TLR4-dependent retinal gene expression in Bacillus cereus endophthalmitis

摘要

Purpose: To evaluate the retinal transcriptional response to B. cereus infection early during the course of experimental murine endophthalmitis and to identify Toll-like receptor (TLR) 4-dependent genes. Methods: The right eyes of male C57BL/6J or TLR4-/- mice were intravitreally injected with 100 CFU of B. cereus ATCC 14579. Left eyes served as uninfected controls. At 4 h post-infection, RNA was isolated from retinas, converted to cDNA, and hybridized to the Affymetrix GeneChip Mouse Genome 430 2.0 array. Data analysis was performed using Parteka??s Genomics Suite software to obtain differential gene expression data. A 5-fold change in gene expression and p 0.05 threshold were selected as the criteria for comparative array analyses. Arrays were performed in duplicate and the results were confirmed by qPCR and ELISA. Results: Genes related to the acute inflammatory response and inflammatory cell recruitment were significantly upregulated 5-fold or greater at 4 h post-infection with B. cereus in C57BL/6J retinas [CXCL1 (KC), CXCL2 (MIP2-?±) CXCL10 (IP-10), CCL2 (MCP1), and CCL3 (MIP1-?±)]. The pro- and anti-inflammatory mediator IL6, the intercellular adhesion molecular ICAM-1, and the inhibitor of cytokine signal transduction, SOCS3, were also significantly upregulated 5-fold or greater. Leukemia inhibitory factor (LIF), crucial for the survival of photoreceptor cells, was also highly induced in response to B. cereus infection. Prostaglandin-endoperoxide synthase 2 (PTGS2/COX-2), which converts arachidonic acid to prostaglandin endoperoxide H2, was upregulated, as was pentraxin 3 (PTX3), which is produced in response to TLR engagement, activates the classical complement pathway, and facilitates pathogen recognition and clearance. Among the genes related to inflammation and infection that are upregulated in wild type retinas at 4h post-infection, only CCL3 was found to be significantly upregulated in B. cereus-infected TLR4-/- retinas. Conclusions: These studies demonstrate that TLR4 regulates cytokine and chemokine genes important for the acute inflammatory response and neutrophil recruitment, as well as genes related to photoreceptor survival and pathogen recognition and clearance. Future studies will evaluate the retinal and global ocular inflammatory responses over the course of B. cereus endophthalmitis to identify pathway-based anti-inflammatory targets, specifically, those that are regulated by TLR4.