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Integrative metabolomics and transcriptomics identifies itaconate as an adjunct therapy to treat ocular bacterial infection

Summary The eye is highly susceptible to inflammation-mediated tissue damage evoked during bacterial infection. However, mechanisms regulating inflammation to protect the eye remain elusive. Here, we used integrated metabolomics and transcriptomics to show that the immunomodulatory metabolite itaconate and immune-responsive gene 1 (Irg1) are induced in bacterial (Staphylococcus aureus)-infected mouse eyes, bone-marrow-derived macrophages (BMDMs), and Müller glia. Itaconate levels are also elevated in the vitreous of patients with bacterial endophthalmitis. Irg1 deficiency in mice led to increased ocular pathology. Conversely, intraocular administration of itaconate protects both Irg1−/− and wild-type mice from bacterial endophthalmitis by reducing inflammation, bacterial burden, and preserving retinal architecture and visual function. Notably, itaconate exerts synergistic effects with antibiotics. The protective, anti-inflammatory effects of itaconate are mediated via activation of NRF2/HO-1 signaling and inhibition of NLRP3 inflammasome. Collectively, our study demonstrates the Irg1/itaconate axis is a regulator of intraocular inflammation and provides evidence for using itaconate, along with antibiotics, to treat bacterial infections.


In brief
Eye infections remain the leading cause of blindness. Singh et al. show that the metabolite itaconate is produced in infected eyes to control aberrant inflammation. Itaconate exerts its antiinflammatory effect by potentiating antioxidant NRF2/HO signaling and as an adjunct therapy reduces antibiotic dose required to treat ocular bacterial infection.

INTRODUCTION
The incidence of intraocular infections, such as bacterial endophthalmitis, is on the rise because of a growing elderly population, which has increased the demand for eye surgeries to treat diseases such as cataracts, age-related macular degeneration (AMD), and glaucoma. 1,2 Treatment for each of these conditions, along with the widespread use of intravitreal injections to manage neovascular AMD and diabetic retinopathy, predisposes individuals to endophthalmitis. [3][4][5] The current treatment for bacterial endophthalmitis involves the intravitreal injection of antibiotics 6 that eliminates the bacteria but fails to suppress inflammation-mediated ocular tissue damage and often results in partial or complete loss of vision. 7,8 Thus, host-directed therapeutics are needed to complement pathogen-targeted approaches to treat ocular infections.
Transcriptomic and metabolomic analyses are powerful highthroughput technologies that have been increasingly used to uncover disease mechanisms and biomarkers, 9 including several pathways involved in the pathobiology of ocular infections. [10][11][12] These analyses have revealed a connection between energy metabolism and ocular innate immunity in bacterial 13 and viral infections. 14,15 Specifically, in bacterial endophthalmitis, the cellular metabolism of both residential (e.g., microglia) and infiltrating cells (e.g., neutrophils and macrophages) exhibited increased glycolysis. 13 Inhibiting glycolysis in bacterial endophthalmitis attenuates intraocular inflammation, 14 suggesting a mechanism that can be targeted for the treatment of ocular infections. Thus, these pre-clinical studies have identified potential mechanisms that enhance protective pathways or limit destructive host responses to attenuate inflammation from bacterial infection in the eye.
In our transcriptomic data from the mouse retina after Staphylococcus aureus infection, 10 we observed marked upregulation of the immune responsive gene 1 (Irg1), which is also called Acod1. Under inflammatory conditions, Irg1 drives the production of itaconate, a product of the Krebs cycle intermediate cisaconitate and has profound immunomodulatory properties. 16 Expression of Irg1 and the subsequent production of itaconate are increased in response to lipopolysaccharide (LPS) challenge 17,18 or bacterial 19 and viral infections. 18,20 Irg1 is essential for neutrophil infiltration, limiting immune-mediated tissue damage in Mycobacterium tuberculosis (Mtb) infection, 21 and promoting trained immunity. 22 In contrast, Irg1 deficiency in immune cells results in reduced antimicrobial activities. 23 The antibacterial properties of itaconate are due to its ability to inhibit isocitrate lyase, a bacterial glyoxylate shunt enzyme. Moreover, it inhibits bacterial growth at supraphysiological concentrations [24][25][26] and is predicted to be an inflammatory marker. 24,27 Thus, because of its broad immunomodulatory properties, the Irg1/itaconate axis 28 has recently become an active area of investigation in the field of immunometabolism.
To determine alterations in gene expression and metabolic pathways that occur during bacterial endophthalmitis, we performed transcriptomic and metabolomic analyses of retinal tissue from infected mouse eyes and the vitreous of patients with bacterial endophthalmitis, which showed increased levels of itaconate. Based on these observations, we hypothesized that the production of itaconate is a protective host response in the eye during bacterial infections. Using Irg1 À/À mice and itaconate analogs, we show that Irg1/itaconate protects the eye from bacterial endophthalmitis by resolving inflammation. In addition, activation of the Irg1/itaconate pathway enhanced the NRF2/ HO1 antioxidant pathway, which abrogates inflammation and reduces retinal cell death. Moreover, itaconate synergized with antibiotics to limit bacterial growth and potentiate anti-inflammatory/antioxidant pathways to prevent tissue damage.

