Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known Gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2-/- mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to108 CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2-/- eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2-/- eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2-/- eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis.

PURPOSE.To develop a mouse model of adenoviral keratitis that will allow further study of viral and host pathogenic mechanisms. METHODS. Corneas of C57BL/6J mice were injected with adenovirus type 37 (Ad37) or virus-free dialysis buffer by a gaspowered microinjection system coupled to a glass micropipette needle. Mouse corneas were examined for signs of inflammation, by clinical examination, immunohistochemistry, and confocal microscopy; assayed for viral and chemokine mRNA expression by real-time PCR; titered to assess viral replication; and subjected to ELISA for chemokine and myeloperoxidase (MPO) protein expression. RESULTS. C57BL/6J mice corneas injected with 10 5 TCID (tissue culture infective dose) Ad37 showed stromal opacification and inflammation beginning from 1 day after injection and continuing for several months, while buffer-injected corneas showed no signs of inflammation. Ad37-injected corneas expressed adenoviral E1A 10S and E1B 19k mRNA but not IIIa, and viral titers had fallen two logs by day 4 after injection. When compared to untouched and buffer-injected corneas, Ad37-injected corneas expressed significantly higher levels of IL-6, KC, and MCP-1 mRNA at 4 hours after injection (P Ͻ 0.05). By ELISA, KC protein was significantly elevated in Ad37-injected corneas at 8 and 16 hours, and MCP-1 protein at 16 hours after injection (P Ͻ 0.05). Ad37-injected corneas showed elevated levels of MPO (P ϭ 0.0024) at 4 days after injection consistent with immunohistochemical evidence for a predominance of neutrophils in the corneal stroma. CONCLUSIONS. Ad37 induces an acute immunopathologic response in the C57BL/6J mouse cornea, despite an absence of viral replication. This new animal model of Ad37 keratitis will facilitate studies of the molecular pathogenesis of the disorder. (Invest Ophthalmol Vis Sci. 2007;48:781-788)

Purpose The purpose of this study was to determine to what extent blood retina barrier (BRB) permeability occurred during experimental Bacillus cereus endophthalmitis and whether tight junction alterations were involved in permeability. Methods Mice were intravitreally injected with 100 CFU B. cereus and eyes were analyzed at specific times postinfection for permeability to fibrin and albumin, quantitation of intraocular plasma constituent leakage, production of inflammatory cytokines, and alterations in tight junction protein localization and expression at the level of the RPE. Results B. cereus-induced leakage of albumin and fibrin into the aqueous and vitreous humor by 8 h postinfection. BRB permeability occurred as early as 4 h and increased 13.30-fold compared to uninfected controls by 8 h. Production of proinflammatory cytokines IL-6, MIP-1α, IL-1β, and KC increased over the course of infection. In the retina, ZO-1 disruption begins by 4 h, followed by decreasing occludin and ZO-1 expression at 4 and 8 h, respectively. Tubulin condensation and RPE65 degradation occurred by 12 h. A quorum sensing mutant B. cereus strain caused BRB permeability comparable to that of wild-type B. cereus. Both wild-type and mutant B. cereus sterile supernatants induced blood ocular barrier permeability similarly to that of wild-type infection. Conclusions These results indicate that BRB permeability occurs during the early stages of experimental B. cereus endophthalmitis, beginning as early as 4 h postinfection. Disruption of tight junctions at the level of the RPE may contribute to barrier breakdown. Quorum-sensing dependent factors may not significantly contribute to BRB permeability.

