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

Intraocular infections are prevalent after traumatic injuries or after common ocular surgeries. Infections cause inflammation that can damage the retina and architecture of the eye, often resulting in poor visual outcomes. Severe cases may result in blindness or require enucleation of the eye. Treatments for intraocular infections include intravitreal antibiotics and corticosteroids or surgical vitrectomy in serious cases. The increase in multidrug-resistant infections calls for novel treatment options. In the present study, a biomimetic erythrocyte-derived nanosponge was tested for the ability to neutralize pore-forming toxins from the most frequent Gram-positive bacterial causes of intraocular infections (, , , and ). Nanosponge pretreatment of supernatants reduced hemolytic activity  In a murine sterile endophthalmitis model, nanosponge pretreatment of injected supernatants resulted in greater retinal function and less ocular pathology compared to that in eyes injected with untreated supernatants from all pathogens except methicillin-resistant  In a murine bacterial endophthalmitis model, treatment with gatifloxacin and gatifloxacin-nanosponges reduced intraocular bacterial burdens, except in the case of methicillin-sensitive  For all pathogens, eyes in both treatment groups showed decreased ocular pathology and inflammation. Overall, reductions in retinal function loss afforded by gatifloxacin-nanosponge treatment were significant for , , and methicillin-resistant  but not for  and methicillin-sensitive  These results suggest that clinical improvements in intraocular infections following nanosponge treatment were dependent on the complexity and types of toxins produced. Nanosponges might serve as an adjunctive therapy for the treatment of ocular infections. Endophthalmitis is a blinding consequence of bacterial invasion of the interior of the eye. Because of increases in the numbers of ocular surgeries and intraocular injections, the incidence of endophthalmitis is steadily increasing. , , , and  are leading causes of infection following ocular procedures and trauma and are increasingly more difficult to treat due to multidrug resistance. Each of these pathogens produces pore-forming toxins that contribute to the pathogenesis of endophthalmitis. Treatment of these infections with antibiotics alone is insufficient to prevent damage to the retina and vision loss. Therefore, novel therapeutics are needed that include agents that neutralize bacterial pore-forming toxins. Here, we demonstrate that biomimetic nanosponges neutralize pore-forming toxins from these ocular pathogens and aid in preserving retinal function. Nanosponges may represent a new form of adjunct antitoxin therapy for serious potentially blinding intraocular infections.

Disarming Pore-Forming Toxins with Biomimetic Nanosponges in Intraocular Infections.

Bacterial infection of the posterior segment of the eye (endophthalmitis) leads to a robust host response that often results in irreversible damage to the layers of the retina, significant vision loss, and in some patients, enucleation of the globe. While a great deal of effort has gone into understanding the role of bacterial virulence factors in disease initiation and propagation, it is becoming increasingly clear that the host response to infection plays a major role in causing the damage associated with endophthalmitis. Researchers have identified the host receptors which detect infecting organisms and initiate the cascade of events that result in inflammation. This inflammation may damage nonregenerative tissues of the eye while attempting to clear the infection. Both Gram-positive and Gram-negative bacteria can cause endophthalmitis. These organisms initiate an immune response by activating toll-like receptor (TLR) pathways. Once an inflammatory response is initiated, the expression of immunomodulators, such as proinflammatory chemokines and cytokines, affect the recruitment of PMNs and other inflammatory cells into the eye. We and others have reported that knockout mice that do not express specific inflammatory pathways and molecules have an attenuated response to infection and retain significant retinal function. These findings suggest that host immune mediators are important components of the response to infections in the posterior segment of the eye, and the timing and level of their production may be related to the severity of the damage and the ultimate visual outcome. If that is the case, a better understanding of the complex and often redundant role of these pathways and inflammatory mediators may identify host molecules as potential anti-inflammatory therapeutic targets. This review highlights potential anti-inflammatory targets during acute inflammation in endophthalmitis, compares and contrasts those with findings in other models of ocular inflammation, and translates current immunomodulatory strategies for other types of infection and inflammation to this blinding disease. Given the poor visual outcomes seen in patients treated with antibiotics alone or in combination with corticosteroids, immunomodulation in addition to antibiotic therapy might be more effective in preserving vision than current regimens.

Targets of immunomodulation in bacterial endophthalmitis.

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.

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

Erin Livingston

Department of Microbiology and Immunology

University of Oklahoma Health Sciences Center

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

Erin Livingston

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology and Immunology

University of Oklahoma Health Sciences Center

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

Department of Microbiology and Immunology