Toxin-Induced Necroptosis Is a Major Mechanism of Staphylococcus aureus Lung Damage

Abstract

Staphylococcus aureus USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of S. aureus toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and S. aureus mutants lacking agr, hla or Hla pore formation, lukAB or psms were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1β production, suggesting a link to the inflammasome. Rip3 -/- mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1β in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.

Humanized Mice Exhibit Increased Susceptibility toStaphylococcus aureusPneumonia

“…While the importance of neutrophils in the response to S. aureus is well established, PVL targeting of macrophages correlated with the pulmonary pathology due to S. aureus infection, as has been demonstrated in other recent studies [19]. This is also consistent with recent studies that demonstrated that S. aureus induction of murine macrophage necroptosis in the early stages of pneumonia resulted in the loss of the immunomodulatory population of macrophages and contributed to the excessive inflammatory responses associated with S. aureus pneumonia [19]. Macrophages can play both proinflammatory and antiinflammatory roles and thus have many functions within the host [40].…”

“…Macrophages can play both proinflammatory and antiinflammatory roles and thus have many functions within the host [40]. Their loss can prevent proper bacterial clearance [19,20], while their signaling cascades can contribute to inflammation and inefficient clearance [19]. Our results from the humanized mice suggest that human macrophages are especially susceptible to PVL-mediated lysis in vivo.…”

“…LukAB is a bicomponent S. aureus toxin with specificity for CD11b and targets human neutrophils [15], as well as monocytes/macrophages by activating NLRP3 [22] and inducing necroptosis [19]. Using the same model of infection in the Δpvl infection of humanized mice, we found no differences in the clearance of WT and lukAB MRSA or in the cell populations recruited ( Figure 7A) or differences in the populations of human immune cells recruited to the lungs ( Figure 7B).…”

“…Also, direct comparison of the immune response between these mice and humans is not possible because of the production of cytokines from both stromal and immune cells in humans. The humanized mice used in these studies, while having excellent reconstitution of T cells, monocytes, and macrophages, all of which are targets of S. aureus toxins [4,14,15,19,21], does not yet fully replicate a functioning human immune system. It has been recognized that neutrophil numbers are not optimal in the peripheral blood of humanized mice, and hence there may be a more limited number of cells to recruit; thus, this is an area to improve in subsequent models [38,39].…”

“…Exactly which immune cell types are involved and what their functions are in pulmonary defenses are difficult to establish, especially if certain leukocyte populations are targeted by human-specific S. aureus toxins. Recent studies suggest that the resident alveolar macrophage populations are especially important in orchestrating staphylococcal clearance [19], whereas dendritic cells [20] and even CD4 + T cells have not been found to be essential in murine models of pneumonia [21]. The human-specific bicomponent toxins PVL and LukAB have been associated with activation of the NLRP3 inflammasome [22][23][24].…”