Irg1/Itaconate pathway is upregulated in bacterial endophthalmitis
In the present study, we integrated metabolomic and transcriptomic approaches to identify key metabolites, genes, and pathways altered during bacterial endophthalmitis. Using retinal tissue from uninfected or S.-aureus-infected mouse eyes at different time points after infection, we performed temporal metabolomic and transcriptomic analyses. Principal component analysis (PCA) of untargeted metabolomics showed distinct clustering of various experimental groups, suggesting alterations in metabolite profiles among the different time points ( Figure 1A). To identify differentially produced metabolites across various time points with respect to controls, we used a partial leastsquares discriminant analysis (PLS-DA), in which each group was divided into a two-dimensional score plot with two principal components (variance of 46.71%). To show the differentially altered metabolites from the PCA analysis, we listed the top 25 metabolites using variable importance in projection (VIP) scores, with the top five metabolites being biopterin, N-acetyl-b-Dglucosamine, itaconate, N-acetylaspartic acid, and nicotinamide mononucleotide, respectively ( Figure 1B). Because of the variability in the experimental groups, we reaffirmed the capability classification of these top altered metabolites by applying the data to a random forest classification model, which showed a prediction error rate of the top metabolites to be less than 8.2% ( Figure 1C). Itaconate was the top altered metabolite identified by this model, confirming its classification as one of the metabolites most affected upon S. aureus infection ( Figure 1D).
To identify the genes associated with altered metabolic pathways, we analyzed transcriptomic data obtained from the retinas of S. aureus-infected mice at 6, 12, and 24 h and retinas of the uninfected control eyes. Among the top 100 differentially expressed genes (DEGs), 63 were differentially expressed across all three time points ( Figure 1E). A heatmap of these DEGs shows the differential changes in expression across the three time points, with Irg1 having the highest variability ( Figure 1F). Given the relationship between Irg1 and itaconate, we correlated the top 63 DEGs and the top 10 altered metabolites and extracted a correlation score for ''itaconate'' and ''Irg1,'' which we plotted in a pairs plot with each of the three time points (6, 12, and 24 h; Figures S1A and S1B). This analysis revealed a positive correlation between itaconate and Irg1. Collectively, our integrated omics analysis revealed that itaconate and Irg1 have the highest concurrent variability across all time points in the context of S. aureus endophthalmitis.
Bacterial infection increases Irg1 and Itaconate levels in mouse and human eyes Although our transcriptomic and metabolomic analyses showed upregulation of Irg1 and elevated levels of itaconate in infected eyes, the functional relevance of this pathway is unknown in bacterial endophthalmitis. Therefore, we performed a time-course study using a mouse model of S. aureus endophthalmitis and cultured cells to validate our multi-omics findings. Our data showed a time-dependent increase in the expression of Irg1 at both the mRNA ( Figure 2A) and protein ( Figure 2B) levels in S. aureus-infected mouse retinas. Macrophages are one of the major populations of innate immune cells infiltrating the eye during infection; therefore, we used bone marrow-derived macrophages (BMDMs) and observed a similar increase in Irg1 expression after S. aureus infection in cultured wild-type (WT) BMDMs ( Figures 2C and 2D) and in the human retinal M€ uller glia cell line MIO-M1, which are the residential glial cells of the retina ( Figures  2E and 2F). Because S. aureus produces several virulence factors that contribute toward the pathogenesis of endophthalmitis, 29 we sought to determine their effects on Irg1 expression. Our results show that, in addition to live S. aureus, heat-killed S. aureus (HK), lipoteichoic acid (LTA), and peptidoglycan (PGN), but not the a-toxin or toxic shock syndrome toxin-1 (TSST-1), induced the Irg1 expression both at transcript (Figure 2G) and protein ( Figure 2H) levels. Because Irg1 is responsible for the synthesis of itaconate during the Krebs cycle (Figure 2I), we used a targeted metabolomics approach to measure itaconate levels after S. aureus infection. Consistent with induced Irg1 expression, the levels of itaconate were noticeably increased in S. aureus-infected mouse vitreous/retinal tissue ( Figure 2J) as well as in BMDMs ( Figure 2K).
To determine whether itaconate is also produced during ocular bacterial infection in humans, we assessed itaconate levels in the vitreous samples from 22 patients who were clinically diagnosed with endophthalmitis, which was confirmed by a positive bacterial culture. The vitreous samples (n = 10) from patients who underwent vitrectomy for other retinal diseases (e.g., retinal detachment) were used as healthy controls (HC). Detailed patient demographics are provided in Table S1. Targeted metabolomics analyses revealed elevated levels of itaconate in the vitreous of patients with bacterial endophthalmitis compared with HC, although no significant difference was  Table S1). These results show that Irg1 expression is induced and itaconate levels are elevated in mouse and human eyes upon infection, indicating a potential role for the Irg1/itaconate signaling in the pathobiology of bacterial endophthalmitis.

