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Direct and Indirect Induction by 1,25-Dihydroxyvitamin D3 of the NOD2/CARD15-Defensin β2 Innate Immune Pathway Defective in Crohn Disease*

Open AccessPublished:November 30, 2009DOI:https://doi.org/10.1074/jbc.C109.071225
      Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D3, robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-κB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin β2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D3 synergistically induced NF-κB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohn’s pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

      Introduction

      Vitamin D has a range of physiological activities (
      • Lin R.
      • White J.H.
      ) and has emerged recently as a direct regulator of immune system function in humans (
      • Liu P.T.
      • Modlin R.L.
      ,
      • White J.H.
      ). It is obtained from limited dietary sources and ultraviolet B-induced conversion of 7-dehydrocholesterol to vitamin D3 in skin (
      • Lin R.
      • White J.H.
      ). Hormonal 1,25-dihydroxyvitamin D3 (1,25D)
      The abbreviations used are: 1,25D
      1,25-dihydroxyvitamin D3
      25D
      25-hydroxyvitamin D3
      AMP
      antimicrobial peptides
      CAMP
      cathelicidin antimicrobial peptide
      CARD15
      caspase-recruitment domain-containing protein 15
      CD
      Crohn disease
      HBD2
      human defensin β2
      HBD3
      human defensin β3
      HIEC
      human crypt intestinal epithelial cells
      IBD
      inflammatory bowel disease
      NOD2
      nucleotide-binding oligomerization domain 2
      MDP
      muramyl dipeptide
      pol II
      RNA polymerase II
      TLR
      toll-like receptor
      TSS
      transcription start site
      VDR
      vitamin D receptor
      VDRE
      vitamin D-response element
      ChIP
      chromatin immunoprecipitation
      3C
      chromosome conformation capture
      BAC
      bacterial artificial chromosome
      qPCR
      quantitative PCR
      RT
      reverse transcription
      LPS
      lipopolysaccharide.
      is produced by hepatic generation of 25-hydroxvitamin D3 (25D), the major circulating metabolite, followed by CYP27B1-catalyzed 1α-hydroxylation in kidney and peripheral tissues (
      • Lin R.
      • White J.H.
      ,
      • Prosser D.E.
      • Jones G.
      ,
      • Holick M.F.
      ). Notably, activated macrophages and dendritic cells express CYP27B1 (
      • Liu P.T.
      • Modlin R.L.
      ,
      • White J.H.
      ), which, unlike the renal enzyme, is regulated primarily by immune inputs. 1,25D signals through its cognate VDR nuclear receptor (
      • Lin R.
      • White J.H.
      ,
      • Rochel N.
      • Wurtz J.M.
      • Mitschler A.
      • Klaholz B.
      • Moras D.
      ), which binds DNA as a heterodimer with retinoid X receptors to vitamin D-response elements (VDREs), direct repeats of PuG(G/T)TCA motifs separated by 3 bp (DR3) (
      • Lin R.
      • White J.H.
      ).
      A molecular basis for regulation by vitamin D of innate immunity emerged with observations by ourselves (
      • Wang T.T.
      • Nestel F.P.
      • Bourdeau V.
      • Nagai Y.
      • Wang Q.
      • Liao J.
      • Tavera-Mendoza L.
      • Lin R.
      • Hanrahan J.W.
      • Mader S.
      • White J.H.
      ) and others (
      • Gombart A.F.
      • Borregaard N.
      • Koeffler H.P.
      ,
      • Weber G.
      • Heilborn J.D.
      • Chamorro Jimenez C.I.
      • Hammarsjo A.
      • Törmä H.
      • Stahle M.
