Cholesterol-dependent cytolysins impair pro-inflammatory macrophage responses

Pushpak Bhattacharjee,Peter A Keyel

doi:10.1038/s41598-018-24955-2

Pushpak Bhattacharjee, Peter A Keyel

Open Access

https://doi.org/10.1038/s41598-018-24955-2

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Abstract

Necrotizing soft tissue infections are lethal polymicrobial infections. Two key microbes that cause necrotizing soft tissue infections are Streptococcus pyogenes and Clostridium perfringens. These pathogens evade innate immunity using multiple virulence factors, including cholesterol-dependent cytolysins (CDCs). CDCs are resisted by mammalian cells through the sequestration and shedding of pores during intrinsic membrane repair. One hypothesis is that vesicle shedding promotes immune evasion by concomitantly eliminating key signaling proteins present in cholesterol-rich microdomains. To test this hypothesis, murine macrophages were challenged with sublytic CDC doses. CDCs suppressed LPS or IFNγ-stimulated TNFα production and CD69 and CD86 surface expression. This suppression was cell intrinsic. Two membrane repair pathways, patch repair and intrinsic repair, might mediate TNFα suppression. However, patch repair did not correlate with TNFα suppression. Intrinsic repair partially contributed to macrophage dysfunction because TLR4 and the IFNγR were partially shed following CDC challenge. Intrinsic repair was not sufficient for suppression, because pore formation was also required. These findings suggest that even when CDCs fail to kill cells, they may impair innate immune signaling responses dependent on cholesterol-rich microdomains. This is one potential mechanism to explain the lethality of S. pyogenes and C. perfringens during necrotizing soft tissue infections.

Highlights

Necrotizing soft tissue infections (NSTIs) are a group of related, potentially fatal bacterial infections, like necrotizing fasciitis and gas gangrene, that quickly spread through and kill soft tissues in the body
We found that the cholesterol-dependent cytolysins (CDCs) Streptolysin O (SLO) and Perfringolysin O (PFO) temporarily impair macrophage responses to LPS and pro-inflammatory cytokines like Interferon γ (IFNγ), as measured by Tumor Necrosis Factor α (TNFα) production and surface expression of activation markers CD69 and CD86 without causing significant cell death
To examine how CDCs could interact with other pathogen-associated molecular patterns that could be present during infection, we challenged murine C57BL/6 (B6) bone-marrow derived macrophages (BMDM) sequentially first with a CDC and with a Toll-like receptors (TLRs) ligand like LPS

Summary

Introduction

Necrotizing soft tissue infections (NSTIs) are a group of related, potentially fatal bacterial infections, like necrotizing fasciitis and gas gangrene, that quickly spread through and kill soft tissues in the body. Following membrane disruption by a CDC like SLO, the cell activates at least two pathways, patch repair and intrinsic repair[16,45,46]. Intrinsic repair is the sequestration and shedding of toxins on microvesicles[16] While these repair mechanisms help the cell by restoring membrane homeostasis, it is not clear if pathogens can exploit this repair process to promote immune evasion. We found that the CDCs SLO and PFO temporarily impair macrophage responses to LPS and pro-inflammatory cytokines like IFNγ, as measured by TNFα production and surface expression of activation markers CD69 and CD86 without causing significant cell death. Mutant toxins that enhanced membrane repair more potently inhibited macrophage responses Overall, these findings suggest one mechanism for the immune evasion caused by C. perfringens and S. pyogenes during NSTI

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