Bacterial gut pathogens such as enteropathogenic Escherichia coli (EPEC) and Salmonella spp. utilise a type III secretion system (T3SS) to translocate effector proteins into host cells to modify cell death signaling for persistent infection and dissemination. Necroptosis is a programmed inflammatory cell death pathway primarily known to be involved in viral defence; however, recent characterisation of EPEC-encoded T3 effector EspL, which directly inhibits necroptosis and associated inflammatory pathways by cleaving shared RHIM domain-containing RIPK1 and RIPK3 adaptors, suggests a novel role in bacterial clearance. Preliminary experiments on Ripk1-/-Ripk3-/-Casp8-/- mice demonstrating heightened susceptibility to gut pathogen Citrobacter rodentium supports this rationale. Preliminary RNA sequencing of colons from these mice also revealed a significant decrease in serum amyloid A2 (SAA2) expression, typically associated with a TH17 response critical for C. rodentium clearance. As the mechanisms of necroptosis in vivo remains unclear, this study sought to clarify the role of RHIM proteins and the underlying pathways mediating exacerbated pathology in the infected Ripk1-/-Ripk3-/-Casp8-/- mice.
Examination of disease in various RHIM-deficient mice found that RIPK3 plays a larger role in mediating pathology in gut infection, whereas RIPK1 restricts bacterial systemic dissemination within the host. Additionally, flow cytometry analysis of colonic lamina propria from infected Ripk1‑/‑Ripk3‑/‑Casp8-/- mice showed significant fold reduction in TH17 cell counts and frequency relative to wildtype controls. This was consistent with the qPCR evaluation of inflammatory cytokine levels where a larger fold reduction in Saa2, Il22 and Il17a expression was exhibited in mice infected with wildtype C. rodentium compared to those with a C. rodentium mutant lacking EspL. Interestingly, these results indicate a link between inactivated RHIM-proteins (innate immunity) and diminished SAA2-mediated TH17 response (adaptive immunity), which prompts further investigation to better inform the significance of necroptosis in bacterial pathogenesis and maintenance of gut homeostasis.