ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in Crohn's disease pathogenesis.
OBJECTIVE
The identification of numerous genes that confer susceptibility to Crohn's disease (CD) indicates that this complex disease might arise from alterations in several genes with related functions. We examined the functional interaction between the CD risk genes ATG16L1 and NOD2 to identify an autophagy-dependent pathway that is altered by disease-associated variants.


METHODS
We assessed Nod2 signaling and autophagy activation in response to muramyl dipeptide (MDP) by immunoblot, confocal microscopy, flow cytometry, reporter gene, and gentamicin protection assays in human epithelial cell lines and primary human macrophages and dendritic cells from healthy individuals. The requirement of Nod2 and ATG16L1 expression and the effects of CD-associated variants in MDP-stimulated autophagy and Nod2-dependent signaling were assessed in cell lines manipulated by RNA interference, inhibitors, or ATG16L1 or NOD2 variants and in primary macrophages and dendritic cells from healthy genotyped donors.


RESULTS
MDP stimulation of epithelial cells, macrophages, and dendritic cells activated autophagy and nuclear factor κB and mitogen-activated protein kinase signaling; it also increased killing of Salmonella. These responses depended on ATG16L1 and Nod2 expression and were impaired by CD-associated NOD2 variants. Nod2-dependent signaling was not impaired in cells with the ATG16L1 T300A genotype, which is associated with CD. However, the ATG16L1 T300A variant blocked the increase in MDP-mediated killing of Salmonella only in epithelial cell lines and not primary macrophages or dendritic cells.


CONCLUSIONS
ATG16L1 and NOD2 are components of an autophagy-mediated antibacterial pathway that is altered in a cell- and function-specific manner by CD-associated mutations.
Homer
Craig R
CR
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Richmond
Amy L
AL
Rebert
Nancy A
NA
Achkar
Jean-Paul
JP
McDonald
Christine
C
D001343:Autophagy; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003424:Crohn Disease / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D003713:Dendritic Cells / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics* / Q000378:metabolism; D010587:Phagocytosis; D012313:RNA / Q000235:genetics*
C518959:ATG16L1 protein, human; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D053473:Nod2 Signaling Adaptor Protein; D012313:RNA