Latest Articles Include:
- Natural killer cells: fighting viruses and much more
- Nat Immunol 12(2):107-110 (2011)
Nature Immunology | Meeting Report Natural killer cells: fighting viruses and much more * Marco Colonna1 Contact Marco Colonna Search for this author in: * NPG journals * PubMed * Google Scholar * Stipan Jonjic2 Search for this author in: * NPG journals * PubMed * Google Scholar * Carsten Watzl3 Contact Carsten Watzl Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume: 12,Pages:107–110Year published:(2011)DOI:doi:10.1038/ni0211-107Published online19 January 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 More than 450 immunologists recently met in Cavtat, Croatia to discuss advances in natural killer (NK) cell biology. The meeting highlighted emerging themes in NK cell responses to viruses, NK cell tolerance and potential use of NK cells in the therapy of malignancies. View full text Author information * Abstract * Author information Affiliations * Marco Colonna is in the Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA * Stipan Jonjic is in the Department for Histology and Embryology and Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia * Carsten Watzl is in the Institute for Immunology, Heidelberg University, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Marco Colonna or * Carsten Watzl Additional data - CCL8 and skin T cells—an allergic attraction
- Nat Immunol 12(2):111-112 (2011)
Nature Immunology | News and Views CCL8 and skin T cells—an allergic attraction * Gudrun F Debes1 Contact Gudrun F Debes Search for this author in: * NPG journals * PubMed * Google Scholar * Malissa C Diehl1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:111–112Year published:(2011)DOI:doi:10.1038/ni0211-111Published online19 January 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 migration of TH2 cells into allergy-affected tissue is key to maintaining the inflammatory response. CCR8-CCL8, a newly identified chemokine receptor–ligand pair, mediates the skin accumulation of TH2 cells with the specific potential to drive chronic eosinophilic inflammation. 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 * Gudrun F. Debes and Malissa C. Diehl are in the Department of Pathobiology at the University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gudrun F Debes 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 - Ldb1, a new guardian of hematopoietic stem cell maintenance
- Nat Immunol 12(2):113-114 (2011)
Nature Immunology | News and Views Ldb1, a new guardian of hematopoietic stem cell maintenance * Eva Welinder1 Search for this author in: * NPG journals * PubMed * Google Scholar * Cornelis Murre1 Contact Cornelis Murre Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:113–114Year published:(2011)DOI:doi:10.1038/ni0211-113Published online19 January 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. Maintenance of hematopoietic stem cells depends on a fine-tuned transcriptional network. A detailed study of the nuclear adaptor Ldb1 provides additional clues as to how hematopoietic stem cell homeostasis is controlled. 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 * Eva Welinder and Cornelis Murre are in the Department of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Cornelis Murre 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 - 2 methylate or not 2 methylate: viral evasion of the type I interferon response
- Nat Immunol 12(2):114-115 (2011)
Nature Immunology | News and Views 2 methylate or not 2 methylate: viral evasion of the type I interferon response * Adolfo García-Sastre1 Contact Adolfo García-Sastre Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume: 12,Pages:114–115Year published:(2011)DOI:doi:10.1038/ni0211-114Published online19 January 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. Recent work highlights the requirement for 2′-O-methylation of capped mRNA to prevent recognition by the antiviral host response. 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 * Adolfo García-Sastre is at the Department of Microbiology, the Department of Medicine, Division of Infectious Diseases, and the Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, New York, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Adolfo García-Sastre 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 - New friends for bone marrow plasma cells
- Nat Immunol 12(2):115-117 (2011)
Nature Immunology | News and Views New friends for bone marrow plasma cells * Robert Brink1 Contact Robert Brink Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume: 12,Pages:115–117Year published:(2011)DOI:doi:10.1038/ni0211-115Published online19 January 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. Long-lived plasma cells require a specialized bone marrow microenvironment in order to survive and produce antibody. Eosinophils make an important contribution to maintaining this survival niche. 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 * Robert Brink is in the Immunology Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Robert Brink 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(2):119 (2011)
Nature Immunology | Research Highlights Research Highlights * Laurie A Dempsey Search for this author in: * NPG journals * PubMed * Google Scholar * Zoltan Fehervari Search for this author in: * NPG journals * PubMed * Google Scholar * Ioana Visan Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume: 12,Page:119Year published:(2011)DOI:doi:10.1038/ni0211-119Published online19 January 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. Apoptosis by incomplete infection Infection with human immunodeficiency virus (HIV) is characterized by progressive apoptosis of CD4+ T cells, and although some of the molecular participants in this process are known, the precise details remain unclear. Greene and colleagues in Cell report the unexpected finding that nonproductive infection of CD4+ T cells can also result in apoptosis. The authors use a human lymphoid aggregation culture system to recapitulate in vitro the events of HIV infection in the lymph node. The addition of HIV to this culture system results in the apoptosis of not only productively infected but also nonpermissive CD4+ cells. Apoptosis of the latter requires fusion with HIV and the initiation but not completion of viral reverse transcription. The accumulation of incomplete HIV reverse transcripts results in activation of caspase-1 and caspase-3 and release of the inflammatory cytokine IL-1β, which ultimately results in apoptosis. This may represent a defense pathway initiated after v! iral infection, but could also underlie the immune exhaustion seen during HIV infection. ZF Cell143, 789–801 (2010) 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 - NLR functions beyond pathogen recognition
- Nat Immunol 12(2):121-128 (2011)
Nature Immunology | Perspective NLR functions beyond pathogen recognition * Thomas A Kufer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Philippe J Sansonetti2, 3 Contact Philippe J Sansonetti Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:121–128Year published:(2011)DOI:doi:10.1038/ni.1985Published online19 January 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 last 10 years have witnessed the identification of a new class of intracellular pattern-recognition molecules—the nucleotide-binding domain and leucine-rich repeat–containing family (NLR). Members of this family garnered interest as pattern-recognition receptors able to trigger inflammatory responses against pathogens. Many studies support a pathogen-recognition function for human NLR proteins and shed light on their role in the broader control of adaptive immunity and various disease states. Other evidence suggests that NLRs function in processes unrelated to pathogen detection. Here we discuss recent advances in our understanding of the biology of the human NLR proteins and their non-pathogen-recognition function in tissue homeostasis, apoptosis, graft-versus-host disease and early development. View full text Figures at a glance * Figure 1: The human NLR family. Organization of the human NLR family according to structural similarities of their proteins. Domain organization of the members of the human NLR family and the NLR-related molecule Apaf-1 is shown. Note that all NLRs differ in the length and composition of their LRR domains (not depicted). * Figure 2: NLR functions in the human body and at the cellular level. () Important cellular functions and subcellular localization of human NLRs are shown in a schematic representation of a cell. () Selected functions of NLRs in the human body are depicted (see also Table 1). Author information * Abstract * Author information Affiliations * Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Köln, Germany. * Thomas A Kufer * Unité de Pathogénie Microbienne Moléculaire et Unité INSERM 786, Institut Pasteur, Paris, France. * Philippe J Sansonetti * Chaire de Microbiologie et Maladies Infectieuses, Collège de France, Paris, France. * Philippe J Sansonetti Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Philippe J Sansonetti Additional data - Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells
- Nat Immunol 12(2):129-136 (2011)
Nature Immunology | Article Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells * LiQi Li1 Search for this author in: * NPG journals * PubMed * Google Scholar * Raja Jothi2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kairong Cui3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Y Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tsadok Cohen4 Search for this author in: * NPG journals * PubMed * Google Scholar * Marat Gorivodsky4 Search for this author in: * NPG journals * PubMed * Google Scholar * Itai Tzchori4 Search for this author in: * NPG journals * PubMed * Google Scholar * Yangu Zhao4 Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra M Hayes5 Search for this author in: * NPG journals * PubMed * Google Scholar * Emery H Bresnick6 Search for this author in: * NPG journals * PubMed * Google Scholar * Keji Zhao3 Search for this author in: * NPG journals * PubMed * Google Scholar * Heiner Westphal4 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul E Love1 Contact Paul E Love Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:129–136Year published:(2011)DOI:doi:10.1038/ni.1978Received05 October 2010Accepted01 December 2010Published online26 December 2010 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 nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non–DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex–binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs. View full text Figures at a glance * Figure 1: Ldb1 is required for hematopoietic specification but is not essential for ESC maintenance. () RT-PCR analysis (left) of the expression of various genes (left margin) in Ldb1+/+ ESCs and Ldb1−/− ESCs (two independently generated clones); flow cytometry of cells in embryoid bodies at day 5 derived from Ldb1+/− or Ldb1−/− ESCs (middle); and frequency of Ldb1+/− or Ldb1−/− ESC-derived embryoid bodies (EB) at day 9 with hematopoietic satellite cells (right; identified by Giemsa staining of cytospin preparations). Numbers