Roger A. Astley

Corneal Cell Survival in Adenovirus Type 19 Infection Requires Phosphoinositide 3-Kinase/Akt Activation

ABSTRACT Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3β and nuclear translocation of the p65 subunit of NF-κB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.

Bacillus cereus-induced permeability of the blood-ocular barrier during experimental endophthalmitis.

PURPOSE The purpose of this study was to determine to what extent blood-retinal 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 colony-forming units of B. cereus, and eyes were analyzed at specific times after infection 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 retinal pigment epithelium. RESULTS B. cereus induced the leakage of albumin and fibrin into the aqueous and vitreous humor by 8 hours after infection. BRB permeability occurred as early as 4 hours and increased 13.30-fold compared with uninfected controls by 8 hours. Production of proinflammatory cytokines IL-6, MIP-1alpha, IL-1beta, and KC increased over the course of infection. In the retina, ZO-1 disruption began by 4 hours and was followed by decreasing occludin and ZO-1 expression at 4 and 8 hours, respectively. Tubulin condensation and RPE65 degradation occurred by 12 hours. A quorum-sensing mutant B. cereus strain caused BRB permeability comparable to that of wild-type B. cereus. 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 hours after infection. Disruption of tight junctions at the level of the retinal pigment epithelium may contribute to barrier breakdown. Quorum-sensing dependent factors may not significantly contribute to BRB permeability.

Histopathology of Salzmann nodular corneal degeneration

Purpose: To define the histopathology of Salzmann nodular degeneration and suggest potential mechanisms involved in its pathogenesis. Methods: Archived corneal biopsy specimens from 5 patients with Salzmann nodular degeneration were evaluated by chemical and immunohistochemical staining to describe the structure of the Salzmann nodules and phenotypes of nodule epithelium and stromal cells. Results: Each Salzmann nodule appeared as a hypercellular mound of extracellular matrix located between a thinned corneal epithelium and a fragmented Bowman layer. Stromal cells within each nodule stained positively for vimentin, consistent with a fibroblast phenotype, whereas the epithelial cells overlying each nodule were positive for matrix metalloproteinase-2 and negative for matrix metalloproteinase-9. Conclusions: The observed epithelial expression of matrix metalloproteinase-2 overlying Salzmann nodules is consistent with chronic epithelial wounding in the disorder but does not identify a cause-effect relationship. Salzmann nodules might develop because of enzymatic disruption of the Bowman layer, anterior migration and proliferation of keratocytes, and secondary deposition of extracellular matrix. Alternatively, desiccation secondary to the elevation of the nodule might induce increased epithelial metalloproteinase expression.

Role of Toll-Like Receptor (TLR) 2 in Experimental Bacillus cereus Endophthalmitis

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.

Structural and cellular architecture of conjunctival lymphoid follicles in the baboon (Papio anubis)

Conjunctival lymphoid follicles (CLFs), present in normal individuals, undergo hyperplasia upon conjunctival infection by a specific array of pathogens; infection-associated enlargement of draining preauricular lymph nodes suggests that conjunctival follicles participate in the afferent limb of acquired immune responses for the ocular surface. The present study was performed to delineate the structural and lymphoid anatomy of CLFs in the baboon (Papio anubis), a non-human primate conjunctival model with close similarity to the human. Conjunctiva from both eyes, along with mesenteric lymph node, spleen, tonsil, and ileum controls were harvested from ten baboons at necropsy, and studied by histochemical and immunohistochemical methods. Baboon conjunctival follicles were identified as dense oval collections of leukocytes in the substantia propria with infiltration into a thinned overlying conjunctival epithelium. Goblet cells were universally absent, the overlying mucin layer was attenuated, and the follicle-associated epithelium (FAE) demonstrated comparatively diminished alkaline phosphatase expression. The basement membrane overlying each follicle appeared discontinuous. CD4-positive T lymphocytes were distributed in parafollicular areas and to a lesser degree in follicle germinal centers. B lymphocytes formed the predominant cell in follicles, and also heavily infiltrated the FAE. B cell IgM expression was prominent in germinal centers, while IgD staining occurred in a horseshoe-shaped distribution in the follicle mantle zone. Although B cell IgA expression was noted in the non-follicular conjunctiva, IgA expression was inconspicuous within conjunctival follicles. S-100- and CD1a-positive dendritic cells were found in FAE, while fascin-positive mature dendritic cells appeared in the deeper areas of each follicle. CD68-positive macrophages were dispersed throughout the follicles. CD35-positive follicular dendritic cells were observed only in germinal centers. CLFs appear highly organized consistent with a role in the adaptive immune response to conjunctival pathogens.

Unexpected Roles for Toll-Like Receptor 4 and TRIF in Intraocular Infection with Gram-Positive Bacteria

ABSTRACT 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.

