ALPK1 controls TIFA/TRAF6-dependent innate immunity against heptose-1,7-bisphosphate of gram-negative bacteria
Fig 5
Sensing of HBP triggers TIFA/TRAF6-dependent innate immunity.
A) TIFA and TRAF6 are not involved in L. monocytogenes-induced IL-8 production. Cells were transfected with control, TIFA- or TRAF6-targeting siRNAs, infected with L. monocytogenes for 3.5 hours and stained for IL-8. Data show the mean +/- SD of 3 independent experiments, ns: p>0.05. B) S. typhimurium infection induces TIFA oligomers. Hela cells were transfected with wild-type TIFA cDNA, infected with L. monocytogenes or S. typhimurium for 45 minutes and stained for TIFA (green) and DNA (blue). Arrows indicate bacteria (red). C) TIFA and TRAF6 are involved in IL-8 expression after S. typhimurium infection. Cells were transfected as in A, infected with S. typhimurium for 3.5 hours and stained for IL-8. Data show the mean +/- SD of 3 independent experiments, p***<0.0005. D) HBP is required for IL-8 induction after S. typhimurium infection. Cells were infected with wt, ΔhldE or ΔgmhB S. typhimurium (green) and stained for IL-8 (red), F-actin (grey) and DNA (blue). E) Quantification of IL-8 after infection with wt, ΔhldE or ΔgmhB S. typhimurium. Data show the mean +/- SD of 3 independent experiments, p*<0.05. F) HBP is required for IL-8 expression after S. flexneri infection. Cells were infected with wt, ΔhldE or ΔgmhB S. flexneri (green) and stained as in D. Scale bars, 20 μm. G) Comparison of the infection rates after infection with wt, ΔhldE or ΔgmhB S. flexneri at multiple MOIs. Data show the mean +/- SD of triplicate wells, graph representative of 3 independent experiments. H) Quantification of IL-8 after infection with wt, ΔhldE or ΔgmhB S. flexneri. Data show the mean +/- SD of triplicate wells, graph representative of 3 independent experiments. I) TIFA oligomerization is HBP-dependent. Cells were transfected with TIFA cDNA and infected with wt, ΔhldE or ΔgmhB S. flexneri (red). Cells were stained for TIFA (green) and DNA (blue). J) IL-8 secretion of S. flexneri-infected Caco-2 cells is largely HBP-dependent. ELISA assay measuring the secretion of IL-8 after infection of Caco-2 cells. Cells were infected for 6 hours with wt (MOI 400), ΔhldE (MOI 4) or ΔwaaC (MOI 4) S. flexneri. Data correspond to the mean +/- SD of 3 independent experiments, p*<0.05.