Cholesterol metabolism drives regulatory B cell IL-10 through provision of geranylgeranyl pyrophosphate.

Animals Antigens, CD19 / metabolism B-Lymphocytes, Regulatory / metabolism Cholesterol / metabolism Class I Phosphatidylinositol 3-Kinases / metabolism Coculture Techniques Hereditary Autoinflammatory Diseases / metabolism Humans Interleukin-10 / metabolism Macrophages / metabolism Metabolic Syndrome / metabolism Mevalonate Kinase Deficiency / metabolism Mice Phosphatidylinositol 3-Kinases / metabolism Polyisoprenyl Phosphates / metabolism Positive Regulatory Domain I-Binding Factor 1 / metabolism Principal Component Analysis Signal Transduction Th1 Cells / metabolism Toll-Like Receptor 9 / metabolism Tumor Necrosis Factor-alpha / metabolism

Journal

Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555

Informations de publication

Date de publication:
08 07 2020
Historique:
received: 15 11 2019
accepted: 04 06 2020
entrez: 10 7 2020
pubmed: 10 7 2020
medline: 9 9 2020
Statut: epublish

Résumé

Regulatory B cells restrict immune and inflammatory responses across a number of contexts. This capacity is mediated primarily through the production of IL-10. Here we demonstrate that the induction of a regulatory program in human B cells is dependent on a metabolic priming event driven by cholesterol metabolism. Synthesis of the metabolic intermediate geranylgeranyl pyrophosphate (GGPP) is required to specifically drive IL-10 production, and to attenuate Th1 responses. Furthermore, GGPP-dependent protein modifications control signaling through PI3Kδ-AKT-GSK3, which in turn promote BLIMP1-dependent IL-10 production. Inherited gene mutations in cholesterol metabolism result in a severe autoinflammatory syndrome termed mevalonate kinase deficiency (MKD). Consistent with our findings, B cells from MKD patients induce poor IL-10 responses and are functionally impaired. Moreover, metabolic supplementation with GGPP is able to reverse this defect. Collectively, our data define cholesterol metabolism as an integral metabolic pathway for the optimal functioning of human IL-10 producing regulatory B cells.

Identifiants

DOI: 10.1038/s41467-020-17179-4 PMID: 32641742 PMC: PMC7343868
pubmed: 32641742
doi: 10.1038/s41467-020-17179-4
pii: 10.1038/s41467-020-17179-4
pmc: PMC7343868
doi:

Substances chimiques

Antigens, CD19 0
CD19 molecule, human 0
IL10 protein, human 0
Polyisoprenyl Phosphates 0
TLR9 protein, human 0
Toll-Like Receptor 9 0
Tumor Necrosis Factor-alpha 0
Interleukin-10 130068-27-8
PRDM1 protein, human 138415-26-6
Cholesterol 97C5T2UQ7J
Positive Regulatory Domain I-Binding Factor 1 EC 2.1.1.-
Class I Phosphatidylinositol 3-Kinases EC 2.7.1.137
PIK3CD protein, human EC 2.7.1.137
geranylgeranyl pyrophosphate N21T0D88LX

Types de publication

Journal Article Research Support, N.I.H., Intramural Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

3412

Subventions

Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 206618/Z/17/Z
Pays : United Kingdom
Organisme : Department of Health
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/M012328/2
Pays : United Kingdom

Références

Rosser, E. C. & Mauri, C. Regulatory B cells: origin, phenotype, and function. Immunity 42, 607–612 (2015).
pubmed: 25902480
Lykken, J. M., Candando, K. M. & Tedder, T. F. Regulatory B10 cell development and function. Int. Immunol. 27, 471–477 (2015).
pubmed: 26254185 pmcid: 4817073
Lino, A. C. et al. LAG-3 inhibitory receptor expression identifies immunosuppressive natural regulatory plasma cells. Immunity 49, 120–133 (2018).
pubmed: 30005826 pmcid: 6057275
Rojas, O. L. et al. Recirculating intestinal IgA-producing cells regulate neuroinflammation via IL-10. Cell 176, 610–624 (2019).
pubmed: 30612739 pmcid: 6903689
Blair, P. A. et al. CD19+CD24hiCD38hi B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic lupus erythematosus patients. Immunity 32, 129–140 (2010).
pubmed: 20079667
Iwata, Y. et al. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood 117, 530–541 (2011).
pubmed: 20962324 pmcid: 3031478
Mauri, C., Gray, D., Mushtaq, N. & Londei, M. Prevention of arthritis by interleukin 10-producing B cells. J. Exp. Med. 197, 489–501 (2003).
pubmed: 12591906 pmcid: 2193864
Shen, P. et al. IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases. Nature 507, 366–370 (2014).
pubmed: 24572363 pmcid: 4260166
Shalapour, S. et al. Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy. Nature 521, 94–98 (2015).
pubmed: 25924065 pmcid: 4501632
Mizoguchi, A., Mizoguchi, E., Smith, R. N., Preffer, F. I. & Bhan, A. K. Suppressive role of B cells in chronic colitis of T cell receptor α mutant mice. J. Exp. Med. 186, 1749–1756 (2002).
Flores-Borja, F. et al. CD19+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci. Transl. Med. 5, 173ra23 (2013).
pubmed: 23427243
Menon, M., Blair, P. A., Isenberg, D. A. & Mauri, C. A regulatory feedback between plasmacytoid dendritic cells and regulatory B cells is aberrant in systemic lupus erythematosus. Immunity 44, 683–697 (2016).
pubmed: 26968426 pmcid: 4803914
Yoshizaki, A. et al. Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions. Nature 491, 264–268 (2012).
pubmed: 23064231 pmcid: 3493692
Stark, A. K. et al. PI3Kδ hyper-activation promotes development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner. Nat. Commun. https://doi.org/10.1038/s41467-018-05674-8 (2018).
Liu, B. S., Cao, Y., Huizinga, T. W., Hafler, D. A. & Toes, R. E. M. TLR-mediated STAT3 and ERK activation controls IL-10 secretion by human B cells. Eur. J. Immunol. 44, 2121–2129 (2014).
pubmed: 24737107
Buck, M. D., Sowell, R. T., Kaech, S. M. & Pearce, E. L. Cell 169, 570–586 (2017).
pubmed: 28475890 pmcid: 5648021
Jung, J., Zeng, H. & Horng, T. Metabolism as a guiding force for immunity. Nat. Cell Biol. 21, 85–93, https://doi.org/10.1038/s41556-018-0217-x (2019).
doi: 10.1038/s41556-018-0217-x pubmed: 30602764
Waters, L. R., Ahsan, F. M., Wolf, D. M., Shirihai, O. & Teitell, M. A. Initial B cell activation induces metabolic reprogramming and mitochondrial remodeling. iScience https://doi.org/10.1016/j.isci.2018.07.005 (2018).
doi: 10.1016/j.isci.2018.07.005 pubmed: 30240649 pmcid: 6123864
Caro-Maldonado, A. et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J. Immunol. 192, 3626–3636 (2014).
pubmed: 24616478 pmcid: 3984038
Jellusova, J. et al. Gsk3 is a metabolic checkpoint regulator in B cells. Nat. Immunol. 18, 303–312 (2017).
