mutations), by measurement of the frequency of B cells in blood. Numbers adjacent to outlined areas () indicate percent CD19+CD3ε− cells (top left) or CD19−CD3ε+ cells (bottom right); each symbol () represents an individual mouse. () Predicted ATP11C protein domain structure: TM, transmembrane domain;! A, actuator domain; P, phosphorylation domain; N, nucleotide binding domain; *, stop codon. Data are representative of one experiment (–) or two (,) experiments (error bars (), s.e.m.). * Figure 7: Intact B lymphopoiesis in fetal liver. (,) Percentage () and absolute number () of B cells of fractions A–D (B220+IgM−) and immature B cells (B220+IgM+; fraction E) in the fetal livers of embryonic siblings from a mating between a male hemizygous (Atp11cemp/Y) mouse and a female heterozygous (Atp11c+/emp) mouse, assessed at embryonic day 18.5. Numbers adjacent to outlined areas () indicate percent cells in each; each symbol () represents an individual embryo. Data are representative of two experiments with eight to fourteen embryos per experiment (error bars, s.e.m.). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Mouse Genome Informatics * 3851764 * 4355241 * 3817458 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Genetics, The Scripps Research Institute, La Jolla, California, USA. * Owen M Siggs, * Carrie N Arnold, * Elaine Pirie, * Yu Xia, * Pei Lin & * Bruce Beutler * Department of Immunology, The Scripps Research Institute, La Jolla, California, USA. * Christoph Huber & * David Nemazee Contributions O.M.S. designed and did experiments, analyzed data and wrote the paper under the guidance of B.B.; C.N.A. and E.P. identified the spelling phenotype and assisted with immunization experiments; Y.X. and P.L. assisted with positional cloning and mutation identification; and C.H. measured immunoglobulin isotypes with reagents that D.N. contributed. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bruce Beutler Author Details * Owen M Siggs Search for this author in: * NPG journals * PubMed * Google Scholar * Carrie N Arnold Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Huber Search for this author in: * NPG journals * PubMed * Google Scholar * Elaine Pirie Search for this author in: * NPG journals * PubMed * Google Scholar * Yu Xia Search for this author in: * NPG journals * PubMed * Google Scholar * Pei Lin Search for this author in: * NPG journals * PubMed * Google Scholar * David Nemazee Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce Beutler Contact Bruce Beutler Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–5 Additional data - ATP11C is critical for the internalization of phosphatidylserine and differentiation of B lymphocytes
- Nat Immunol 12(5):441-449 (2011)
Nature Immunology | Article ATP11C is critical for the internalization of phosphatidylserine and differentiation of B lymphocytes * Mehmet Yabas1 * Charis E Teh1 * Sandra Frankenreiter1 * Dennis Lal1 * Carla M Roots2 * Belinda Whittle3 * Daniel T Andrews2 * Yafei Zhang3 * Narci C Teoh4 * Jonathan Sprent5 * Lina E Tze2 * Edyta M Kucharska1 * Jennifer Kofler2 * Geoffrey C Farell4 * Stefan Bröer6 * Christopher C Goodnow2, 7 * Anselm Enders1, 7 * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:441–449Year published:(2011)DOI:doi:10.1038/ni.2011Received08 November 2010Accepted21 February 2011Published online20 March 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses, but few genetic tools are available to test their function. Here we describe a previously unknown X-linked B cell–deficiency syndrome in mice caused by mutations in Atp11c, which encodes a member of the P4 ATPase family thought to serve as 'flippases' that concentrate aminophospholipids in the cytoplasmic leaflet of cell membranes. Defective ATP11C resulted in a lower rate of phosphatidylserine translocation in pro-B cells and much lower pre-B cell and B cell numbers despite expression of pre-rearranged immunoglobulin transgenes or enforced expression of the prosurvival protein Bcl-2 to prevent apoptosis and abolished