Pseudomonas aeruginosa (PA) is an opportunistic pathogen that induces a rapidly developing and destructive disease of the cornea and is a global cause of visual impairment and blindness. It is also the most commonly recovered causative organism in contact lens-related disease in developed countries. Of most concern, continued emergence of antibiotic-resistant bacterial strains poses a serious challenge for effective disease management and adjunctive treatments are required. Therefore, the long-term objective of the studies proposed is to test the regulatory role of microRNAs (miRNAs), a newly recognized, important level of gene- expression regulation, in bacterial keratitis, identify new therapeutic targets and provide alternative treatment strategies. To support this goal, preliminary published studies showed that the miR-183/96/182 cluster, which produces miR-183, miR-96 and miR-182, is expressed in the cornea and in innate immune cells, including macrophages (M?) and polymorphonuclear neutrophils (PMN), in both mouse and human. Inactivation of this cluster in mice led to decreased pro-inflammatory chemotactic cytokines (e.g., MCP1, MIP2 and IL-1?) in the cornea and a decreased severity of PA-induced keratitis in miR-183/96/182 cluster knockout (ko) mice. Consistent with reduced chemotactic cytokines in ko mice, PMN number was decreased early (1 day post infection, dpi) in disease, and bacterial load increased. Yet later in disease (5 dpi) PMN number was similar in both groups, but bacterial load was significantly decreased in the ko animals. Other preliminary data showed that PMN from ko mice had enhanced phagocytic and killing abilities, consistent with in vivo data showing reduction of bacterial load in the cornea, despite similar PMN number. Regarding potential treatment strategies, a pilot experiment with prophylactic subconjunctival and topical application of anti-miRs in PA-infected wild- type mice, provided information that down-regulation of miR-183/96/182 cluster function successfully decreased the severity of PA keratitis. Therefore, to achieve our long-term objectives, the following aims are proposed:
Aim 1 will test that inactivation of the miR-183/96/182 cluster in corneal resident M? (myeloid cells) decreases production of pro-inflammatory chemotactic cytokines, specifically, MCP1, IL-1? and MIP2, contributing to a decreased infiltration of PMN and M? to the infected cornea.
Aim 2 will test that restoration of miR-183/96/182 cluster expression in myeloid cells of ko mice by breeding them to myeloid specific Cre transgenic animals, is sufficient to reverse the corneal response to PA infection in vivo, as well as phagocytosis and intracellular killing by infiltrating cells.
Aim 3 will test that DAP12 is a direct target of the miR- 183/96/182 cluster in PMN and M?, and mediates its regulation of these cells.
Aim 4 will test that local knockdown of miR-183/96/182 cluster function in the cornea is therapeutic for PA-induced keratitis. It is anticipated that these studies will reveal novel molecular mechanisms of miR-183/96/182 cluster regulation of innate immune responses in bacterial keratitis and provide a new target for its treatment.
Pseudomonas aeruginosa (PA)-induced keratitis is one of the most rapidly developing and destructive diseases of the cornea and a global cause of visual impairment and blindness. It is also one of the most common causative organisms of contact lens-related disease in developed countries. The proposed research will test the role of the miR-183/96/182 cluster and the mechanisms by which it regulates the innate immune response to PA keratitis as well as the potential of knockdown of the cluster function by anti-miRs to protect the cornea from the adverse events of this disease.