pubmed: 28114292 pmcid: 5310963
O’Neill, L. A. J., Kishton, R. J. & Rathmell, J. A guide to immunometabolism for immunologists. Nat. Rev. Immunol. 16, 553–565 (2016).
pubmed: 27396447 pmcid: 5001910
Perucha, E. et al. The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells. Nat. Commun. https://doi.org/10.1038/s41467-019-08332-9 (2019).
Akula, M. K. et al. Control of the innate immune response by the mevalonate pathway. Nat. Immunol. 17, 922–929 (2016).
pubmed: 27270400 pmcid: 4955724
Sjogren, A. K. M. et al. GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer. J. Clin. Invest. 117, 1294–1304 (2007).
pubmed: 17476360 pmcid: 1857236
ter Haar, N. M. et al. The phenotype and genotype of mevalonate kinase deficiency: a series of 114 cases from the eurofever registry. Arthritis Rheumatol. 68, 2795–2805 (2016).
pubmed: 27213830
Favier, L. A. & Schulert, G. S. Mevalonate kinase deficiency: current perspectives. Appl. Clin. Genet. https://doi.org/10.2147/TACG.S93933 (2016).
Wang, M. & Casey, P. J. Protein prenylation: unique fats make their mark on biology. Nat. Rev. Mol. Cell Biol. 17, 110–122 (2016).
pubmed: 26790532
Maurer-Stroh, S. et al. Towards complete sets of farnesylated and geranylgeranylated proteins. PLoS Comput. Biol. https://doi.org/10.1371/journal.pcbi.0030066 (2007).
Martin, M., Rehani, K., Jope, R. S. & Michalek, S. M. Toll-like receptor—mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat. Immunol. 6, 777–784 (2005).
pubmed: 16007092 pmcid: 1933525
Lambert, S. A. et al. The human transcription factors. Cell 172, 650–665 (2018).
pubmed: 29425488 pmcid: 29425488
Pro, S. C. et al. Global landscape of mouse and human cytokine transcriptional regulation. Nucleic Acids Res. 46, 9321–9337 (2018).
Heine, G. et al. Autocrine IL-10 promotes human B-cell differentiation into IgM- or IgG-secreting plasmablasts. Eur. J. Immunol. 44, 1615–1621 (2014).
pubmed: 24643722
Minnich, M. et al. Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation. Nat. Immunol. 17, 331–343 (2016).
pubmed: 26779602 pmcid: 5790184
Balow, J. E. et al. Microarray-based gene expression profiling in patients with cryopyrin-associated periodic syndromes defines a disease-related signature and IL-1-responsive transcripts. Ann. Rheum. Dis. 72, 1064–1070 (2013).
pubmed: 23223423
Doughty, C. A. et al. Antigen receptor-mediated changes in glucose metabolism in B lymphocytes: role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth. Blood 107, 4458–4465 (2006).
pubmed: 16449529 pmcid: 1895797
Zeng, H. et al. MTORC1 couples immune signals and metabolic programming to establish Treg-cell function. Nature 499, 485–490 (2013).
pubmed: 23812589 pmcid: 3759242
Chellappa, S. et al. The PI3K p110δ isoform inhibitor idelalisib preferentially inhibits human regulatory T cell function. J. Immunol. 202, 1397–1405 (2019).
pubmed: 30692213
Gbelcová, H. et al. Isoprenoids responsible for protein prenylation modulate the biological effects of statins on pancreatic cancer cells. Lipids Health Dis. https://doi.org/10.1186/s12944-017-0641-0 (2017).
doi: 10.1186/s12944-017-0641-0 pubmed: 29262834 pmcid: 5738693
Ali, B. R., Nouvel, I., Leung, K. F., Hume, A. N. & Seabra, M. C. A novel statin-mediated ‘prenylation block-and-release’ assay provides insight into the membrane targeting mechanisms of small GTPases. Biochem. Biophys. Res. Commun. 397, 34–41 (2010).