pre-B cell population expansion in response to a transgene encoding interleukin 7. The only other abnormalities we noted were anemia, hyperbilirubinemia and hepatocellular carcinoma. Our results identify an intimate connection between phospholipid transport and B lymp! hocyte function. View full text Figures at a glance * Figure 1: Identification and initial characterization of strains with fewer B lymphocytes after N-ethyl-N-nitrosourea–induced mutagenesis. () Flow cytometry of B220+ B cells in the blood of male Atp11c+/0 wild-type (WT), Atp11camb/0 ambrosius (Amb) and 18NIH30a mice. Numbers adjacent to outlined areas (left) indicate percent B220+CD3− cells (top left) or B220−CD3+ cells (bottom right). Far right, B220+ cells as a percentage of total lymphocytes. () Primary antibody response 14 d after immunization with inactivated B. pertussis or alum-precipitated CGG (far left) presented as absorbance at 650 nm (A650); and antibody to CGG (middle left), and the hapten ABA (middle right) or nitrophenyl-Ficoll (NP-Ficoll; far right) 6 d after booster immunization (arbitrary units). () Lymphocytes, neutrophils, reticulocytes and erythrocytes in the blood of Atp11camb/0 mice and their Atp11c+/0 littermates. () Unconjugated (Uconj) and conjugated (Conj) bilirubin in the plasma of Atp11c+/0 and Atp11camb/0 mice. () Typical dysplastic focus on a hematoxylin and eosin–stained liver section from an Atp11camb/0 mouse at 6 months. ! Scale bar, 200 μm. Each symbol (–) represents a single mouse. Data are from more than five experiments () or are representative of two experiments (–) or one experiment () with four to ten mice per group in each. * Figure 2: Identification of a splice-site mutation in Atp11c by next-generation DNA sequencing. () Read depth from a single sequencer lane across the tiled region between 54 and 59 megabases on the X chromosome; box outlines Atp11c. Below: blue lines, all annotated exons on the sense and antisense strands; red lines, exons with a read depth of less than 5; black lines, capture baits used for enrichment of exons from genomic DNA. () Enlargement of Atp11c outlined in , with read depth and chromosomal coordinates (National Center for Biotechnology Information build 37.1). () Atp11c exon 27 splice donor at single-nucleotide resolution, read depth across these nucleotides and sequence of first 30 reads. Chr, chromosome; bp, base pairs. Data are representative of two independent experiments. * Figure 3: The Atp11camb mutation results in smaller B cell subsets except marginal zone B cells. () Flow cytometry of bone marrow cells from male Atp11c+/0 and Atp11camb/0 mice. Numbers adjacent to outlined areas indicate percent B220+ B cells (left column); IgD+IgM+ mature B cells (top right), IgD−IgM+ immature B cells (bottom right) and IgD−IgM− pro- and pre-B cells (bottom left) in the B220+ subset (middle column); and CD24hiCD43− pre-B cells (top left), CD24intCD43+ pro-B cells (top right) and CD24−CD43+ pre-pro-B cells (bottom right) among cells gated on B220+ IgM− IgD− cells (right column). FSC, forward scatter. (,) Leukocytes () and B cells and B cell subsets () in bone marrow from Atp11c+/0 and Atp11camb/0 mice. Imm, immature; Mat, mature. () Flow cytometry of B cell subpopulations in spleens from male Atp11c+/0 and Atp11camb/0 mice. Numbers adjacent to outlined areas indicate percent B220+ B cells (top left) and CD3+ T cells (bottom right; left column), or CD21hiCD23− marginal zone B cells (left) and CD21intCD23+ follicular B cells (right) in th! e B220+CD93− mature B cell subset (right column); and numbers above bracketed lines indicate percent CD93− mature B cells (left) and CD93+ immature B cells (right) among B220-gated cells (middle column). (,) Leukocytes () and lymphocyte subsets () in the spleen. Each symbol (,,,) represents a single mouse (bars and symbols in , as in ,). Data are representative of at least five independent experiments with two to five mice per group in each (mean and individual values in ,,,). * Figure 4: Effects of transgenes