Oral administration of S-nitroso-l-glutathione (GSNO) provides anti-inflammatory and cytoprotective effects during ocular bacterial infections

Abstract

Bacterial endophthalmitis is a severe complication of eye surgeries that can lead to vision loss. Current treatment involves intravitreal antibiotic injections that control bacterial growth but not inflammation. To identify newer therapeutic targets to promote inflammation resolution in endophthalmitis, we recently employed an untargeted metabolomics approach. This led to the discovery that the levels of S-nitroso-l-glutathione (GSNO) were significantly reduced in an experimental murine Staphylococcus aureus (SA) endophthalmitis model. In this study, we tested the hypothesis whether GSNO supplementation via different routes (oral, intravitreal) provides protection during bacterial endophthalmitis. Our results show that prophylactic administration of GSNO via intravitreal injections ameliorated SA endophthalmitis. Therapeutically, oral administration of GSNO was found to be most effective in reducing intraocular inflammation and bacterial burden. Moreover, oral GSNO treatment synergized with intravitreal antibiotic injections in reducing the severity of endophthalmitis. Furthermore, in vitro experiments using cultured human retinal Muller glia and retinal pigment epithelial (RPE) cells showed that GSNO treatment reduced SA-induced inflammatory mediators and cell death. Notably, both in-vivo and ex-vivo data showed that GSNO strengthened the outer blood-retinal barrier during endophthalmitis. Collectively, our study demonstrates GSNO as a potential therapeutic agent for the treatment of intraocular infections due to its dual anti-inflammatory and cytoprotective properties.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-023-04963-w.

Introduction

Infectious diseases can impact ocular health, with intraocular infections such as bacterial endophthalmitis potentially leading to blindness, if not treated promptly [1]. The incidence of such infections continues to rise due to the increasing number of ocular surgeries for diseases such as cataracts, glaucoma, and age-related macular degeneration (AMD), all of which are becoming more prevalent due to an increase in the aging population [2, 3]. The current treatment for bacterial endophthalmitis involves intravitreal injection of antibiotics and vitrectomy in severe cases of endophthalmitis [4, 5]. In addition to intravitreal antibiotics, systemic antibiotics are administered in most endogenous endophthalmitis cases to control the pathogen spread [6]. Intravitreal corticosteroids are often used to limit intraocular inflammation, their use in endophthalmitis remains controversial owing to their immunosuppressive and deleterious long-term consequences [7]. The intravitreal antibiotics, while destroying the bacteria, may release various bacterial cell wall components [8, 9], which induce an inflammatory response, as reported in studies from ours [10] and other laboratories [11, 12]. Moreover, frequent intravitreal injections for the treatment of retinal diseases can predispose individuals to develop endophthalmitis [13, 14]. Therefore, there is a pressing need for therapeutics that can effectively target both the pathogen and the host inflammatory responses during endophthalmitis [15].

Given the complexity of host–pathogen interactions, our lab has adopted high-throughput technologies such as transcriptomics and metabolomics along with systems biology approaches to study the pathobiology of ocular infections [16, 17]. This has uncovered potential crosstalk between various metabolic pathways and the innate immune responses during ocular bacterial [18] and viral [19] infections. We reported increased glycolytic response in both residential and infiltrating immune cells (e.g. macrophages) during Staphylococcus aureus (SA) infection in the eye [18] and where intraocular administration of the glycolytic inhibitor, 2-deoxy-glucose (2DG) attenuated intraocular inflammation [20]. Similarly, our metabolomics studies led to the discovery of a Krebs cycle metabolite, itaconate, in promoting anti-inflammatory and antioxidant responses during SA endophthalmitis [21]. Collectively, these studies indicate the role of metabolic pathways in orchestrating protective innate responses during intraocular infections. Similarly, other studies have utilized metabolomics in unravelling host–pathogen interactions and identifying biomarkers and potential therapeutic targets in different diseases [22–24].

Among the various altered metabolites, we found that levels of S-nitroso-l-glutathione (GSNO) were reduced in SA-infected mouse retina. GSNO, a S-nitrosothiol (SNO) is a key signaling molecule in various stress responses [25]. It is a naturally occurring molecule with the primary function to regulate NO levels in the body by acting as its reservoir. It is well known that NO signaling plays a critical role in a wide range of physiological processes, including blood vessel dilation, neurotransmitter release, and immune system functions. The interaction of NO with many macromolecules is facilitated by its radical nature and capacity to traverse lipid membranes [26]. During microbial infection, NO is produced as a host defense mechanism [27] by increased activity of NO synthases i.e., eNOS, nNOS, or iNOS [28]. However, excessive, and prolonged NO production can trigger nitrosative stress causing host tissue damage [29]. Several strategies have been employed to increase the therapeutic efficacy of NO due to its extremely short half-life [30]. GSNO, formed by the combination of NO and a key antioxidant, glutathione (GSH) may perpetuate the in vivo functions of locally produced NO [31]. GSNO is the most abundant endogenous NO donor and carrier [32, 33] that can spontaneously release NO at different rates [34]. In addition, GSNO is a potent S-nitrosating agent and can regulate activities of transcription factors and cytokines. By altering these proteins, GSNO has the ability to reduce excessive inflammation or strengthen immune defenses by amplifying the magnitude and the duration of immunological responses [25]. This makes GSNO a crucial mediator of cellular responses to oxidative stress and inflammation. The optimal levels of GSNO are maintained by GSNOR (S-nitrosoglutathione reductase) by controlling its degradation. Thus, GSNOR ensures balanced and beneficial effects of NO under various physiological conditions and influencing disease outcomes [35].

Given the importance of GSNO in diverse cellular functions and its potential therapeutic applications in infectious [36, 37] and inflammatory diseases [38–40], we hypothesized that GSNO may improve the disease outcomes of ocular bacterial infections. Here, we report that GSNO administration via different routes attenuates ocular inflammation and exerts cytoprotective effects in a mouse model of SA endophthalmitis.

