PMC

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.

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.

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.

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).

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).

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.