title: Crohn's disease-associated ATG16L1 polymorphism modulates pro-inflammatory cytokine responses selectively upon activation of NOD2.
abstract: OBJECTIVE
  Autophagy has recently been shown to modulate the production of pro-inflammatory cytokine production and to contribute to antigen processing and presentation through the major histocompatibility complex. Genetic variation in the autophagy gene ATG16L1 has been recently implicated in Crohn's disease pathogenesis. The mechanisms underlying this association are not yet known, although experimental models suggest an inhibitory effect of autophagy on interleukin 1β (IL-1β) responses. Here, the effect of ATG16L1 genetic variation on cytokine responses has been assessed in humans.
  
  
  METHODS
  Peripheral blood mononuclear cells from healthy individuals and patients with Crohn's disease with different ATG16L1 genotypes were stimulated with ligands for Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerisation domain 2 (NOD2), with or without the autophagy inhibitor 3-methyladenine. Induction of cytokine production and related factors were measured at the mRNA and protein level. Furthermore, protein levels of ATG16L1 were assessed by western blot.
  
  
  RESULTS
  The present study demonstrates that cells isolated from individuals bearing the ATG16L1 Thr300Ala risk variant, which is shown to affect ATG16L1 protein expression upon NOD2 stimulation, display increased production of the pro-inflammatory cytokines IL-1β and IL-6, specifically after stimulation with NOD2 ligands. In contrast, no differences were found when cells were stimulated with TLR2 or TLR4 agonists. These findings were confirmed in two independent cohorts of volunteers and in a group of patients with Crohn's disease. The increased production could be ascribed to increased mRNA expression, while processing of pro-IL-1β by caspase-1 activation was not affected. The effect of the ATG16L1 polymorphism was abrogated when autophagy was blocked.
  
  
  CONCLUSIONS
  The present study is the first to link the ATG16L1 polymorphism with an excessive production of IL-1β and IL-6 in humans, which may explain the effects of this polymorphism on the inflammatory process in Crohn's disease.
authors.affiliation: Department of Medicine, Radboud University, Nijmegen Medical Centre, Internal postal code 463, P.O. Box 9101, Geert Grooteplein 8, 6500 HB Nijmegen, The Netherlands.
authors.forename: Theo S
authors.initials: TS
authors.lastname: Plantinga
authors.forename: Tania O
authors.initials: TO
authors.lastname: Crisan
authors.forename: Marije
authors.initials: M
authors.lastname: Oosting
authors.forename: Frank L
authors.initials: FL
authors.lastname: van de Veerdonk
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: de Jong
authors.forename: Dana J
authors.initials: DJ
authors.lastname: Philpott
authors.forename: Jos W M
authors.initials: JW
authors.lastname: van der Meer
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Girardin
authors.forename: Leo A B
authors.initials: LA
authors.lastname: Joosten
authors.forename: Mihai G
authors.initials: MG
authors.lastname: Netea
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000494:pharmacology; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D020170:Caspase 1 / Q000378:metabolism; D002478:Cells, Cultured; D003424:Crohn Disease / Q000235:genetics* / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000276:immunology; D015870:Gene Expression; D020022:Genetic Predisposition to Disease; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D053583:Interleukin-1beta / Q000096:biosynthesis / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000276:immunology; D053473:Nod2 Signaling Adaptor Protein / Q000502:physiology*; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000235:genetics
chemical_list: C518959:ATG16L1 protein, human; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D016207:Cytokines; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger; C025946:3-methyladenine; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D020170:Caspase 1; D000225:Adenine