Hydrogen Sulfide-Releasing Compounds Attenuate Lipopolysaccharide-Induced Inflammation in Porcine Anterior Uveal Explants, Ex Vivo

Abstract

In the present study, we investigated the pharmacological actions of both fast- and slow-releasing H₂S compounds, as well as polysulfides, on acute inflammation induced by the bacterial lipopolysaccharide (LPS) in an ex vivo porcine model of anterior uveitis. Isolated porcine iris-ciliary bodies (ICB) were maintained in oxygenated Krebs-Henseleit solution and then incubated in separate wells of RPMI 1640 supplemented media with an antibiotic. ICBs were exposed to sodium hydrosulfide (NaHS), GYY4137, S-allyl-cysteine (SAC), and polysulfides, diallyl disulfide (DADS), and diallyl tetrasulfide (DATTS) four hours before the end of incubation. We observed that LPS (5–200 ng/ml) produced increases in the release of pro-inflammatory mediators (TNF-α, IL-6, and PGE₂) and a corresponding decrease in the anti-inflammatory marker, IL-10, in the ICB explants. Both fast- (NaHS, 0.001–100 μM) and slow-releasing H₂S compounds (GYY4137, 0.001–10 μM) and the organosulfur H₂S-releasing compounds, DADS (0.001–300 μM), SAC (0.1–1000 nM), and DATTS (0.001 pM − 1 μM), caused a concentration-dependent attenuation of LPS-induced increases in the levels of TNF-α, IL-6, and PGE₂. DATTS (0.001 pM – 10 nM) reversed the LPS (25 ng/ml)-induced decrease in IL-10 production in the explants. We conclude that both fast- and slow-releasing H₂S compounds, as well as polysulfides, were effective in reducing LPS-induced acute inflammation in porcine anterior uveal explants, ex vivo. Inhibition of endogenous H₂S biosynthesis exacerbates LPS-induced production of an inflammatory mediator, a response that was reversed by an H₂S-releasing compound.

Background

Inflammation is a complex component of the immune system's response to harmful agents, resulting in the release of proinflammatory mediators such as tumor necrosis factor-alpha (TNF-α), prostaglandin E₂ (PGE₂), interleukin-6 (IL-6), and anti-inflammatory mediators such as interleukin-10 (IL-10) [1, 2]. Inflammation is a common feature of many diseases, such as sepsis, cardiovascular diseases (atherosclerosis), diabetes, arthritis, and uveitis [3–7]. Uveitis is an inflammatory condition involving the anterior uvea, as well as the adjacent structures in the eye [8, 9]. This disease accounts for 5–10% of visual impairment and is a significant cause of blindness around the world [3, 10]. While the etiology of uveitis is yet to be fully understood, infections, autoimmune disorders, and exposure to toxins have been reported to cause this disease [11, 12]. Although current strategies for the therapy of uveitis include the use of drugs such as corticosteroids, antimetabolites, alkylating agents, and biological agents, some side effects have been reported [13]. Consequently, the identification of alternate therapeutic targets for treating uveitis is warranted.

Hydrogen sulfide (H₂S) is a gaseous molecule with extensive physiological and pathological actions (e.g., neuromodulation, cardio-protection, cell proliferation, apoptosis, etc.) in several tissues and organs [14]. The four major enzymes that are involved in the biosynthesis of H₂S in the eye include cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfur transferase (3-MST) with cysteine aminotransferase (CAT) [15]. These enzymes are also present in ocular tissues such as the cornea, iris, ciliary body, and retina [15–17]. Evidence from our laboratory also confirmed the endogenous production of H₂S in ocular tissues [18] and the pharmacological actions of H₂S-releasing compounds (such as decreases in intraocular pressure and relaxation of the vasculature) in eyes from several species [18–31]. H₂S has been reported to play a crucial role in inflammation in several mammalian tissues and organ systems [32, 33]. At low concentrations of H₂S, this gas has been shown to possess anti-inflammatory actions [34–37]. However, there is some evidence that high concentrations of this gas can have pro-inflammatory actions in some systems [32, 38]. H₂S-releasing compounds employed in biological studies are classified based on the rate of release of H₂S gas. The most common class is the fast H₂S-releasing compounds, which provide direct and instantaneous release of H₂S and include the sulfide salts such as sodium hydrosulfide (NaHS) and sodium sulfide (Na₂S) [39]. Use of synthetic compounds that release H₂S slowly, including the Lawesson’s (phosphorodithioate) derivative such as GYY4137 have been widely used to evaluate the therapeutic potential of exogenous H₂S delivery to tissues and/or organs [39]. Garlic (Allium sativum) is widely used due to its numerous health benefits, which can be attributed to its rich source of organosulfur compounds [40]. These naturally occurring compounds extracted from garlic include allicin, S-allyl cysteine, diallyl mono- and polysulfides, as well as methylated forms of allyl mono- and polysulfides are known to release H₂S [41, 42]. Since ocular tissues can produce H₂S [18, 43, 44], and there is evidence that this gas is involved in the inflammatory process [32, 38], the aim of the present study was: (1) to investigate the pharmacological actions of H₂S (using exogenously administered as gas-releasing compounds) on LPS-induced inflammatory effects in a porcine ex vivo model of acute uveitis, and (2) to examine the role of endogenous H₂S in the LPS-induced release of the inflammatory mediator, prostaglandin E₂ in the absence and presence of the garlic-derived, H₂S-releasing compound, S-allyl-cysteine (SAC) in this model. Some of the preliminary data presented in this paper have been published as Abstracts [45, 46].