IRG1 deficiency exacerbates bacterial endophthalmitis
To investigate the role of IRG1 and itaconate in ocular infection, we induced S. aureus endophthalmitis in WT and Irg1 À/À mice. We previously showed that WT mouse eyes challenged with an infective dosage of 500 colony forming units (CFUs) of S. aureus resolve the infection within 48-72 h (resolving condition), whereas a dosage of 5,000 CFUs causes severe endoph-thalmitis (non-resolving condition) leading to blindness. 13,30 Thus, the bacterial dosage is an important determinant in disease outcomes in endophthalmitis. 31,32 Given the anti-inflammatory role of Irg1/itaconate, we hypothesized that IRG1 deficiency would delay the resolution of inflammation and worsen disease outcomes. As such, we decided to assess the effects of the 500 CFUs/eye dosage, which is typically resolved in WT mice. In comparison to WT mice, endophthalmitis was exacerbated in Irg1 À/À mice, as demonstrated by increased corneal haze, opacity, and hypopyon ( Figure 3A, top panel). Histopathological analysis showed increased retinal tissue damage, retinal folding, and heavy cellular infiltrate in the eyes of Irg1 À/À mice ( Figure 3A, bottom panel), and this was associated with reduced retinal function as determined by electroretinography (ERG) showing a decline in a-and b-wave amplitudes ( Figure 3B). Moreover, The data represented are the culmination of two independent experiments and are shown as means ± SD. Statistical analysis was performed using one-way ANOVA (panels A, C, E, G, and L) or unpaired t test (J and K). ns; non-significant, *p < 0.05, **p < 0.001, ***p < 0.0001. See also  Figure 3C) was significantly higher in Irg1 À/À mice with respect to their WT counterparts, which coincided with elevated levels of the inflammatory mediators, interleukin 1 beta (IL-1b), IL-6, and tumor necrosis factor alpha (TNF-a) ( Figure 3D). The anti-inflammatory role of Irg1 in response to S. aureus infection was also confirmed in BMDMs isolated from Irg1 À/À mice, which showed increased levels of inflammatory mediators ( Figure 3E). The effect of Irg1 deficiency on itaconate production was revealed by drastically low levels of itaconate in Irg1 À/À BMDMs ( Figure 3F). Together, these findings indicate that Irg1 exerts protective effects in the eye by reducing intraocular inflammation.
To determine whether the increased susceptibility of Irg1 À/À mice to bacterial endophthalmitis at an even lower infectious dose of S. aureus was due to defective production of itaconate, we performed a rescue experiment; in which, Irg1 À/À mice were supplemented with 4-octyl itaconate (OI), an itaconate derivative, via intravitreal injection 6 h after S. aureus infection (Figure S2A). Mice that received OI had reduced corneal haze/opacity and hypopyon, and histopathological analysis showed the retinal architecture was preserved ( Figure S2B). ERG analysis showed that itaconate treatment significantly retained a-and b-wave amplitudes as compared with the untreated disease group ( Figure S2C). Similarly, the bacterial burden ( Figure S2D) and inflammatory cytokines ( Figure S2E) were significantly reduced in itaconate-treated eyes. These results indicate that exogenous supplementation of itaconate protected Irg1 À/À mice from bacterial endophthalmitis. The percentage of a-and b-wave amplitudes from scotopic ERG that were recorded from S. aureus-infected WT and Irg1 À/À mice (n = 6 for each group) and compared with PBS-injected control eyes whose wave amplitudes were adjusted to 100%. (C) The intraocular bacterial burden in eye lysates from mice treated in (A) was determined by serial dilution and plate count method and represented as CFUs/eye (n = 6) (D) The indicated inflammatory cytokines in whole eye lysates were quantified by ELISA. (E) WT and Irg1 À/À BMDMs (n = 4/condition) were either left uninfected 'C' or infected with S. aureus RN6390 'SA' (MOI 10:1) for 8 h. The inflammatory cytokines (IL-1b, IL-6, and TNF-a) were quantified from the conditioned media by ELISA. (F) Itaconate levels in WT and Irg1 À/À BMDMs (n = 3) that were infected with S. aureus (MOI 10:1) for 8 h and quantified by LC-MS. The data represented are the culmination of two to three independent experiments and are shown as means ± SD. Statistical analysis was performed using twoway ANOVA (panels B, D, E, and F) with Tukey's multiple comparison test or by unpaired t test (panel C). ns; non-significant, *p < 0.05, **p < 0.001, ***p < 0.0001. C, cornea; AC, anterior chamber; L, lens; VC, vitreous chamber; R, retina. See also Figure

