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Comparative Study
. 2010 Nov;139(5):1630-41, 1641.e1-2.
doi: 10.1053/j.gastro.2010.07.006. Epub 2010 Jul 14.

ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in Crohn's disease pathogenesis

Craig R Homer  1 , Amy L Richmond, Nancy A Rebert, Jean-Paul Achkar, Christine McDonald
Affiliations
Comparative Study

ATG16L1 and NOD2 interact in an autophagy-dependent antibacterial pathway implicated in Crohn's disease pathogenesis

Craig R Homer et al. Gastroenterology. 2010 Nov.

Abstract

Background & aims: 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.

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Conflict of interest statement

Disclosures: The authors disclose no conflicts of interest.

Figures

Figure 1
Figure 1
MDP stimulates autophagy in epithelial and dendritic cells. (A) Immunoblot of HCT116 cells treated with 10µg/mL MDP. LC3-II accumulation quantified by LC3-II/GAPDH ratio. p-p38, phosphorylated p38. (B) Assay performed as in A. with primary human dendritic cells. (C) Confocal micrographs of HCT116 expressing EGFP-LC3 (green) in media or MDP-stimulated (20µg/mL) for 1h. (D) Quantification of cells with punctuate EGFP-LC3, mean ±SD, n≥4 fields with >50 cells/field. (E) Electron micrograph of MDP-stimulated HCT116:LC3 cells (10µg/mL for 1h). Arrows indicate autophagosomes (F) Immunoblot of MDP-LL-stimulated (10µg/mL) HCT116 cells.
Figure 2
Figure 2
MDP enhances Salmonella killing via an ATG16L1-dependent mechanism in gentamycin protection assays. (A) Infected HCT116 cells treated with media, MDP (10µg/mL), or MDP-LL (10µg/mL), mean ±SD. (B) HCT116 cells transfected with control or ATG16L1 RNAi constructs and infected ±MDP (10µg/mL), mean ±SD. (C) Assay performed as in A. using primary human macrophages or dendritic cells, mean ±SD.
Figure 3
Figure 3
Nod2 is necessary and sufficient for MDP-enhanced Salmonella killing by autophagy. (A) HCT116 cells transfected with control or Nod2 RNAi and infected ±MDP (10µg/mL), mean ±SD. (B) Gentamycin protection assay performed with HEK293T cells transfected with indicated amounts of Nod2 plasmid, mean ±SD. (C) Control or RICK RNAi transfected HCT116 cells and infected ±MDP (10µg/mL), mean ±SD. (D) Gentamycin protection assay performed with HEK293T cells transfected with vector, Nod2 or NLRP3 expression plasmid and control or ATG16L1 RNAi construct, mean ±SD. (E) Autophagosomal maturation assessed by flow cytometric analysis of GFP-mCherry-LC3 in HEK293T cells transfected with vector, Nod2 or NLRP3 expression vectors. Histogram of GFP MFI of the mCherry-positive population (left). Quantification of GFP/mCherry MFI ratio normalized to vector transfected sample, mean ±SD (right, n=4).
Figure 4
Figure 4
CD-associated NOD2 mutants are impaired in signaling, anti-bacterial killing, and autophagy. (A) NF-κB luciferase reporter assay in HEK293T cells transfected with Nod2 plasmids and stimulated with MDP (1µg/mL) for 18h. Luciferase values normalized to β-gal transfection control (nLuc), mean ±SD. (B) Gentamycin protection assay of HEK293T cells transfected with Nod2 plasmids, mean ±SD. (C) NF-κB luciferase reporter assay in HEK293T cells transfected with Nod2 and IKK dominant negative (DN) constructs, mean ±SD. (D) Gentamycin protection assay of HEK293T cells transfected with Nod2 and IKK DN plasmids, mean ±SD. (E) Autophagosomal maturation assessed by flow cytometric analysis of GFP-mCherry-LC3 in HEK293T cells transfected with vector, Nod2 or Nod2 L1007fs plasmids. Histogram of GFP MFI of the mCherry-positive population (left). Quantification of GFP/mCherry MFI ratio normalized to vector transfected sample, mean ±SD (right, n=7). (F) LC3 immunoblots of HEK293T cells transfected with Nod2 or Nod2 L1007fs plasmids treated with MDP (1µg/mL) or rapamycin (25µg/mL) for the indicated times (hours). Quantification of LC3-II amount indicated by LC3-II/tubulin ratio (right).
Figure 5
Figure 5
Inhibition of autophagy impairs activation of Nod2 signaling. NF-κB luciferase reporter assays were performed in HCT116 cells in the presence of autophagy inhibitors. Luciferase values normalized to β-gal transfection control (nLuc), mean ±SD. (A) Steps of autophagosome formation and maturation with key molecules and action of inhibitors indicated. (B) Cells stimulated with MDP and indicated amounts of 3-methyl adenine (3-MA). (C) Cells transfected with either control or Beclin-1 RNAi constructs. (D) Cells transfected with control, ATG16L1, or Nod2 RNAi constructs. (E) Cells stimulated with MDP and indicated amounts of chloroquine.
Figure 6
Figure 6
ATG16L1 T300A impairs MDP-enhanced bacterial killing in a cell type specific manner. (A) TNFα secreted by MDP-stimulated PBMCs (0, 0.1 or 1µg/mL for 18h) with the indicated genotypes, median values of the group (n=6) indicated by bars. WT, wild-type ATG16L1; HT, heterozygous ATG16L1; T300A, homozygous ATG16L1 T300A. (B) Immunoblot analysis of MDP-stimulated, (10µg/mL), genotyped, dendritic cells. Autophagy induction quantified by LC3-II accumulation indicated by LC3-II/GAPDH ratio, with similar results seen by alternative quantification of LC3-I/LC3-II ratios. p-p38, phosphorylated p38. (C) Gentamycin protection assay of genotyped macrophages performed in media ± MDP (10µg/mL). Bars indicate the geometric mean values (WT, n=7; HT, n=7; T300A, n=14) with the 95% confidence limits shown by the boxes. The range of response is indicated by the vertical bars. (D) Gentamycin protection assay of HT29 or HT29GR cells performed in media ± MDP (20µg/mL), mean ±SD. (E) NF-κB luciferase reporter assay (left) and gentamycin protection assay (right) in HT29GR cells transfected with vector or Nod2 plasmid and treated with MDP (10µg/mL), mean ±SD. (F) Gentamycin protection assay of Nod2-expressing HT29GR cells transfected with vector, ATG16L1 or ATG16L1 T300A plasmids and control or ATG16L1 RNAi performed in media ± MDP (20µg/mL), mean ±SD.
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Comment in

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