Fig. 5: Enhanced susceptibility of ATG16L1 T316A variant in response to Eh.

Previously shown Crohn’s disease associated ATG16L1 T300A variant (in mice corresponding SNP is at 316 position) is highly susceptible to Eh interaction. a BMDM cells from WT and ATG16L1 T316A variant were stimulated with different Eh to macrophage ratio for 10 min. b HEK293T cells were overexpressed with HA-tagged WT and ATG16L1 T300A variant and immunoprecipitated with anti-HA antibody. Immunoprecipitants were incubated 16 h at 37 °C with active recombinant caspase-6 in absence or presence of inhibitor Z-VEID-fmk (50 μM) and ATG16L1 cleavage was assessed by western blot with anti-ATG16L1 antibody and quantified by densitometric analysis. Direct cell lysate of WT (lane 5) and T300A variant (lane 9) were used as a control. mRNA expression of different pro-inflammatory cytokines and chemokines from Eh inoculated closed colonic loop tissues of WT and ATG16L1 T316A variant mice c, IL-1β and IL-6, d. KC and MCP-1. e Multiplex cytokine array by Luminex showed significantly increased TNF-α, KC and MCP-1 secretion in T316A BMDM cells stimulated with Eh for 3 h at 1:20 ration. Cells without treatment is symbolized as (−ve). Data are representative of at least three independent experiments (n = 3) and for statistical significance one-way ANOVA followed by post hoc Bonferroni test was done. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represent mean ± SEM.