Inflammation caused by infection with Gram-positive bacteria is typically initiated by interactions with Toll-like receptor 2 (TLR2). Endophthalmitis, an infection and inflammation of the posterior segment of the eye, can lead to vision loss when initiated by a virulent microbial pathogen. Endophthalmitis caused by Bacillus cereus develops as acute inflammation with infiltrating neutrophils, and vision loss is potentially catastrophic. Residual inflammation observed during B. cereus endophthalmitis in TLR2 ؊/؊ mice led us to investigate additional innate pathways that may trigger intraocular inflammation. We first hypothesized that intraocular inflammation during B. cereus endophthalmitis would be controlled by MyD88-and TRIF-mediated signaling, since MyD88 and TRIF are the major adaptor molecules for all bacterial TLRs. In MyD88 ؊/؊ and TRIF ؊/؊ mice, we observed significantly less intraocular inflammation than in eyes from infected C57BL/6J mice, suggesting an important role for these TLR adaptors in B. cereus endophthalmitis. These results led to a second hypothesis, that TLR4, the only TLR that signals through both MyD88 and TRIF signaling pathways, contributed to inflammation during B. cereus endophthalmitis. Surprisingly, B. cereus-infected TLR4 ؊/؊ eyes also had significantly less intraocular inflammation than infected C57BL/6J eyes, indicating an important role for TLR4 in B. cereus endophthalmitis. Taken together, our results suggest that TLR4, TRIF, and MyD88 are important components of the intraocular inflammatory response observed in experimental B. cereus endophthalmitis, identifying a novel innate immune interaction for B. cereus and for this disease. Bacillus cereus is a Gram-positive, spore-forming, and beta-hemolytic soil bacterium (1). Commonly identified as a causative agent of foodborne illnesses, B. cereus is also associated with a multitude of clinical conditions, such as central nervous system infections (2), pneumonia (3), endocarditis (4), and gas-gangrene-like cutaneous infections (5). B. cereus also causes a virulent form of endophthalmitis, an intraocular inflammatory condition resulting from the introduction of microorganisms into the posterior segment of the eye following surgery or injury or from a distant site of infection. This infection causes irreversible damage to the retina, often leading to vision loss within 1 or 2 days (6). Typically, B. cereus endophthalmitis occurs following a penetrating eye injury (posttraumatic) with retained intraocular foreign bodies (7, 8) but has also been reported in postoperative patients (9-11). Fewer than 30% of posttraumatic B. cereus endophthalmitis patients retained useful vision, and out of these, only 9% retained 20/70 vision or better (7, 12). Moreover, 48% of B. cereus and other Bacillus species infections required evisceration or enucleation of the eye despite therapeutic intervention (7, 12). Intraocular inflammation that occurs during B. cereus endophthalmitis interferes with the clarity of the visual axis, contributing to disruption...

B. cereus possesses flagella which allow the organism to migrate within the eye during a blinding form of intraocular infection called endophthalmitis. Because flagella is a ligand for Toll-like receptor 5 (TLR5), we hypothesized that TLR5 contributed to endophthalmitis pathogenesis. Endophthalmitis was induced in C57BL/6J and TLR5−/− mice by injecting 100 CFU of B. cereus into the mid-vitreous. Eyes were analyzed for intraocular bacterial growth, retinal function, and inflammation by published methods. Purified B. cereus flagellin was also injected into the mid-vitreous of wild type C57BL/6J mice and inflammation was analyzed. TLR5 activation by B. cereus flagellin was also analyzed in vitro. B. cereus grew rapidly and at similar rates in infected eyes of C57BL/6J and TLR5−/− mice. A significant loss in retinal function in both groups of mice was observed at 8 and 12 hours postinfection. Retinal architecture disruption and acute inflammation (neutrophil infiltration and proinflammatory cytokine concentrations) increased and were significant at 8 and 12 hours postinfection. Acute inflammation was comparable in TLR5−/− and C57BL/6J mice. Physiological concentrations of purified B. cereus flagellin caused significant inflammation in C57BL/6J mouse eyes, but not to the extent of that observed during active infection. Purified B. cereus flagellin was a weak agonist for TLR5 in vitro. These results demonstrated that the absence of TLR5 did not have a significant effect on the evolution of B. cereus endophthalmitis. This disparity may be due to sequence differences in important TLR5 binding domains in B. cereus flagellin or the lack of flagellin monomers in the eye to activate TLR5 during infection. Taken together, these results suggest a limited role for flagellin/TLR5 interactions in B. cereus endophthalmitis. Based on this and previous data, the importance of flagella in this disease lies in its contribution to the motility of the organism within the eye during infection.