pubmed: 20471365 pmcid: 2908739
Khan, O. M. et al. Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice. J. Clin. Invest. 121, 628–639 (2011).
pubmed: 21266780 pmcid: 3026725
López-Posadas, R. et al. Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation. J. Clin. Invest. 126, 611–626 (2016).
pubmed: 26752649 pmcid: 4731169
Lighaam, L. C. et al. In vitro-induced human IL-10+ B cells do not show a subset-defining marker signature and plastically co-express IL-10 with pro-inflammatory cytokines. Front. Immunol. https://doi.org/10.3389/fimmu.2018.01913 (2018).
Dil, N. & Marshall, A. J. Role of phosphoinositide 3-kinase p110δ in TLR4- and TLR9-mediated B cell cytokine production and differentiation. Mol. Immunol. 46, 1970–1978 (2009).
pubmed: 19362372
Kramer, P. R., Winger, V. & Reuben, J. PI3K limits TNF-α production in CD16-activated monocytes. Eur. J. Immunol. 39, 561–570 (2009).
pubmed: 19180470 pmcid: 2734280
Fukao, T. et al. P13K-mediated negative feedback regulation of IL-12 production in DCs. Nat. Immunol. 3, 875–881 (2002).
pubmed: 12154357
Hill, E. V. et al. Glycogen synthase kinase-3 controls IL-10 expression in CD4+ effector T-cell subsets through epigenetic modification of the IL-10 promoter. Eur. J. Immunol. 45, 1103–1115 (2015).
pubmed: 25627813 pmcid: 4405077
Neumann, C. et al. Role of Blimp-1 in programing Th effector cells into IL-10 producers. J. Exp. Med. 211, 1807–1819 (2014).
pubmed: 25073792 pmcid: 4144744
Wang, Y. H. et al. Blimp-1 contributes to the development and function of regulatory B cells. Front. Immunol. https://doi.org/10.3389/fimmu.2019.01909 (2019).
Alexander, L. E. M. M. et al. Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival. Mol. Immunol. https://doi.org/10.1016/j.molimm.2017.08.016 (2017).
Wang, R.-X. et al. Interleukin-35 induces regulatory B cells that suppress autoimmune disease. Nat. Med. 20, 633–641 (2014).
pubmed: 24743305 pmcid: 4048323
Matsumoto, M. et al. Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation. Immunity 41, 1040–1051 (2014).
pubmed: 25484301
Jenks, S. A. et al. Distinct effector B cells induced by unregulated Toll-like receptor 7 contribute to pathogenic responses in systemic lupus erythematosus. Immunity 49, 725–739 (2018).
pubmed: 30314758 pmcid: 6217820
Munoz, M. A. et al. Defective protein prenylation is a diagnostic biomarker of mevalonate kinase deficiency. J. Allergy Clin. Immunol. 140, 873–875 (2017).
pubmed: 28501347
Park, Y. H., Wood, G., Kastner, D. L. & Chae, J. J. Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS. Nat. Immunol. 17, 914–921 (2016).
pubmed: 27270401 pmcid: 4955684
Bekkering, S. et al. Metabolic induction of trained immunity through the mevalonate pathway. Cell 172, 135–146 (2018).
pubmed: 29328908
Lin, X. et al. IL-10-producing regulatory B cells restrain the T follicular helper cell response in primary Sjögren’s syndrome. Cell. Mol. Immunol. 16, 921–931 (2019).
pubmed: 30948793
Lal, G. et al. IL-10 from marginal zone precursor B cells controls the differentiation of Th17, Tfh and Tfr cells in transplantation tolerance. Immunol. Lett. https://doi.org/10.1016/j.imlet.2016.01.002 (2016).
Okkenhaug, K. et al. Impaired B and T cell antigen receptor signaling in p110δ PI 3-kinase mutant mice. Science 297, 1031–1034 (2002).
pubmed: 12130661

