Adjunctive Thymosin Beta-4 Treatment Influences MΦ Effector Cell Function to Improve Disease Outcome in Pseudomonas aeruginosa-Induced Keratitis.

Our previous work has shown that topical thymosin beta 4 (Tß4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tß4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tß4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tß4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tß4 alone and adjunctive Tß4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tß4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study.

Activation of Conjunctiva-Associated Lymphoid Tissue in Patients With Infectious Keratitis Using In Vivo Confocal Microscopy.

Purpose: We aimed to evaluate activation of conjunctiva-associated lymphoid tissue (CALT) in patients with keratitis using in vivo confocal microscopy (IVCM) and conjunctival impression cytology (CIC). Methods: In addition to anterior segment photography and corneal fluorescein staining, IVCM revealed the palpebral conjunctiva in all subjects, and CIC and immunofluorescence staining were performed. Results: Diffuse lymphoid tissue cell density in the eyes of patients with keratitis was significantly greater compared with healthy volunteers (P < 0.001). Similar trends were found in perifollicular lymphocyte density (P < 0.001), follicular density (P = 0.029), follicular center reflection intensity (P = 0.011), and follicular area (P < 0.001). Immunofluorescence staining showed that the proportions of CD4+ (61.7% ± 8.0% vs. 17.3% ± 10.2%, respectively, P < 0.001) and CD8+ (46.9% ± 10.0% vs. 19.6% ± 11.5%, respectively, P < 0.001) cells in patients with keratitis was greater compared with healthy volunteers. Interestingly, we also observed changes in the contralateral eye in subjects with keratitis. Conclusions: Our research suggests that CALT, as an ocular immune structure, is activated and plays an important role in the pathogenesis of keratitis. This has been overlooked previously. CALT is also active in the contralateral eye of subjects with keratitis.

Establishing novel roles of bifidocin LHA, antibacterial, antibiofilm and immunomodulator against Pseudomonas aeruginosa corneal infection model.

Bifidocin LHA, a novel bacteriocin, was extracted from bee honey B. adolescentis and purified. Bifidocin LHA was characterized as a protein in nature, without lipid or carbohydrate moieties, the molecular weight was 16,000 Da protein, heat-stable and active at a wide range of pH values, bactericidal effect, detergent, and solvents did not affect bifidocin activity and can be classified as type II bacteriocin. In vitro, the antibacterial activity of purified bifidocin LHA was significantly higher than crude bifidocin LHA (P < 0.05) against Pseudomonas aeruginosa (P. aeruginosa). The antibiofilm activity of bifidocin LHA was significantly higher than the antibiofilm activity of Amikacin (P < 0.05). In vivo, bifidocin LHA demonstrates a significant decreased in the number of P. aeruginosa in the eye, while complete clearance of P. aeruginosa comparing with the control (P < 0.05) when treating with Bifidobacterium adolescentis and bifidocin LHA together. Bifidobacterium adolescentis and bifidocin LHA treatment together induced substantial elevation of IL10 and IL-12 concentrations (P < 0.01) that helped to prevent damage caused by the inflammatory response. Succeeded to eradicate P. aeruginosa infection improved by histological patterns of the eye tissues. This study indicated Bifidobacterium adolescentis and bifidocin LHA consider as crucial strategies for the practical treatment of eye infection in the future.

Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillus immune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild-type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The ΔinhA1 and ΔinhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the ΔinhA1-3 mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of WT B. thuringiensis or InhA mutants. Eyes were analyzed for intraocular Bacillus and myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with the ΔinhA1 or ΔinhA2 mutant strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with the WT strain. Eyes infected with the ΔinhA1-3 mutant cleared the infection. Quantitative real-time PCR (qRT-PCR) results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.

Molecular Mimicry between Betaine Aldehyde Dehydrogenase of Leptospira and Retinal Dehydrogenase 1 of Human Lens: A Potential Trigger for Cataract Formation in Leptospiral Uveitis Patients.