Itaconate treatment ameliorates bacterial endophthalmitis
The elevated levels of itaconate observed in the vitreous of patients with bacterial endophthalmitis, who are unlikely to have a deficiency of IRG1, suggest itaconate may harbor protective properties. To test that possibility, we used a non-resolving infection model (i.e., a higher infective dose of 5,000 CFUs/ eye) to mimic the severe bacterial endophthalmitis that leads to blindness in humans and tested the therapeutic efficacy of two itaconate derivatives, dimethyl itaconate (DMI) and OI. Mice were treated with a single intravitreal injection of DMI or OI 6 h after infection with a high dose of S. aureus ( Figure 4A). Disease progression was evaluated 24 h after itaconate treatment by performing ophthalmic exams (slit-lamp microscopy) and histopathological analysis, assessing retinal function, and estimating intraocular bacterial burden and inflammation. In mice infected with S. aureus, DMI or OI treatments markedly reduced the corneal haze/opacity and hypopyon as compared with those without treatment ( Figure 4B, top panel). Histopathological analyses showed a striking reduction in retinal tissue damage and cellular infiltration in mice treated with the itaconate derivatives ( Figure 4B, bottom panel). Additionally, both DMI and OI treatment significantly reduced bacterial burden in the eyes of infected mice, with DMI treatment having a greater anti-bacterial effect ( Figure 4C).
In addition to direct retinal damage as a result of bacterial toxins, 29 the pathogenesis of bacterial endophthalmitis leads to ocular injury from excessive activation of inflammatory pathways. 33 Thus, we assessed intraocular inflammation and found that itaconate treatment (either DMI or OI) drastically reduced expression of the inflammatory cytokines IL-1b and IL-6 at the gene ( Figure 4D) and protein ( Figure 4E) levels. Surprisingly, the levels of TNF-a remained unchanged, irrespective of treatment, indicating differential effects of itaconate on inflammatory molecules. Similar to our in vivo observation, DMI/OI treatment significantly reduced inflammatory cytokines (IL-1b and IL-6) at (F and G) BMDMs from B6 WT mice (n = 4) were pretreated with DMI or OI (125 mM) for 2 h, followed by infection with S. aureus (MOI 10:1) for 8 h. The inflammatory cytokines were measured by qPCR (F) and represented as relative fold change by normalizing gene expression with that of endogenous b-actin (G). Quantification of protein levels of the indicated inflammatory cytokines in conditioned media from BMDMs, as determined by ELISA (n = 4). The data represented are the culmination of two to four independent experiments and are shown as means ± SD. Statistical analysis was performed using oneway ANOVA with Tukey's multiple comparison test by comparing S.-aureus-infected samples with or without DMI/OI treatment. ns; non-significant, *p < 0.05, **p < 0.001, ***p < 0.0001. C, cornea; AC, anterior chamber; L, lens; VC, vitreous chamber; R, retina. See also Figure Figure 4G) in WT BMDMs. In addition, DMI/OI treatment reduced the generation of reactive oxygen species (ROS) in S. aureus-infected BMDMs ( Figure S3A), indicating its ability to diminish oxidative stress. 34 Collectively, these results indicate that itaconate exerts both antibacterial and anti-inflammatory properties during bacterial endophthalmitis.
As in similar studies, we used the cell-permeable derivatives of itaconate and demonstrated their anti-inflammatory role in the eye. However, some recent reports show that the itaconate derivatives (DMI and OI), exert different mechanisms compared with endogenous itaconate in regulating inflammation. 35,36 Thus, we tested the effect of pH-buffered (pH 7.0) itaconic acid (ITA), along with OI, in our disease model. We found that ITA treatment also reduced corneal haze/opacity and hypopyon ( Figure S3B) and bacterial burden ( Figure S3C) in S. aureus-infected eyes. Similarly, ITA was found to attenuate the expression of the inflammatory cytokines at transcript ( Figure S3D) and protein ( Figure S3E) levels. However, the comparative analysis revealed that OI was superior to ITA in reducing intraocular inflammation, supporting its use to treat ocular infections. Itaconate synergizes with antibiotics to reduce inflammation and disease severity Intravitreal injection of antibiotics (vancomycin and ceftazidime) is currently the only standard treatment for managing bacterial endophthalmitis. 37 However, emerging resistance to antibiotics among common ocular pathogens necessitates an increased and continuous evaluation of the best antibiotic treatment for this condition. 38 The anti-inflammatory effects of itaconate suggest that it could be used as an adjunct therapeutic in combination with antibiotics to ameliorate bacterial endophthalmitis. To test that, we first determined the minimum inhibitory concentrations (MICs) of vancomycin (V), DMI, and OI against S. aureus strain RN6390 ( Figure S4), which were found to be 2.5 mg/mL, 10 mg/mL, and 12.5 mg/mL, respectively. Next, sub-MIC levels of vancomycin were used either alone or in a combination with DMI or OI (e.g., V+DMI or V+OI) to treat S. aureus-infected eyes. As expected, treatment with vancomycin drastically reduced bacterial burden, yet the combined treatment of both V+DMI or V+OI further reduced viable bacterial counts ( Figure 5A). Moreover, the combination therapy protected the retina from tissue damage ( Figure 5B) and preserved retinal function with better retention of a-and bwave amplitudes relative to treatment with V, DMI, or OI alone ( Figure 5C). Similarly, the levels of inflammatory mediators were significantly diminished in eyes treated with combination therapy as compared with V, DMI, and OI treatments alone (Figure 5D). These results suggest the potential use of itaconate as Itaconate exerts its anti-inflammatory effect by potentiating NRF2/HO-1 signaling To understand the protective mechanisms evoked by itaconate, we performed RNA sequencing (RNA-seq) analysis of WT BMDMs infected with S. aureus in the presence or absence of OI. Itaconate modulated the expression of a total 355 genes (212[; and 143Y), including those which regulate antioxidant (e.g., Nrf2 and Ho-1), innate immunity, immune activation, and inflammasome pathways ( Figure 6A). We analyzed the top 20 DEGs based on the proteins they encode to generate a protein-protein interaction map ( Figure 6B). This revealed that itaconate may affect genes encoding proteins associated with the inflammasome (group 1) and that are involved in antioxidant pathways (group 2). Prior studies have shown that itaconate exerts its anti-inflammatory effects via Nrf2, 39 which, along with its downstream target gene Ho-1, was differentially expressed in our analysis. Therefore, we set out to determine the role of Nrf2 and Ho-1 in our disease model ( Figure S5A).
We found S. aureus infection induced the expression of Nrf2 and Ho-1 in mouse retina and DMI or OI treatment potentiated their expression both at the transcript ( Figure 6C) and protein ( Figures 6D and 6E) levels. The induction and potentiation of Nrf2 and Ho-1 at the transcript ( Figure 6F) and protein ( Figures  6G and 6H) levels was also observed in S. aureus-infected and OI-treated WT BMDMs. Like DMI/OI, itaconic acid (ITA) also induced the expression of Nrf2 and Ho-1 in S. aureus-infected mouse retina ( Figure S5B), albeit at lower levels than OI induced, suggesting the involvement of Nrf2 in both endogenous and exogenously administered itaconate derivatives in the eye. We also assessed Nrf2/Ho-1 signaling in BMDMs from Irg1 À/À mice and found no significant induction of Nrf2 and Ho-1 transcripts ( Figure S5C) or protein ( Figure S5D), in response to S. aureus infection alone. However, DMI/OI treatment markedly induced NRF2 and HO-1 expression. These results indicate that Irg1 deficiency (i.e., reduced endogenous itaconate) impaired NRF2/HO-1 antioxidant pathway in ocular infections.
We also assessed the effect of DMI and OI on Nrf2 and Ho-1 expression in the human retinal M€ uller glia cell line MIO-M1. Cells that were infected with S. aureus and treated with either DMI or OI showed increased expression of Nrf2 and Ho-1 at both the mRNA (data not shown) and protein levels ( Figures S5E and  S5F). Because DMI and OI were found to synergize with vancomycin in reducing intraocular inflammation ( Figure 5), we determined whether the combination therapy also boosted antioxidant pathways. Our data show that in vivo, the combination of vancomycin and DMI or OI significantly increased expression of Nrf2 and Ho-1 transcripts as compared with vancomycin or itaconate alone (Figures S5G and S5H).
The ability of itaconate to induce NRF2 and HO-1 expression in both in vivo and in vitro models suggests that NRF2/HO-1 signaling regulates the inflammatory response in bacterial endophthalmitis. Therefore, to test that hypothesis, we used small hairpin RNAs (shRNAs) to silence their expression in WT BMDMs. Knockdown of NRF2 and HO-1 at the protein level was confirmed by western blot ( Figure S6A) and that led to increased expression of S. aureus-induced inflammatory mediators ( Figure S6B). To further validate the anti-inflammatory role of NRF2 signaling in our model, we used Nrf2 À/À mice. As expected, OI treatment significantly reduced corneal haze, opacity, and hypopyon in WT mice; however, that effect was diminished in Nrf2 À/À mice ( Figure 6I). A similar trend of relatively lower antibacterial ( Figure 6J) and anti-inflammatory ( Figure 6K) properties of OI was observed in Nrf2 À/À versus WT mice. ERG analysis also revealed that, although OI treatment significantly retained retinal function (i.e., amplitudes of a-and b-waves) in both WT and Nrf2 À/À mice compared with the untreated eye, the percentage retention was more in the WT mice. (Figure S6C). In vitro studies showed significant induction of NRF2 and HO-1 in DMI or OI treated WT BMDMs but not in Nrf2 À/À BMDMs ( Figure S6D). Like in vivo observation, reduced anti-inflammatory effects of OI and DMI were observed in Nrf2 À/À BMDMs compared with WT cells ( Figure S6E). Collectively, these results indicate that OI/ DMI treatment was more effective in protecting WT as compared with Nrf2 À/À mouse eyes, indicating the role of NRF2/HO-1 signaling in regulating ocular inflammation in bacterial endophthalmitis.