above outlined areas (middle) indicate percent Flk1+ cells. SSC, side scatter. *P < 0.01 (Student's t-test). Data represent one of two experiments (error bars, s.d.). () Frequency (above) and absolute number (below) of CD45.2+, LSK, granulocyte (Gr-1+) and B cells in the bone marrow and mature T cells in the lymph nodes and mature B cells (B220+) in the spleen of 8-week-old adult Ldb1+/− ESC and Ldb1−/− ESC chimeric mice. Numbers adjacent to outlined areas (above) indicate percent cells in gate; numbers above bars (below) indicate! number of cells for bars not visible. Ig, immunoglobulin. *P < 0.01 (Student's t-test). Data are representative of three experiments with six Ldb1+/− mice and three Ldb1−/− mice. * Figure 2: Ldb1−/− hematopoietic progenitors are present in chimeric fetal livers but are unable to reconstitute hematopoiesis in irradiated recipients. () LSK cells in chimeric embryos at E13.5, generated by injection of Ldb1+/+ or Ldb1−/− ESCs into B6 (Ldb1+/+) blastocysts (left); ESC-derived (Ly9.1+) LSK cells were detected by staining for Ly9.1. Right, staining of gated Ly9.1+ (ESC-derived) thymocytes from chimeras at E18.5 with anti-CD4 plus anti-CD8 or anti-CD3. Numbers adjacent to outlined areas (left) and numbers in top right quadrants (right) indicate percent cells in gate. Data are representative of five experiments. () Flow cytometry of donor fetal liver (FL) cells at E15.5 and recipient bone marrow (BM) cells: Ldb1+/− or Ldb1−/− ESCs or Ldb1−/− ESCs reconstituted with a bacterial artificial chromosome containing Ldb1 (Ldb1−/− BAC) (Ly9.1+) were injected into Rag2−/− (Ly9.1−) blastocysts to generate chimeric embryos, followed by collection of fetal liver cells at E15.5 and injection into irradiated Rag2−/− (Ly9.1−) mice; 16 weeks later, bone marrow from recipient mice was analyzed for! the presence of ESC-derived (Ly9.1+) LSK cells. Numbers adjacent to outlined areas (left) indicate percent Ly9.1+ LSK cells. Right, summary of data at left. Data are representative of four independent experiments (error bars, s.d.). () Flow cytometry of fetal liver cells from chimeric embryos at E13.5–E16.5, generated by injection of Ldb1+/+ or Ldb1−/− (Ly9.1+) ESCs into B6 (Ldb1+/+, Ly9.1−) blastocysts, assessing c-Kit versus Flt3 profiles on gated ESC-derived (Ly9.1+) LSK cells. Numbers adjacent to outlined areas indicate percent Flt3− LSK cells (HSC) or Flt3+ LSK cells (MPP). Data are representative of seven experiments with a total of two to three fetal livers per each time point. * Figure 3: Ldb1−/− fetal hematopoietic progenitor (LSK) populations do not contain LTR-HSCs. () Fetal liver LSK cells (left) in embryos at E12.5: Tie2-Cre Ldb1fl/Δ, 1.87 × 104 ± 0.61 × 104; control (Ldb1fl/Δ, Ldb1+/fl and Tie2-Cre Ldb1+/fl littermates), 1.68 × 104 ± 0.57 × 104 (mean ± s.d.). P = 0.52 (Student's t-test). Middle, flow cytometry of Linlo–neg fetal liver cells from Tie2-Cre Ldb1+/fl and Tie2-Cre Ldb1fl/Δ littermates at E12.5, stained for c-Kit and Sca-1 (left), followed by analysis of Flt3 expression (right) in the gate outlined at left (LSK cells; arrows). Numbers below outlined areas indicate percent LSK cells (left plots) or percent HSCs (left area; Flt3−) and MPPs (right area; Flt3+) among LSK cells (right plots). Far right, flow cytometry of fetal liver LSK cells at E12.5; numbers adjacent to outlined areas indicate percent CD48−CD150+ LSK cells. Data are from one representative of two experiments. () Flow cytometry of donor-derived (CD45.2+) cells in irradiated Rag2−/− (CD45.1) hosts 16 weeks after adoptive transfer of total fe! tal liver cells from Tie2-Cre Ldb1+/fl or Tie2-Cre Ldb1fl/Δ mice at E12. Numbers adjacent to outlined areas indicate percent in each gate. Data are from one representative of two experiments. * Figure 4: Ldb1 is continuously required for the maintenance of adult HSCs. () LSK profile (left) of bone marrow cells from mice injected with poly(I:C) on days 1, 3 and 5, assessed on day 12 by staining of gated Linlo–neg bone marrow cells for c-Kit and Sca-1. Control, Ldb1fl/fl, Ldb1+/fl and Mx1-Cre Ldb1+/fl mice. Numbers above outlined areas indicate percent LSK cells; numbers above plots indicate mice with a phenotype similar to that shown in the plot/total mice. Right, total bone marrow and LSK cells from control mice (n = 14) and Mx1-Cre Ldb1fl/fl mice (n = 11). *P < 0.05 (Student's t-test). Data are from one representative of five experiments (error bars, s.d.). () Flt3 expression (left) by LSK cells from adult Ldb1fl/fl or Mx1-Cre Ldb1fl/fl littermates injected with poly(I:C) on days 1, 3 and 5, assessed on day 6. Numbers adjacent to outlined areas indicate percent Flt3− LSK cells (HSC) or Flt3+ LSK cells (MPP). Right, total bone marrow and LSK cells from control mice (n = 3) and Mx1-Cre Ldb1fl/fl mice (n = 3). *P < 0.05 and **P < 0.01 (! Student's t-test). Data are representative of two experiments (error bars, s.d.). () Flow cytometry (left) of bone marrow cells and thymocytes from irradiated Rag2−/− (CD45.1) recipient mice 6 months after injection of 50:50 mixtures of bone marrow cells from Mx1-Cre Ldb1fl/fl (CD45.2) mice or littermate control (CD45.2) mice and B6 (CD45.1) mice that had been injected with poly(I:C) as described in . Numbers adjacent to outlined areas indicate percent cells in each gate. Right, summary of data at left. Data are from one representative of three independent experiments (error bars, s.d.). () CD45.2+ cells among bone marrow cells from premade bone marrow chimeras generated with 50:50 mixtures of bone marrow cells from Mx1-Cre Ldb1fl/fl (CD45.2) mice or control Mx1-Cre Ldb1+/fl (CD45.2) mice and bone marrow cells from B6 (CD45.1) mice after injection of poly(I:C) (three times, every other day). CMP, common myeloid progenitor, GMP, granulocyte macrophage progenitor. Data ar! e from one representative of two experiments. * Figure 5: Ldb1 regulates the expression of many transcription factors required for HSC maintenance. () Expression of transcripts encoding Ldb1 and subunits of the Ldb1 complex in hematopoietic progenitors. T, total adult bone marrow; LK, sorted Linlo–negc-Kit+ bone marrow; LSK, sorted LSK bone marrow. Data are from one representative of two independent experiments. () Real-time RT-PCR analysis of various transcripts (horizontal axis) in bone marrow cells obtained from adult Mx1-Cre Ldb1fl/fl mice or control Mx1-Cre Ldb1+/+ mice 2 d after the first of two injections of poly(I:C) (day 3), then enriched for hematopoietic progenitors (lineage depletion). Results in and are presented relative to expression of mRNA for the endogenous reference gene Actb. Below, genes with (+) or without (−) Ldb1-, Scl- and/or GATA-2-binding sites in the gene body or within 5 kb of the transcription start site, assessed by ChIP-Seq. *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three independent experiments (error bars, s.d.). * Figure 6: Ldb1 complex–binding sites are present in a high percentage of genes critical for HSCs. () Selected genes involved in HSC maintenance with Ldb1 complex–binding sites in the promoter and/or gene body, as determined by ChIP-Seq analysis with control antibody to immunoglobulin G (IgG), anti-Ldb1, anti-Scl and anti-GATA-2. Numbers in plots indicate positions of binding sites at known distal regulatory elements. Sequence conservation track is shown at the bottom of each browser shot. Data are representative of two experiments. () Ldb1 complex–binding site fragments in the HSC-maintenance gene set (left) or in known regulatory elements near these genes (right) with conserved GATA motifs and/or E-box (CANNTG) motifs. Data are representative of two experiments. () Consensus sequence motif of sites containing a GATA-binding sequence in Ldb1 complex–binding sites at the promoter, gene body and/or known enhancers of HSC maintenance genes. Letter size indicates nucleotide frequency, scaled to the information content (measure of conservation) at each position; colors ! distinguish the nucleotides. Data are representative of two experiments. () Prevalence of Scl or GATA-2 binding at HSC gene sites identified by ChIP-Seq with anti-Ldb1. Data set includes the 53 DNA fragments in Supplementary Tables 1 and 3. Data are representative of two experiments. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE26031 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Section on Cellular & Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA. * LiQi Li, * Jan Y Lee & * Paul E Love * Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA. * Raja Jothi * Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. * Kairong Cui & * Keji Zhao * Section on Mammalian Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA. * Tsadok Cohen, * Marat Gorivodsky, * Itai Tzchori, * Yangu Zhao & * Heiner Westphal * Department of Microbiology & Immunology, State University of New York Upstate Medical University, Syracuse, New York, USA. * Sandra M Hayes * Wisconsin Institutes for Medical Research, Paul Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA. * Emery H Bresnick Contributions L.L. designed and (with assistance from P.E.L.) did all of the experiments; L.L. and P.E.L. designed the study and wrote the manuscript; K.C. and K.Z. assisted in designing and doing the ChIP-Seq experiments; R.J. did the statistical analysis of ChIP-Seq data; E.H.B. provided reagents and input for the ChIP-Seq experiments; J.Y.L., T.C., M.G., I.T., Y.Z. and S.M.H. assisted with specific mouse experiments and provided input on experimental design; and H.W. provided the Ldb1 mouse strains. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paul E Love Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–9, Supplementary Tables 1–4 and Supplementary Methods Additional data - Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5
- Nat Immunol 12(2):137-143 (2011)