Purpose: Rapidly progressing cataract is one of the ocular manifestations in leptospiral uveitis patients. We examined whether molecular mimicry between the leptospira antigens and lens proteins exists that could result in cataract in these patients.Methods: Immunoblot analysis using patient sera was done with proteins from normal lens and cataract lens from leptospiral uveitis patients and the cross-reacting lens proteins were identified by mass spectrometry analysis.Results: Retinal dehydrogenase 1 and crystallins (α-B, α-A2, ß-B2), were recognized by the antibodies in the serum of leptospiral uveitis patients. And, retinal dehydrogenase 1 is homologous to the leptospiral protein, betaine aldehyde dehydrogenase.Conclusions: Leptospiral uveitis patient serum contains antibodies that cross-react with multiple lens proteins that have a role in maintaining lens transparency. And, these antibodies could act as a potential trigger for cataractogenesis.

Expression of Cytokines in Porcine Iris, Retina and Choroidal Tissues Stimulated by Microbe-associated Molecular Patterns.

PURPOSE: The innate immune system is strongly implicated in the pathogenesis of uveitis. This study was designed to clarify the responses of the innate immune system in uveal tissues. MATERIALS AND METHODS: We utilized quantitative, real-time RT-PCR to measure mRNA of innate immune system receptors from porcine iris, choroid, and retina tissues. We used RT-PCR for cytokines to evaluate the responses of these tissues to specific ligands or extracts of whole bacteria that activate the innate immune system. We used ELISA for IL-6 on selected choroidal supernatants to confirm that the mRNA measurement correlated with protein levels. RESULTS: In each of the studied tissues, we detected the expression of important receptors belonging to the innate immune system including dectin-1, TLR4, TLR8, and NOD2. Relative mRNA expression was generally lower in the retina compared to iris or choroid. All three tissues demonstrated upregulation of cytokine mRNA in response to a range of ligands that activate the innate immune system. The measurement of IL-6 protein was consistent with results based on mRNA. Notably, the expression of mRNA for IL-23 was more pronounced than IL-12 in all three tissues after stimulation with various innate immune system ligands. CONCLUSIONS: These data provide evidence of a potent innate immune response intrinsic to uveal tissues. Specific innate immune system ligands as well as bacterial extracts enhanced the production of several inflammatory cytokines. Furthermore, the observation of higher upregulation of IL-23 mRNA, compared to IL-12 in response to innate immune stimuli, suggested that a local TH17 response might be more robust than a local TH1 response in uveal tissues. Our results expand the understanding as to how the innate immune system may contribute to uveitis.

Innate Immune Interference Attenuates Inflammation In Bacillus Endophthalmitis.

Purpose: To explore the consequences of innate interference on intraocular inflammatory responses during Bacillus endophthalmitis. Methods: Bacillus endophthalmitis was induced in mice. Innate immune pathway activation was interfered by injecting S layer protein-deficient (∆slpA) B. thuringiensis or by treating wild-type (WT)-infected mice with a TLR2/4 inhibitor (WT+OxPAPC). At 10 hours postinfection, eyes were harvested and RNA was purified. A NanoString murine inflammation panel was used to compare gene expression in WT-infected, WT+OxPAPC, ∆slpA-infected, and uninfected eyes. Results: In WT-infected eyes, 56% of genes were significantly upregulated compared to uninfected controls. Compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ∆slpA-infected eyes, respectively. Expression of 61 genes that were upregulated in WT-infected eyes was decreased in WT+OxPAPC and ∆slpA-infected eyes. Innate interference resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate pathway genes (TLRs 2, 4, 6, and 8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2). Innate interference also reduced the expression of several inflammatory cytokines (CSF2, CSF3, IL-6, IL-1ß, IL-1α, TNFα, IL-23α, TGFß1, and IL-12ß) and chemokines (CCL2, CCL3, and CXCLs 1, 2, 3, 5, 9, and 10). All of the aforementioned genes were significantly upregulated in WT-infected eyes. Conclusions: These results suggest that interfering with innate activation significantly reduced the intraocular inflammatory response in Bacillus endophthalmitis. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.