Itaconate inhibits the expression of bacterial-induced NLRP3 and CaspaseÀ1
Thus far, our studies showed that S. aureus induces the production of IL-1b in both the mouse retina and in cultured BMDMs and that itaconate attenuated that response. Because activation of the NLRP3 inflammasome is primarily responsible for the cleavage of pro-IL-1b into active IL-1b via activation of caspase 1, we sought to determine the effect of itaconate on NLRP3 activation. Using WT and Nrf2 À/À BMDMs, our data showed that S. aureus induced the expression of Nlrp3 ( Figure 7A) and caspase-1 (Figure 7B) transcripts, whereas DMI or OI treatment significantly inhibited their expression in both cell types, albeit more in WT BMDMs. This observation was confirmed by western blot for NLRP3 ( Figures 7C and 7E) and cleaved caspase 1 P20 ( Figures  7D and 7F), which showed inhibition of NLRP3 and Casp-1 in DMI-or OI-treated WT BMDMs. These findings indicate that itaconate attenuates the NLRP3-Casp-1-IL-1b cascade to downregulate ocular inflammation, and Nrf2 is partially involved in that phenomenon. (E and F) Densitometry analysis was performed using ImageJ, and data are expressed as relative fold changes normalized to the loading control, HSP90. The data represented are the culmination of two to four independent experiments and are shown as means ± SD. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test by comparing S. aureus-infected samples with or without DMI or OI treatment. (# indicates comparison between WT versus Nrf2 À/À ; A, B, E, and F). ns; non-significant, # p < 0.05, **p < 0.001, ***p < 0.0001.