encoding Bcl-2, IL-7 or the BCR on the development of ATP11C-mutant B cells. (–) Flow cytometry showing the frequency of B cell subpopulations in the bone marrow of mice with wild-type or mutant ATP11C and no transgene (), the Vav-Bcl2 transgene (), the H2Ea-Il7 transgene () or the MD4 transgene (). Numbers adjacent to outlined areas indicate percent cells in each. () Pro-B cells, pre-B cells and immature B cells in the bone marrow and B cells in the spleen of mice as in –, presented relative to those in Atp11c+/0 wild-type control mice with the same transgene or of the same line. Each symbol represents an individual mouse. Data are representative of two to five experiments (–) or are pooled from two to three experiments () with one to five mice of each genotype in each (mean and individual values in ). * Figure 5: Less transition of pro-B cells from immunoglobulin-negative to immunoglobulin-positive cells and absence of a response to the H2Ea-Il7 transgene despite higher expression of IL-7Rα. () Flow cytometry analysis of the surface expression of CD24 and intracellular expression of IgM (cμ), gated on mIgM−B220lo7 B cells in bone marrow from nontransgenic (Non-TG) or H2Ea-Il7-transgenic mice with wild-type or mutant ATP11C, or from Cd79a−/− or Rag1−/− mice. Numbers in quadrants indicate percent gated cells in each. () IgM+CD24+ cells gated as in (top) and CD24 staining on those cells (bottom), presented as geometric mean fluorescence intensity (MFI). () Flow cytometry of the cell cycle of pre-pro-B cells, pro-B cells and pre-B cells. Numbers adjacent to bracketed lines indicate percent cycling cells in G0-G1 (left), S (middle) or G2-M (right). () Frequency of pre-pro-B cells, pro-B cells and pre-B cells in S or G2-M phase. () Absolute number of cells after 7 d of culture of sorted pro-B cells in the presence of IL-7 (25 ng/ml). () Flow cytometry analysis of mIgM on live B220+ cells after culture as in . Numbers above outlined areas indicate percent mI! gM+ cells among all live B220+ cells (). Flow cytometry of bone marrow from MD4 mice, assessing surface expression of IgM and CD24 (top) or CD19 (bottom) by B220loIgD− cells. Numbers in quadrants indicate percent cells in each. () Frequency of CD24+mIgM+ cells (left) or CD19+mIgM+ cells (right) among the cells in . () Expression of CD127 (IL-7Rα) on CD24intCD43+CD19+IgM−B220lo pro-B cells (top left) from Atp11camb/0 mice (black line) and wild-type mice (shaded histogram) and CD127 fluorescence on pro-B cells (top right). P value, Student's t-test. Below, flow cytometry of CD24 versus CD127 on CD19+IgM−B220lo7-AAD− cells in bone marrow from nontransgenic or H2Ea-Il7-transgenic mice with wild-type or mutant ATP11C, or from Cd79a−/− and Rag1−/− mice. Numbers adjacent to outlined areas indicate percent cells of the CD24int pro-B cell subset. Each symbol (,–,,) represents an individual mouse. Data are representative of two to five () or two () experiments, thr! ee separate experiments with one to four mice per group in eac! h (,,,) or two separate experiments with two to five mice per group in each (), or are combined from three separate experiments with one to three mice per group in each (,). * Figure 6: The Atp11camb mutation results in less translocation of PS into pro-B cells. () NBD-PS fluorescence profiles of pro-B cells from mice with wild-type or mutant ATP11C lacking (Non-TG) or carrying the Vav-Bcl2 transgene (black lines) and of the corresponding CD45.1+ wild-type pro-B cells (shaded histograms), after 1 or 12 min of incubation. () NBD-PS in pro-B cells of mice (genotypes as in ) after 0–12 min of incubation, presented as geometric mean fluorescence intensity. () NBD-PS in bone marrow pro-B cells or monocytes from Atp11camb/0 mice, presented as fluorescence intensity relative to that of CD45.1+ wild-type pro-B cells or monocytes. Each symbol represents an individual mouse; small horizontal lines indicate the mean. () NBD-PS fluorescence profiles in pre-pro-B cells, pro-B cells, B220loIgM+ cells, B220hiIgM+ cells, NK1.1+ cells, TCRβ+ cells, Mac-1+Gr-1+ cells and Ter119+ cells from ATP11C-mutant bone marrow (black lines), assessed after 1 min incubation and presented relative to that of the corresponding CD45.1+ wild-type bone marrow cells! (shaded histograms). () Apoptotic pro-B cells or pre-B cells from mice with wild-type or mutant ATP11C with or without the Vav-Bcl2 transgene, assessed as positive staining for annexin V (AnnV) and 7-AAD. Each symbol represents an individual mouse. Data are representative of two to three experiments (–) or are from two experiments (nontransgenic) or one experiment (Vav-Bcl2) with two to five mice per group in each (). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 1wpg * 1wpg Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Christopher C Goodnow & * Anselm Enders Affiliations * Ramaciotti Immunization Genomics Laboratory, Department of Immunology, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia. * Mehmet Yabas, * Charis E Teh, * Sandra Frankenreiter, * Dennis Lal, * Edyta M Kucharska & * Anselm Enders * Department of Immunology, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia. * Carla M Roots, * Daniel T Andrews, * Lina E Tze, * Jennifer Kofler & * Christopher C Goodnow * Australian Phenomics Facility, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia. * Belinda Whittle & * Yafei Zhang * Gastroenterology and Hepatology Unit, Australian National University Medical School, The Canberra Hospital, Canberra, Australia. * Narci C Teoh & * Geoffrey C Farell * Immunology Program, Garvan Institute of Medical Research, Darlinghurst, Australia, and World Class University–Integrative Biosciences and Biotechnology Program, Pohang University of Science and Technology, Pohang, Korea. * Jonathan Sprent * Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, Australia. * Stefan Bröer Contributions M.Y. did and analyzed most of the experiments; C.E.T., S.F., D.L. E.M.K., J.K. and A.E. contributed to experiments; C.M.R. and A.E. identified the ATP11C-mutant strains; B.W., D.T.A., Y.Z. and A.E. mapped and identified the mutation in the ambrosius strain; N.C.T. and G.C.F. analyzed liver histology and clinical chemistry; S.B. helped with the flippase assay; C.C.G. and A.E. conceived of the research and directed the study; and M.Y., C.C.G. and A.E. prepared the figures and wrote the paper in consultation with all authors. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Christopher C Goodnow or * Anselm Enders Author Details * Mehmet Yabas Search for this author in: * NPG journals * PubMed * Google Scholar * Charis E Teh Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra Frankenreiter Search for this author in: * NPG journals * PubMed * Google Scholar * Dennis Lal Search for this author in: * NPG journals * PubMed * Google Scholar * Carla M Roots Search for this author in: * NPG journals * PubMed * Google Scholar * Belinda Whittle Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel T Andrews Search for this author in: * NPG journals * PubMed * Google Scholar * Yafei Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Narci C Teoh Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan Sprent Search for this author in: * NPG journals * PubMed * Google Scholar * Lina E Tze Search for this author in: * NPG journals * PubMed * Google Scholar * Edyta M Kucharska Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Kofler Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey C Farell Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Bröer Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher C Goodnow Contact Christopher C Goodnow Search for this author in: * NPG journals * PubMed * Google Scholar * Anselm Enders Contact Anselm Enders Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–10 Additional data - The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells
- Nat Immunol 12(5):450-459 (2011)
