Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Harvard Medical School

The potential role of commensals as triggering factors that promote inflammation in dry eye disease has not been explored. The objective of this study was to evaluate whether ocular microbiota changes with the onset of dry eye disease in thrombospondin-1-deficient (TSP-1(-/-)) mice, a strain that develops Sjögren's syndrome-like disease.

TSP-1 Deficiency Alters Ocular Microbiota: Implications for Sjögren's Syndrome Pathogenesis.

The ocular surface is continuously exposed to the environment and, therefore, it is surprising that it harbors only few commensals with low degree of diversity. This unique aspect of the ocular surface physiology prompts the question whether there are core ocular commensal communities and how they affect ocular immunity. The purpose of this review is to provide an overview of what is known about the ocular surface commensals in health and disease and what we would like to learn in the near future. In addition, we discuss how microbiota at sites other than the eye may influence ocular immune responses. The information discussed in the review has been gathered using PubMed searches for literature published from January 1982 to December 2015.

Impact of Microbiome on Ocular Health.

The existence of the ocular microbiota has been reported but functional analyses to evaluate its significance in regulating ocular immunity are currently lacking. We compared the relative contribution of eye and gut commensals in regulating the ocular susceptibility to Pseudomonas aeruginosa-induced keratitis. We find that in health, the presence of microbiota strengthened the ocular innate immune barrier by significantly increasing the concentrations of immune effectors in the tear film, including secretory IgA and complement proteins. Consistent with this view, Swiss Webster (SW) mice that are typically resistant to P. aeruginosa-induced keratitis become susceptible due to the lack of microbiota. This was exemplified by increased corneal bacterial burden and elevated pathology of the germ free (GF) mice when compared to the conventionally maintained SW mice. The protective immunity was found to be dependent on both eye and gut microbiota with the eye microbiota having a moderate, but significant impact on the resistance to infection. These events were IL-1ß-dependent as corneal IL-1ß levels were decreased in the infected GF and antibiotic-treated mice when compared to the SPF controls, and neutralization of IL-1ß increased the ocular bacterial burden in the SPF mice. Monocolonizing GF mice with Coagulase Negative Staphylococcus sp. isolated from the conjunctival swabs was sufficient to restore resistance to infection. Cumulatively, these data underline a previously unappreciated role for microbiota in regulating susceptibility to ocular keratitis. We predict that these results will have significant implications for contact lens wearers, where alterations in the ocular commensal communities may render the ocular surface vulnerable to infections.

Impact of Microbiota on Resistance to Ocular Pseudomonas aeruginosa-Induced Keratitis.

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense.

An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal γδ T Cells.

The purpose of this study was to evaluate mechanisms controlling secretory IgA (SIgA) production, thereby ensuring maintenance of ocular surface health.

Role of Microbiota in Strengthening Ocular Mucosal Barrier Function Through Secretory IgA.

Pseudomonas aeruginosa-induced corneal keratitis is a sight-threatening disease. The rise of antibiotic resistance among P. aeruginosa keratitis isolates makes treatment of this disease challenging, emphasizing the need for alternative therapeutic modalities. By comparing the responses to P. aeruginosa infection between an outbred mouse strain (Swiss Webster, SW) and a susceptible mouse strain (C57BL6/N), we found that the inherent neutrophil-killing abilities of these strains correlated with their susceptibility to infection. Namely, SW-derived neutrophils were significantly more efficient at killing P. aeruginosa in vitro than C57BL6/N-derived neutrophils. To interrogate whether the distinct neutrophil killing capacities were dependent on endogenous or exogenous factors, neutrophil progenitor cell lines were generated. The in vitro differentiated neutrophils from either SW or C57BL6/N progenitors retained the differential killing abilities, illustrating that endogenous factors conferred resistance. Consistently, quantitative LC-MS/MS analysis revealed strain-specific and infection-induced alterations of neutrophil proteomes. Among the distinctly elevated proteins in the SW-derived proteomes were α-mannosidases, potentially associated with protection. Inhibition of α-mannosidases reduced neutrophil bactericidal functions in vitro. Conversely, topical application of α-mannosidases reduced bacterial biofilms and burden of infected corneas. Cumulatively, these data suggest novel therapeutic approaches to control bacterial biofilm assembly and improve bacterial clearance via enzymatic treatments.

Frontline Science: Employing enzymatic treatment options for management of ocular biofilm-based infections.

Multidrug-resistant (MDR) bacterial infections are a leading cause of mortality, affecting approximately 250,000 people in Canada and over 2 million people in the United States, annually. The lack of efficacy of antibiotic-based treatments is often caused by inability of the drug to penetrate bacterial biofilms in sufficient concentrations, posing a major therapeutic challenge. Here, we review the most recent information about the architecture of  biofilms in vivo and describe how advances in imaging and mass spectroscopy analysis bring about novel therapeutic options and challenge existing dogmas.

Tasked with a Challenging Objective: Why Do Neutrophils Fail to Battle  Biofilms.

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1), an actin bundling protein, resulted in an ocular commensal overgrowth, characterized with increased presence of conjunctival  spp. The commensal overgrowth correlated with susceptibility to -induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the -infected corneas, altered inflammatory responses, and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre ( ) and S100A8 Cre ( ) promoters had a similar phenotype to the LPL KOs mice. In contrast, infected  mice did not display elevated susceptibility to infection, implicating the myeloid L-plastin-sufficient cells (e.g., macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically, the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to , augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively, our data highlight the importance of neutrophils in controlling ocular paucibacteriality, reveal molecular and cellular events involved in the process, and suggest a link between commensal exposure and resistance to infection.

Abirami Kugadas

Department of Medicine,
Division of Infectious Diseases,
Brigham and Women's Hospital,
Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital

Harvard Medical School

Conjunctival Commensal Isolation and Identification in Mice

Abirami Kugadas

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medicine,Division of Infectious Diseases,Brigham and Women's Hospital,Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital

Harvard Medical School

Conjunctival Commensal Isolation and Identification in Mice

Department of Medicine,
Division of Infectious Diseases,
Brigham and Women's Hospital,
Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital