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 ...

University of Oklahoma Health Sciences Center

Dean McGee Eye Institute

 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.

Some tissues of the eye are susceptible to damage due to their exposure to the outside environment and inability to regenerate. Immune privilege, although beneficial to the eye in terms of homeostasis and protection, can be harmful when breached or when an aberrant response occurs in the face of challenge. In this review, we highlight the role of the PMN (polymorphonuclear leukocyte) in different bacterial ocular infections that invade the immune privileged eye at the anterior and posterior segments: keratitis, conjunctivitis, uveitis, and endophthalmitis. Interestingly, the PMN response from the host seems to be necessary for pathogen clearance in ocular disease, but the inflammatory response can also be detrimental to vision retention. This "Pyrrhic Victory" scenario is explored in each type of ocular infection, with details on PMN recruitment and response at the site of ocular infection. In addition, we emphasize the differences in PMN responses between each ocular disease and its most common corresponding bacterial pathogen. The in vitro and animal models used to identify PMN responses, such as recruitment, phagocytosis, degranulation, and NETosis, are also outlined in each ocular infection. This detailed study of the ocular acute immune response to infection could provide novel therapeutic strategies for blinding diseases, provide more general information on ocular PMN responses, and reveal areas of bacterial ocular infection research that lack PMN response studies.

A Pyrrhic Victory: The PMN Response to Ocular Bacterial Infections.

Bacillus cereus (B. cereus) endophthalmitis is a devastating intraocular infection primarily associated with post-traumatic injuries. The majority of these infections result in substantial vision loss, if not loss of the eye itself, within 12-48 h. Multifactorial mechanisms that lead to the innate intraocular inflammatory response during this disease include the combination of robust bacterial replication, migration of the organism throughout the eye, and toxin production by the organism. Therefore, the window of therapeutic intervention in B. cereus endophthalmitis is quite narrow compared to that of other pathogens which cause this disease. Understanding the interaction of bacterial and host factors is critical in understanding the disease and formulating more rational therapeutics for salvaging vision. In this review, we will discuss clinical and research findings related to B. cereus endophthalmitis in terms of the organism's virulence and inflammogenic potential, and strategies for improving of current therapeutic regimens for this blinding disease.

The cereus matter of Bacillus 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.

 produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections.  also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to  virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we reported that PlcR-deficient  was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for  in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of  endophthalmitis, and future analysis of the  virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence.

Expression of  Virulence-Related Genes in an Ocular Infection-Related Environment.

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

Article	Year
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
A Pyrrhic Victory: The PMN Response to Ocular Bacterial Infections. Microorganisms\| PubMed ID: 31703354	2019
The cereus matter of Bacillus endophthalmitis. Experimental eye research\| PubMed ID: 32032628	2020
S-Layer-Mediated Innate Interactions During Endophthalmitis. Frontiers in immunology\| PubMed ID: 32117322	2020
Expression of Virulence-Related Genes in an Ocular Infection-Related Environment. Microorganisms\| PubMed ID: 32331252	2020
Innate Immune Interference Attenuates Inflammation In Bacillus Endophthalmitis. Investigative ophthalmology & visual science\| PubMed ID: 33180117	2020

Md Huzzatul Mursalin

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