DISCUSSION
The incidence of bacterial endophthalmitis is on the rise, and current treatments are often inadequate in preventing adverse outcomes, necessitating the discovery of alternative therapeutics that alleviate the underlying pathophysiology of this condition. However, the immune-privilege status of the eye poses significant challenges in treating ocular infections. Ophthalmologists have generally been reluctant to prescribe adjunctive anti-inflammatory drugs, such as corticosteroids, because of their immunosuppressive effects. [40][41][42] Thus, there is significant interest in identifying biologic agents and immunomodulatory therapies that target ocular inflammation without major disruption to the normal immune response in the eye. 33,43 Here, we used an integrated transcriptomic 10 and untargeted metabolomics 11 approach to identify genes and metabolites involved in experimental bacterial endophthalmitis. Using the vitreous of human patients and mouse eyes during bacterial endophthalmitis, we identified an increase in the expression of Irg1 and the concentration of its metabolite, itaconate, and that there was a strong pairwise correlation between the two upon infection with S. aureus. Given the anti-inflammatory role of itaconate, 44 we hypothesized that activation of the Irg1/itaconate axis promotes resolution of inflammation during bacterial endophthalmitis. Using Irg1 À/À mice and through shRNA-mediated gene silencing, we demonstrate that itaconate exerts protective effects by modulating NRF2/HO1 signaling. Most notably, intraocular administration of itaconate either alone or in combination with antibiotic treatment drastically improved disease outcomes.
Collectively, our study reveals an essential role of IRG1 and itaconate in regulating protective responses during bacterial infection in the eye and suggests a model wherein intraocular administration of itaconate reduces inflammation and bacterial burden to ameliorate bacterial endophthalmitis. Thus, our study demonstrates considerable promise for the translation of an itaconatebased treatment strategy to mitigate ocular bacterial infections.
An increasing number of studies have shown that under inflammatory stimuli (e.g., LPS challenge), Irg1 is expressed, and consequently, itaconate is produced in macrophages. 23,45,46 However, the role of Irg1/itaconate remains largely unknown in the pathobiology of eye diseases, especially in ocular bacterial infections. Here, we show that Irg1 is upregulated both in vivo (mouse retina) and in vitro (BMDMs and human retinal M€ uller glia) during S. aureus infection. Moreover, induced Irg1 expression coincided with an increased accumulation of itaconate in infected mouse eyes, which was also consistent with elevated levels observed in the vitreous of patients with culture-positive bacterial (both Gram-positive and Gram-negative) endophthalmitis. The increased itaconate levels in Gram-negative infection are likely due to LPS, which is known to induce Irg1 and is abundantly present in Gram-negative bacterial cell walls. 47,48 Here, we show that among the S. aureus virulence factors, cell-wall components, but not the toxins, are the inducers of Irg1 expression, which is consistent with our prior study showing their inflammatory role in the eye. 29,49 Together, these findings provide the evidence for the involvement of the Irg1/itaconate pathway in ocular infection.
In the absence of Irg1, we found mice are more susceptible to developing bacterial endophthalmitis, even when subjected to a lower infectious dose of bacteria that is usually resolved in WT mice. Irg1 À/À mice had a higher bacterial burden, increased inflammatory mediators, and more tissue damage, culminating in the loss of retinal/visual function. This is in agreement with other studies showing increased infection and inflammation in Irg1 À/À mice in response to Mtb, leading to greater mortality rates. 50 As anticipated, BMDMs from Irg1 À/À mice had reduced itaconate levels, and supplementing those mice with itaconate derivatives ameliorated bacterial endophthalmitis by reducing intraocular inflammation and preserving retinal tissue, suggesting itaconate could serve as a therapeutic option for humans with this condition. However, humans with this condition are likely not commonly deficient for IRG1. Therefore, we assessed the therapeutic efficacy of itaconate in WT mice with S. aureus endophthalmitis and observed profound protection. This suggests interventions that enhance Irg1 expression and/or itaconate levels within the eye could present potential therapeutic strategies for the treatment of ocular bacterial infections.
The clinical outcomes of bacterial endophthalmitis depend upon both the virulence of the infecting organism and the time at which appropriate therapy is initiated. Because of the poor penetration of systemic antibiotics, intravitreal injections of antibiotics remain the most widely accepted treatment option to manage the condition. 51 Although these intraocular antibiotics eliminate the bacterial infection, they often liberate bacterial cell wall components, 52,53 contributing to bystander inflammatory damage to ocular tissue. 29 Given the anti-inflammatory and antimicrobial role of itaconate 24,46,54 and its elevated levels in patients with endophthalmitis, we tested its therapeutic potential when co-administered with antibiotics. Notably, co-administration of itaconate with sub-MIC concentrations of vancomycin significantly reduced ocular inflammation and improved disease outcomes. Thus, our study provides evidence that the combination of antibiotics and itaconate function synergistically to ameliorate endophthalmitis and reduce ocular inflammation. Moreover, including itaconate as an anti-inflammatory therapy could reduce the overall dosage of antibiotics required to treat bacterial endophthalmitis.
Macrophages 13 and M€ uller glia 34,55 are known to have essential roles in orchestrating retinal innate responses in bacterial endophthalmitis, and our RNA-seq analysis in S. aureus-infected BMDMs indicated that NRF2/HO-1 and NLRP3 signaling are differentially modulated by itaconate treatment. Both itaconate derivatives, DMI and OI, potentiated the expression of NRF2 and its downstream signaling molecule HO1 in S. aureus-infected BMDMs and human retinal M€ uller glia. Moreover, levels of NRF2 and HO1 were increased in the mouse retina treated with itaconate. The reduction in inflammatory mediators upon itaconate treatment in S. aureus-infected cultured cells and its reversal by silencing Nrf2 indicate that itaconate likely exerts its anti-inflammatory effects in the eye by regulating NRF2/ HO1 signaling. The mechanisms for anti-inflammatory effects of itaconate have been reported to involve the inhibition of succinate dehydrogenase, 26,56 blockade of IkBz translation, 57 and activation of NRF2. 39 Although our data support the idea of itaconate-induced Nrf2/Ho-1 antioxidant signaling in exerting protective effects, 39,58,59 recent studies report distinct mechanisms for endogenous itaconate versus its derivatives in regulating inflammation. 35,36 The itaconate derivative OI reduces inflammation by triggering electrophilic stress, which is compensated by activation of the antioxidant Nrf2/Ho-1 axis, whereas endogenous itaconate evokes NRF2-independent mechanisms. 35 However, a more recent study using 13 C 5 -labeled OI showed that exogenously added OI can directly generate endogenous itaconate and reduce inflammation by carboxypropylation of NLRP3 and prohibiting its interaction with NEK7, which is essential for inflammasome activation. 60 Therefore, those authors argued that OI could be used as a surrogate to study itaconate because it can be taken up and converted to intracellular itaconate by macrophages. We also found that OI treatment reduced NLRP3 coinciding with attenuated IL-1b secretion.
In view of recent studies and to clarify whether DMI-or OImediated protection is dependent on Nrf2 signaling, we used Nrf2 À/À mice. Our data showed that (1) in comparison to WT mice, infected Nrf2 À/À mouse eyes had higher inflammation and bacterial burden; and (2) the protective and anti-inflammatory effects of OI were reduced but not completely abolished in Nrf2 À/À mice. Previous studies investigating the Irg1/itaconate axis have used LPS-stimulated macrophages. In contrast, we used live bacteria (which possess multiple virulence factors), and the observed differences in Nrf2 dependency could be attributed to the nature of the inflammatory stimuli. Nonetheless, we conclude that Nrf2 signaling, in part, contributes to OI-mediated protection, and Nrf2 deficiency exacerbates S. aureus endophthalmitis. Our data show that S. aureus induced NLRP3/ Casp1 expression and IL-1b secretion in WT BMDMs, and Nrf2 Cell Reports Medicine 2, 100277, May 18, 2021 11 Article ll OPEN ACCESS silencing or knockout (Nrf2 À/À ) potentiated IL-1b levels. However, in Nrf2 À/À BMDMs, although, reduced by OI or DMI treatments, NLRP3/Casp1 levels did not change by S. aureus alone, indicating Nrf2-independent regulation of NLRP3 inflammasome. Some potential mechanisms by which S. aureus could activate NLRP3/Casp-1 in an Nrf2-independent manner include (1) induction of oxidative (i.e., ROS generation; Figure S3A) and endoplasmic reticulum (ER) stress, 61 which can directly activate NLRP3/Casp1/IL-1b cascade; 62,63 and (2) the production of saturated fatty acids, such as palmitate, under inflammatory conditions in the retina, 64,65 which could also trigger NLRP3 activation, leading to IL-1b and IL-18 production. 66 We acknowledge that further studies using Irg1 and Nrf2; Nlrp3 and Nrf2 double knockout mice and cells are needed to determine the role of endogenous itaconate and to dissect Irg1-Nrf2-Nlrp3 crosstalk in ocular infection.
Controlling intraocular inflammation is an effective approach to improve the outcome of ocular infections. In support of that, our results provide evidence of the effectiveness of itaconate as adjunctive therapy with antibiotics to treat bacterial endophthalmitis by reducing the bacterial burden and correcting the dysregulated inflammatory response, thus preventing ocular tissue damage.