Nature Immunology | Article Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5 * Roland Züst1, 10, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Luisa Cervantes-Barragan1, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Habjan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Reinhard Maier1 Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin W Neuman2 Search for this author in: * NPG journals * PubMed * Google Scholar * John Ziebuhr3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Kristy J Szretter5 Search for this author in: * NPG journals * PubMed * Google Scholar * Susan C Baker6 Search for this author in: * NPG journals * PubMed * Google Scholar * Winfried Barchet7 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael S Diamond5 Search for this author in: * NPG journals * PubMed * Google Scholar * Stuart G Siddell8 Search for this author in: * NPG journals * PubMed * Google Scholar * Burkhard Ludewig1, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Volker Thiel1, 9 Contact Volker Thiel Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:137–143Year published:(2011)DOI:doi:10.1038/ni.1979Received02 November 2010Accepted02 December 2010Published online09 January 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 5′ cap structures of higher eukaryote mRNAs have ribose 2′-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2′-O-methylation of mRNA remains elusive. Here we show that 2′-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2′-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2′-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2′-O-methylation of mRNA suggests that RNA modifications such as 2′-O-methylation provide a molecular signature for the discrimination of self and non-self mR! NA. View full text Figures at a glance * Figure 1: Conservation of viral 2′-O-methyltransferases. () Human and mouse coronavirus genomes, including viral open reading frames (boxes; labels above and below identify gene products). The replicase gene, including conserved domains and viral proteinase cleavage sites (upward arrowheads) that separate nsp1–nsp16, is enlarged. CoV nsp16 MTase, nsp16-associated 2′-O-methyltransferase. () ClustalW2 alignment of coronavirus nsp16 amino acid sequences, representative of α-, β- and γ-coronaviruses. Keys (bottom right) identify sequence conservation (amino acid identity), amino acid substitutions and phenotypes of mutant proteins. FCoV, feline coronavirus; MHV, MHV strain A59; SARS-CoV, severe acute respiratory syndrome coronavirus; IBV, infectious bronchitis virus. () Comparison of the sequences of viral and cellular methyltransferase motifs. WBV, white bream virus (order Nidovirales); DENV, dengue virus (family Flaviviridae); VSV, vesicular stomatitis virus (order Mononegavirales); VACV, vaccinia virus (family Poxviridae); F! BL, fibrillarin (Homo sapiens). Right, key for amino acid similarity (single-letter codes) and conservation according to default ClustalX. * Figure 2: The HCoV 2′-O-methyltransferase mutant has altered replication kinetics and induction of and sensitivity to type I interferon. () Plaque assay of HCoV-229E and HCoV-D129A. () Replication kinetics of wild-type HCoV-229E (WT) and the mutant HCoV-D129A (D129A) in MRC-5 cells infected at an MOI of 0.1, presented as viral titer in plaque-forming units (PFU). () Incorporation of 3H into poly(A)-containing RNA derived from mock-infected cells (Self RNA) or cells infected with HCoV-229E or HCoV-D129A (Non-self RNA) after in vitro 2′-O-methylation with VP39. NS, not significant (P > 0.05); *P < 0.01 (unpaired Student's t-test). () Enzyme-linked immunosorbent assay of IFN-β in supernatants of human blood–derived macrophages 24 h after infection with HCoV-229E or HCoV-D129A (MOI = 1). Each symbol represents an individual donor (n = 9); thick horizontal lines indicate the mean (thin horizontal lines, s.d.). *P < 0.005 (Wilcoxon matched-pairs test). () Plaque assay of viral titers in human blood–derived macrophages pretreated with increasing doses of IFN-α (horizontal axis) 4 h before infection with HCoV! -229E or HCoV-D129A (MOI = 1), assessed 24 h after infection. ND, not detected. Data are representative of three experiments () or represent two (,) or three (,) independent experiments (average and s.e.m. of triplicates in ; mean and s.d. in ; error bars, s.d. of six samples in ). * Figure 3: MHV 2′-O-methyltransferase mutants induce IFN-β in an Mda5-dependent manner. () Ethidium bromide staining (left) of poly(A)-containing RNA (300 ng) from cells infected with wild-type MHV-A59 (WT), MHV-Y15A (Y15A) or MHV-D130A (D130A), separated by electrophoresis through a 1% agarose gel; right margin (1–7), genomic and subgenomic mRNA; left margin, size in kilobases (kb). Right, incorporation of 3H into poly(A)-containing RNA from mock-infected cells or cells infected with MHV-A59, MHV-Y15A or MHV-D130A after in vitro 2′-O-methylation with VP39. () Replication kinetics of MHV-A59, MHV-Y15A and MHV-D130A in 17Cl1 cells after infection at an MOI of 1 or 0.0001. (–) Enzyme-linked immunosorbent assay of IFN-β in supernatants of wild-type (WT; ), IFNAR-deficient (Ifnar−/−; ) or Mda5-deficient (Mda5−/−; ) macrophages (MΦ; 1 × 106) infected with wild-type or mutant MHV or Sendai virus (SeV) at an MOI of 1 and assessed 15 h after infection. (,) Quantitative RT-PCR analysis of the kinetics of IFN-β mRNA expression in wild-type () or IFNAR-d! eficient () macrophages (1 × 106) infected with MHV-A59, MHV-Y15A or MHV-D130A (MOI = 1), presented relative to its expression in uninfected cells. *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired Student's t-test). Data represent seven (), two (,,) or three (–) independent experiments (mean and s.d. of seven () or six (–) samples, or mean and s.e.m. of six samples ()). * Figure 4: MHV 2′-O-methyltransferase mutants induce the nuclear localization of IRF3 in an Mda5-dependent manner. () IRF3 in IFNAR-deficient or Mda5-deficient mouse macrophages infected with MHV-A59, MHV-Y15A or MHV-D130A at an MOI of 1 and stained at 3 h after infection for IRF3 (red) and with the DNA-intercalating dye DAPI (blue). Original magnification, ×20. Data are representative of three experiments. () Frequency of cells (infected as in ) with nuclear IRF3. *P < 0.01 and **P < 0.001 (unpaired Student's t-test). Data are representative of three experiments (mean and s.d. of five random fields with approximately 50–250 cells each). * Figure 5: Differences in the effect of type I interferon on the replication of MHV 2′-O-methyltransferase mutants. (,) Kinetics of the replication of MHV-A59, MHV-Y15A or MHV-D130A in wild-type and Mda5-deficient mouse macrophages (1 × 106) after infection at an MOI of 1 () or 0.0001 (). Data represent two independent experiments (mean ± s.e.m. of five samples). (,) Titer of MHV-A59, MHV-Y15A or MHV-D130A at 24 h after infection (MOI = 1) of wild-type () or Mda5-deficient () macrophages (1 × 105) pretreated with IFN-α (dose, horizontal axis) 4 h before infection. Data represent two independent experiments (mean and s.d. of four samples). * Figure 6: Restoration of MHV-D130A replication in IFIT1-deficient macrophages. Kinetics of the replication of MHV-A59, MHV-Y15A or MHV-D130A after infection (MOI = 0.01) of wild-type mice () or IFIT1-deficient () macrophages (5 × 105), assessed by plaque assay of viral titers in supernatants. Data represent two independent experiments (mean ± s.e.m. of four samples). * Figure 7: Deficiency in 2′-O-methyltransferase affects the recognition of virus by the innate immune system in vivo. Viral titers in the spleen () and liver () of wild-type mice and mice deficient in IFNAR, Mda5 or TLR7 or both Mda5 and TLR7, assessed 48 h after infection with MHV-A59, MHV-Y15A or MHV-D130A (500 plaque-forming units injected intraperitoneally). Data represent two independent experiments (mean and s.d. of six samples). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * IPR002877 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Roland Züst & * Luisa Cervantes-Barragan Affiliations * Institute of Immunobiology, Kantonal Hospital St. Gallen, St. Gallen, Switzerland. * Roland Züst, * Luisa Cervantes-Barragan, * Matthias Habjan, * Reinhard Maier, * Burkhard Ludewig & * Volker Thiel * School of Biological Sciences, University of Reading, Reading, UK. * Benjamin W Neuman * Centre for Infection and Immunity, Queen's University Belfast, Belfast, UK. * John Ziebuhr * Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany. * John Ziebuhr * Department of Medicine, Department of Molecular Microbiology, and Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, USA. * Kristy J Szretter & * Michael S Diamond * Department of Microbiology and Immunology, Loyola University Stritch School of Medicine, Maywood, Illinois, USA. * Susan C Baker * Institute for Clinical Chemistry and Pharmacology, University Hospital, University of Bonn, Bonn, Germany. * Winfried Barchet * Department of Cellular and Molecular Medicine, School of Medical and Veterinary Sciences, University of Bristol, Bristol, UK. * Stuart G Siddell * Vetsuisse Faculty, University of Zürich, Zürich, Switzerland. * Burkhard Ludewig & * Volker Thiel * Present address: Singapore Immunology Network, Agency for Science, Technology and Research, Singapore. * Roland Züst Contributions R.Z., L.C.-B., M.H., R.M. and K.J.S. did most of the experiments; B.W.N. did phylogenetic analyses; B.W.N. and S.C.B. did electron microscopy; J.Z., S.C.B., W.B., M.S.D., S.G.S. and B.L. contributed reagents and expertise; and S.G.S., B.W.N., B.L. and V.T. conceived of and designed the project and wrote and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Volker Thiel Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (396K) Supplementary Figures 1–2 and Supplementary Table 1 Additional data - IL-1β-driven neutrophilia preserves antibacterial defense in the absence of the kinase IKKβ
- Nat Immunol 12(2):144-150 (2011)
Nature Immunology | Article IL-1β-driven neutrophilia preserves antibacterial defense in the absence of the kinase IKKβ * Li-Chung Hsu1, 2, 9 Contact Li-Chung Hsu Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Enzler2, 3, 8, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Jun Seita4 Search for this author in: * NPG journals * PubMed * Google Scholar * Anjuli M Timmer5 Search for this author in: * NPG journals * PubMed * Google Scholar * Chih-Yuan Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ting-Yu Lai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Guann-Yi Yu2 Search for this author in: * NPG journals * PubMed * Google Scholar * Liang-Chuan Lai6 Search for this author in: * NPG journals * PubMed * Google Scholar * Vladislav Temkin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ursula Sinzig8 Search for this author in: * NPG journals * PubMed * Google Scholar * Thiha Aung8 Search for this author in: * NPG journals * PubMed * Google Scholar * Victor Nizet5, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Irving L Weissman4 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Karin2 Contact Michael Karin Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 12,Pages:144–150Year published:(2011)DOI:doi:10.1038/ni.1976Received27 September 2010Accepted19 November 2010Published online19 December 2010 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 Transcription factor NF-κB and its activating kinase IKKβ are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKβ–NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKβ (IkkβΔ mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in IkkβΔ mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, IkkβΔIl1r1−/− mice, unlike IkkβΔ mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKβ–NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severel! y compromises innate immunity. View full text Figures at a glance * Figure 1: IkkβΔ mice develop neutrophilia. () Neutrophil counts in the blood of wild-type (WT) and IkkβΔ mice, collected retro-orbitally at various time points (horizontal axis) after injection of poly(I:C). *P < 0.05, **P < 0.02 and ***P < 0.01 (Student's t-test). Data are from one experiment with 12 mice per genotype (average ± s.d.). () Flow cytometry of peripheral blood cells collected from wild-type and IkkβΔ mice and stained with fluorescein isothiocyanate–conjugated antibody to CD3 (anti-CD3) and phycoerythrin-conjugated anti-Ly6G (top) or with allophycocyanin-conjugated anti-B220 and phycoerythrin-conjugated anti-Ly6G (middle). Numbers adjacent to outlined areas indicate percent (± s.d.) CD3−Ly6G+ cells (top) or