Methods

Chemicals and Reagents

Lipopolysaccharide (LPS, Escherichia Coli Serotype O55:B5), Hanks’ balanced salt solution (HBSS), sodium hydrosulfide (NaHS), indomethacin, and enzyme-linked immunoassay kits for TNF-α and IL-6 were purchased from Sigma Chemical (St. Louis, MO). GYY4137, flurbiprofen, aminoaxyxacetic acid (AOAA), and enzyme-linked immunoassay kits for PGE₂, as well as stock solutions of diallyl disulfide (DADS), S-allyl cysteine (SAC), and diallyl tetrasulfide (DATTS), were purchased from Cayman Chemical (Ann Harbor, MI). Roswell Parkland Institute Medium 1640 (RPMI 1640), Penicillin-Streptomycin, and enzyme-linked immunoassay kits for IL-10 were purchased from ATCC, Corning, and Thermofisher, respectively. All test agents were freshly prepared immediately before use in the series of experiments. Stock solutions of NaHS and GYY4137 were prepared in deionized water, while stock solutions of indomethacin and flurbiprofen were prepared in 100% DMSO.

Tissue Preparation and Tissue Culture

Porcine eyes were obtained within 24 hours of euthanasia from First Vision Tech (Dallas, TX) and transported to the laboratory on ice. Eyeballs were dissected, and the irises with the ciliary body attached were carefully isolated. The methodology utilized for the preparation of tissues for ex vivo studies was essentially similar to that described by Brito et al. [47] using human iris-ciliary bodies, with some modifications. Briefly, isolated iris-ciliary body (ICB) explants were cut into four equivalent quadrants, maintained in oxygenated Krebs-Henseleit solution, and incubated in separate wells of a 24-well culture plate containing 500 μl of RPMI 1640 supplemented with 1% Penicillin-Streptomycin. Quadrants of the porcine ICB were randomly assigned to the control and treatment groups. LPS was reconstituted in HBSS to make a stock solution of 100 μg/ml, aliquoted, and stored at −20°C. Final concentrations of LPS, H₂S-releasing compound (SAC), and the inhibitor of H₂S biosynthesis (AOAA) were prepared in RPMI 1640 medium. Explants were treated with varying concentrations of LPS (5–200 ng/mL). When used, explants were treated with the fast H₂S-releasing compound, NaHS, for 30 minutes before the end of the incubation period. For slow H₂S-releasing compounds, GYY4137, DADS, SAC, and DATTS, the explants were treated for up to four hours before the end of the incubation period. In some experiments, explants were pre-treated with an inhibitor of H₂S biosynthesis, AOAA (1 μM), or inhibitors of cyclooxygenase (COX), indomethacin (10 μM) and flurbiprofen (30 μM), for two hours before treatment with LPS. These inhibitors remained in culture media until the end of incubation. All explants were incubated at 37°C for 20 hours, and the culture media were collected, centrifuged, and assayed for their content of mediators, TNF-α, IL-6, PGE₂, and IL-10 by enzyme-linked immunosorbent assay (ELISA) using commercially available kits according to the manufacturer’s instructions.

Statistical Analysis

Results were expressed as either cytokine concentration per milligram soluble protein or as the percentage reduction in cytokine level, with values reported as means ± standard errors of the mean (mean ± S.E.M). The percentage reduction/increase for each cytokine was calculated based on the mediator level in tissues treated with H₂S-releasing compounds in the presence of LPS when compared with tissues stimulated with the same concentration of LPS. IC₅₀ values were calculated as the concentration of H₂S-releasing compound that caused 50% inhibition of LPS-stimulated production of inflammatory mediators. Statistical analysis was performed using one-way ANOVA with Dunnett's test or two-way ANOVA with Tukey's post hoc analysis. Differences with P values < 0.05 were denoted as statistically significant. All data were analyzed using GraphPad Prism, version 10.2.3 (GraphPad Software Inc.).

