Pore-forming toxins impair pro-inflammatory macrophage responses

Abstract

Abstract Necrotizing soft tissue infections (NSTIs) are lethal microbial infections. One virulence factor common to most bacteria responsible for NSTIs such as Streptococcus pyogenes is a pore-forming toxin (PFT). PFTs promote immune evasion through unknown mechanisms and lyse cells by forming pores. One mechanism by which cells resist these PFTs is intrinsic membrane repair which is sequestering and shedding toxin pores on microvesicles. It is possible that intrinsic membrane repair is hijacked by bacteria to promote immune evasion. We hypothesized that PFTs promote immune evasion by inducing immune receptor shedding during intrinsic membrane repair. We tested this hypothesis by challenging murine bone marrow derived macrophages with sublytic toxin doses prior to LPS or IFNγ challenge. We measured surface receptor expression and intracellular TNFα by flow cytometry in live cells. We found that toxins from one family of PFTs, cholesterol-dependent cytolysins (CDCs), suppressed LPS or IFNγ-stimulated TNFα production and CD69 and CD86 upregulation in a cell-intrinsic manner independently of cell death. CDCs further induced partial shedding of TLR4 and IFNγR, which might contribute to macrophage dysfunction. In contrast, we found that a toxin from a second PFT family, aerolysin, prevented TNFα production by inducing cell death in macrophages at equivalent hemolytic doses. Overall, these results suggest that different PFT families use different strategies to impair macrophages. CDCs may hijack intrinsic membrane repair to impair TNFα production, while aerolysin kills macrophages at similar doses. These findings might explain the immune suppression and lethality observed during NSTIs.