Results

Prophylactic but not therapeutic intraocular administration of GSNO protects mice from endophthalmitis

Our analysis of metabolomics data showed that GSNO levels decreased during endophthalmitis. The temporal analysis revealed that GSNO levels increased slightly at 6 h post SA infection, but declined by 50% at 12 h, 75% at 24 h, and ~ 90% both at 48 and 72 h as compared to the control (Fig. S1). Since GSNO is known for its anti-inflammatory and anti-apoptotic properties, we hypothesized that GSNO protects the eye during bacterial infections. To test this hypothesis, we first investigated whether intravitreal injection of GSNO could protect the infected mouse eye. Our results indicated that a low dose (1 μg/eye) of GSNO administered via intravitreal injection at 6 h post SA infection, did not reduce corneal haze and opacity (Fig. S2A) or bacterial burden (Fig. S2B). However, a higher dose (10 μg/eye) reduced bacterial load but not the inflammatory mediators (Fig. S2C). We then examined whether prophylactically increasing GSNO levels could provide protection. Interestingly, we found that even the lower dose (1 μg/eye) of GSNO when administered 12 h before SA infection, significantly reduced corneal haze and anterior chamber opacity (Fig. 1A). To assess retinal health and function, we conducted an electroretinogram (ERG) test, which revealed better retention of the A-wave but not the B-wave in GSNO-treated eyes (Fig. 1B, C). Furthermore, in contrast to post-treatment, GSNO pretreatment significantly reduced bacterial burden (Fig. 1D) which was accompanied by reduced levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and chemokines (CXCL-1 and CXCL-2) (Fig. 1E). It is pertinent to note that the only GSNO-treated group did not show any adverse effect including corneal haze or anterior chamber inflammation (data not shown). These results indicate that a single intraocular injection of GSNO exerts protection when administered prophylactically, but not therapeutically.

Fig. 1.

Prophylactic ocular administration of GSNO during SA endophthalmitis. Eyes (n = 10) of C57BL/6 mice were treated with intravitreal injections of GSNO (1 μg/eye) or PBS (control) 12 h before being infected with S. aureus (SA, 5000 cfu/eye). A At 24 h post-infection, all eyes were examined using slit-lamp examination, and representative photomicrographs are shown from each group showing similar pathology. B Scotopic electroretinogram (ERG) analysis was performed to assess retinal function. C A bar graph was created to show the retention of a- and b-wave amplitude percentages relative to the control eyes, which were set at 100%. D Enucleated eyes were homogenized to determine the bacterial load via serial dilution and plating on TSA plates. E ELISA was used to quantify the levels of indicated inflammatory cytokines and chemokines in the whole eye lysates. Statistical analysis was performed using ANOVA, with significance levels of (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001, and ns indicating non-significance. Comparisons were made between uninfected control (C) vs. SA and SA vs. GSNO-treated samples. The results are cumulative of two independent experiments.

Systemic administration of GSNO improves the outcome of endophthalmitis

In clinical scenarios, patients usually come to the clinic when they experience pain or blurry vision, indicative of disease manifestation. Therefore, prophylactic use of GSNO, although effective, may not be of clinical use in these situations. However, we reported that antifungal therapy when given systemically i.e., orally (OR) or intravenously (IV) was found to be as effective as intravitreal (IVT) or the combination of systemic and intraocular routes in the treatment of experimental fungal endophthalmitis [41]. Thus, we decided to check the therapeutic efficacy of GSNO via different routes and using multiple doses (Fig. 2A; also see methods for details). After the induction of SA endophthalmitis and indicated GSNO treatments, all experiments were terminated at 72 h post-SA infection. As expected, SA infection resulted in increased corneal haze, anterior chamber opacity, and hypopyon formation. In contrast, except for IVT alone, GSNO treatment via various routes remarkably decreased the clinical symptoms of endophthalmitis (Fig. 2B). Additionally, the bacterial burden was significantly reduced by GSNO treatment via all routes, with no significant differences among various routes (Fig. 2C). Coinciding with these observations, the levels of inflammatory mediators were significantly reduced in all treatment groups with OR and IV routes being relatively lower than the others (Fig. 2D). These results indicated the effectiveness of systemic administration of GSNO in ameliorating bacterial endophthalmitis.

Fig. 2.

Therapeutic administration of GSNO via systemic routes during SA endophthalmitis. A Schematic representation of GSNO treatment administered through different routes (IVT, intravitreal; IV, intravenous; OR, oral) and using multiple doses at indicated times post-infection (p.i). Endophthalmitis was induced by IVT injection of S. aureus (SA) in B6 mice (n = 8 eyes) were analyzed at 24 h p.i. B Slit-lamp examination was performed, and representative photomicrographs are shown from each group showing similar pathology. C Eyes were enucleated, homogenized, and bacterial load was determined by serial dilution and plating. D ELISA was performed on whole eye lysates from control, infected, and GSNO-treated eyes to quantify levels of indicated inflammatory mediators. Data are represented as mean ± SD. Statistical analysis was performed using ANOVA (**) p < 0.01 (***) p < 0.001 (****) p < 0.0001, ns non-significant. Comparisons were made between uninfected control (C) vs SA and SA vs GSNO-treated samples. The results are cumulative of two independent experiments

Oral administration of GSNO attenuates SA endophthalmitis

According to the Infectious Disease Society of America (IDSA) and the European Society of Cataract and Refractive Surgeons (ESCRS), oral antimicrobials are recommended as an adjunct therapy to intravitreal injections for the treatment of severe endophthalmitis [42, 43]. Our study found that oral administration of GSNO was effective in reducing intraocular inflammation. Therefore, we conducted a time course study to evaluate the therapeutic efficacy of GSNO via the oral route. We observed that oral gavage of either PBS (vehicle control) or GSNO (drug control) did not cause any clinical signs or symptoms, including opacity, in mouse eyes. As expected, SA infection caused a time-dependent increase in corneal haze and opacity in mice which were given PBS by oral gavage. However, mice administered with oral gavages of GSNO had relatively reduced clinical symptoms of endophthalmitis at time points as evidenced by reduced corneal haze and opacity (Fig. 3A). The drug control group showed a similar phenotype as the PBS injected and uninfected eyes (data not shown).

Fig. 3.