Limitations of study
Despite the importance of these findings, there are a few limitations to our study that should be considered. First, elevated levels of itaconate were detected in both Gram-positive and Gram-negative bacterial endophthalmitis, whereas we only tested the effect of itaconate in a S. aureus (e.g., Gram-positive) infection model. In a recent study, itaconate was found to exacerbate Pseudomonas lung infection. 67 Therefore, additional studies are needed to evaluate the effect of itaconate in Gram-negative ocular infections. Second, we only evaluated the therapeutic efficacy of itaconate at 6 h after infection. However, patients may not seek treatment until they experience pain and blurry vision, indicative of an advanced disease state. Hence, the timing (e.g., too early or very late) of treatment and optimal dose of anti-inflammatory therapy must be further studied because excessive suppression of inflammation could reduce the ability of the host to mount a sufficient antimicrobial response. Corresponding with that, the efficacy of itaconate as an adjunct therapeutic needs to be compared with ocular corticosteroids and evaluated with other types of antibiotics used to treat ocular infections. Although we observed reduced inflammation and protection in itaconic-acid-treated eyes, the effect was not as profound as that of OI. As reported recently, alteration of the itaconate structure in DMI or OI is responsible for extra effects that may not be attributed to itaconate. 35 Therefore, itaconate derivatives used in this study may not fully recapitulate the endogenous itaconate mode of action and warrant further investigation. We also acknowledge that the amount of itaconate (4OI [$41 mM] and DMI [$63 mM]) administered was greater than the endogenous itaconate levels ($55-120 pM at 24 h after infection) detected in the mouse eyes. Additional time kinetics studies are needed to determine the physiological range of itaconate in both human and mouse eyes.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
This study is supported by the National Institutes of Health grants R01EY026964, R01EY027381, and R21AI140033 (A.K.) and an unrestricted grant to the Department of Ophthalmology, Visual, and Anatomical Sciences from Research to Prevent Blindness (RPB). The immunology resource core is supported by NIH center grant P30EY004068. S.G. is supported by National Institutes of Health grants NS112727 and AI144004. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. The authors are grateful to Prof. Luke A. O'Neill, Trinity College Dublin, Ireland, for his kind gift for 4-octyl itaconate. We are thankful for the pharmacology and metabolomics core, Karmanos Cancer Institute, Wayne State University, for the estimation of itaconate. The authors are also thankful to Robert Wright for the technical support and other members of the laboratory for their helpful discussion and editing of the final manuscript.

AUTHOR CONTRIBUTIONS
A.K. conceived the idea and designed experiments and provided direction and funding for the project. S.S. and P.K.S. designed and performed the experiments, analyzed the data, and prepared the figures. A.J. analyzed metabolomics and transcriptomic data. P.N. and J.J. collected human vitreous samples and performed itaconate measurements in those samples. S.S., P.K.S., and

Materials availability
This study did not generate new unique reagents.

Data and code availability
The data generated from this study is deposited in the NIH Gene Expression Omnibus. The accession number for the raw and processed data is GSE168928.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mice and ethics statement Wild-type (WT) C57BL/6, Nrf2 À/À and Irg1 À/À mice (both male and female, aged 6-8 weeks) were purchased from the Jackson Laboratory and/or bred in-house in the Division of Laboratory Animal Resources facility at Kresge Eye Institute. Mice were maintained in a 12h light/dark cycle at 22 C and fed LabDiet rodent chow (Labdiet Pico lab Laboratory, St Louis, MO) and water ad libitum. All experimental procedures were performed in compliance with the Animals in Ophthalmic and Vision Research (ARVO) statement for the use of animals and were approved by the institutional animal care and use committee (IACUC) of Wayne State University.

Patient vitreous collection
A total of 22 patients clinically diagnosed and treated for infectious endophthalmitis (12 Gram-positive and 10 Gram-negative), who presented to the L V Prasad Eye Institute, Hyderabad, India, retina clinic and underwent diagnostic vitreous biopsy/vitrectomy between November 2018 and February 2019 were recruited for the study. Patient's demographical and clinical details are provided in the Table S1. In the control group, 10 patients with uninflamed eyes undergoing vitrectomy for non-infectious retinal disorders during the same period were included. In the test group, we excluded cases where the diagnosis was suspect or where the clinical characteristics were ambiguous. All patients diagnosed underwent complete ophthalmological examinations, including slit-lamp microscopy, visual acuity recordings, and later underwent pars plana vitrectomy. All cases were culture positive and had clinical features such as intense anterior chamber inflammation, extensive vitreous exudates, corneal infiltrates, or a lens abscess suggestive of infectious endophthalmitis. Written informed consent was obtained from all subjects and the study was approved by the Institutional Review Board, (LEC 09-18-125) L V Prasad Eye Institute, Hyderabad, India. The microbiological processing of the vitreous samples from the study group included direct microscopy and culture. A small portion of the vitreous sample ($100mL) was immediately stored at À80 C for itaconate measurement.