B220−Ly6G+ cells (middle) among all nucleated cells. Below, quantification of the results above. *P < 0.01 (Student's t-test). Data are representative of two experiments with three separate measurements per genotype (error bars, s.d.). () Hematoxylin and eosin–stained sections of wild-ty! pe and IkkβΔ bone marrow (BM), spleen and liver. Original magnification, ×40 (spleen and liver) or ×100 (bone marrow). Data are representative of two experiments with three mice per genotype. () Flow cytometry of wild-type and IkkβΔ bone marrow and spleen cells stained with phycoerythrin-conjugated anti-Ly6G and fluorescein isothiocyanate–conjugated anti-CD11b, assessing gated Ly6GloCD11b+ immature granulocytes (top). Numbers adjacent to outlined areas indicate percent (± s.d.) Ly6GloCD11b+ cells relative to total Ly6G+CD11b+ cells. ND, not detected. Below, flow cytometry of bone marrow cells stained with anti-Ly6G and antibody to intracellular myeloperoxidase (MPO). Numbers in plots indicate percent (± s.d.) Ly6G+MPO+ cells. Data are representative of three experiments. * Figure 2: Neutrophilia in IKKβ-deficient mice is transplantable. () Neutrophil counts in the peripheral blood of lethally irradiated wild-type mice 3 months after transplantation with wild-type (WTWT) or IkkβΔ (IkkβΔWT) bone marrow cells. *P < 0.01 (Student's t-test). Data are from one experiment with six mice per genotype (error bars, s.d.). () Bioluminescence-based imaging of irradiated wild-type mice 30 d after adoptive transfer of wild-type or IkkβΔ bone marrow cells transduced with a luciferase reporter. Right (heat map), bioluminescence in photons per second per cm2 per steradian (p/s/cm2/sr): minimum, 3 × 103; maximum, 17 × 103. Data are representative of three experiments per group. () Peripheral IkkβΔ neutrophils in lethally irradiated CD45.1+ C57BL/6 wild-type mice given transplantation of 5 × 106 bone marrow cells from CD45.2+IkkβΔ mice plus 5 × 106 bone marrow cells from CD45.1+ C57BL/6 wild-type mice, or 1 × 107 CD45.2+IkkβΔ bone marrow cells alone (positive control); results were calculated on the basis of d! ifferential blood counts and on flow cytometry with labeled anti-CD45.1 (wild-type cells) or anti-CD45.2 (IkkβΔ cells) and anti-Ly6G. Data are representative of three experiments with three mice per group (mean ± s.d.). * Figure 3: Ablation of IL-1R1 prevents neutrophilia in IkkβΔ mice. () Serum concentrations of IL-1β and tumor necrosis factor (TNF) in wild-type and IkkβΔ mice 6 months after injection of poly(I:C). () Enzyme-linked immunosorbent assay of IL-1β in supernatants of neutrophils (Neut), monocytes (Mono) and macrophages (Mac) from wild-type and IkkβΔ mice given intraperitoneal injection of thioglycollate, followed by collection of peritoneal and peripheral blood cells, purification with magnetic beads and culture for 24 h at a density of 1 × 105 cells per 100 μl. () Peripheral neutrophil counts in mice of various genotypes (horizontal axis) 6 months after injection of poly(I:C). () Spleen weights 2 months after injection of poly(I:C) (n = 3 mice per genotype). () Hematoxylin and eosin–stained spleen sections from the mice in . Original magnification, ×40. () Peripheral neutrophil counts of wild-type mice treated with PBS, anakinra or ML120B alone or anakinra plus ML120B. Each symbol represents an individual count; small horizontal bar! s indicate the median. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t test). Data are representative of two experiments with three mice per genotype () or two experiments with six mice per group (; mean and s.d. in –). * Figure 4: Larger granulocyte progenitor populations in IkkβΔ mice. () Colony count (left) and diameter (right) of wild-type and IkkβΔ bone marrow cells grown for 10 d in Methocult progenitor cell medium at a density of 3.3 × 103 cells per ml. Diameter results are presented relative to those of wild-type cells, set as 1. *P < 0.05 (Student's t-test). Data are representative of three experiments with three plates per mouse and three mice per genotype (left) or 27 colonies per genotype (right; mean and s.d.). () Colony count of wild-type and IkkβΔ bone marrow cells cultured as in with or without recombinant IL-1β (100 ng/ml). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of two experiments (mean ± s.d.). () Size of CMP, GMP, pre-granulocyte (Pre-Gra) and granulocyte (Gra) populations from the bone marrow of wild-type and IkkβΔ mice, assessed by flow cytometry and presented as absolute number per bilateral hind limbs. Data are representative of two experiments with three mice per genotype (mean and s.d.). () Inco! rporation of EdU and DNA content of progenitor cell populations from the bone marrow collected from mice 3 h after injection of EdU (200 μg). Numbers in plots indicate percent EdU+ cells (mean ± s.d.). Data are from one representative of three independent experiments. () Actual number of cells in the S-G2-M portion of the cell cycle in . P values, Student's t-test. Data are from one representative of three independent experiments (error bars, s.d.). * Figure 5: Effects of IKKβ ablation on neutrophil lifespan and gene expression. () Flow cytometry of cultured Ly6G+ peritoneal neutrophils stained with propidium iodide. *P < 0.05 and **P < 0.01, compared with wild-type (Student's t-test). () Microarray analysis of gene expression in neutrophils isolated as in ; genes are grouped as encoding molecules relevant for apoptosis, proliferation or survival (one mouse per 'lane'): red indicates genes with an intensity greater than the mean intensity of the genes presented here; green indicates genes with an intensity lower than that mean intensity. () Quantitative real-time PCR analysis of mRNA expression in peritoneal neutrophils (genes encoding the mRNA, horizontal axis). *P < 0.05 and **P < 0.01 (Student's t-test). () Immunoblot analysis of proteins involved in cell survival and proliferation, as well as actin (loading control), in lysates of peritoneal Ly6G+ neutrophils (genotype, above blot; one mouse per lane). () Quantitative real-time PCR analysis of Bcl-xL mRNA expression among total RNA from wild-typ! e or IkkβΔ Ly6G+ peritoneal neutrophils incubated for 4 h with or without recombinant IL-1β (100 ng/ml); results were normalized to cyclophilin mRNA and are presented relative to expression in wild-type cells. *P < 0.05 (Student's t-test). () Immunoblot analysis of total and phosphorylated (p-) STAT3 and Jak2 in lysates of wild-type or IkkβΔ Ly6G+ peritoneal neutrophils (one mouse per lane). () Quantitative real-time PCR analysis of Bcl-xL mRNA expression in wild-type Ly6G+ peritoneal neutrophils incubated for 4 h in the presence or absence of AG490 (30 μM) or recombinant IL-1β (100 ng/ml); results were normalized to cyclophilin mRNA and are presented relative to expression in wild-type cells. *P < 0.05 (Student's t-test). Data are representative of three (,) or two (,) independent experiments in triplicate (error bars, s.d.), or are from one experiment each with many mice (,,). * Figure 6: Inactivation of signaling via IL-1–IL-1R1 and IKKβ–NF-κB compromises antimicrobial immunity. () Killing of bacteria by peritoneal neutrophils collected from wild-type and IkkβΔ mice 4 h after thioglycollate injection, then incubated with GAS at a multiplicity of infection (MOI) of 0.5 or 0.1; results are presented as colony-forming units (CFU) of live bacteria. () Lesion size in mice given subcutaneous (back) injection of 1 × 108 GAS. () Live bacteria in lesions of mice in that remained alive 4 d after infection. Each symbol represents an individual mouse. () Hematoxylin and eosin staining of skin lesions collected from the mice in at 4 d after infection. Original magnification, ×10. *P < 0.05 and **P < 0.01, compared with wild type (Student's t-test). Data are representative of three independent experiments in triplicate (; mean ± s.d.) or two experiments with five mice per genotype (–; error bars, s.d.). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE25211 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Li-Chung Hsu & * Thomas Enzler Affiliations * Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan. * Li-Chung Hsu, * Chih-Yuan Lee & * Ting-Yu Lai * Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology and Department of Pathology, School of Medicine, University of California, San Diego, La Jolla, California, USA. * Li-Chung Hsu, * Thomas Enzler, * Guann-Yi Yu, * Vladislav Temkin & * Michael Karin * Department of Medicine, Stanford University School of Medicine, Stanford, California, USA. * Thomas Enzler * Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA. * Jun Seita & * Irving L Weissman * Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California, USA. * Anjuli M Timmer & * Victor Nizet * Graduate Institute of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan. * Liang-Chuan Lai * Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA. * Victor Nizet * Present address: Department of Hematology and Oncology, Universitaetsmedizin Goettingen, Goettingen, Germany. * Thomas Enzler, * Ursula Sinzig & * Thiha Aung Contributions J.S. and A.M.T. contributed equally to this work. L.-C.H. and T.E. designed and did most of the experiments; M.K. helped in designing experiments; M.K., T.E. and L.-C.H. wrote the paper; J.S. and I.L.W. planned and did most of the progenitor cell analyses; A.M.T. and V.N. planned and did the bacterial killing experiments; and C.-Y.L., T.-Y.L., G.-Y.Y., L.-C.L., V.T., U.S. and T.A. helped with some of the experiments. Competing financial interests M.K. holds several US and international patents on the development and use of IKKβ inhibitors; I.L.W. has stock in Amgen, is a cofounder of Cellerant and Stem Cells and is also a consultant for Stem Cells. Corresponding authors Correspondence to: * Li-Chung Hsu or * Michael Karin Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–10, Supplementary Table 1 and Supplementary Methods Additional data - Eosinophils are required for the maintenance of plasma cells in the bone marrow
- Nat Immunol 12(2):151-159 (2011)
Nature Immunology | Article Eosinophils are required for the maintenance of plasma cells in the bone marrow * Van Trung Chu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anja Fröhlich1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gudrun Steinhauser1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias Scheel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Toralf Roch1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Simon Fillatreau1 Search for this author in: * NPG journals * PubMed * Google Scholar * James J Lee3 Search for this author in: * NPG journals * PubMed * Google Scholar * Max Löhning1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia Berek1 Contact Claudia Berek Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:151–159Year published:(2011)DOI:doi:10.1038/ni.1981Received16 November 2010Accepted06 December 2010Published online09 January 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 Plasma cells are of crucial importance for long-term immune protection. It is thought that long-lived plasma cells survive in specialized niches in the bone marrow. Here we demonstrate that bone marrow eosinophils localized together with plasma cells and were the key providers of plasma cell survival factors. In vitro, eosinophils supported the survival of plasma cells by secreting the proliferation-inducing ligand APRIL and interleukin-6 (IL-6). In eosinophil-deficient mice, plasma cell numbers were much lower in the bone marrow both at steady state and after immunization. Reconstitution experiments showed that eosinophils were crucial for the retention of plasma cells in the bone marrow. Moreover, depletion of eosinophils induced apoptosis in long-lived bone marrow plasma cells. Our findings demonstrate that the long-term maintenance of plasma cells in the bone marrow requires eosinophils. View full text Figures at a glance * Figure 1: Bone marrow eosinophils provide plasma cell survival factors. () Flow cytometry of bone marrow cells; numbers adjacent to outlined areas indicate percent Gr-1loF4/80+ cells. () Staining of intracellular APRIL (I-APRIL) in Gr-1lo (top) and F4/80+ (bottom) bone marrow cells (left) and immunohistology showing colocalization of staining for APRIL and F4/80 in the bone marrow (right). Numbers adjacent to outlined areas (left) indicate percent cells in each. Scale bar, 75 μm. () Flow cytometry identifying three different CD11b+ subsets (R1, R2 and R3) among CD11c−FcεRIα−Gr-1lo–neg cells. Numbers in plots indicate percent cells in each outlined area (left) or subset (right). () Hematoxylin and eosin staining of bone marrow cell subsets sorted and prepared by cytospins. Eos, eosinophil; Neu, neutrophil; EosP, immature eosinophil; PerC MΦ, peritoneal cavity macrophage (control); Mo & MΦ, monocyte-macrophage. Scale bar, 15 μm. () Intracellular expression of APRIL (top) or IL-6 (bottom) in bone marrow cells gated on Gr-1lo–neg cells! (left) and frequency of R1, R2 and R3 cells in the fraction of Gr-1loAPRILhi or Gr-1loIL-6+ cells (right). Numbers adjacent to outlined areas indicate percent cells in each. () Immunofluorescence of sorted eosinophils (Eos (R1)), immature eosinophils (EosP (R2)) and monocytes-macrophages (Mo & MΦ (R3)) stained with antibodies specific for APRIL, IL-6 or MBP; nuclei were counterstained with the DNA-intercalating dye DAPI. Scale bar, 45 μm. Data are representative of three to five experiments. * Figure 2: Eosinophils support the survival of plasma cells through the secretion of soluble factors. () IL-4 secretion by eosinophils from the bone marrow of naive BALB/c mice and immunized BALB/c mice (days 0 and 6 of the secondary (2°) response). () APRIL and IL-6 mRNA (left), intracellular APRIL staining (top right) and IL-6 secretion (bottom right) by eosinophils from the bone marrow of naive and immunized BALB/c mice at day 6 of the secondary response (n = 4 mice per group). RU, relative units (relative to expression in naive bone marrow eosinophils); MFI, mean fluorescence intensity (presented relative to control). () Enzyme-linked immunospot analysis of the frequency of plasma cells (PC) cultured with medium alone (Med) or various numbers of eosinophils from day 6 of the secondary response. (ratio, PC/Eos horizontal axis) () Frequency of plasma cells cultured at a ratio of 1:1 with eosinophils from naive and immunized mice (day 6 of secondary response). () Frequency of plasma cells cultured at a ratio of 1:1 with eosinophils alone from immunized mice (Eos) or along ! with various inhibitors (horizontal axis). α-IL-6, antibody to IL-6. () Plasma cells negative for annexin V (AnnV−) after culture at a ratio of 1:1 with eosinophils from immunized animals in Transwells with (+) or without (−) inserts. () Plasma cells negative for annexin V after culture as in . NS, not significant. P values, Student's t-test. Data are from one of two experiments (error bars, s.d. of triplicate cultures). * Figure 3: Colocalization of eosinophils and plasma cells in the bone marrow. () Microscopy of bone marrow sections from day 6 of secondary response, double stained with fluorescence-labeled antibodies (overlays). Scale bar, 15 μm. () Immunohistochemical staining (left) and confocal microscopy of immunofluorescence staining (right) of bone marrow sections from day 60 of secondary response. Scale bars, 20 μm (left) or 10 μm (right). () Microscopy of bone marrow tissue sections from day 6 of secondary response, triple stained with antibodies specific for plasma cells (IgG) and eosinophils (MBP) and APRIL (left) or monocytes-macrophages (F4/80) (right). Scale bar, 20 μm. Right, frequency of IgG+ plasma cells localizing together with APRIL+, MBP+ or F4/80+ cells. () Microscopy of bone marrow tissue sections from day 6 of the secondary response, stained for VCAM-1 and MBP (left), VCAM-1 and APRIL (middle), VCAM-1 and IL-6 (right). Scale bar, 20 μm. () Microscopy of a bone marrow tissue section from day 6 of the secondary response, stained with antibod! ies specific for VCAM-1, IgG and MBP. Outlined areas at left are enlarged at right, visualizing cell clusters of eosinophils, plasma cells and stromal cells. Scale bar, 75 μm (main image) or 15 μm (right). Below, frequency of IgG+ plasma cells localizing together with MBP+ eosinophils and VCAM-1+ stromal cells after secondary immunization. Data are representative of five experiments with five mice per group (error bars, s.d.). * Figure 4: Lower expression of APRIL and IL-6 in the bone marrow of eosinophil-deficient mice. () Flow cytometry of eosinophils (R1), immature eosinophils (R2) and monocytes-macrophages (R3) from BALB/c and ΔdblGATA-1 mice, with gating on bone marrow F4/80+Gr-1lo cells. Numbers adjacent to outlined areas indicate percent cells in each. () Immunohistological staining of bone marrow tissue sections from BALB/c and ΔdblGATA-1 mice for MBP, APRIL and IL-6. Scale bar, 150 μm. () Expression of APRIL mRNA (top) and IL-6 mRNA (middle) and IL-6 secretion (bottom) by total bone marrow cells from BALB/c and ΔdblGATA-1 mice. RU, relative units (relative to expression in bone marrow cells from ΔdblGATA-1 mice). *P < 0.002 (Student's t-test). Data are representative of four experiments with four mice per genotype (error bars, s.d.). * Figure 5: Impaired accumulation of plasma cells in the bone marrow of eosinophil-deficient mice. () Frequency of CD138+ plasma cells (left), number of IgG+ plasma cells (middle) and IgG secretion (right), assessed for total bone marrow (BM) cells of unimmunized BALB/c mice (n = 3) and ΔdblGATA-1 mice (n = 4). Concentration of IgG in supernatants of total bone marrow cell cultures. A405, absorbance at 405 nm. (,) Frequency of CD138hi plasma cells () and antigen-specific plasma cells () in the bone marrow of phOx-immunized BALB/c and ΔdblGATA-1 mice during the primary (1°) and secondary (2°) response (n = 4 mice per group per time point). (,) Immune response in the bone marrow of BALB/c and ΔdblGATA-1 mice 7 months after LCMV infection. () Frequency of CD138+ plasma cells. Numbers in plots indicate percent cells in outlined area. () Enzyme-linked immunospot analysis of IgG+ plasma cells specific for glycoprotein 1 GP1. Right (,), data summaries. P values, Student's t-test and two-way ANOVA. Data are representative of three experiments with six mice per group (error b! ars, s.d.). * Figure 6: Eosinophils are required for the retention of plasma cells in the bone marrow. () Flow cytometry analysis of the frequency of Siglec-F+CD11bint eosinophils and Siglec-FintCD11bhi immature eosinophils from BALB/c mice, ΔdblGATA-1 mice and ΔdblGATA-1 mice 6 and 12 d after reconstitution with eosinophils (ΔdblGATA-1 + Eos). () MBP staining of bone marrow tissue sections for analysis of eosinophils at day 6 (n = three mice per group). Scale bar, 150 μm. (,) Frequency of CD138+ plasma cells (left) and number of antigen (Ag)-specific IgG plasma cells (right) in the bone marrow () and spleen () 6 and 12 d after the secondary immune response to phOx (n = three mice per group). Numbers above outlined areas indicate percent cells in each. P values, Student's t-test. Data are representative of two experiments (error bars, s.d.). * Figure 7: The long-term survival of plasma cells is dependent on eosinophils. () MBP+ eosinophils in tissue sections from bone marrow and spleen and in cytospins from blood samples of control BALB/c mice (Rat IgG2a) or BALB/c mice depleted of eosinophils (α-Siglec-F; n = 3 mice per group). Scale bar, 300 μm. Below, data summary. (–) Frequency of CD138+TACI+ plasma cells (), absolute number of antigen-specific plasma cells () and frequency of annexin V–positive (AnnV+) apoptotic plasma cells () in the bone marrow and spleen of mice as in (n = 6 mice per group). Below, data summaries. P values, Student's t-test. Data are representative of two experiments (error bars, s.d.). Author information * Abstract * Author information * Supplementary information Affiliations * Deutsches Rheuma-Forschungszentrum, Institut der Leibniz-Gemeinschaft, Berlin, Germany. * Van Trung Chu, * Anja Fröhlich, * Gudrun Steinhauser, * Tobias Scheel, * Toralf Roch, * Simon Fillatreau, * Max Löhning & * Claudia Berek * Experimental Immunology, Department of Rheumatology and Clinical Immunology, Charite-University Medicine, Berlin, Germany. * Anja Fröhlich & * Max Löhning * Department of Biochemistry and Molecular Biology, Division of Pulmonary Medicine, Mayo Clinic Arizona, Scottsdale, Arizona, USA. * James J Lee * Present address: Centre for Biomaterial Development, Helmholtz-Zentrum Geesthacht, Berlin, Germany. * Toralf Roch Contributions V.T.C., C.B. and M.L. designed experiments and analyzed the data; V.T.C. did most of the experiments; A.F. and M.L. analyzed the immune response to LCMV; T.S. analyzed the GC response; J.J.L. contributed the PHIL mouse and eosinophil-specific antibodies; T.R. and G.S. helped with data acquisition; and V.T.C., C.B., M.L., A.F. and S.F. prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Claudia Berek Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–11 and Supplementary Table 1 Additional data - The splicing regulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of AID to switch-region DNA
- Nat Immunol 12(2):160-166 (2011)