The miR-183/96/182 Cluster Regulates the Functions of Corneal Resident Macrophages.

Tissue-resident macrophages (ResMϕ) play important roles in the normal development and physiological functions as well as tissue repair and immune/inflammatory response to both internal and external insults. In cornea, ResMϕ are critical to the homeostasis and maintenance, wound healing, ocular immune privilege, and immune/inflammatory response to injury and microbial infection. However, the roles of microRNAs in corneal ResMϕ are utterly unknown. Previously, we demonstrated that the conserved miR-183/96/182 cluster (miR-183/96/182) plays important roles in sensory neurons and subgroups of both innate and adaptive immune cells and modulates corneal response to bacterial infection. In this study, we provide direct evidence that the mouse corneal ResMϕ constitutively produce both IL-17f and IL-10. This function is regulated by miR-183/96/182 through targeting Runx1 and Maf, key transcriptional regulators for IL-17f and IL-10 expression, respectively. In addition, we show that miR-183/96/182 has a negative feedback regulation on the TLR4 pathway in mouse corneal ResMϕ. Furthermore, miR-183/96/182 regulates the number of corneal ResMϕ. Inactivation of miR-183/96/182 in mouse results in more steady-state corneal resident immune cells, including ResMϕ, and leads to a simultaneous early upregulation of innate IL-17f and IL-10 production in the cornea after Pseudomonas aeruginosa infection. Its multiplex regulations on the simultaneous production of IL-17f and IL-10, TLR4 signaling pathway and the number of corneal ResMϕ place miR-183/96/182 in the center of corneal innate immunity, which is key to the homeostasis of the cornea, ocular immune privilege, and the corneal response to microbial infections.

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment.

Mass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil-associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by P. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Murine model of ocular infection Basic Protocol 2: Murine model sample collection Basic Protocol 3: Protein extraction from eye wash Basic Protocol 4: Protein extraction from corneal tissue Basic Protocol 5: Mass spectrometry-based proteomics and bioinformatics from eye wash and corneal tissue samples.

ISG15 Acts as a Mediator of Innate Immune Response to Pseudomonas aeruginosa Infection in C57BL/6J Mouse Corneas.

Purpose: IFN-stimulated gene (ISG) 15 is a type 1 IFN-induced protein and known to modify target proteins in a manner similar to ubiquitylation (protein conjugation by ISG15 is termed ISGylation). We sought to determine the role of ISG15 and its underlying mechanisms in corneal innate immune defense against Pseudomonas aeruginosa keratitis. Methods: ISG15 expression in cultured human corneal epithelial cells (HCECs) and mouse corneas was determined by PCR and Western blot analysis. Gene knockout mice were used to define the role of ISG15 signaling in controlling the severity of P. aeruginosa keratitis, which was assessed with photographing, clinical scoring, bacterial counting, myeloperoxidase assay, and quantitative PCR determination of cytokine expression. Integrin LFA-1 inhibitor was used to assess its involvement of ISG15 signaling in P. aeruginosa-infected corneas. Results: Heat-killed P. aeruginosa induced ISG15 expression in cultured HCECs and accumulation in the conditioned media. Isg15 deficiency accelerated keratitis progress, suppressed IFNγ and CXCL10, and promoted IL-1ß while exhibiting no effects on IFNα expression. Moreover, exogenous ISG15 protected the corneas of wild-type mice from P. aeruginosa infection while markedly reducing the severity of P. aeruginosa keratitis in type 1 IFN-receptor knockout mice. Exogenous ISG15 increased bacteriostatic activity of B6 mouse corneal homogenates, and inhibition of LFA-1 exacerbated the severity of and abolished protective effects of ISG15 on P. aeruginosa keratitis. Conclusions: Type 1 INF-induced ISG15 regulates the innate immune response and greatly reduces the susceptibility of B6 mouse corneas to P. aeruginosa infection in an LFA-1-dependent manner.

Glycolytic inhibitor 2-deoxyglucose suppresses inflammatory response in innate immune cells and experimental staphylococcal endophthalmitis.

Previously, we have shown that Staphylococcus (S) aureus induces a glycolytic response in retinal residential (microglia) and infiltrated cells (neutrophils and macrophages) during endophthalmitis. In this study, we sought to investigate the physiological role of glycolysis in bacterial endophthalmitis using a glycolytic inhibitor, 2-deoxyglucose (2DG). Our data showed that 2DG treatment attenuated the inflammatory responses of mouse bone marrow-derived macrophages (BMDM) and neutrophils (BMDN) when challenged with either live or heat-killed S. aureus (HKSA). Among the inflammatory mediators, 2DG caused a significant reduction in levels of cytokines (TNF-α, IL-1ß, IL-6) and chemokines (CXCL1 and CXCL2). Western blot analysis of 2DG treated cells showed downregulation of bacterial-induced MEK/ERK pathways. In vivo, intravitreal administration of 2DG both pre- and post-bacterial infection resulted in a significant reduction in intraocular inflammation in C57BL/6 mouse eyes and downregulation of ERK phosphorylation in retinal tissue. Collectively, our study demonstrates that 2DG attenuates inflammatory response in bacterial endophthalmitis and cultured innate immune cells via inhibition of ERK signaling. Thus glycolytic inhibitors in combination with antibiotics could mitigate inflammation-mediated tissue damage in ocular infections.

Periocular necrotizing soft tissue infection in Greater Copenhagen.

PURPOSE: Necrotizing soft tissue infection, also known as necrotizing fasciitis (NF), is a fast-spreading life-threatening infection that most commonly affects the lower limbs, groin, or abdomen. Periocular necrotizing fasciitis (PNF) is rare. Limited data exist on PNF immune cell subset; hence, this study aims to determine the representation of immune cell subsets in patients diagnosed with PNF using immunohistochemical stainings. METHODS: All patients diagnosed with PNF at Copenhagen University Hospital from 2008 to 2018 were included. Their electronic medical records and pathology reports were assessed, and available tissue specimens were reviewed and stained with monoclonal antibodies for CD1a+ Langerhans' cells, CD3+ T lymphocytes, CD15+ granulocytes, CD44+ lymphohematopoietic cells, CD68+ histiocytes, CD79α+ B lymphocytes, and FXIIIa+ dendritic macrophages and Langerhans' cells. The number of positive cells was counted, and an average score was calculated. The location of immune cells and bacteria was assessed. RESULTS: The specimens were characterized by acute inflammation and necrosis of the fascia, while striated muscle involvement was less frequent. Haemolytic group A streptococci and Staphylococcus aureus were identified and mainly located in the deep dermis and subcutis in close relation to the fascia. Only few areas harboured both bacteria and inflammatory cells. Granulocytes, histiocytes and CD44+ lymphohematopoietic cells were demonstrated to be abundant in all patients, while B and T lymphocytes, dendritic macrophages and Langerhans' cells were less frequent. CONCLUSION: The immune cell subsets found in this study of PNF were consistent with those identified in the literature on NF in other anatomical locations. This study concludes that immune cells are abundant and exhibit a typical pattern in PNF.

Targets of immunomodulation in bacterial endophthalmitis.

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.

Immune-mediated tuberculous uveitis - A rare association with papulonecrotic tuberculid.

The tuberculids are a group of distinct clinicopathological form of skin lesions representing hypersensitivity reaction to hematogenous dissemination of Mycobacterium tuberculosis or its antigen from an underlying active or a silent focus of tuberculosis elsewhere in the body in an individual with a strong antituberculous cell-mediated immunity and by definition do not show bacilli on special stains and are culture-negative. Ocular involvement can occur in tuberculosis, both due to direct invasion by the bacilli as well as an immune-mediated reaction; however, immune-mediated tuberculous uveitis occurring as a hypersensitivity response in association with PNT has hardly been reported in the literature. Here we report one such rare case.

Manifestations and outcomes of nocardia infections: Comparison of immunocompromised and nonimmunocompromised adult patients.

Nocardia is a ubiquitous environmental pathogen that causes infection primarily following inhalation into the lungs. It is generally thought to cause infection primarily in immunocompromised patients, but nonimmunocompromised individuals are also at risk of infection. We sought to compare risk factors, clinical manifestations, diagnostic approach, treatment, and mortality in immunocompromised and nonimmunocompromised adults with nocardiosis.We studied all adults with culture-proven Nocardia infection at a tertiary care hospital from 1994 to 2015 and compared immunocompromised with nonimmunocompromised patients. The immunocompromised group included patients who had a solid organ transplant, hematopoietic cell transplant (HCT), hematological or solid tumor malignancy treated with chemotherapy in the preceding 90 days, inherited immunodeficiency, autoimmune/inflammatory disorders treated with immunosuppressive agents, or high-dose corticosteroid therapy for at least 3 weeks before the diagnosis of nocardiosis.There were 112 patients, mean age 55 ±â€Š17 years; 54 (48%) were women. Sixty-seven (60%) were immunocompromised, and 45 (40%) were nonimmunocompromised. The lung was the site of infection in 54 (81%) immunocompromised and 25 (55%) nonimmunocompromised patients. Pulmonary nocardiosis in immunocompromised patients was associated with high-dose corticosteroids, P = .002 and allogeneic HCT, P = .01, and in nonimmunocompromised patients with cigarette smoking, bronchiectasis, and other chronic lung diseases, P = .002.Cavitation occurred only in the immunocompromised group, P < .001. Disseminated infection was more common in the immunocompromised, P = .01, and was highest in solid organ transplant recipients, P = .007. Eye infection was more common in nonimmunocompromised patients, P = .009. Clinical signs and symptoms did not differ significantly between the 2 groups. The initial treatment for most patients in both groups was trimethoprim-sulfamethoxazole with or without a carbapenem. All-cause 1-year mortality was 19%; 18 (27%) immunocompromised and 3 (7%) nonimmunocompromised patients died, P = .01.Immunocompromised patients with nocardiosis had more severe disease and significantly higher mortality than nonimmunocompromised patients, but clinical presentations did not differ.

Antibodies to Conserved Surface Polysaccharides Protect Mice Against Bacterial Conjunctivitis.

Purpose: Bacterial conjunctivitis is a major problem in ocular health. Little is known about protective immune effectors in the conjunctiva. We evaluated whether opsonic antibody to the conserved surface/capsular polysaccharide poly-N-acetyl glucosamine (PNAG) expressed by Streptococcus pneumoniae and Staphylococcus aureus was protective against bacterial conjunctivitis, as well as an antibody to the Pseudomonas aeruginosa surface polysaccharide alginate. Methods: Bacteria were injected directly into the conjunctivae of either A/J mice or into conjunctivae of wild type C57Bl/6 mice for comparisons to responses of recombination activating gene 1-knock out (RAG 1 KO) or germ-free mice in the C57Bl/6 genetic background. Human IgG1 monoclonal antibodies (MAb) to either PNAG or alginate were administered as follows: direct injection of 10 µg into the conjunctivae or topical application onto the cornea 4, 24, and 32 hours post infection; or intraperitoneal injection of 200 µg 18 hours prior to and then 4, 24, and 32-hours postinfection. After 48 hours, eyes were scored for pathology, mice were euthanized, and CFU/conjunctiva was determined. Results: All methods of antibody administration reduced S. pneumoniae, S. aureus, or P. aeruginosa pathology and bacterial levels in the conjunctivae. Histopathologic analysis showed severe inflammatory cell infiltrates in conjunctivae of mice treated with control MAb, whereas immune mice showed only very mild cellular infiltration. The protective effect of MAb to PNAG was abolished in RAG 1 KO and germ-free mice. Conclusions: Antibodies to both PNAG and alginate demonstrated therapeutic efficacy in models of S. pneumoniae, S. aureus, and P. aeruginosa conjunctivitis, validating the protective capacity of antibodies to surface polysaccharides in distinct ocular tissues.