Macrophages activate a MLK3/MEK signaling axis to trigger Annexin mediated membrane repair after cholesterol-dependent cytolysin damage

Abstract

Abstract The innate immune system is critical to promoting host survival during bacterial necrotizing soft tissue infections (NSTIs). NSTI causing pathogens like Streptococcus pyogenes and Clostridium perfringens use pore-forming toxins to kill innate immune cells like macrophages. Toxins cause rapid influx of Ca2+, which triggers repair responses. Ca2+ sensitive proteins like Annexins repair via clogging the injured site and are shed with toxin pores on microvesicles during repair. However, the molecular mechanism orchestrating this repair is unknown. Since Mitogen Activated Protein Kinase (MAPK) pathways initiate long-term inflammatory responses, short-term signaling could drive membrane repair. We hypothesized that Mixed Lineage Kinase 3 (MLK3) activates a prosurvival kinase cascade (MEK/ERK) to activate membrane repair by microvesicle shedding in macrophages. We tested cell viability and repair responses to toxins after blocking proteins in the MAPK pathways using inhibitors or RNAi in primary murine macrophages and cell lines. We found MLK3 or MEK inhibition significantly reduced toxin resistance. Protective MLK3/MEK activity was downstream of Ca2+ and accounted for ~ 60% of all Ca2+ dependent repair. MEK inhibition led to increased cytosolic Ca2+ flux after toxin damage and reduced ANXA2, but not ANXA1 or ANXA6, translocation to damaged site. MEK blockade reduced shedding of toxin pores. Overall, these findings suggest that a Ca2+ dependent MLK3/MEK axis protects macrophages and other cells from pore-forming toxins by recruiting ANXA2 to the cell membrane to promote repair. Modulation of this axis could promote resistance to NSTIs.