Oral GSNO treatment ameliorated SA endophthalmitis. Eyes of C57BL/6 mice (n = 8 eyes) were intravitreally injected with 5000 cfu/eye of S. aureus (SA), followed by oral administration of GSNO at 6, 24, and 48 h post-infection. PBS-treated mice were used as a control (C). A Slit-lamp examination and photomicrographs were taken at each time interval. Representative photomicrographs are shown from each group showing similar pathology. B Retinas were harvested at the indicated time points post-infection, lysed, and analyzed for the expression of inflammatory pathway proteins by western blot, with β-actin used as the endogenous control. C Densitometric analysis was performed using ImageJ software and the results were expressed as relative fold changes normalized to the respective loading control, β-actin. D Whole eye lysates from control, infected and GSNO-treated eyes at indicated time points were subjected to ELISA to quantify levels of inflammatory cytokines and chemokines. The data are represented as mean ± SD. Statistical analysis was performed using ANOVA (∗) p < 0.05 (∗∗) p < 0.01 (∗∗∗) p < 0.001 (****) p < 0.0001, ns non-significant. Significance was compared between control, C vs SA samples and SA vs GSNO-treated samples. The results are cumulative of two independent experiments

Next, we assessed the activation of signaling pathways involved in the inflammatory response (Fig. 3B). Our data showed that retinal tissues from GSNO-treated mice had reduced activation (i.e., phosphorylation) of NF-kB p65 at 48 and 72 h post-infection. Similarly, the levels of NLRP3 inflammasome and cleaved Caspase 1 levels were reduced in GSNO-treated mouse retina compared to the PBS group (Fig. 3C). The ELISA assay from whole eye lysates showed a time-dependent increase in levels of inflammatory mediators in the PBS-treated (control) treated group. However, this response was attenuated in all GSNO-treated mice (Fig. 3D). Taken together, these results indicated that oral administration of GSNO was effective in mitigating SA endophthalmitis in mice.

GSNO treatment inhibits iNOS levels during SA endophthalmitis

Although inducible nitric oxide (iNOS) contributes to the host defense responses against invading pathogens, excessive production of NO radicals leads to uncontrolled inflammation, ultimately causing damage to host tissue [29, 44]. GSNO is a slow NO donor, and thus its role in regulating iNOS levels in SA-infected retinas was determined following GSNO oral treatments. Immunofluorescence assays revealed that SA infection induced the expression of iNOS in the mouse retina, and its levels were reduced in the GSNO-treated group (Fig. 4A). The expression of iNOS was also evaluated by western blot analysis of retinal tissue lysates, and it was observed that GSNO treatment inhibited its expression in SA-infected eyes (Fig. 4B, C). To further test the effect of GSNO on iNOS, we used a cultured human retinal Müller glia cell line, MIO-M1. As anticipated, Müller glia challenged with SA showed a significant increase in iNOS levels, whereas GSNO-treated cells had a drastic reduction (Fig. 4D, E). In addition to Müller glia, myeloid cells such as neutrophils and macrophages infiltrate the eye during endophthalmitis. Thus, we tested the effect of GSNO on mouse bone marrow-derived macrophages (BMDMs) and observed that GSNO-treated BMDMs had reduced expression of iNOS in response to SA infection (Fig. S3).

Fig. 4.

Effect of GSNO treatment on iNOS expression during SA infection. A Eyes of SA-infected and oral GSNO (72 h time point) treated B6 mice were fixed in paraformaldehyde, embedded in OCT, and 10-micron cryo-sections were subjected to immunofluorescence staining for iNOS (red) and nuclear stain DAPI (blue), Scale bar; 100 μm. B Retinal lysates were subjected to immunoblotting to check iNOS expression and quantified by densitometric analysis (C). In another experiment, human retinal Müller glia (MIO-M1 cell line) were treated with GSNO (200 μg/mL) for 1 h, followed by infection with S. aureus (SA) at MOI 10:1 for 6 h. PBS-treated cells served as control (C). C iNOS expression was assessed by western blot (D) and quantified by densitometric analysis (E). Data are represented as mean ± SD. Statistical analysis was performed using ANOVA (∗) p < 0.05 (∗∗) p < 0.01, ns non-significant. Significance was compared between control, C vs SA samples, SA vs GSNO-treated samples, and C vs GSNO-treated samples. The results are represented from two independent experiments with three technical replicates each

To check whether GSNO treatment increases NO levels, we performed a Griess assay, which measures stable nitrites in the cell-free culture supernatants. Our data showed that GSNO-treated Muller glia had a time-dependent increase in NO levels (Fig. S4) without exhibiting significant cytotoxicity especially at the lower (< 0.5 mg/mL) concentrations (Fig. S5). These observations indicated that GSNO, at these concentrations is well tolerated by the retinal cells.

GSNO treatment reduces inflammatory responses in retinal Müller glia

Müller glia are the primary resident glial cells in the retina [45] and play a role in evoking retinal innate response during endophthalmitis via Toll-like receptor signaling [46, 47]. However, overactivation of Muller glia can cause photoreceptor damage, leading to retinal degeneration [3]. Therefore, we assessed the effect of GSNO on the cultured human Müller glial cell line, MIO-M1. We performed a dose–response study by exposing Müller glia to various concentrations (50–500 μg/mL) of GSNO prior to SA infection. Our results showed that SA-induced expressions of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) were reduced at both mRNA (Fig. 5A) and protein (Fig. 5B) levels in GSNO pre-treated cells. Furthermore, the response was found to be dose-dependent, with drastic reductions at 250 and 500 μg/mL concentrations. To ensure that the observed effects were not due to GSNO-mediated cellular toxicity, we performed an MTT assay using various concentrations of GSNO (0.1, 0.2, 0.5, 1, 2, and 3 mg/mL) for 16 h. Our data showed that GSNO had no significant toxicity at lower concentrations (100, 200, and 500 μg/mL), as indicated by > 90% cellular viability. Moreover, the higher doses also had ~ 80% cell survival (Fig. S5). Because GSNO releases NO, which can contribute to direct antimicrobial activity, we determined its minimum inhibitory concentration (MIC) against SA and found it to be 2.5 mg/mL (Fig. S6). It is important to note that the doses used in our study were significantly lower than the MIC. These results indicated that GSNO exerted anti-inflammatory properties independent of its potential antimicrobial activity.

Fig. 5.

