Pseudomonas aeruginosa, Streptococcus pneumoniae and Staphylococcus aureus are important causes of microbial keratitis worldwide. Neutrophils play an important role in corneal infections and in trauma and corneal burn injuries, not only for microbial killing and tissue damage, but also as a major source of pro-inflammatory cytokines. We have shown that IL-1? is a pivotal cytokine in the host immune response required to regulate bacterial keratitis. IL-1? lacks a signal sequence for release by the classical secretory pathway and thus requires a non-conventional pathway for export. A key mediator of non-canonical IL-1? release is gasdermin D (GSDMD), a cytosolic protein which is cleaved by caspase-1 during inflammasome signaling to generate fragments that form macropores in the plasma membrane; these can directly mediate efflux of IL-1? but also induce pyroptotic lysis. Our preliminary data show that GSDMD in neutrophils is required for IL-1? secretion and is cleaved by caspase-1. However, in contrast to macrophages, neutrophils do not undergo pyroptosis or accumulate GSDMD pores in their plasma membrane instead, cleaved GSDMD appears to associate with intracellular organelles, including autophagosomes, to possibly facilitate IL-1? release by secretory autophagy. We hypothesize that this redirection of N-GSDMD trafficking serves to preserve neutrophil viability for direct bacterial killing during keratitis, while still permitting the IL-1? release which sustains local inflammation until bacteria are cleared. Although neutrophils resist pyroptosis during initial inflammasome activation, other preliminary data and recent reports suggest that GSDMD can mediate neutrophil-specific cell death pathways during sustained inflammasome signaling. We hypothesize that these alternative GSDMD cell death pathways serve to decrease neutrophil viability, dampen inflammation, and increase neutrophil clearance during the resolving phase of bacterial keratitis required for restoration of corneal clarity. We have also identified a population of IL-1? inflammatory monocytes in bacteria-infected corneas of mice that likely contribute significantly to the outcome of infection. To characterize and extend our new findings, we propose three aims.
Aim 1 will test the hypothesis that infiltrating inflammatory monocytes regulate bacterial keratitis by production of IL-1?, regulation and clearance of neutrophils, and restoration of corneal clarity.
Aim 2 will test the hypothesis that GSDMD-dependent IL-1? secretion in neutrophils is mediated by specific trafficking proteins in the secretory autophagy pathway.
Aim 3 will test the hypothesis that serine proteases released from azurophilic granules in response to S. aureus can cleave GSDMD to mediate neutrophil cell death pathways in coordination with caspase-1-independent IL-1? production. Results of the proposed studies will identify the function of autophagy and GSDMD in neutrophils and monocytes during bacterial keratitis. We anticipate that these data will also identify targets that can be used for pharmacological intervention of corneal infections.
Bacterial infections of the cornea (keratitis), including Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae are a major cause of blindness and visual impairment in the USA and worldwide, with the predominant risk factors being ocular trauma and contact lens wear. Using mouse models of keratitis, we reported that neutrophils are rapidly recruited to infected corneas where they are required for microbial clearance; however they are also responsible for corneal damage by release of proteases and promoting inflammation by producing cytokines, especially IL-1?. We propose to regulate inflammation by inhibiting IL-1? release at the molecular level. In the current proposal, we focus on a newly identified protein termed Gasdermin D (GSDMD), which we show is critical for production of IL-1? and for controlling bacterial keratitis. Proposed studies will examine the molecular basis for GSDMD regulation of IL-1? to identify potential targets for anti-inflammatory therapies in these blinding infections.
Showing the most recent 10 out of 35 publications