Role of TLR5 and Flagella in Bacillus Intraocular Infection

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.

CXCL1, but not IL‐6, significantly impacts intraocular inflammation during infection

During intraocular bacterial infections, the primary innate responders are neutrophils, which may cause bystander damage to the retina or perturb the clarity of the visual axis. We hypothesized that cytokine IL‐6 and chemokine CXCL1 contributed to rapid neutrophil recruitment during Bacillus cereus endophthalmitis, a severe form of intraocular infection that is characterized by explosive inflammation and retinal damage that often leads to rapid vision loss. To test this hypothesis, we compared endophthalmitis pathogenesis in C57BL/6J, IL‐6−/−, and CXCL1−/− mice. Bacterial growth in eyes of CXCL1−/−, IL‐6−/−, and C67BL/6J mice was similar. Retinal function retention was greater in eyes of IL‐6−/− and CXCL1−/− mice compared with that of C57BL/6J, despite these eyes having similar bacterial burdens. Neutrophil influx into eyes of CXCL1−/− mice was reduced to a greater degree compared with that of eyes of IL6−/− mice. Histology confirmed significantly less inflammation in eyes of CXCL1−/− mice, but similar degrees of inflammation in IL6−/− and C57BL/6J eyes. Because inflammation was reduced in eyes of infected CXCL1−/− mice, we tested the efficacy of anti‐CXCL1 in B. cereus endophthalmitis. Retinal function was retained to a greater degree and there was less overall inflammation in eyes treated with anti‐CXCL1, which suggested that anti‐CXCL1 may have therapeutic efficacy in limiting inflammation during B. cereus endophthalmitis. Taken together, our results indicate that absence of IL‐6 did not affect overall pathogenesis of endophthalmitis. In contrast, absence of CXCL1, in CXCL1−/− mice or after anti‐CXCL1 treatment, led to an improved clinical outcome. Our findings suggest a potential benefit in targeting CXCL1 to control inflammation during B. cereus and perhaps other types of intraocular infections.

Modeling intraocular bacterial infections.

Bacterial endophthalmitis is an infection and inflammation of the posterior segment of the eye which can result in significant loss of visual acuity. Even with prompt antibiotic, anti-inflammatory and surgical intervention, vision and even the eye itself may be lost. For the past century, experimental animal models have been used to examine various aspects of the pathogenesis and pathophysiology of bacterial endophthalmitis, to further the development of anti-inflammatory treatment strategies, and to evaluate the pharmacokinetics and efficacies of antibiotics. Experimental models allow independent control of many parameters of infection and facilitate systematic examination of infection outcomes. While no single animal model perfectly reproduces the human pathology of bacterial endophthalmitis, investigators have successfully used these models to understand the infectious process and the host response, and have provided new information regarding therapeutic options for the treatment of bacterial endophthalmitis. This review highlights experimental animal models of endophthalmitis and correlates this information with the clinical setting. The goal is to identify knowledge gaps that may be addressed in future experimental and clinical studies focused on improvements in the therapeutic preservation of vision during and after this disease.

TLR4 Contributes to the Host Response to Klebsiella Intraocular Infection

Abstract Purpose/Aim: Klebsiella pneumoniae causes a blinding infection called endogenous endophthalmitis. The role of innate immune recognition of K. pneumoniae in the eye during infection is not known. We hypothesized that intraocular recognition of K. pneumoniae was mediated by Toll-like receptor (TLR)-4 and may be dependent on MagA-regulated hypermucoviscosity. Materials and Methods: Experimental endophthalmitis was induced in C57BL/6J or TLR4−/− mice by intravitreal injection of 100 CFU of wild type or ΔmagA K. pneumoniae. Infection and inflammation were quantified by determining viable K. pneumoniae per eye, retinal responses via electroretinography, myeloperoxidase activity of infiltrating neutrophils and the proinflammatory cytokine and chemokine response. Results: C57BL/6J and TLR4−/− mice could not control intraocular wild-type K. pneumoniae growth. TLR4−/− mice were less able than C57BL/6J to control the intraocular growth of ΔmagA K. pneumoniae. Retinal function testing suggested that infection with ΔmagA K. pneumoniae resulted in less retinal function loss. There was a TLR4-dependent delay in initial neutrophil recruitment, regardless of the infecting organism. The proinflammatory cytokine/chemokine data supported these results. These findings were not due to an inability of TLR4−/− neutrophils to recognize or kill K. pneumoniae. Conclusions: These studies suggest that TLR4 is important in the early intraocular recognition and host response to K. pneumoniae. However, the role of MagA in TLR4-mediated intraocular recognition and subsequent inflammation is less clear.