GSNO treatment attenuates SA-induced inflammatory responses in retinal Müller glia. Human retinal Müller glial cells (MIO-M1 cell line) were pretreated with indicated concentrations (μg/mL) of GSNO for 1 h, followed by infection with S. aureus (SA) at MOI of 10 for 6 h. PBS treated cells served as control (C). Cells were harvested for qPCR analysis of inflammatory cytokines (A) and culture supernatants were used to quantify the protein levels by ELISA (B). Data are represented as mean ± SD. Statistical analysis was performed using ANOVA ∗) p < 0.05 (∗∗) p < 0.01 (∗∗∗) p < 0.001 (****) p < 0.0001, ns non-significant. Significance was compared between control, C vs SA samples and SA vs GSNO-treated samples. The results are cumulative of three independent experiments with three technical replicates each

GSNO exerts cytoprotective effects during SA infection

During endophthalmitis, the increased retinal cell death, including Müller glia, is associated with vision loss and impaired retinal function [48, 49]. Our data showed that GSNO-treated mice had better retention of ERG response, and cultured cells appeared healthier under the microscope during in vitro infection studies. This led us to investigate the effect of GSNO on SA-induced cell death using the Incucyte SX5, a live cell imaging system. We used the MIO-M1 cell line pre-treated with various concentrations of GSNO for 1 h. Afterward, cells were challenged with SA, and 7AAD dye was added to monitor cell death in real time for the next 13 h. Cell death was visualized by the accumulation of 7AAD dye (red) and automatically quantified. Our data showed that the percentage of cell death was inversely proportional to GSNO concentrations (Fig. 6A). Even the lowest concentration (100 μg/mL) of GSNO showed a significant reduction in Müller glia death compared to untreated cells infected with SA at any given time point. The highest concentration (400 μg/mL) of GSNO had comparable cell death to the mock-treated control cells (Fig. 6B). The video recording showed that SA infection triggered cell death as early as 6 h, whereas the GSNO-treated cells entered the cell death phase at a relatively slower rate (Supplementary Movie S1, S2, and S3). These results indicated that GSNO treatment exerted cytoprotective effects and reduced bacterial-induced cell death during endophthalmitis.

Fig. 6.

GSNO treatment reduced SA-induced cell death in retinal Müller glia. Human retinal Müller glial cells (MIO-M1 cell line) were pretreated with indicated concentrations (μg/mL) of GSNO for 1 h, followed by infection with S. aureus (SA) at MOI of 5 for 13 h. PBS-treated cells served as control (C). Real-time cell death analysis was performed by using 7AAD dye in the live imaging Incucyte system. A Line graphs show time-dependent increase in the intensity of 7AAD staining, an indicator of cell death. B Representative microscopic images (orange channel and phase contrast) were taken at 13 h post infection at 200 μg/mL GSNO dose. Scale bar, 200 μm. The results are cumulative of two independent experiments with three technical replicates each

GSNO preserves outer blood-retinal barrier properties during SA infection

The breakdown of the blood-retinal barriers (BRB) can contribute to the loss of retinal architecture and function during endophthalmitis [50–52]. SA virulence factors have been shown to cause vascular permeability by disrupting junction proteins in the retinal pigment epithelium (RPE), which form the outer BRB and protect the eye from blood-borne pathogens [10, 51]. Additionally, RPE is involved in phagocytosis, express PRRs and thereby, produces inflammatory mediators during infection [3]. To investigate the effect of GSNO on maintaining barrier integrity, we evaluated the expression of Zonula occludens tight junction proteins (ZO-1 and ZO-2), tight junction proteins, in cultured human RPE (ARPE-19 cell line). Immunofluorescence assays showed consistent ZO-1 expression in mock-treated control cells, whereas SA infection caused a significant loss in ZO-1 staining in vehicle-treated cells, but not in GSNO-treated cells (Fig. 7A). Western blot analysis of the tight junction protein ZO-2 showed its levels were significantly preserved in GSNO-treated cells, while its levels were reduced in SA-infected untreated cells (Fig. 7B, C). We further evaluated the effect of GSNO on barrier properties using transepithelial permeability assays. Our data showed that GSNO treatment significantly reduced the permeability of FITC dextran through the monolayer of ARPE-19 cells, which was increased due to SA infection (Fig. 7D). To complement these in vitro findings, we performed an ex vivo model of SA infection using mouse eyecups. Immunostaining of ZO-1 in RPE/choroid flat mounts showed significant RPE degeneration in response to SA infection, whereas GSNO treatment partially maintained RPE integrity, as evidenced by preserved ZO-1 staining patterns (Fig. 7E). Immunoblotting of retinal lysates, including the RPE layer, showed almost undetectable levels of ZO-2 in SA-infected samples, but slightly higher levels in GSNO-treated mouse eyes (Fig. 7F, G). These results indicated GSNO treatment preserves BRB integrity during SA endophthalmitis.

Fig. 7.

GSNO treatment prevents the disruption of outer blood-retinal barrier. A–D Human retinal RPE cells (ARPE-19 cell line) were treated with GSNO (200 μg/mL) or PBS (control, C) for 1 h, followed by infection with S. aureus (SA) at MOI of 10 for 6 h. A The cells were fixed and immunostained with ZO-1 antibody (green) and nuclear stain DAPI (blue) and visualized under fluorescence microscope. Scale bar; 50 μm. B In another experiment cells were subjected to immunoblotting to quantify ZO-2 expression and qualification by densitometry analysis (C). The barrier properties were assessed by the FITC-dextran trans-epithelial permeability assay (D). The integrity of outer BRB was assessed using ex-vivo model and staining of RPE layer in choroid/RPE flat mount. E Eye cups from B6 mice were incubated in DMEM, followed by GSNO (200 μg/mL) treatment for 1 h and SA infection for additional 4 h. Following PFA fixation, eye cups were stained with ZO-1 and flat mounted to visualize under confocal microscope (blue, DAPI nuclear stain; red, ZO-1), Scale bar; 100 μm. The retinal lysates from oral GSNO treated (72 h time point) mice were subjected to immunoblotting to check ZO-2 expression (F) and quantitation by densitometric analysis using ImagJ (G) Densitometric analysis was done data are represented as mean ± SD. Statistical analysis was performed using ANOVA (*) p < 0.05 (**) p < 0.01 (***) p < 0.001 (****) p < 0.0001, ns; non-significant. Significance was compared between control, C vs SA samples and SA vs GSNO-treated samples. The results are represented from at least two independent experiments

Oral GSNO synergizes with an intravitreal antibiotic to alleviate SA endophthalmitis

Intravitreal antibiotic injections are the current standard treatment for bacterial endophthalmitis, with vitrectomy as an alternative option [53]. Additionally, oral antibiotics may be recommended to reduce disease severity [42, 54]. Given our findings of the anti-inflammatory and cytoprotective effects of GSNO, we investigated whether oral GSNO could serve as an adjunct therapy alongside intravitreal vancomycin (VAN) injections. We found that a single intravitreal injection of sub-MIC (0.5 µg/eye) of VAN significantly reduced corneal haze (Fig. 8A) and bacterial burden (Fig. 8B) by several log at 72 h p.i. Interestingly, the combination of a single intravitreal injection of VAN and three oral doses of GSNO further reduced anterior chamber opacity but did not significantly reduce the bacterial growth compared to VAN treatment alone. The retention of A- and B-waves in the ERG response indicated better preservation of retinal function with either vancomycin alone or in combination with GSNO, with slightly better results in the combination treatment group (Fig. 8C, D). The most significant effect of the combination therapy was observed on intraocular inflammation, with a drastic reduction in SA-induced inflammatory mediators in mice treated with both oral GSNO and intravitreal VAN, compared to VAN alone (Fig. 8E). These results suggested that oral GSNO administration synergized with intravitreal antibiotic treatment in ameliorating SA endophthalmitis.