      ) that 1,25D induces expression of genes encoding antimicrobial peptides (AMPs), vanguards of responses against microbial pathogens (
      • White J.H.
      ,
      • Jenssen H.
      • Hamill P.
      • Hancock R.E.
      ). 1,25D modestly induced defensin β2 (DEFB2/HBD2) and strongly stimulated cathelicidin antimicrobial peptide (CAMP) gene expression through consensus VDREs (
      • Wang T.T.
      • Nestel F.P.
      • Bourdeau V.
      • Nagai Y.
      • Wang Q.
      • Liao J.
      • Tavera-Mendoza L.
      • Lin R.
      • Hanrahan J.W.
      • Mader S.
      • White J.H.
      ). Neither VDRE is conserved in mice, and the element in the CAMP gene is imbedded in a human/primate-specific Alu repeat (
      • Gombart A.F.
      • Borregaard N.
      • Koeffler H.P.
      ). Notably, human macrophages stimulated through toll-like receptors (TLRs) strongly induced CYP27B1 and VDR expression, rendering cells responsive to circulating levels of 25D (
      • Liu P.T.
      • Stenger S.
      • Li H.
      • Wenzel L.
      • Tan B.H.
      • Krutzik S.R.
      • Ochoa M.T.
      • Schauber J.
      • Wu K.
      • Meinken C.
      • Kamen D.L.
      • Wagner M.
      • Bals R.
      • Steinmeyer A.
      • Zügel U.
      • Gallo R.L.
      • Eisenberg D.
      • Hewison M.
      • Hollis B.W.
      • Adams J.S.
      • Bloom B.R.
      • Modlin R.L.
      ). This study showed that the magnitude of the downstream induction of CAMP by 25D in TLR-stimulated macrophages was strongly dependent on variations in 25D levels within the physiological range (
      • Liu P.T.
      • Stenger S.
      • Li H.
      • Wenzel L.
      • Tan B.H.
      • Krutzik S.R.
      • Ochoa M.T.
      • Schauber J.
      • Wu K.
      • Meinken C.
      • Kamen D.L.
      • Wagner M.
      • Bals R.
      • Steinmeyer A.
      • Zügel U.
      • Gallo R.L.
      • Eisenberg D.
      • Hewison M.
      • Hollis B.W.
      • Adams J.S.
      • Bloom B.R.
      • Modlin R.L.
      ).
      At higher latitudes, solar ultraviolet B is insufficient for cutaneous vitamin D3 synthesis for periods of several months around winter. This, coupled with vitamin D-poor diets, leads to seasonal variations in circulating 25D levels and widespread vitamin D deficiency (
      • White J.H.
      ,
      • Holick M.F.
      ), which has been linked to certain cancers and to infectious and autoimmune diseases. Clinical and preclinical studies (e.g. Refs.
      • Lim W.C.
      • Hanauer S.B.
      • Li Y.C.
      and
      • Liu N.
      • Nguyen L.
      • Chun R.F.
      • Lagishetty V.
      • Ren S.
      • Wu S.
      • Hollis B.
      • DeLuca H.F.
      • Adams J.S.
      • Hewison M.
      ) have suggested that 1,25D deficiency may contribute to the pathogenesis of inflammatory bowel diseases (IBD), including Crohn disease (CD), a chronic incurable inflammatory condition, which is believed to arise from defective innate immune regulation of intestinal bacterial load (
      • Lim W.C.
      • Hanauer S.B.
      • Li Y.C.
      ,
      • Cho J.H.
      ). North-South gradients in rates of CD have been described in Europe and North America (
      • Lim W.C.
      • Hanauer S.B.
      • Li Y.C.
      ,
      • Loftus Jr., E.V.
      • Sandborn W.J.
      ,
      • Loftus Jr., E.V.
      ), although data concerning seasonal variations in CD relapse rates are conflicting (
      • Zeng L.
      • Anderson F.H.
      ,
      • Lewis J.D.
      • Aberra F.N.
      • Lichtenstein G.R.
      • Bilker W.B.
      • Brensinger C.
      • Strom B.L.
      ,
      • Aratari A.
      • Papi C.
      • Galletti B.
      • Angelucci E.
      • Viscido A.
      • D'Ovidio V.
      • Ciaco A.
      • Abdullahi M.