Nature Immunology | Article The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells * Yasutaka Motomura1, 2 * Hiroshi Kitamura3 * Atsushi Hijikata3 * Yuko Matsunaga4 * Koichiro Matsumoto4 * Hiromasa Inoue4 * Koji Atarashi5 * Shohei Hori6 * Hiroshi Watarai7 * Jinfang Zhu8 * Masaru Taniguchi7 * Masato Kubo1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:450–459Year published:(2011)DOI:doi:10.1038/ni.2020Received04 October 2010Accepted10 March 2011Published online03 April 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The immunoregulatory cytokine interleukin 10 (IL-10) is expressed mainly by T helper type 2 (TH2) cells but also by TH1 cells during chronic infection. Here we observed plasticity in the expression of IL-10 and IL-13 after chronic TH1 stimulation; furthermore, the expression of Il10 and Il13 was regulated by the transcription factor E4BP4. Chronically stimulated E4BP4-deficient (Nfil3−/−; called 'E4bp4−/−' here) TH1 cells, regulatory T cells (Treg cells) and natural killer T cells (NKT cells) had attenuated expression of IL-10 and IL-13. Enforced expression of E4bp4 initiated the production of IL-10 and IL-13 by conventional TH1 cells. E4bp4−/− TH2 cells showed impairment of IL-10 production with no effect on IL-13. Our results indicate that E4BP4 has multiple functions in controlling the plasticity of IL-13 in TH1 cells and IL-10 in TH1 cells, TH2 cells, Treg cells and NKT cells. View full text Figures at a glance * Figure 1: IL-13 production induced by repetitive antigen stimulation in TH1 cells. () Enzyme-linked immunosorbent assay (ELISA) of IFN-γ, IL-4, IL-5 and IL-13 in cells generated from naive CD4+ T cells (from DO11.10 mice on a recombination-activating gene 2–deficient background) repeatedly stimulated with OVA peptide in the presence of BALB/c APCs every week under TH1-skewing conditions, then restimulated with mAb to TCRβ at 1–4 weeks after initial stimulation. () ELISA of cytokines in cells generated from naive CD4+ T cells obtained from Il4r+/+ (DO11.10) or Il4r−/− (DO11.10 IL-4R-KO) DO11.10 mice (on the BALB/c background) and stimulated with OVA peptide in the context of APCs (far left and middle left), or generated from naive CD4+ T cells obtained from wild-type (WT) or Stat6−/− (STAT6-KO) mice and stimulated by mAb to TCRβ and mAb to CD28 under TH1-skewing conditions (far right and middle right). () ICS detection of cells producing IFN-γ, IL-13 and IL-4 among cells generated from CD4+ T cells (from DO11.10 mice on a recombination-activ! ating gene 2–deficient or Il4r−/− background) stimulated with OVA peptide in the context of APCs every week under TH1-skewing conditions, followed by restimulation with mAb to TCRβ. Numbers in or below quadrants indicate percent cells in each throughout. Data are from three independent experiments (,; mean ± s.e.m.) or are representative of three experiments with similar results (). * Figure 2: E4BP4 regulates IL-13 expression in TH1 cells. () Quantitative RT-PCR analysis of the expression of 16 candidate genes identified by comparative transcriptome analysis in TH1 cells given one round (R1) or four rounds (R4) of simulation and then restimulated with mAb to TCRβ, and also in 23-1-8 cells, a TH1 cell clone corresponding to R4 TH1 cells (primer sequences, Supplementary Table 1). () ELISA of IL-4 and IL-13 in supernatants of cultures of BALB/c CD4+ T cells given initial stimulation with mAb to TCRβ and mAb to CD28, then transduced for 7 d with retroviral plasmid encoding green fluorescent protein (GFP) alone (Vector) or plasmid encoding GFP plus E4BP4, c-Maf, JunB or NR2C1, followed by restimulation of GFP+ cells for 24 h with mAb to TCRβ. () ICS detection (left) of IL-13-producing cells among BALB/c CD4+ T cells given initial stimulation with mAb to TCRβ and mAb to CD28, then transduced for 7 d with retroviral plasmid alone or plasmid encoding E4BP4, followed by restimulation of GFP+ cells for 6 h with mAb ! to TCRβ; right, frequency of IL-13-producing cells among all CD4+ T cells. () Quantitative RT-PCR analysis of the expression of E4bp4, Tbx21 and Gata3 in R1–R4 TH1 cells and in TH2 cells (primers, Supplementary Table 1). () ICS