Fig. 8.

GSNO synergizes with intravitreal vancomycin in mitigating severity of SA endophthalmitis. C57BL/6 mice were intravitreally injected (n = 4 eyes) with 5000 cfu/eye of S. aureus or PBS (control, C). After 6 h, eyes were treated with intravitreal injections of vancomycin (0.5 μg/eye) alone or in combination with oral GSNO (1 mg/kg). Two additional oral GSNO doses were given at 24 h and 48 h p.i, and various assays were performed at 72 h p.i. A Slit-lamp examination was performed, and representative photomicrographs are shown from each group showing similar pathology. B Scotopic electroretinogram (ERG) analysis was performed to assess retinal function. C Bar graph showing percent a- and b-wave amplitude retained with respect to control eyes set at 100%. D Eyes were enucleated, homogenized, and the bacterial burden was estimated via serial dilution and plating. E Whole eye lysates were subjected to ELISA to quantify inflammatory mediators. Data are represented as mean ± SD. Statistical analysis was performed using ANOVA (*) p < 0.05 (**) p < 0.01 (***) p < 0.001 (****) p < 0.0001, ns; non-significant. Comparisons were made between uninfected control, C vs SA and SA vs drug-treated samples. The results are cumulative of two independent experiments

Discussion

Bacterial endophthalmitis remains a severe and potentially blinding intraocular infection caused by bacterial invasion of the eye during surgery or trauma [1]. One of the main challenges in treating bacterial endophthalmitis is the limited penetration of antibiotics into the eye [55]. This is due to the blood-ocular barrier, which limits the entry of systemic antibiotics into the eye. Therefore, high doses of antibiotics are required to achieve therapeutic levels in the eye, which can result in systemic toxicity [56]. Another challenge is the emergence of antibiotic-resistant bacteria, which makes the treatment of bacterial endophthalmitis even more difficult [57]. In addition, bacterial biofilms can form in the eye, which provide a protective environment for bacteria to survive and grow, making them more resistant to antibiotics [58]. In this study, we show the therapeutic benefits of oral administration of GSNO alone or in combination with an antibiotic for the treatment of bacterial endophthalmitis. The mechanisms underlying GSNO-mediated protective effects involve the inhibition of inflammatory signaling, reduction in bacterial-induced retinal cell death, and the preservation of the outer blood-retinal barrier.

In the last decade, multiomics studies are being extensively used to better understand host–pathogen interactions, including in the eye, to identify therapeutic targets [21, 23]. In our recent untargeted metabolomics analysis, we observed a temporal reduction in GSNO levels in mouse retinal tissues during SA endophthalmitis. Given the anti-inflammatory and antioxidant properties of GSNO [59], we hypothesized that GSNO promotes inflammation resolution during endophthalmitis. First, using a prophylactic approach, we observed that intravitreal injection of a low dose (1 μg/eye) of GSNO injection reduced bacterial burden as well as inflammation in SA-infected mouse eyes. However, the same dose was ineffective when administered post-bacterial infection. This necessitated the use of a higher dose (10 μg/eye) to decrease the severity. Several studies have suggested multiple doses of systemic drugs to help supplement intravitreal injections of antibiotics in bacterial [60–63] and fungal [41, 64] endophthalmitis. This prompted us to test the therapeutic efficacy of GSNO via systemic routes either alone or in combination with intravitreal antibiotics. Interestingly, we found that oral administration of GSNO was the most effective route to ameliorate SA endophthalmitis as evidenced by a significant reduction in inflammatory mediators and clinical symptoms. These findings corroborate an earlier report showing a reduction in IL-1β and TNF-α levels by oral GSNO treatment in experimental autoimmune uveitis [40].

To determine the potential anti-inflammatory mechanisms of GSNO, we assessed its effect on classical inflammatory signaling pathways including NF-ĸB [65], whose p65 subunit regulates a plethora of inflammatory response genes when phosphorylated [66]. GSNO has been shown to inhibit NF-ĸB activation via S-nitrosylation of the p65 subunit [38]. Similarly, in our model, the phosphorylated NF-kB levels were drastically reduced by oral GSNO treatment in mice retinal tissue. We recently reported the essential role of the NLRP3 inflammasome in regulating intraocular inflammation via the production of IL-1β [67] and its inhibition at the later stages of endophthalmitis exerts protection [21]. Therefore, we assessed the effect of GSNO on the NLRP3/Caspase-1 signaling axis [68] and observed a significant reduction in levels of both NLRP3 and cleaved caspase-1 p20. These results indicate that the anti-inflammatory effects of GSNO could be mediated in part by the inhibition of NLRP3 inflammasome signaling.

GSNO is a potent antioxidant due to it being composed of both nitric oxide (NO) and glutathione (GSH). During microbial infection NO is generated via the activation of iNOS which assists in the regulation of host’s innate and adaptive immune responses [27]. Although iNOS-mediated NO production is crucial for antimicrobial activity, excessive activation of iNOS can trigger nitrosative and oxidative stress, culminating in host tissue damage via different cellular processes such as apoptosis, neurodegeneration, and uncontrolled inflammation [69, 70]. Indeed, iNOS-induced NO secretion from stimulated retinal Müller glial cells causes neuronal cell death [71]. Studies have reported that GSNO downregulates iNOS expression under various disease conditions [29, 39]. Our data also shows that GSNO treatment reduced SA-induced iNOS expression in both mouse retinal tissues and cultured retinal Müller glia and mouse BMDMs. Interestingly, we observed that GSNO treatment increased NO (measured as stable product, nitrites) levels in uninfected cells but reduced iNOS levels in presence of bacteria. GSNO-mediated iNOS inhibition during infection might exert a protective response to minimize nitrosative stress, indicating a dual effect [29].