Nature Immunology | Article The splicing regulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of AID to switch-region DNA * Urszula Nowak1 Search for this author in: * NPG journals * PubMed * Google Scholar * Allysia J Matthews1 Search for this author in: * NPG journals * PubMed * Google Scholar * Simin Zheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jayanta Chaudhuri1, 2 Contact Jayanta Chaudhuri Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:160–166Year published:(2011)DOI:doi:10.1038/ni.1977Received20 September 2010Accepted01 December 2010Published online26 December 2010 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 During immunoglobulin class-switch recombination (CSR), the cytidine deaminase AID induces double-strand breaks into transcribed, repetitive DNA elements called switch sequences. The mechanism that promotes the binding of AID specifically to switch regions remains to be elucidated. Here we used a proteomic screen with in vivo biotinylation of AID to identify the splicing regulator PTBP2 as a protein that interacts with AID. Knockdown of PTBP2 mediated by short hairpin RNA in B cells led to a decrease in binding of AID to transcribed switch regions, which resulted in considerable impairment of CSR. PTBP2 is thus an effector of CSR that promotes the binding of AID to switch-region DNA. View full text Figures at a glance * Figure 1: AID interacts with PTBP2. () AID expression construct (biotag-DM-AID). *, lysine residue biotinylated by BirA. The H56R and E58Q substitutions inactivate the DNA-deaminase activity of AID. () Immunoblot analysis of protein extracts of stimulated CH12(BirA) or CH12(BirA–biotag-DM-AID) cells, probed with anti-AID (α-AID). Left margin, molecular size in kilodaltons (kDa). () Immunoblot analysis of bound proteins in extracts of CH12(BirA) or CH12(BirA–biotag-DM-AID) cells incubated with streptavidin-agarose beads, probed with anti-AID (α-AID; top) or streptavidin coupled to horseradish peroxidase (SA-HRP; bottom). () Modified ChIP analysis of crosslinked DNA-protein complexes from unstimulated (–CIT) or CIT-stimulated (+CIT) CH12(BirA–biotag-DM-AID) cells, with steptavidin-agarose (SA-ChIP) instead of the antibodies used in conventional ChIP, followed by PCR of threefold dilutions of DNA (wedges) bound to streptavidin-agarose to assess the presence of Sμ or the μ-chain promoter. (,) Immunoass! ay of whole-cell extracts of wild-type (WT) or AID-deficient (Aicda−/−) mouse splenic B cells stimulated with anti-CD40 plus IL-4, immunoprecipitated with anti-AID () or anti-PTBP2 () and analyzed by immunoblot with anti-PTBP2 () or anti-AID (). E1 and E2 are two elutions of bound proteins. Data are representative of two independent experiments. * Figure 2: Knockdown of PTBP2 impairs CSR. () Immunoblot analysis of PTBP2 in whole-cell extracts of CH12 cells infected by lentivirus expressing PTBP2-specific shRNA (PTBP2-1 or PTBP2-2) or a nonspecific control shRNA (Scramble), then left unstimulated (U) or stimulated with CIT (S), probed with anti-PTBP2 or anti-GAPDH (control). () Flow cytometry analysis of switching to IgA in CH12 cells infected as in and stimulated for 72 h with CIT. Numbers above bracketed lines indicate percent IgA+ cells. () Quantification of CSR in cells in which PTBP2 was knocked down with PTBP2-1 or PTBP2-2, presented relative to CSR in control cells expressing control shRNA, set as 100. P values, Student's t-test. () RT-PCR amplification of circle transcripts (Iα-Cμ) in cells infected as in . Wedges indicate threefold dilutions of cDNA generated by reverse transcription; – indicates template without reverse transcriptase. Actb (below), transcripts of the gene encoding β-actin (loading control). () Immunoblot analysis of PTBP2 in ext! racts of CH12 cells expressing scrambled or PTBP2-2 shRNA, transduced with empty vector or vector containing PTBP2 cDNA. () Flow cytometry analysis of CSR to IgA in cells expressing scrambled or PTBP2-2 shRNA, transduced with empty lentiviral vector or vector containing PTBP2 cDNA and stimulated with CIT; results are presented relative to CSR in control cells expressing control shRNA, set as 100. Data are representative of five (,) or three (–) experiments (mean and s.d. in ,) or are from one representative of five experiments (). * Figure 3: Germline transcription is unaffected in cells with knockdown of PTBP2. Real-time quantitative PCR analysis of germline transcripts of μ-chain (μ-GLT; ) and α-chain (α-GLT; ) among RNA from cells expressing PTBP2-specific shRNA; results were normalized to Actb and are presented relative to expression in control cells expressing scrambled shRNA, set as 1. Data are representative of three independent experiments (mean and s.d.). * Figure 4: The expression and nuclear localization of AID are not altered in cells with knockdown of PTBP2. () Real-time quantitative RT-PCR analysis of the expression of AID mRNA in CIT-stimulated cells expressing PTBP2-specific shRNA; results were normalized to Actb and are presented relative to expression in control cells expressing scrambled shRNA, set as 1. () Immunoblot analysis of whole-cell extracts (100 μg) of unstimulated cells (U) or CIT-stimulated cells (S) expressing scrambled or PTBP2-specific shRNA. Expt, experiment. () Immunoblot analysis of nuclear protein (~50 μg) from cells expressing scrambled or PTBP2-specific shRNA, probed with anti-AID or anti-XRCC1 (loading control). () AID activity of whole-cell extracts (WCE) or nuclear extracts (NE; ~25 μg each) of cells expressing scrambled or PTBP2-specific shRNA, assessed by measurement of the conversion of cytidine to uridine (deamination) on an ssDNA substrate with a uracil-release assay16. () Fraction of nuclear AID activity as a percentage of total AID activity, calculated from the values in . Data are represen! tative of three experiments (; mean and s.d.) or are from two independent experiments (–; mean and s.d. of three independent assays in ). * Figure 5: PTBP2 binds S transcripts in vitro. RNA-binding ability of histidine-tagged PTBP2 immobilized on nickel-agarose beads and incubated with in vitro–transcribed radiolabeled RNA, then washed, followed by measurement of the retention of radioactivity on the beads with a scintillation counter; results were subtracted from the retention by a control protein (Gen1) and are presented relative to total input counts. Controls 1–4 are transcripts derived from four protein-encoding genes: 1, protein kinase A Cα subunit; 2, protein kinase A RIα subunit; 3, Ku70; 4, AID. Smu1 and Smu2 contain 1-kb and 3-kb Sμ RNA sequence in the sense orientation, respectively; SmuR is a 1-kb antisense Sμ transcript. P values, Student's t-test with RNA 1 as the non–S region control. Data are representative of three independent experiments (mean and s.d.). * Figure 6: PTBP2 promotes the binding of AID to S-region DNA. () ChIP analysis of cells expressing PTBP2-specific or control shRNA, stimulated for 48 h with CIT and immunoprecipitated with anti-AID, antibody to histone H3 or IgG (control), followed by PCR analysis of threefold dilutions of DNA (wedges) to assess the presence of Sμ or Iμ (control). Data are representative of three experiments. () Quantitative real-time PCR analysis of Sμ in cells expressing PTBP2-specific or control shRNA, immunoprecipitated by ChIP with anti-AID; results were normalized to the cycling threshold values of input DNA, followed by subtraction of the cycling threshold values for ChIP with IgG (background) and presented relative to results in control cells, set as 100. P value, Student's t-test. Data are from three independent experiments (mean and s.d.). * Figure 7: Knockdown of PTBP2 impairs CSR and the binding of AID to activated S regions in primary B cells. () Immunoblot analysis of extracts of activated splenic B cells transduced with control or PTBP2- or AID-specific shRNA. () Flow cytometry analysis of CSR to IgG1 in cells transduced with PTBP2- or AID-specific shRNA, presented relative to CSR in cells transduced with control shRNA, set as 100. () RT-PCR analysis of Iγ-1Cμ circle transcripts in cells transduced with control, PTBP2- or AID-specific shRNA. Wedges indicate threefold dilutions of cDNA generated by reverse transcription; – indicates template without reverse transcriptase. Actb (below), loading control. (,) Binding of AID to Sμ () or Sγ1 () in primary B cells transduced with control, PTBP2- or AID-specific shRNA, then stimulated for 48 h with anti-CD40 and IL-4 and analyzed by ChIP with anti-AID or IgG (control), followed by quantitative real-time PCR analysis of Sμ or Sγ1; results were normalized to the cycling threshold values of input DNA, followed by subtraction of the cycling threshold values for ChIP! with IgG (background) and are presented relative to results in control cells, set as 100. P values, Student's t-test. Data are representative of three experiments (,), seven independent experiments (; mean and s.d.) or are from three independent experiments (,; mean and s.d.). Author information * Abstract * Author information * Supplementary information Affiliations * Graduate Program in Immunology and Microbial Pathogenesis, Weill-Cornell Medical School, New York, New York, USA. * Urszula Nowak, * Allysia J Matthews, * Simin Zheng & * Jayanta Chaudhuri * Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Jayanta Chaudhuri Contributions U.N. and J.C. designed and did experiments, analyzed the data and wrote the manuscript; and A.J.M. did the shRNA knockdown for the AID-localization studies and with S.Z. established shRNA knockdown techniques in CH12 cells. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jayanta Chaudhuri Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–19 and Supplementary Table 1 Additional data - Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ TH2 cells
- Nat Immunol 12(2):167-177 (2011)
Nature Immunology | Article Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ TH2 cells * Sabina A Islam1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel S Chang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard A Colvin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mike H Byrne1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michelle L McCully2 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernhard Moser2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sergio A Lira3 Search for this author in: * NPG journals * PubMed * Google Scholar * Israel F Charo4 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew D Luster1 Contact Andrew D Luster Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:167–177Year published:(2011)DOI:doi:10.1038/ni.1984Received18 October 2010Accepted09 December 2010Published online09 January 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 Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (TH2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of TH2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5–enriched TH2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of TH2 cell homing that drives IL-5–mediated chronic allergic inflammation. View full text Figures at a glance * Figure 1: Mouse CCL8 RNA and protein are detected in normal mouse skin. () Relative positioning of the six MCP-cluster chemokine genes on human chromosome 17 and mouse chromosome 11, modified from the Ensembl site. Black, known orthologs; blue, non-orthologs; red, presumed orthologs (focus of our study here). () Percent identity/percent similarity of amino acids using the GenBank sequence extending from the start codon to the stop codon, calculated using the EMBOSS Needleman-Wunsch algorithm. m, Mouse; h, human. () RNA hybridization blot comparing mRNA expression of MCP-family chemokines and CCL11 (eotaxin-1) in pooled organs of normal BALB/c mice, conducted once. () Representative immunofluorescence staining of normal wild-type (WT) C57BL/6 mouse skin and Ccl8−/−Ccl12−/− C57BL/6 mouse skin with a primary polyclonal antibody to mouse CCL8 and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Data are reflective of at least six independent experiments from three or more mice. * Figure 2: Mouse CCL8 induces migration and calcium flux in TH2-R2A cells. () Migration of mouse BMDMs to mouse CCL8 and CCL2. () Migration of NK cells to mouse CCL8, CCL2, CCL4 and CXCL11. () Migration of lymph node CD4+ and CD8+ T cells to mouse CCL8 and CXCL12. () Representative cytokine profiles and assays of TH1-R1 cell migration to mouse CCL8. Migration to mouse CXCL11 and CXCL12 was a positive control. () Representative cytokine profiles and assays of TH2-R1 cell migration to mouse CCL8. Migration to mouse CCL22 and CXCL12 was a positive control. Data in – are representative of three or more experiments. () Dose-response migration of TH2-R2A cells to mouse CCL8 (representative of more than 10 experiments) and PTX-mediated inhibition of mouse CCL8–induced migration (one of three independent experiments shown). Results in – are shown as mean ± s.e.m. () Analysis of TH2-R2A cells for calcium flux to 40 nM mouse CCL8 (downward arrow indicates time of addition); representative of eight separate experiments. () Analysis of dose-response of ! TH2-R2A cells to mouse CCL8 in calcium flux assays; representative of two independent experiments. Arrows pointing up indicate onset of control calcium flux response to ionomycin (1 μg/ml) at each concentration of chemokine. * Figure 3: CCR8 is required for mouse CCL8-induced TH2-R2A cell migration. () CC-chemokine receptor mRNA enrichment measured by QPCR in TH2-R2A cells that migrated to mouse CCL8 in Transwell assays relative to medium alone; representative of three experiments. () Kinetics of mouse Ccr8 and Ccl1 mRNA induction in TH2-R2A cells generated by repeat rounds of polarization and subsequent activation with antibodies to CD3 and CD28. Data are normalized to β2-microglobulin and presented on a scale of 0 to 100; representative of three experiments. () Comparison of Ccr8 mRNA expression in leukocyte subsets assayed for mouse CCL8 migration; representative of two to five experiments. () Comparison of chemotaxis of wild-type (WT), Ccr8-, Ccr2- and Ccr5-deficient TH2-R2A cells to mouse CCL8 or mouse CCL1; control migration to the CCR4 agonist CCL22 is also shown. One of three experiments is shown. () Dose-dependent inhibition of wild-type TH2-R2A cell migration to mouse CCL8 using a neutralizing polyclonal mouse CCR8 antibody (nAb), and migration to control CCL! 22 chemokine at various concentrations of antibody. One of two experiments is shown. Results in and are shown as mean ± s.e.m. * Figure 4: Mouse CCL8 is a specific agonist of mouse and human CCR8. () Dose-response chemotaxis assay of mouse Ccr8–transfected Baf/3 cells to mouse CCL8 and CCL1. Untransfected Baf/3 cells migrated only to CXCL12 (data not shown). () Calcium flux of mouse Ccr8–transfected cells to mouse CCL8 but not to the CCR4 agonist mouse CCL22. Mouse CCL8 did not induce calcium flux of untransfected Baf/3 cells, but positive control CXCL12 did. () Dose-response calcium flux of mouse Ccr8–transfected cells to mouse CCL8. () Cross-desensitization of mouse Ccr8–transfected cells to mouse CCL8 and CCL1. () Dose-response chemotaxis assay of human CCR8 receptor–transfected 4DE4 cells to mouse CCL8 and CCL1. Nontransfected 4DE4 cells did not migrate to either ligand (data not shown). () Specificity of mouse CCL8–induced calcium flux signaling in human CCR8–transfected cells, as shown by desensitization to repeat signaling by mouse CCL8; lack of flux of untransfected 4DE4 cells to mouse CCL8. () Human CCL8 did not induce calcium flux in CCR8-trans! fected cells. () Cross-desensitization of human CCR8–transfected cells by mouse CCL8 and human CCL1. Data are representative of at least three independent experiments (error bars (–), s.e.m). * Figure 5: Ccr8−/− and Ccl8−/−Ccl12−/− mice have decreased skin inflammation in a model of chronic atopic dermatitis. Histological analysis of skin was done on day 50, 24 h after the last of three 1-week rounds of topical sensitization with PBS or OVA. () Hematoxylin and eosin (H&E) staining of wild-type (WT) and Ccr8−/− mice sensitized with PBS or OVA. Far right, immunohistochemical analysis of CD3+ T cells in sensitized skin of wild-type and Ccr8−/− mice. Data are representative of three to seven experiments. () H&E staining of wild-type and Ccl8−/−Ccl12−/− mice sensitized with PBS or OVA. Far-right panels are at higher magnification, showing eosinophils and characteristic spongiosis of keratinocytes in wild-type sensitized mice; representative of three experiments. () H&E staining of OVA-sensitized Ccr4−/− and Ccl12−/− mice; representative of two experiments. () Skin thickness and leukocyte counts in wild-type and Ccr8−/− mice shown as mean ± s.e.m.; n = 6–8 mice per group. *P < 0.00001, **P = 0.004, ***P = 0.002, ****P = 0.02 and *****P = 0.005 for OVA-sen! sitized Ccr8−/− versus wild-type mice. HPF, high-powered field. () Skin thickness and leukocyte counts in OVA-sensitized wild-type, Ccl8−/−Ccl12−/−, Ccl12−/− and Ccr4−/− mice. *P = 0.0004, **P = 0.02, ***P < 0.00001 and ****P < 0.00001 for gene-deficient versus wild-type mice. Data are shown as mean ± s.e.m.; n = 6–9 mice per group. () H&E-stain of OVA-sensitized wild-type mice treated with CCL1-neutralizing antibody (left) or isotype control (right) during last week of epicutaneous sensitization. () Skin thickness and leukocyte counts in mice from ; n = 4–5 mice per group in two independent experiments. Scale bar = 100 μm in all photomicrographs except for far-right panels in , where scale bar = 20 μm. * Figure 6: Ccr8−/− mice have decreased production of IL-5, IL-25 and eosinophil-active chemokines in allergen-sensitized skin. () QPCR measurements of transcripts for eosinophil-attracting chemokines. () Summary of QPCR measurements of chemokine mRNA expression in skin biopsies of wild-type (WT; *P = 0.01, **P = 0.0004 and ***P = 0.003 for PBS versus OVA) and Ccr8−/− (*P = 0.04 and **P = 0.03 for PBS versus OVA) mice after topical sensitization. () QPCR measurements of transcripts for TH2 cytokines. NS, not significant. () Serum OVA-specific IgE and IgG1 concentrations measured by enzyme-linked immunosorbent assay (ELISA) in sensitized mice. () QPCR of transcripts for CCR8 ligands. () Representative immunofluorescence analysis of mouse CCL8 protein expression in PBS-sensitized epidermis (epi) and dermis (derm) and OVA-sensitized epidermis and dermis of wild-type and Ccr8−/− mice from two experiments; n = 4 mice per group. (,) QPCR measurements of CCR4 and CCR10 ligands in skin biopsies of wild-type and Ccr8−/− mice after topical sensitization. () TH2 cytokine production by DLN cells afte! r ex vivo stimulation with OVA protein and CD3, measured by ELISA (n = 8–10 mice per group). Pooled data from three experiments are shown. Results in and are shown as mean ± s.e.m. All data were obtained 50 d after the initiation of sensitization and are reflective of at least three experiments, except for . * Figure 7: Competitive in vivo homing of adoptively transferred OVA-specific wild-type and Ccr8−/− TH2 and TH1 cells in OVA-sensitized mice. OVA(323–339) peptide–specific TCR-transgenic (OTII) Thy1.1 and Ccr8−/− OTII Thy1.2 TH2 cells were transferred into OVA-sensitized Thy1.1 × Thy1.2 mice on day 41. Separate mice received OTII Thy1.1 TH1 cells and Ccr8−/− OTII Thy1.2 TH1 cells. Twenty-four hours later, recipient mice underwent topical sensitization with OVA for 96 h, and sensitized skin, DLN and spleen cells were analyzed by flow cytometry. () CD4+ T cells recovered from organs at time of collection. Top, mice that received TH2 cells; bottom, mice that received TH1 cells. () Homing index was calculated as the ratio of wild-type Thy1.1 to Ccr8−/− Thy1.2 cells, corrected for input ratio at time of transfer. Summary of competitive in vivo homing of wild-type and Ccr8−/− TH2 (left; n = 13–16 mice) and TH1 (right; n = 6 mice) cells from three or more experiments. () In vivo proliferation of transferred OTII Thy1.1 wild-type and OTII Thy1.2 Ccr8−/− TH2 cells in response to antigen was asses! sed by injecting sensitized recipient mice with BrdU intraperitoneally 24 h before organ collection in experiments set up as above. Left and middle, representative BrdU staining of wild-type and Ccr8−/− T cells isolated from lymph nodes. Right, summary of three experiments comparing in vivo proliferation of wild-type and Ccr8−/− T cells measured by BrdU uptake; n = 12 mice. () Amounts of TH1 cell–active (Cxcl9 and Cxcl10) and TH2–cell active (Ccl1, Ccl8, Ccl17 and Ccl22) chemokine mRNA in unsensitized and OVA-sensitized wild-type day-50 skin DLNs and spleen; n = 6 mice. ND, not detected. () Comparison of steady-state mRNA expression of Ccl1, Ccl8 and Ccl21 in lymph nodes draining various organs in normal 6- to 8-week-old mice; pooled data from n = 3–7 mice are shown as mean ± s.e.m. () Ccl1 and Ccl8 mRNA expression in organ library of representative C57BL/6 mouse. * Figure 8: Mouse CCL8–responsive TH2-R2A cells are enriched for IL-5, IL-25R, TNF and OX40. () Flow cytometry of IL-4 and IL-5 by ICS in TH2-R1, TH2-R2, and TH2-R2A cells; representative of six experiments. Below each plot is the chemotactic index of the various TH2 cells to mouse CCL8 compared to medium alone in migration assays. () Gata3 mRNA expression in paired TH2-R1 and TH2-R2 cells from four independent experiments; *P = 0.02. () Correlation of Ccr8 and Tnfrsf4 (OX40) mRNA expression in TH2-R2A cells. () Flow cytometry of TNF and IL-9 by ICS in TH2-R2A cells; representative of three to six experiments. () Enrichment of TH2-associated cytokines and receptors in TH2-R2A cells that migrated to mouse CCL8 and CXCL12 relative to medium in Transwell assay; schematic of assay shown on right. Representative of at least three experiments. () QPCR analysis of Tnf and Tnfrsf4 mRNA expression in skin biopsies of wild-type and Ccr8−/− mice after topical sensitization. Pooled data from two to three independent experiments with n = 5–9 mice per group are shown; *P = ! 0.03 and **P = 0.03 in OVA-sensitized wild-type versus Ccr8−/− mice. () Flow cytometry of IL-5 and IL-4 by ICS after phorbol myristate acetate–ionomycin activation of peripheral blood CD4+ T cells from healthy human donors. Data from same sample were gated on CCR8+CD4+ T cells (left) and bulk total CD4+ T cells (right); reflective of data from seven donors. () Summary of IL-5 and IL-4 cytokine production by circulating fresh CD4+ T cell subsets from healthy human donors. Results are shown as mean ± s.e.m., reflecting data from n = 3–7 donors. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * BC103566 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Sabina A Islam, * Daniel S Chang, * Richard A Colvin, * Mike H Byrne & * Andrew D Luster * Department of Infection, Immunity and Biochemistry, Cardiff University, Cardiff, UK. * Michelle L McCully & * Bernhard Moser * The Immunology Institute, Mount Sinai School of Medicine, New York, New York, USA. * Sergio A Lira * Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California, USA. * Israel F Charo Contributions A.D.L. screened the EST database to identify mouse Ccl8, designed the construct to express functional recombinant mouse CCL8 protein, conducted the RNA blot analysis, designed experiments and wrote the paper. D.S.C. did mouse experiments, immunohistochemistry and QPCR of skin RNA preps. R.A.C. helped devise a strategy to clone Ccr8 and helped generate Ccr2 transient transfectants. M.H.B. helped clone Ccr8. M.L.M. and B.M. generated and provided the biotinylated monoclonal antibody to human CCR8. S.A.L. provided the Ccr8−/− mice, and I.F.C. provided the Ccl8−/−Ccl12−/− and Ccl12−/− mice. S.A.I. carried out all other in vitro and in vivo experiments, designed research, analyzed data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Andrew D Luster Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–8 and Supplementary Table 1 Additional data - ECM1 controls TH2 cell egress from lymph nodes through re-expression of S1P1