Auteurs

Jack A Bibby (JA)

Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial Sciences, King's College London, London, SE1 1UL, UK. jack.bibby@nih.gov.
Complement and Inflammation Research Section (CIRS), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA. jack.bibby@nih.gov.
ORCID: 0000-0002-0875-446X

Harriet A Purvis (HA)

Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial Sciences, King's College London, London, SE1 1UL, UK.
ORCID: 0000-0001-5271-4863

Thomas Hayday (T)

Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial Sciences, King's College London, London, SE1 1UL, UK.

Anita Chandra (A)

Division of Immunology, Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK.
ORCID: 0000-0002-9061-879X

Klaus Okkenhaug (K)

Division of Immunology, Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, UK.
ORCID: 0000-0002-9432-4051

Sofia Rosenzweig (S)

Inflammatory Disease Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
ORCID: 0000-0003-2243-5315

Ivona Aksentijevich (I)

Inflammatory Disease Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Michael Wood (M)

National Amyloidosis Centre, Division of Medicine, University College London and Royal Free Hospital London NHS Foundation Trust, London, NW3 2PF, UK.

Helen J Lachmann (HJ)

National Amyloidosis Centre, Division of Medicine, University College London and Royal Free Hospital London NHS Foundation Trust, London, NW3 2PF, UK.

Claudia Kemper (C)

Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial Sciences, King's College London, London, SE1 1UL, UK.
Complement and Inflammation Research Section (CIRS), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany.
ORCID: 0000-0003-4196-1417

Andrew P Cope (AP)

Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial Sciences, King's College London, London, SE1 1UL, UK. andrew.cope@kcl.ac.uk.
Centre for Rheumatic Diseases, King's College London, London, SE1 1UL, UK. andrew.cope@kcl.ac.uk.
ORCID: 0000-0003-0470-1827

Esperanza Perucha (E)

Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial Sciences, King's College London, London, SE1 1UL, UK. esperanza.perucha@kcl.ac.uk.
Centre for Rheumatic Diseases, King's College London, London, SE1 1UL, UK. esperanza.perucha@kcl.ac.uk.
ORCID: 0000-0002-7802-0875

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Classifications MeSH

questionsmedicales.fr › Eucaryotes › Animaux › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Facteurs biologiques › Marqueurs biologiques › Antigènes de différenciation › Antigènes de différenciation des lymphocytes B › Antigènes CD19 › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Systèmes sanguin et immunitaire › Système immunitaire › Leucocytes › Agranulocytes › Lymphocytes › Lymphocytes B › Sous-populations de lymphocytes B › Lymphocytes B régulateurs › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Lipides › Lipides membranaires › Stérols › Cholestérol › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines et peptides de signalisation intracellulaire › Phosphatidylinositol 3-kinases › Phosphatidylinositol-4-phosphate 3-kinase › Phosphatidylinositol 3-kinases de classe I › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Techniques d'investigation › Techniques in vitro › Techniques de culture › Techniques de coculture › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Maladies de la peau et du tissu conjonctif › Maladies de la peau › Maladies génétiques de la peau › Maladies auto-inflammatoires héréditaires › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Facteurs biologiques › Protéines et peptides de signalisation intercellulaire › Cytokines › Interleukines › Interleukine-10 › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Systèmes sanguin et immunitaire › Système immunitaire › Phagocytes › Macrophages › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Maladies métaboliques et nutritionnelles › Maladies métaboliques › Syndrome métabolique X › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Maladies du système immunitaire › Syndromes immunoprolifératifs › Déficit en mévalonate kinase › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Rodentia › Muridae › Murinae › Souris › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines et peptides de signalisation intracellulaire › Phosphatidylinositol 3-kinases › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Composés chimiques organiques › Composés organiques du phosphore › Organophosphates › Polyisoprényl-phosphates › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Facteurs de transcription › Protéines de répression › Facteur-1 liant le domaine de régulation positive I › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Techniques d'investigation › Analyse en composantes principales › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Phénomènes physiologiques cellulaires › Transduction du signal › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Systèmes sanguin et immunitaire › Système immunitaire › Leucocytes › Agranulocytes › Lymphocytes › Lymphocytes T › Sous-populations de lymphocytes T › Lymphocytes T auxiliaires › Lymphocytes auxiliaires Th1 › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines membranaires › Récepteurs de surface cellulaire › Récepteurs immunologiques › Récepteurs de reconnaissance de motifs moléculaires › Récepteurs de type Toll › Récepteur-9 de type Toll-like › Cholesterol metabolism drives regulatory B...
questionsmedicales.fr › Facteurs biologiques › Protéines et peptides de signalisation intercellulaire › Cytokines › Facteurs de nécrose tumorale › Facteur de nécrose tumorale alpha › Cholesterol metabolism drives regulatory B...