Studies have indicated that GSNO may have a potential role in the prevention and treatment of various eye diseases, including glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy (DR). In an experimental glaucoma model, GSNO was found to lower intraocular pressure (IOP) by relaxing the trabecular meshwork, thus increasing the outflow of aqueous humor from the eye [72, 73]. In addition, GSNO protected RGCs from apoptosis by activating the protein kinase B (Akt) signaling pathway, which promotes cell survival [74]. In AMD pathobiology, GSNO was shown to protect RPE cells from oxidative stress and inflammation and promote regeneration of photoreceptors by activating the Wnt/β-catenin signaling. Similarly, GSNO has been found to protect retinal neurons and glial cells from apoptosis and inflammation, which occur in DR [29]. Collectively, these studies indicate the cytoprotective effects of GSNO in ocular tissues. Our real-time imaging of Müller glia showed that cells pretreated with GSNO had less cell death in response to SA infection. Moreover, GSNO-treated mice had better preservation of ERG response, indicating reduced damage to photoreceptors. This led us to conclude that GSNO protect retinal cells from bacterial-induced cytopathic effects.

The progression of bacterial endophthalmitis is associated with a loss of blood ocular barrier integrity [75], affecting the tight junction proteins in the RPE layer [76]. As GSNO has been reported to inhibit blood–brain barrier disruption during brain injury and stroke [77, 78], we sought to determine its effect in our model. We observed that SA infection resulted in the loss of both Zonula occludens proteins, ZO-1, and ZO-2 in cultured RPE cells, mice retina, and in RPE flat mount (ex vivo model). In contrast, GSNO treatment maintained or restored the expression of the tight junction proteins in response to bacterial infection. This observation was supported by FITC-dextran transepithelial permeability assay in RPE cells showing reduced permeability in cells treated with GSNO.

The standard therapy for bacterial endophthalmitis includes intravitreal antibiotic injections, such as vancomycin and ceftazidime [53]. Additionally, systemic antimicrobials, either intravenously or orally are thought to be beneficial in treating endogenous endophthalmitis and have been widely used as prophylactic therapy for open-globe injuries [6]. Steroids have also been considered to reduce inflammation; however, most ophthalmologists refrain from prescribing them as adjunct anti-inflammatories during intraocular infections, owing to their immunosuppressive properties [79, 80]. Our data using oral GSNO along with vancomycin was found to be effective in reducing intraocular infection during SA endophthalmitis. Thus, we propose that GSNO can be potentially used as an adjunct anti-inflammatory therapy. However, further studies are warranted to evaluate the role of GSNO in the eye. One of the limitations of our study is to quantify intraocular levels of NO following oral administration of GSNO. There are analytical challenges in differentiating NO release from endogenous versus exogenous supplemented GSNO. Due to the short half-life of GSNO, different nano formulations have been recently developed to protect it from degradation and for its sustained release at a physiological concentration [81]. In our laboratory, we are also developing various nanoformulations for delivery of therapeutic modalities to treat endophthalmitis.

In summary, using metabolomics analysis, our study uncovered the role of GSNO in the pathobiology of bacterial endophthalmitis. GSNO treatment was found to protect the eye during endophthalmitis by attenuating intraocular inflammation and protecting retinal cells from infection-induced cell death. Moreover, GSNO was found to maintain BRB by regulating the expression of tight junction proteins. Finally, we showed the synergistic effect of oral GSNO with standard intravitreal antibiotic injections to ameliorate SA endophthalmitis. Thus, GSNO has shown promising benefits in our experimental model of intraocular bacterial infection. The potential of GSNO as a therapeutic agent in the treatment of other ocular infections needs further investigation.

Materials and methods

Bacterial strains

The bacterium utilized in this study is Staphylococcus aureus strain RN6390 [82], known to cause consistent endophthalmitis when injected into mice eyes [10, 21, 83]. The strain is routinely cultured in Tryptic Soy medium (TSA or TSB; Sigma, St. Louis, MO, USA) at 37 °C.

Mice and ethics statement

Both male and female C57BL/6 (B6) mice (JAX stock #000664) [84], aged 6 to 8 weeks, were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and were housed in a DLAR facility with restricted access at the Kresge Eye Institute. The mice were maintained under a 12:12 light/dark cycle and were provided with rodent chow (Labdiet; Pico Laboratory, St. Louis, MO, USA) and water ad libitum. All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All standard operating procedures (SOPs) were approved by the Institutional Animal Care and Use Committee (IACUC) of Wayne State University under protocol #IACUC-22-04-4557.

Bacterial endophthalmitis and GSNO treatment

Bacterial endophthalmitis was induced in B6 mice by intravitreal injection of 5000 cfu of bacteria per eye, as previously described [83]. Eyes injected with sterile PBS or only GSNO were used as controls. Bacterial cultures grown overnight were washed and diluted in 1X PBS to achieve the desired inoculum per eye. Before infection, mice were anesthetized with ketamine and xylazine, and 2 μl of inoculum was injected into the vitreous using a 34-gauge needle under a microscope, as previously reported [18, 85, 86]. For the treatment group, GSNO (S-nitroso-l-glutathione #82240; Cayman Chemical Company, MI, USA) was administered at various doses via different routes (described below), and vancomycin was given intravitreally at a sub-MIC dose of 0.5 μg per eye 6 h post-infection (p.i).

To test the therapeutic efficacy of GSNO, GSNO was administered intravitreally (IVT), intravenously (IV) through the tail vein or orally (OR) using an oral gavage-feeding needle. Uninfected and PBS (vehicle) treated mice were used as mock controls. The treatment group includes:

Group I IVT administration.

Mice received one IVT injection of GSNO (10 μg/eye) 6 h p.i and a second IVT injection of the same dose at 24 h p.i.

Group II IV administration.

Mice received daily IV injections of GSNO (1 mg/kg), for a total of three injections. The first dose was given at 6 h p.i, 2nd dose at 24 h p.i. and 3rd dose at 48 h p.i.

Group III OR administration.

Mice received oral gavage (1 mg/kg) once daily, for a total of three doses. The first dose was given at 6 h p.i, 2nd dose at 24 h p.i. and 3rd dose at 48 h p.i.

