Thursday, November 21, 2024
11/21/2024
PROJECT ABSTRACT Diabetic patients are known to have a higher incidence of infection, with increased disease severity and an increased rate of multi-drug resistance. In the diabetic (DM) cornea, this results in increased susceptibility to and the rapid progression of microbial keratitis. To date, the mechanisms underlying this are unknown. To address this critical and understudied question, Pseudomonas (P.) aeruginosa and Staphylococcus (S.) aureus keratitis models with Streptozotocin-induced type 1 (T1) and db/db type 2 (T2) DM mice were established. In our models, higher inocula of both P. aeruginosa and S. aureus were required to infect mouse corneas and bacterial keratitis progressed faster in DM, compared to normoglycemia B6 mice, mimicking human diabetic infectious keratitis. High-throughput RNA sequencing (RNA-seq), in combination with comprehensive bioinformatics, was used to identify differentially expressed genes (DEGs) and biological processes associated with the increased susceptibility and severity of diabetic BK. Our preliminary data show that P. aeruginosa infection resulted in many DEGs, some of which have not been linked previously to the pathogenesis of bacterial keratitis. Gene Ontology enrichment analysis revealed that programmed cell death (PCD) pathways, known to play a key role in homeostasis and in the pathogeneses of many human diseases, including DM and sepsis, were altered during P. aeruginosa infection. Our preliminary data showed that while noninflammatory apoptosis was decreased, lytic PCDs, including caspase-8, receptor-interacting protein family of serine/threonine protein kinases (RIPK)-1 and -3, mediated necroptosis, NETosis, a biological process to generate neutrophil extracellular traps (NETs), as well as efferocytosis that removes cell corpses, were elevated in DM, compared to NL corneas in response to P. aeruginosa infection. The hypothesis that DM causes lytic PCD pathways and impairs efferocytosis, resulting in increased susceptibility and severity of BK will be tested with three specific aims: (1) To test the hypothesis that DM skews PCD from apoptosis to necroptosis, resulting in hyperinflammation and tissue damage. This can be tested by assessing the levels of cleaved (activated) caspase-8 (apoptosis), phospho-RIPK1, and 3 (necroptosis), and by targeting Casp8 and RIPK1, 3 in both T1 and T2 DM mice. (2) To test the hypothesis that DM primes PMNs for NETosis, increasing bacterial burden, and keratitis severity in B6 mouse corneas. This can be tested by assessing the generation of Neutrophil extracellular traps containing neutrophil granule proteins and host DNA that were modified by peptidyl arginine deiminase (PAD)-4 and nuclear neutrophil elastase and blockade of DAD4 in T1 and T2DM mice. (3) To test the hypothesis that DM impairs efferocytosis, resulting in secondary necrosis and tissue deterioration in bacterial infected B6 mouse corneas. This can be tested by measuring macrophage efferocytosis of dead corneal epithelial cells and neutrophils and assessing the effectiveness of promoting efferocytosis on bacterial clearance and tissue preservation. This proposed study will allow for a better understanding of cornea immunity, identify factors and pathways responsible for the increased susceptibility of diabetic corneas to microbial infection, and lead to the identification of mechanism-based therapies for treating microbial keratitis, for both T1 and T2DM patients. CDC (1/22/2023) reported one death, three (five more recently) with permanent vision loss, and at least 50 people infected with antibiotic-resistant P. aeruginosa, linked to the use of EzriCare eye drops. Hence, our proposal is timely, necessary, and relevant.
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