      • Caprilli R.
      ). In addition, VDR gene polymorphisms correlate with susceptibility to CD and to ulcerative colitis (
      • Simmons J.D.
      • Mullighan C.
      • Welsh K.I.
      • Jewell D.P.
      ,
      • Martin K.
      • Radlmayr M.
      • Borchers R.
      • Heinzlmann M.
      • Folwaczny C.
      ). Associations between VDR signaling and IBD are also supported by animal models, where vitamin D sufficiency is associated with reduced frequency of onset and improved IBD symptoms in animals with established disease (
      • Cantorna M.T.
      • Munsick C.
      • Bemiss C.
      • Mahon B.D.
      ,
      • Froicu M.
      • Weaver V.
      • Wynn T.A.
      • McDowell M.A.
      • Welsh J.E.
      • Cantorna M.T.
      ).
      Here we provide further evidence for the key role of vitamin D signaling in regulating innate immunity in humans and its links to inflammatory bowel disease. We find that 1,25D is a direct and robust inducer of expression of the gene encoding pattern recognition receptor NOD2/CARD15/IBD1 in cells of monocytic and epithelial origin. NOD2/CARD15 detects muramyl dipeptide (MDP), a lysosomal breakdown product of bacterial peptidoglycan common to Gram-minus and Gram-positive bacteria (
      • Girardin S.E.
      • Boneca I.G.
      • Viala J.
      • Chamaillard M.
      • Labigne A.
      • Thomas G.
      • Philpott D.J.
      • Sansonetti P.J.
      ,
      • Inohara N.
      • Ogura Y.
      • Fontalba A.
      • Gutierrez O.
      • Pons F.
      • Crespo J.
      • Fukase K.
      • Inamura S.
      • Kusumoto S.
      • Hashimoto M.
      • Foster S.J.
      • Moran A.P.
      • Fernandez-Luna J.L.
      • Nuñez G.
      ). We also find that pretreatment of cells with 1,25D leads to a synergistic induction of genes encoding HBD2 and CAMP upon introduction of MDP in cells expressing functional NOD2 but not in cells from patients with Crohn disease homozygous for an inactivating mutation of the NOD2 gene. These studies provide a key link between vitamin D signaling and the genetics of Crohn disease as attenuated NOD2 or HBD2 function has been linked to the CD pathogenesis (
      • Lim W.C.
      • Hanauer S.B.
      • Li Y.C.
      ,
      • Hugot J.P.
      • Chamaillard M.
      • Zouali H.
      • Lesage S.
      • Cézard J.P.
      • Belaiche J.
      • Almer S.
      • Tysk C.
      • O’Morain C.A.
      • Gassull M.
      • Binder V.
      • Finkel Y.
      • Cortot A.
      • Modigliani R.
      • Laurent-Puig P.
      • Gower-Rousseau C.
      • Macry J.
      • Colombel J.F.
      • Sahbatou M.
      • Thomas G.
      ,
      • Ogura Y.
      • Bonen D.K.
      • Inohara N.
      • Nicolae D.L.
      • Chen F.F.
      • Ramos R.
      • Britton H.
      • Moran T.
      • Karaliuskas R.
      • Duerr R.H.
      • Achkar J.P.
      • Brant S.R.
      • Bayless T.M.
      • Kirschner B.S.
      • Hanauer S.B.
      • Nuñez G.
      • Cho J.H.
      ,
      • Fellermann K.
      • Stange D.E.
      • Schaeffeler E.
      • Schmalzl H.
      • Wehkamp J.
      • Bevins C.L.
      • Reinisch W.
      • Teml A.
      • Schwab M.
      • Lichter P.
      • Radlwimmer B.
      • Stange E.F.
      ), and show that NOD2 regulation by 1,25D markedly affects the amplitude of downstream AMP gene regulation induced by MDP.

      Acknowledgments

      We are grateful to Dr. J. F. Beaulieu, Université de Sherbrooke, for kindly providing HIEC cells, to Dr. Josée Dostie (Department of Biochemistry, McGill University) for advice with 3C assays, and to Marianna Orlova (McGill University) for assistance with NOD2 genotyping.

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