Group IV IVT + IV administration.

Mice received a single IVT injection of GSNO (10 μg/eye) and by Intravenous injection (1 mg/kg) both at 6 h p.i, followed by two more doses at 24 h and 48 h p.i.

Group V IVT + OR administration.

Mice received a single IVT injection of GSNO (10 μg/eye) and by oral gavage (1 mg/kg) both at 6 h p.i, followed by two more OR doses at 24 h and 48 h p.i.

Cell and culture conditions

The human retinal pigment epithelial cell line, ARPE-19, was maintained in Dulbecco’s modified Eagle’s medium Nutrient mixture F-12 K (DMEM F-12 K) with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin antibiotic solution, and 10 μg/mL l-glutamine at 37 °C in 5% CO2. The human Müller glial cell line, MIO-M1, was cultured in DMEM GlutaMAX supplemented with 10% FBS, 1% penicillin–streptomycin antibiotic solution, and 10 μg/mL l-glutamine. Bone-marrow derived macrophages were isolated from mouse as described previously [48]. However, prior to infection, all cells were cultured overnight in antibiotic- and serum-free media. They were then pre-treated with the drugs for 1 h before challenging with S. aureus (MOI 10:1) for the specified duration.

Cellular toxicity assay

The cell viability assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Invitrogen) reagent, as previously described [87]. Briefly, cultured cells were seeded in DMEM in a 96-well plate overnight in a 37 °C incubator with 5% CO₂. The following day, the cells were incubated with varying concentrations of GSNO for 16 h, washed with 1X PBS three times, and then fresh DMEM was added. Next, MTT reagent (5 mg/mL in PBS) was added to each well and kept for 4 h at 37 °C. The supernatant was then removed, and 100 μl of cell lysis buffer (20% SDS in 50% DMF) was added for an hour. Finally, the absorbance was measured using a microplate reader (Synergy multi-mode reader, BioTek, Winooski, VT, USA), and the cell viability was expressed as a percentage by calculating the mean OD of treated cells divided by the mean OD of untreated control cells, multiplied by 100.

RNA extraction, cDNA synthesis and qPCR

Total RNA was extracted from cultured retinal cells using TRIzol reagent following the manufacturer's protocol (Invitrogen, Carlsbad, CA) and earlier study [88]. cDNA synthesis was carried out using 1 μg of RNA and the Maxima first-strand cDNA synthesis kit (Thermo Scientific, Rockford, IL, USA). qRT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using gene-specific PCR primers obtained from Integrated DNA Technologies (Coralville, IA, USA). The data were analyzed using the comparative ΔΔCT method and the fold-change differences in gene expression in test samples were calculated with respect to the control. Normalization of gene expression was done using beta actin as housekeeping control.

Cytokine and chemokine ELISA

At the designated time points, the supernatants of cell cultures were collected, and the levels of inflammatory mediators were determined by ELISA using commercially available kits from R&D Systems (Minneapolis, MN, USA). To determine in vivo cytokine (IL-1β, IL-6, and TNF-α) and chemokine (CXCL-1 and CXCL-2) levels, whole eyes from mice were enucleated, homogenized in PBS using a Tissue Lyser (Qiagen, Valencia, CA, USA), and centrifuged to obtain the supernatants, which were then subjected to ELISA. Prior to performing ELISA, eye lysates were quantified using the Thermo Scientific™ Micro BCA™ Protein Assay Kit to ensure equal protein concentrations were used.

Bacterial burden determination

Bacterial growth in the infected eyes of B6 mice was evaluated using the standard serial dilution and bacterial plate count method. The eyes were enucleated and homogenized in sterile PBS using a Tissue Lyser (Qiagen, Valencia, CA, USA), followed by serial dilution and plating on tryptic soy agar (TSA) plates. Colony-forming units (cfu)/eye were determined by counting the colonies on the plates, and the data were expressed as the mean ± SD.

Minimum inhibitory concentration (MIC) determination

The MIC for GSNO against S. aureus was determined using a microbroth dilution method [21]. Bacterial cultures were inoculated in a 96-well plate at a concentration of 10⁵ cfu/well and treated with a two-fold serial dilution of the drug. After overnight incubation, the optical density (A₆₀₀) of each well was measured using a spectrophotometer. MICs were determined based on the optical density of growth in control wells and the lowest drug concentration that inhibited S. aureus growth compared to media alone.

Retinal function assessment

The scotopic electroretinogram (ERG) was used to assess retinal function during endophthalmitis [20]. Briefly, bilateral mydriasis and overnight dark adaptation were performed, and ERGs were recorded using the Celeris ERG system (Diagnosis LLC, Lowell, MA, USA) following the manufacturer's instructions. The ERG a-wave was measured as the amplitude between the baseline and the first negative peak, while the ERG b-wave was measured as the amplitude between the first negative peak and the first positive peak. Data were compared to values obtained from control eyes.

Immunoblotting

At the desired time points of infection, two retinas were pooled and homogenized in RIPA buffer by sonication. Clear lysates were obtained, and protein quantification was performed using the BCA method. Samples were then separated on SDS polyacrylamide gels and transferred to 0.45 μm nitrocellulose membranes using a BioRad wet blot transfer system. The membranes were blocked with 5% skim milk in TBST (20 mM Tris HCl [pH 7.6], 0.15 M sodium chloride, and 0.5% Tween 20) for 1 h at room temperature and incubated with primary antibodies (obtained from Cell Signaling, USA or Santa Cruz Biotechnology, USA) overnight at 4 °C. The membranes were then washed and incubated with appropriate secondary antibodies (anti-mouse or anti-rabbit Ig) for 2 h. Finally, the blots were washed and developed using the SuperSignal™ West Femto substrate from Thermo Fisher Scientific. For in vitro experiments, cells were harvested at desired time points, lysed by scraping, and subjected to the same procedures for western blot.