Results

In 2004, Brito and colleagues reported that in an explant culture of the human iris-ciliary body, LPS can induce the release of IL-6 and TNFα, a response that was inhibited by anti-toll-like receptor-4 monoclonal antibody, suggesting that the sensitivity of the anterior uvea to bacterial endotoxin could be used as a standard animal model of acute endotoxin-induced uveitis [47]. In the present study, we modified the experimental conditions employed by Brito et al. [47] using porcine iris-ciliary body explants, and indeed, obtained similar results when the explants were exposed to LPS.

Effect of Lipopolysaccharide on the Release of Pro-inflammatory/Anti-inflammatory Mediators in Porcine Iris-ciliary Body Explants

In a series of experiments, we examined the effect of different concentrations of LPS on the release of inflammatory mediators, IL-6, TNF-α, and PGE₂ in the porcine ICB explants. As shown in Fig. 1(A–C), incubation of porcine ICB explants with LPS (5 ng/ml – 200 ng/ml) caused a concentration-dependent significant (p < 0.05 to p < 0.001) increase in the release of IL-6, TNF-α, and PGE₂ into the media of the porcine ICB explants after 20 hours of incubation. The basal concentrations of TNF-α, IL-6, and PGE₂ were 258.6 ± 10.7 pg/ml/mg protein, 3695.6 ± 121.8 pg/ml/mg protein, and 144.3 ± 5.6 pg/ml/mg protein, respectively.

Figure 1.

Effect of LPS (5 – 200 ng/ml) on the Release of Pro-inflammatory and Anti-inflammatory Mediators in Cultured Porcine Iris-ciliary Body Explants. Each value represents the mean ± SEM for (a) TNF-α, (b) IL-6, (c) PGE_2, and (d) IL-10. *p < 0.05, **p < 0.01, ****p < 0.001, significantly different from basal concentrations (n = 9 – 12).

In another series of experiments, we measured the concentration of IL-10, an anti-inflammatory mediator, in the media containing porcine ICB explants. LPS (5 ng/ml – 100 ng/ml) produced a concentration-dependent decrease in IL-10 concentration that reached a maximum at 25 ng/ml. (Fig. 1D).

Effects of Fast- and Slow-H₂S-Releasing Compounds on Lipopolysaccharide-induced Release of Pro-inflammatory Mediators

To assess the effects of fast- and slow-releasing H₂S compounds on the production of pro-inflammatory mediators, porcine ICB explants were incubated with a fast (NaHS) and a slow (GYY4137) H₂S-releasing compound. Incubation with NaHS (0.001 μM − 100 μM) caused a concentration-dependent reduction in TNF-α release induced by LPS (25 ng/ml), with the highest reduction achieved at a concentration of 5 μM (29.7% ± 1.5%) (Fig. 2A). Similarly, GYY4137 (0.001 μM − 10 μM) produced a concentration-related decrease in TNF-α release induced by LPS (25 ng/ml), with the highest reduction achieved at 1 μM (27.4% ± 2.8%) (Fig. 2B).

Figure 2.

Effects of Fast- and Slow-Releasing H₂S Compounds on LPS-induced TNF-α Production in Iris-ciliary Body Explants. Each value represents the mean ± SEM for (a) NaHS (0.001 μM - 100 μM), (b) GYY4137 (0.001 μM −10 μM). *p < 0.05; **p < 0.01, significantly different from stimulated TNF-α release by LPS 25 ng/ml (n = 12).

The fast-releasing H₂S compound, NaHS (0.1 μM − 100 μM), also produced a concentration-dependent decrease in IL-6 production in the presence of LPS 25 ng/ml, with the most reduction observed at 50 μM (45.4% ± 2.7%) (Fig. 3A). Likewise, in the presence of LPS 25 ng/ml, GYY4137 (0.01 μM −100 μM) elicited a concentration-dependent decline in IL-6 release, reaching a maximum at 3 μM (55.4% ± 6.5%) (Fig. 3B).

Figure 3.

Effects of Fast- and Slow-Releasing H₂S Compounds on LPS-induced IL-6 Production in Iris-ciliary Body Explants. Each value represents the mean ± SEM for (a) NaHS (0.1 μM - 100 μM) (b) GYY4137 (0.01 μM −100 μM). *p < 0.05; **p < 0.01, significantly different from stimulated IL-6 release by LPS 25 ng/ml (n = 9 – 12).

