intravitreal-injection-quantitation-infection-parameters-mouse-model

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune ...

Dean McGee Eye Institute

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-3beta and nuclear translocation of the p65 subunit of NF-kappaB, 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.

Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation.

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.

Modeling intraocular bacterial infections.

To report a case of polymicrobial keratitis caused by  and coagulase-negative  in a patient who cleaned their extended wear contact lenses with only tap water for 2 weeks.

Contact Lens-related Polymicrobial Keratitis from  and .

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.

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

Bacterial endophthalmitis is a potentially blinding intraocular infection. The bacterium Bacillus cereus causes a devastating form of this disease which progresses rapidly, resulting in significant inflammation and loss of vision within a few days. The outer surface of B. cereus incites the intraocular inflammatory response, likely through interactions with innate immune receptors such as TLRs. This study analyzed the role of B. cereus pili, adhesion appendages located on the bacterial surface, in experimental endophthalmitis. To test the hypothesis that the presence of pili contributed to intraocular inflammation and virulence, we analyzed the progress of experimental endophthalmitis in mouse eyes infected with wild type B. cereus (ATCC 14579) or its isogenic pilus-deficient mutant (ΔbcpA-srtD-bcpB or ΔPil). One hundred CFU were injected into the mid-vitreous of one eye of each mouse. Infections were analyzed by quantifying intraocular bacilli and retinal function loss, and by histology from 0 to 12 h postinfection. In vitro growth and hemolytic phenotypes of the infecting strains were also compared. There was no difference in hemolytic activity (1:8 titer), motility, or in vitro growth (p > 0.05, every 2 h, 0-18 h) between wild type B. cereus and the ΔPil mutant. However, infected eyes contained greater numbers of wild type B. cereus than ΔPil during the infection course (p ≤ 0.05, 3-12 h). Eyes infected with wild type B. cereus experienced greater losses in retinal function than eyes infected with the ΔPil mutant, but the differences were not always significant. Eyes infected with ΔPil or wild type B. cereus achieved similar degrees of severe inflammation. The results indicated that the intraocular growth of pilus-deficient B. cereus may have been better controlled, leading to a trend of greater retinal function in eyes infected with the pilus-deficient strain. Although this difference was not enough to significantly alter the severity of the inflammatory response, these results suggest a potential role for pili in protecting B. cereus from clearance during the early stages of endophthalmitis, which is a newly described virulence mechanism for this organism and this infection.

The role of pili in Bacillus cereus intraocular infection.

 is a common pathogen of the eye, capable of infecting external tissues such as the tear duct, conjunctiva, and the cornea, as well the inner and more delicate anterior and posterior chambers.  produces numerous toxins and enzymes capable of causing profound damage to tissues and organs, as well as modulating the immune response to these infections. Unfortunately, in the context of ocular infections, this can mean blindness for the patient. The role of α-toxin in corneal infection (keratitis) and infection of the interior of the eye (endophthalmitis) has been well established by comparing virulence in animal models and α-toxin-deficient isogenic mutants with their wild-type parental strains. The importance of other toxins, such as β-toxin, γ-toxin, and Panton-Valentine leukocidin (PVL), have been analyzed to a lesser degree and their roles in eye infections are less clear. Other toxins such as the phenol-soluble modulins have yet to be examined in any animal models for their contributions to virulence in eye infections. This review discusses the state of current knowledge of the roles of  toxins in eye infections and the controversies existing as a result of the use of different infection models. The strengths and limitations of these ocular infection models are discussed, as well as the need for physiological relevance in the study of staphylococcal toxins in these models.

An Eye on  Toxins: Roles in Ocular Damage and Inflammation.

Bacillus causes a sight-threating infection of the posterior segment of the eye. The robust intraocular inflammatory response in this disease is likely activated via host innate receptor interactions with components of the Bacillus cell envelope. S-layer proteins (SLPs) of some Gram-positive pathogens contribute to the pathogenesis of certain infections. The potential contributions of SLPs in eye infection pathogenesis have not been considered. Here, we explored the role of a Bacillus SLP (SlpA) in endophthalmitis pathogenesis.

S-layer Impacts the Virulence of Bacillus in Endophthalmitis.

 endophthalmitis is a severe intraocular infection. Hallmarks of  endophthalmitis include robust inflammation and rapid loss of vision. We reported that the absence of  surface layer protein (SLP) significantly blunted endophthalmitis severity. Here, we further investigated SLP in the context of retinal cell interactions and innate immune pathways to explore the mechanisms by which SLP contributes to intraocular inflammation. We compared phenotypes of Wild-type (WT) and SLP deficient Δ)  by analyzing bacterial adherence to and phagocytosis by human retinal Muller cells and phagocytosis by mouse neutrophils. Innate immune receptor activation by the  envelope and purified SLP was analyzed using TLR2/4 reporter cell lines. A synthetic TLR2/4 inhibitor was used as a control for this receptor activation. To induce endophthalmitis, mouse eyes were injected intravitreally with 100 CFU WT or Δ. A group of WT infected mice was treated intravitreally with a TLR2/4 inhibitor at 4 h postinfection. At 10 h postinfection, infected eyes were analyzed for viable bacteria, inflammation, and retinal function. We observed that  SLPs contributed to retinal Muller cell adherence, and protected this pathogen from Muller cell- and neutrophil-mediated phagocytosis. We found that  envelope activated TLR2 and, surprisingly, TLR4, suggesting the presence of a surface-associated TLR4 agonist in . Further investigation showed that purified SLP from  activated TLR4, as well as TLR2 . Growth of WT  was significantly higher and caused greater inflammation in untreated eyes than in eyes treated with the TLR2/4 inhibitor. Retinal function analysis also showed greater retention of A-wave and B-wave function in infected eyes treated with the TLR2/4 inhibitor. The TLR2/4 inhibitor was not antibacterial , and did not cause inflammation when injected into uninfected eyes. Taken together, these results suggest a potential role for  SLP in host-bacterial interactions, as well as in endophthalmitis pathogenesis via TLR2- and TLR4-mediated pathways.

 S-Layer-Mediated Innate Interactions During Endophthalmitis.

To explore the consequences of innate interference on intraocular inflammatory responses during Bacillus endophthalmitis.

Article	Year
Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation. Journal of virology\| PubMed ID: 16160160	2005
Modeling intraocular bacterial infections. Progress in retinal and eye research\| PubMed ID: 27154427	2016
Contact Lens-related Polymicrobial Keratitis from and . American journal of ophthalmology case reports\| PubMed ID: 27158679	2016
CXCL1, but not IL-6, significantly impacts intraocular inflammation during infection. Journal of leukocyte biology\| PubMed ID: 27286792	2016
The role of pili in Bacillus cereus intraocular infection. Experimental eye research\| PubMed ID: 28336259	2017
An Eye on Toxins: Roles in Ocular Damage and Inflammation. Toxins\| PubMed ID: 31248125	2019
S-layer Impacts the Virulence of Bacillus in Endophthalmitis. Investigative ophthalmology & visual science\| PubMed ID: 31479113	2019
S-Layer-Mediated Innate Interactions During Endophthalmitis. Frontiers in immunology\| PubMed ID: 32117322	2020
Innate Immune Interference Attenuates Inflammation In Bacillus Endophthalmitis. Investigative ophthalmology & visual science\| PubMed ID: 33180117	2020

Roger Astley

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