Cell and culture conditions
The immortalized human M€ uller glia cell line MIO-M1 was cultured in DMEM at 37 C in 5% CO2 supplemented with 10% FBS, 1% penicillin-streptomycin and 10 mg/mL L-glutamine. Prior to infection cells were grown overnight in serum and antibiotic-free media and infected with S. aureus (MOI, 10:1) for various time points.

METHOD DETAILS
Bacterial burden estimation Mouse eyes were enucleated following infection and 24h post-drug treatment. Whole-eye lysates were prepared in 250 mL of PBS by homogenization using stainless steel beads in a tissue lyser (QIAGEN, Valencia, CA). A small portion (50 mL) of the tissue homogenate was serially diluted and plated on Tryptic Soy Agar (TSA) to enumerate bacterial burden. The following day, colonies were counted, and results were expressed as the mean number of CFUs/eye ± SD.

Isolation of bone marrow-derived macrophages
Mouse bone marrow-derived macrophages (BMDMs) were isolated as described previously. 14,69 Briefly, mice were euthanized, and bone marrow was flushed from tibias and femurs with RPMI media containing 10% FBS and 0.2 mM EDTA. Cells were pelleted by centrifugation at 400 x g for 5 minutes at 4 C. Red blood cells (RBCs) were lysed by adding 0.2% NaCl solution for 20-30 s, followed by the addition of 1.6% NaCl and centrifugation. Following RBC lysis, cell pellets were washed with RPMI media by centrifugation. Cells were re-suspended and seeded in RPMI media supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin and 10 ng/ml M-CSF for macrophage differentiation at 37 C in 5% CO 2 . Six days post differentiation 4 3 10 6 BMDMs/mL were seeded in 65 mm Petri-dishes for in vitro experiments. For drug treatments, BMDMs were pretreated with DMI or OI (125 mM) for 2 h followed by challenge with S. aureus (multiplicity of infection (MOI) 10:1), for 8h.
MeOH (ice bath) by vortexing thoroughly and centrifuged at 28,672 x g for 10min at 4 C. Supernatant from two rounds of extraction was combined and dried in a CentriVap Refrigerated Centrifugal Concentrator. The residue was reconstituted with 50 mL of ddH 2 O, and a series of 20-time dilution samples were prepared for paralleled quantification by Waters Acquity H-class UPLC system connected to a Xevo TQ-xS triple LC-MS/MS system. The itaconate levels in the patient vitreous were measured by LC-MS/MS as indicated above at the Indian Institute of Chemical Technology, Hyderabad, India.
Preprocessing and annotation of metabolomic and transcriptomic data The metabolomics raw data were acquired from LC-QTOF/MS and were converted to a mzData format using Mass Hunter Qualitative Analysis Software (Agilent) and then imported to xcms R package for preprocessing. The default parameters in xcms software were used. The preprocessed data were obtained with three features of the dataset, including the retention time, the mass-to-charge ratio (m/z), and peak intensity. 71 The annotation was done using the CAMERA R package to annotate isotope peaks, adducts, and fragments. 72 Lastly, the data were normalized for each sample as described in the statistical analysis section.
For transcriptomic data analysis, the original Affymetrix data was pre-processed using an oligo R package and later normalized and log-transformed using a multi-array average (RMA) method. Further, the CDF package was used for probe annotation of Affymetrix data. The probes of the normalized data were successfully mapped to Entrez Gene IDs and Gene Symbols by annotation package (annotate) in R. Wherever multiple probes matched a single gene symbol, we calculated the median values of those probes as the expression value for that gene.

Metabolomic and transcriptomic data analysis
Multivariate data analysis of Metabolomics data was performed using MetaboAnalyst R package. 73 Identification of clusters to locate metabolites across various time points was performed using a supervised partial least-squares discriminant analysis (PLS-DA) method. 74 The metabolites were ranked for PC1 and PC2, and in an absence of internal validation, the PLS-DA method avoided overfitting of metabolites. The PLS-DA found differences in metabolites between the various time points versus controls. Variable importance was calculated as a coefficient for the selection of each variable. In addition to the supervised method, the nonparametric Kruskal-Wallis test was also performed to measure the importance of each metabolite. The R package ''caret'' was used for the random forest method to classify key altered metabolites. All default values of the caret package were used. The top metabolites were obtained based on the ''Gini'' score ranking. For Affymetrix gene expression data, all statistical analyses were performed using the multiple R Bioconductor packages (http://www.Bioconductor.org). For analysis, the P-values were adjusted for multiple testing with the Benjamini and Hochberg's method to control for false discovery rate (FDR). Probe sets showing at least a ± 2.5-fold change and an FDR < 0.05 were considered significant for the analysis. ggplot2 was used to generate the plots, R package VennDigram was used to generate the Venn diagram and the heatmap.2 package was used for heatmap generation from fold changes value.

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses were performed using Prism version 8.1 (Graph Pad, San Diego, CA). Data were expressed as mean ± SD unless indicated otherwise and statistical significance was determined using either unpaired Student's t test or two-way ANOVA or one-way ANOVA with multiple comparisons as indicated in the figure legends. A confidence interval of 95% was used for all statistical tests. A p < 0.05 was considered statistically significant.