Immunostaining

Immunostaining procedures were performed following a previously published protocol [89]. Briefly, ARPE-19 human RPE cells and murine primary BMDMs were cultured in four-well chamber slides (Fisher Scientific, Rochester, NY, USA), treated with 200 μg/mL of GSNO and infected with SA at an MOI of 10. At the desired time points, SA-infected and GSNO-treated cells were fixed overnight with 4% PFA at 4 °C, followed by PBS washes. The cells were then permeabilized and blocked with 1% (wt/vol) BSA and 0.5% Triton X-100 for 1 h, and incubated with the desired primary monoclonal antibodies (1:100) (Cell Signaling, USA) overnight at 4 °C. After extensive washing with PBS, the cells were incubated with anti-rabbit Alexa Fluor 485/594-conjugated secondary antibodies (1:200) at RT for 1 h. Finally, the cells were given a final wash, slides were mounted in Vectashield antifade mounting medium (Vector Laboratories, Burlingame, CA, USA), and visualized using the BZ-X810 Keyence microscope (Keyence, Itasca, IL, USA).

For the in vivo experiment, mouse eyes were enucleated and fixed in 4% PFA, followed by embedding in OCT cryo-matrix after passing through a sucrose gradient. Thin 10-µm sections were made using a cryostat and mounted onto lysine-coated microscope slides. Immunostaining was performed by fixing retinal sections in 4% PFA for 20 min at room temperature and washing them four times with PBS for 10 min each time. Permeabilization was achieved using 0.5% PBST and blocking was carried out using 10% normal goat serum with 0.5% Triton X-100 for 2 h at room temperature. The sections were incubated with primary antibodies (1:100) overnight, followed by rigorous washing with PBS and incubation with anti-rabbit Alexa Fluor 594-conjugated secondary antibody (1:200) for 2 h at room temperature the next day. After washing the eye sections again, the slides were mounted in Vectashield antifade mounting medium (Vector Laboratories, Burlingame, CA), and the eye sections were visualized using the BZ-X810 Keyence microscope (Keyence, Itasca, IL).

In vitro permeability assay

The in vitro transepithelial permeability assay was conducted with slight modifications as previously described [90]. ARPE-19 cells were seeded at a density of 7 × 104 cells/insert on trans-well tissue culture inserts (6.5 mm diameter, 0.4 µm pore size; Corning Costar, CLS3470) coated with Attachment factor solution (Cell Applications Inc., San Diego, CA). The inserts were placed on a 24-well tissue culture plate, and complete DMEM F-12K growth medium was added to both upper and lower chambers. The cells were allowed to form a monolayer and the junctions were allowed to mature by culturing the cells for three days in complete media. The ARPE-19 cells were then pre-treated with 200 µg/mL GSNO or mock-treated in serum-free media for 1 h before being infected with S. aureus at MOI 10. At the desired time point, the media in both the upper and lower chambers were removed, and FITC-dextran 40 000 MW (FD40S; Sigma-Aldrich) (150 µL of 1 mg/mL in 1X Hank’s balanced salt solution, HBSS, Invitrogen) was added to the upper chamber and 600 µL of plain 1X HBSS was added to the lower chamber, followed by incubation for 1 h at 37 °C. The amount of FITC-dextran permeated in the lower chamber medium was measured using absorbance at 490 nm excitation and 530 nm emission with a Biotek Synergy LX multi-mode reader. Dextran permeability was expressed as a percentage increase over the basal permeability observed in the mock-infected monolayer at the respective time points.

Ex-vivo model of bacterial infection

Eyes from 8-week-old B6 mice were enucleated and the cornea, lens, and neuroretina were carefully removed. The posterior eye cups containing the RPE and choroid layers were transferred to DMEM F-12 K medium supplemented with 2% FBS. The cups were then treated with 200 µg/ml of GSNO and infected with bacteria (MOI 10:1) for 4 h. After infection, the eye cups were washed in PBS and fixed in 4% paraformaldehyde. The RPE flatmounts were prepared according to a previously described protocol [91]. Following permeabilization and blocking, they were stained with rabbit polyclonal antibodies against mouse ZO-1 (1:200, Invitrogen) and visualized with Alexa 594 before flat mounting with Vectashield antifade medium on lysine-coated microscope slides. All images were obtained using a confocal scanning laser microscope (Zeiss LSM 780).

Nitric oxide estimation

The NO levels were measured following a previously described protocol [92]. Müller glia cells (MIO-M1 cell line) were seeded in a 24-well plate and cultured overnight in antibiotic and serum-free media. The next day, 200 µg/mL of GSNO was added to each well and culture supernatants were collected at desired time intervals. The supernatants were then centrifuged at 12,000×g for 10 min at 4 °C to remove any cell debris. Culture supernatants were evaluated for the presence of nitrites using the Griess reagent [93] by mixing 100 µL of the supernatant with the Griess reagent followed by a 15 min incubation at room temperature in dark. Nitrite levels were quantified by measuring absorbances at 550 nm and data was analyzed using a standard curve for sodium nitrite.

Real-time cell death assay

The live cell imaging SX5 system by Incucyte (Sartorius, Germany) was utilized to perform the mammalian cell death assay. Muller glia cells (MIO-M1) were seeded in a 96-well tissue culture plate and subjected to serum starvation prior to exposure to varying concentrations of GSNO for 1 h, followed by infection with SA. Non-viable cells were detected by the fluorescent compound 7-AAD in the culture media over a period of 13 h post-infection. Data were analyzed and exported using the Incucyte software, including images, videos, and graphs.

Statistical analysis

Data analysis was performed using GraphPad Prism version 9.3.1 (GraphPad, San Diego, CA, USA). Statistical significance was determined using either unpaired Student's t tests or ANOVA with multiple comparisons, as indicated in the figure legends. A confidence interval of 95% was maintained for all experimental values, and a p value < 0.05 was considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

AK conceived the idea, provided direction and funding for the project, and finalized the manuscript. SD designed and performed the experiments, analyzed the data, prepared manuscript, and figures, and revised the final manuscript. ZA, SS and SS helped with few experiments and edited the manuscript. RW performed sectioning and imaging and edited the manuscript. SG helped in conceptualization of the idea, provided critical feedback in experimental design, and edited the final manuscript.

Funding

This study was supported by National Institute of Health (NIH) Grants R01 EY027381, R01EY026964, and R21AI135583 awarded to AK. We would like to acknowledge the Research to Prevent Blindness (RPB) for their unrestricted grant to the Kresge Eye Institute/Department of Ophthalmology, Visual, and Anatomical Sciences. The immunology core is supported by an NEI vision center grant P30EY004068. The funders had no role in the design of the study, data collection, data analysis, interpretation of the results, or in the decision to submit the work for publication.

Data availability

The data generated during the current study are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare no conflict of interest. The authors have no relevant financial or non-financial interests to disclose.