detection (left) of IL-13- and IFN-γ-producing cells among R4 TH1 cells from DO11.10 mice; immunocytochemistry analysis (middle) of E4BP4 and IL-13 in DO11.10 T cells; and frequency of IL-13+ cells among E4BP4+ or E4BP4− cells (right). Original magnification (middle), ×640. DAPI, DNA-intercalating dye. *P < 0.01 (Student's t-test). Data are representative of two experiments with 100 cells expressing both IFN-γ and IL-13 () or are from three independent experiments (–; mean and s.e.m.). * Figure 3: E4bp4 overexpression induces the expression of IL-10 and IL-13 by CD4+ T cells. () Intracellular staining to detect transgene expression by CD4+ T cells purified from the spleens of a wild-type mouse or C57BL/6 mice expressing a transgene (TG) encoding Flag-tagged E4BP4 (three independent lines, TG (1)–TG (3)), assessed with anti-Flag. () Concentration of cytokines and chemokines in wild-type and E4bp4-transgenic CD4+ T cells stimulated for 7 d with mAb to TCRβ and mAb to CD28, then restimulated with mAb to TCRβ. *P < 0.05 (Student's t-test). () Intracellular staining to detect T cells producing IL-4, IL-10 and IL-13 among Flag-positive cells (TG+) or Flag-negative cells (TG−) generated from E4bp4-transgenic CD4+ T cells stimulated for 7 d as in , then restimulated with mAb to TCRβ. *P < 0.05 (Student's t-test). () Intracellular staining to detect T cells producing IL-10 and IFN-γ (left) among GFP+ T cells generated from C57BL/6 CD4+ T cells transduced with plasmid encoding GFP alone or GFP plus E4BP4 and activated under TH1 conditions; right, f! requency of IL-10+ cells. () Production of IL-10 and IL-13 in the GFP+ T cells isolated in . *P < 0.01 (Student's t-test). Data are representative of two experiments () or are from three independent experiments (–; mean and s.e.m.). * Figure 4: E4BP4 regulates the expression of Il10 and Il13 in TH1 cells. () ICS analysis (top) and ELISA (bottom) of IL-13, IL-10 and IFN-γ in cells generated from C57BL/6 CD4+ T cells stimulated four times with mAb to TCRβ and mAb to CD28 under TH1 conditions. Data are representative of three independent experiments (mean ± s.e.m.). () Immunoblot analysis (top left) of E4BP4 and STAT6 in CD4+ T cells stimulated for 48 h under TH2 conditions and transduced with retrovirus encoding control shRNA (Ctrl) or E4bp4-specific shRNA (E4BP4), assessed 1 week later by probing with anti-E4BP4, followed by densitometry analysis (above; normalized to STAT6 values and presented relative to E4BP4 in cells transduced with the control shRNA, set as 100); and ELISA of IFN-γ in R4 TH1 cells (top right) or of IL-10 and IL-13 in TH1 cells stimulated 1–4 weeks (R1–R4; bottom). *P < 0.01 (Student's t-test). Data are from three independent experiments (mean ± s.e.m.). () ICS analysis (top) and ELISA (bottom) of IL-13, IL-10 and IFN-γ in CD4+ T cells derived fr! om E4bp4−/− mice (KO) and their wild-type littermates (WT), then stimulated for 4 weeks under TH1-skewing conditions. *P < 0.01 (Student's t-test). Data are representative of three independent experiments (ICS) or are from three independent experiments (mean ± s.e.m.). * Figure 5: Impaired production of IL-10 and IL-13 in E4bp4-deficient T cells. (–) Intracellular staining analysis (,) and ELISA () of IL-4, IL-10 and IL-13 in CD4+ T cells obtained from E4bp4−/− mice (KO) and their E4bp4+/+ littermates (WT) and stimulated under TH2 conditions. *P < 0.01 (Student's t-test). Data are representative of () or from (,) three independent experiments (mean and s.e.m. in ,). () Intracellular staining analysis of IL-4, IL-10 and IL-13 in cells generated from naive CD4+ T cells obtained from Cd4-Cre mice or E4bp4f/fCd4-Cre mice (E4BP4-CKO), then stimulated with mAb to TCRβ and mAb to CD28 under TH2 conditions or TH1 conditions. Data are representative of three independent experiments. () Intracellular staining analysis of IL-10 in cells generated from activated CD4+ T cells derived from E4bp4−/− mice (E4BP4-KO) and their E4bp4+/+ littermates (WT) and transduced with empty plasmid or