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Staphylococcus aureustriggers inflammation through inflammasome activation and recruitment of neutrophils, responses that are critical for pathogen clearance but are associated with substantial tissue damage. We postulated that necroptosis, cell death mediated by the RIPK1/RIPK3/MLKL pathway, would function to limit pathological inflammation. In models of skin infection or sepsis, Mlkl−/− mice had high bacterial loads, an inability to limit interleukin-1b (IL-1b) production, and excessive inflammation. Similarly, mice treated with RIPK1 or RIPK3 inhibitors had increased bacterial loads in a model of sepsis. Ripk3−/−mice exhibited increased staphylococcal clearance and decreased inflammation in skin and systemic infection, due to direct effects of RIPK3 on IL-1b activation and apoptosis. In contrast to Casp1/4−/− mice with defective S. aureuskilling, the poor outcomes of Mlkl−/−mice could not be attributed to impaired phagocytic function. We conclude that necroptotic cell death limits the pathological inflammation induced by S. aureus.

ABSTRACTSkin is the most common site of Staphylococcus aureusinfection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureusis able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureusisolated from chronically infected patients with atopic dermatitis, we noted 22% had an agrmutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agrmutants of methicillin-resistant S. aureus(MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agror RNAIII mutants (P< 0.0001) of an inoculum of wild-type (WT) MRSA from within wortmannin-treated keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureusand keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms.IMPORTANCEHuman skin is a major site of staphylococcal infection, and keratinocytes actively participate in eradication of these pathogens. We demonstrate that methicillin-resistant Staphylococcus aureus(MRSA) is ingested by keratinocytes and activates caspase-1-mediated clearance through pyroptosis. Toxin-deficient MRSA mutants are selected within keratinocytes that fail to induce caspase-1 activity and keratinocyte-mediated clearance. These intracellular staphylococci induce autophagy that enhances their intracellular survival by diminishing inflammasome components. These findings suggest that S. aureusmutants, by exploiting autophagy, can persist within human keratinocytes.