- Nat Immunol 12(2):178-185 (2011)
Nature Immunology | Article ECM1 controls TH2 cell egress from lymph nodes through re-expression of S1P1 * Zhenhu Li1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuan Zhang1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiduo Liu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaodong Wu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuhan Zheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiyun Tao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kairui Mao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jie Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Guomei Lin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lin Tian1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yongyong Ji1 Search for this author in: * NPG journals * PubMed * Google Scholar * Meiling Qin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shuhui Sun2 Search for this author in: * NPG journals * PubMed * Google Scholar * Xueliang Zhu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bing Sun1, 3 Contact Bing Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 12,Pages:178–185Year published:(2011)DOI:doi:10.1038/ni.1983Received20 July 2010Accepted08 December 2010Published online09 January 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 Type 2 helper T cells (TH2) are critically involved in allergies and asthma. Here we demonstrate that extracellular matrix protein-1 (ECM1) is highly and selectively expressed in TH2 cells. ECM1 deficiency caused impaired TH2 responses and reduced allergic airway inflammation in vivo. Functional analysis demonstrated that although the TH2 polarization of ECM1-deficient cells was unimpaired, these cells had a defect in migration and were retained in peripheral lymphoid organs. This was associated with reduced expression of KLF2 and S1P1. We also found that ECM1 could directly bind the interleukin-2 (IL-2) receptor to inhibit IL-2 signaling and activate S1P1 expression. Our data identify a previously unknown function of ECM1 in regulating TH2 cell migration through control of KLF2 and S1P1 expression. View full text Figures at a glance * Figure 1: ECM1 is specifically expressed by TH2 cells. (,) Real-time PCR of Ecm1 mRNA () and immunoblot of ECM1 protein () expression in CD62LhiCD44−CD4+ T cells from C57BL/6 mice stimulated under TH1 or TH2 conditions for 5 d. (–) Real-time PCR of Ecm1 mRNA (), immunoblot of ECM1 protein () and ELISA of secreted ECM1 () in CD62LhiCD44−CD4+ T cells differentiated to different TH subsets for 4 d. () Expression of Ecm1 mRNA in sorted CD11c+, CD19+, CD8+ and CD4+ cells stimulated with lipopolysaccharide (dendritic cells (DC) and B cells) or antibodies to CD3 and CD28 (T cells). Graphs show mean ± s.e.m. of three independent experiments. Immunoblot results are representative of two independent experiments. * Figure 2: ECM1 expression is regulated by STAT6 and GATA-3. () Immunoblots from TH1 and TH2 cells of wild-type (WT) and STAT6-knockout (KO) mice cultured for 4 d. () Immunoblots from T cells transfected with retrovirus expressing STAT6-CA plasmid and harvested after sorting and restimulation for 24 h. p-STAT6, phosphorylated STAT6. () Immunoblots from TH2 cells transfected with retrovirus expressing GATA-3 siRNA (RNAi). ′RNAi ctrl′ denotes a control siRNA. () Chromatin immunoprecipitation (ChIP) from day 2 and day 3 TH2 cell samples. Four GATA-boxes are shown on the Ecm1 5′ regulatory sequence. Real-time PCR primers were designed for these GATA-box sites. Data in – are representative of three independent experiments; graph shows mean and s.e.m. * Figure 3: Proliferation and lineage commitment are unaltered in T cells from ECM1-deficient mice. () Immunoblots from CD4+ T cells of wild-type (WT) and Ecm1−/− (knockout, KO) mice activated by anti-CD3 and anti-CD28 under TH1 or TH2 conditions. () Flow cytometry analysis of T cells collected on day 4 after treatment with phorbol 12-myristate 13-acetate and ionomycin for 6 h and brefeldin A for 1 h. Numbers in quadrants represent ratios of cytokine-secreting cells. () T cell proliferation detected after 24 h restimulation of TH1 and TH2 cells. [3H]thymidine was added to the culture 84 h after TCR induction. Samples were collected 12 h later and [3H]thymidine was measured. () ELISAs from supernatant collected on day 4 under TH1 or TH2 conditions. Data in – are representative of three independent experiments; graphs show mean and s.e.m. * Figure 4: ECM1[BM]-deficient mice show impaired TH2 function owing to defective TH2 cell migration in vivo. Wild-type (WT) or Ecm1−/− (knockout, KO) bone marrow cells (1 × 107) were transferred into irradiated C57BL/6 mice. Two months later, mice were immunized with OVA and alum and challenged with aerosolized OVA as described in Online Methods. Mice immunized with PBS served as a negative control. () Immune cells counted in bronchoalveolar lavage. () ELISA showing OVA-specific IgE levels in serum. () Lung tissue sections stained with hematoxylin and eosin. Scale bar, 200 μm. (,) Numbers of different subsets of cells in spleen and LLN. CD4+, CD8+, TH1 (IFN-γ+), TH2 (IL-4+), TH17 (IL-17A+) and Treg (Foxp3+) cells were measured by flow cytometry. () ELISA showing cytokine production from supernatants of draining lymph node cells restimulated with OVA for 96 h. *P < 0.05. Results in – are representative of three independent experiments; graphs show mean and s.e.m. * Figure 5: ECM1 functions in control of TH2 cell migration. Shown are numbers of CFSE+ TH1 cells or CFSE+ TH2 cells in spleen, blood, inguinal lymph nodes (iLN) and mesenteric lymph node (MLN), 24 h after TH1 and TH2 cells were cultured for 4 d, labeled with CFSE and injected intravenously into C57BL/6 mice. WT, wild-type mice; KO, ECM1-knockout mice. *P < 0.05. Graphs show mean and s.e.m. from three independent experiments. * Figure 6: ECM1 deficiency reduces expression of S1P1 in TH2 cells. () mRNA levels determined by real-time PCR of TH1 and TH2 cells from wild-type (WT) and ECM1-knockout (KO) mice cultured for 4 d. Sell encodes L-selectin. () mRNA levels for the transcription factors KLF2 and Foxo1 detected by real-time PCR. () mRNA levels and immunoblots from WT and KO TH2 cells infected by ECM1-expressing retrovirus as described in Online Methods; data were obtained 4 d after TCR signaling treatment. () Chemotactic response of TH2 cells infected with a control retrovirus vector or one encoding Ecm1 from WT and KO mice. Shown is the percentage of the input cell population that responded to each concentration of S1P. () Numbers of CFSE+ WT TH2 cells or CFSE+ KO TH2 cells in spleen, blood, inguinal lymph nodes (iLN) and mesenteric lymph node (MLN) of injected mice. WT or KO TH2 cells were transfected with Ecm1, Klf2, S1pr1 or control plasmid, cultured for 4 d and injected intravenously into C57BL/6 mice; 24 h later, 100 μg antibody to L-selectin was injected! intravenously, and CFSE+ cell numbers were measured 1 d later. *P < 0.05; **P < 0.01. Data in – are representative of three independent experiments; graphs show mean ± s.e.m. * Figure 7: Secreted ECM1 functions through extracellular pathways. () Separation of co-cultured wild-type (WT) and ECM1-knockout (KO) TH2 cells by flow cytometry. WT and KO TH2 cells were labeled with 5 μM CFSE 3 d after anti-CD3/CD28 treatment, and WT TH2 cells with no CFSE were co-cultured with KO TH2 cells for 24 h. (,) Chemotactic response (, measured as in Fig. 6d) and mRNA levels (, measured by real-time PCR) of WT and KO cells cultured separately or co-cultured as in . () Numbers of CFSE+ cells in spleen, blood, iLN and MLN of mice injected with separately cultured or co-cultured cells. Three days after TCR engagement, WT or KO TH2 cells were labeled with different concentrations of CFSE (WT, 0.5 μM; KO, 5 μM) and co-cultured for 24 h. Cells were injected intravenously into C57BL/6 mice. After 24 h, 100 μg anti-L-selectin was injected intravenously. CFSE+ cell numbers were measured 1 d later. *P < 0.05. Data in – are representative of three independent experiments; graphs show mean ± s.e.m. * Figure 8: ECM1 promotes S1P1 expression by binding to IL-2R and inhibiting IL-2 signaling. () mRNA levels determined by real-time PCR in wild-type (WT) or ECM1-knockout (KO) TH2 cells cultured for a total of 4 d, with the inhibitors LY294002 and rapamycin (Rap) added 24 h before termination of culture. Med, medium alone (negative control). (,) Immunoblots () and mRNA levels measured by real-time PCR () from WT or KO TH2 cells cultured for 4 d with mouse IL-2 (mIL-2) neutralizing antibody added after 2 d to block human IL-2 (hIL-2) signaling. () Immunoprecipitation (IP) and immunoblot (IB) analysis of lysates of HEK293T cells expressing ECM1 and various IL-2 receptor units. Ctrl, positive control; V, vector; E, ECM1; WCL, whole-cell lysis. () CD25, CD122 and CD132 expression determined by flow cytometry of TH2 cells. (,) Immunoblots () and mRNA levels measured by real-time PCR () from TH2 cells prepared as in , and subjected to ECM1 co-culture as in Figure 7. ECM1 neutralizing antibody was used for blocking secreted ECM1 protein. Ut, untreated; Co-cu, co-culture. *! P < 0.05. Data in – are representative of three experiments; graphs show mean and s.e.m. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE19707 * GSM491757 * GSM491758 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Zhenhu Li & * Yuan Zhang Affiliations * Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. * Zhenhu Li, * Yuan Zhang, * Zhiduo Liu, * Xiaodong Wu, * Yuhan Zheng, * Zhiyun Tao, * Kairui Mao, * Jie Wang, * Guomei Lin, * Lin Tian, * Yongyong Ji, * Meiling Qin, * Xueliang Zhu & * Bing Sun * Fudan University School of Medicine, Shanghai, China. * Shuhui Sun * Molecular Virus Unit, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China. * Bing Sun Contributions Z. Li designed (with help from Z. Liu) and performed mouse experiments and in vitro cell culture experiments, analyzed data and wrote the manuscript; Y. Zhang did all western blotting and real-time PCR experiments and helped in mouse experiments and analyzing data; X.W., K.M. and Y. Zheng helped in mouse experiments; Z.T. bred mice and confirmed the Ecm1−/− genotype; J.W. helped with immunoprecipitation experiments; G.L., L.T. and Y.J. made two ECM1 antibodies; M.Q. supplied reagents; S.S. performed flow cytometry; X.Z. helped design experiments and edit the manuscript; and B.S. conceived of the research, directed the study and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bing Sun Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (632K) Supplementary Figures 1–10 Additional data
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