plasmid encoding GATA-3 (top), or derived from Gata3f/f (GATA-3-KO) mice or C57BL/6 mice (WT) and transduced with plasmid encoding GFP a! nd E4BP4 (E4BP4) or plasmid encoding human CD8 (Vector; bottom), followed by restimulation with mAb to TCRβ. GATA-3-deficient T cells (bottom) were isolated by gating on cells positive for human CD8. Data are representative of three independent experiments. () ICS analysis (top) of IL-10 and IL-13 in CD4+ T cells prepared from Cd4-CreGata3f/f mice (GATA-3-CKO) and Cd4-Cre mice, then stimulated for 4 weeks under TH1-skewing conditions; and frequency of IL-10+ and IL-13+ cells (bottom). *P < 0.01 (Student's t-test). Data are representative of three independent experiments (ICS) or are from three independent experiments (mean and s.e.m.). * Figure 6: Impaired production of IL-10 and IL-13 by CD4+ T cell subsets lacking E4BP4. () ICS detection of IL-13- and IL-10-producing cells among whole BALB/c splenocytes stimulated for 48 h with the α-GalCer–CD1d dimer (CD1d dimer). () Quantitative RT-PCR analysis of gene expression among total RNA from IL-17RB+ NKT cells generated from IL-17RB+α-GalCer–CD1d+TCRβ+ cells sorted from spleen cells and cultured for 24 h in the presence (IL-25+) or absence (IL-25−) of IL-25 together with bone marrow–derived DCs induced by granulocyte-macrophage colony-stimulating factor. () Flow cytometry analysis of NKT cells from the spleens of E4bp4−/− mice and their E4bp4+/+ littermates. Numbers adjacent to outlined areas indicate percent α-GalCer–CD1d+TCRβ+ cells. () ELISA of cytokine production in IL-17RB+ NKT cells derived from E4bp4−/− mice and their E4bp4+/+ littermates (C57BL/6 background) and stimulated for 24 h with various concentrations of IL-25 (top) or α-GalCer (bottom) in the presence of bone marrow–derived DCs induced by granulocyte-macr! ophage colony-stimulating factor. (,) ELISA of cytokines in culture supernatants of Foxp3+CD4+ (CD25− or CD25hi) T cells () and naive (Foxp3−CD62LhiCD44lo) and memory (Foxp3−CD62LloCD44hi) CD4+ T cells () purified from reporter mice expressing Foxp3 from human CD2 (hCD2), on an E4bp4+/+ or E4bp4−/− background, then stimulated for 48 h with mAb to TCRβ and mAb to CD28. *P < 0.01 (Student's t-test). Data are representative of two experiments () or three experiments (–; mean and s.e.m.). * Figure 7: E4BP4 protein binds specifically to the Il13 promoter and in the Il10 locus. () Methylation of genomic DNA (bottom) isolated from naive CD4+ T cells and from TH1 cells and TH2 cells given 1 week of stimulation (R1), analyzed by sequencing with primers specific for the Il13 promoter (top) after bisulfite treatment. () ChIP analysis of the enrichment of histone H3 trimethylated at Lys4 (H3K4me3) in R1 TH1 cells, R1 TH2 cells or R3 TH1 cells (top) and of E4BP4 in R1 TH1 and R4 TH1 cells (bottom), both at the Il13 locus (middle). CNS1, conserved noncoding sequence 1; HSS, hypersensitive site; Rad50, gene encoding a protein involved in the repair of DNA double-strand breaks. () Electrophoretic mobility-shift assay of nuclear extracts (NE) of TH1 and TH2 cells, with an oligonucleotide from a specific region of the Il13 promoter as the probe (far left); supershift analysis of nuclear extracts from TH2 cells with anti-E4BP4 (Ab; middle left); and competition analysis with 25-, 50- or 100-fold excess competitor (middle right) or mutant oligonucleotides (M1–! M5) and the Il13 promoter (pIL-13; far right). () ChIP analysis of the enrichment of histone H3 acetylated at Lys9 and Lys14 (H3K9ac-H3K14ac; top) or of E4BP4 (bottom) at the Il10 locus (above) in chromatin fractions extracted from TH1 or TH2 cells; results are presented relative to those obtained with input DNA prepared from untreated chromatin. () ChIP analysis of the enrichment of histone H3 acetylated at Lys9 and Lys14 at the Il10 locus (above) in TH2 cells from E4bp4−/− and wild-type mice; results presented as in . *P < 0.01 (Student's t-test). Data are representative of two experiments (–) or are from three independent experiment (,; mean and s.e.m.). * Figure 8: Exacerbated colitis and EAE in E4bp4−/− mice. () Flow cytometry analysis of the expression of human CD2 (as a marker of Foxp3) and CD4 (left), and ELISA of IL-10 (right) in Foxp3+ Treg cells induced from naive T cells from Foxp3hCD2 reporter mice on the E4bp4−/− or wild-type background by culture with transforming growth factor-β. () Body weight of mice deficient in recombination-activating gene 1 (n = 10 per group) given intravenous transfer of naive CD4+ T cells (4 × 105) from the spleens of E4bp4−/− mice (n = 10) or wild-type mice (n = 10), presented relative to initial body weight on day 0. † indicates death (two of ten mice died). () Hematoxylin and eosin staining of colon tissue sections at day 34 in the recipient mice in . Scale bars, 200 μm (left) or 50 μm (right). () ELISA of IL-10 in CD4+ T cells isolated at day 34 from the lymph nodes (LN) and colons of the recipient mice in , then stimulated for 48 h with mAb to TCRβ and mAb to CD28. () EAE clinical scores of E4bp4−/− mice (n = 5) and wild! -type mice (n = 5) immunized at day 0 with a peptide of myelin oligodendrocyte glycoprotein (amino acids 35–55) in complete Freund's adjuvant. () ELISA of cytokines in CD4+ T cells isolated at day 35 from the lymph nodes of mice immunized as in , then stimulated for 48 h with mAb to TCRβ and mAb to CD28. *P < 0.01 (Student's t-test). Data are from three (,,,) or ten () independent experiments (mean and s.e.m. in ,,–). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory for Signal Network, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan. * Yasutaka Motomura & * Masato Kubo * Division of Molecular Pathology, Research Institute for Biological Science, Tokyo University of Science, Chiba, Japan. * Yasutaka Motomura & * Masato Kubo * Laboratory for Immunogenomics, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan. * Hiroshi Kitamura & * Atsushi Hijikata * Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. * Yuko Matsunaga, * Koichiro Matsumoto & * Hiromasa Inoue * Department of Immunology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. * Koji Atarashi * Laboratory for Immune Homeostasis, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan. * Shohei Hori * Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan. * Hiroshi Watarai & * Masaru Taniguchi * National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. * Jinfang Zhu Contributions Y. Motomura built the initial constructs, generated mouse lines and confirmed them in vivo, and did most of the experiments; H.K. and A.H. did microarray and bioinformatics analysis; Y. Matsunaga, K.M. and H.I. did airway hyper-responsiveness experiments; K.A. provided Il10 reporter mice; S.H. analyzed Treg cells; H.W. and M.T. analyzed NKT cells; J.Z. provided materials; and M.K. designed and coordinated experiments, supervised the project and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Masato Kubo Author Details * Yasutaka Motomura Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroshi Kitamura Search for this author in: * NPG journals * PubMed * Google Scholar * Atsushi Hijikata Search for this author in: * NPG journals * PubMed * Google Scholar * Yuko Matsunaga Search for this author in: * NPG journals * PubMed * Google Scholar * Koichiro Matsumoto Search for this author in: * NPG journals * PubMed * Google Scholar * Hiromasa Inoue Search for this author in: * NPG journals * PubMed * Google Scholar * Koji Atarashi Search for this author in: * NPG journals * PubMed * Google Scholar * Shohei Hori Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroshi Watarai Search for this author in: * NPG journals * PubMed * Google Scholar * Jinfang Zhu Search for this author in: * NPG journals * PubMed * Google Scholar * Masaru Taniguchi Search for this author in: * NPG journals * PubMed * Google Scholar * Masato Kubo Contact Masato Kubo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–10 and Supplementary Table 1 Additional data
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