The fast-releasing H₂S compound, NaHS (1 μM −100 μM) caused a concentration-related decrease in LPS (50 ng/ml)-induced PGE₂ release in the porcine ICB explants, reaching a maximum at 10 μM (44.1% ± 2.0%) (Fig. 4A). Furthermore, GYY4137 (0.01 μM − 10 μM) elicited a concentration-dependent decrease in LPS (50 ng/ml)-induced PGE₂ production, with the most reduction observed at a concentration of 1 μM (52.8% ± 2.2%) (Fig. 4B).

Figure 4.

Effects of Fast- and Slow-Releasing H₂S Compounds on LPS-induced PGE₂ Production in Iris-ciliary Body Explants. Each value represents the mean ± SEM for (a) NaHS (1 μM - 100 μM) (b) GYY4137 (0.01 μM −10 μM). *p < 0.05; **p < 0.01, significantly different from stimulated PGE₂ release by LPS 50 ng/ml (n = 9 – 12).

Effects of Organosulfur HS-Releasing Compounds on Lipopolysaccharide-induced Release of PGE in Porcine Iris-ciliary Body Explants

Garlic-derived H₂S-releasing compounds have been shown to have anti-inflammatory properties in non-ocular tissues. To assess the effects of these H₂S-releasing compounds on the release of PGE2, porcine ICB explants were incubated with diallyl disulfide (DADS), S-allyl cysteine (SAC), and diallyl tetrasulfide (DATTS) in the presence of LPS (50 ng/ml). DADS (0.001 μM − 300 μM) produced a concentration-related decrease in PGE₂ release from the explants, with a maximum reduction reached at a concentration of 10 μM (55.1% ± 1.2%) (Fig. 5A). Similarly, SAC (0.1 nM − 1000 nM) and DATTS (0.001 pM − 1 μM) produced significant concentration-dependent decreases in PGE₂ release, with the highest reduction achieved at 300 nM (60.9% ± 1.1%) and 0.1 nM (54.6% ± 7.3%) shown in Fig. 5B and Fig. 5C respectively.

Figure 5.

Effects of Organosulfur H₂S-Releasing Compounds on LPS-induced PGE₂ Production in Iris-ciliary Body Explants. Each value represents the mean ± SEM for (a) DADS (0.001 μM - 300 μM) (b) SAC (0.1 nM - 1000 nM) (c) DATTS (0.001 pM - 1 μM). *p < 0.05; **p < 0.01; ***p < 0.001, significantly different from stimulated PGE₂ release by LPS 50 ng/ml (n = 9 – 12).

When compared to fast- and slow-releasing H₂S compounds, organosulfur compounds derived from garlic also inhibited the release of PGE₂ stimulated by LPS. The IC₅₀ values of the different H₂S-releasing compounds in inhibiting the release of PGE₂ stimulated by LPS are shown in Table 1.

Table 1.

IC₅₀ Values of H₂S-Releasing Compounds in the Inhibition of LPS-induced PGE₂ Production in Iris-ciliary Body Explants.

H2S-Releasing Compound	IC50 VALUES (nM)
NaHS	107.6 ± 79.5
GYY4137	74.4 ± 4.2
DADS	72.6 ± 5.8
SAC	1.19 ± 0.6
DATTS	0.015 ± 0.01

Each IC₅₀ value represents the mean ± SEM of the concentration of H₂S-releasing compounds that caused 50% inhibition of LPS-stimulated PGE₂ production (n = 9–12).

Effects of a Polysulfide on the Release of an Anti-inflammatory Mediator in Lipopolysaccharide-treated Porcine Iris-ciliary Body Explants

We next assessed the effects of a polysulfide, DATTS, on the production of an anti-inflammatory mediator, IL-10, in the presence of LPS (50 ng/ml) in the porcine ICB explants. DATTS (0.001 pM – 10 nM) produced significant concentration-related reversal of LPS-induced decrease in IL-10 levels in the explants, reaching a maximum at a concentration of 0.1 nM (Fig. 6).

Figure 6.

Effects of DATTS on LPS (25 ng/ml)-induced inhibition of IL-10 Production in Iris-ciliary Body Explants. Basal and in the presence of DATTS (0.001 pM – 10 nM). Each value represents the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, significantly different from inhibited IL-10 release by LPS 25 ng/ml (n = 9).

Effect of inhibition of CBS/CSE on LPS-induced PGE production in Porcine Iris-ciliary Body Explants

In a series of experiments, the effect of inhibition of CBS/CSE was examined on the LPS-induced inflammatory response. At a concentration of AOAA (1 μM) that had no significant action on basal PGE₂ production from porcine ICB explants, LPS induced a significant (p < 0.001) increase in PGE₂ release compared to LPS (50 ng/ml) alone (Fig. 7).

Figure 7.

Effect of AOAA on LPS-Induced PGE₂ Production in Cultured Porcine Iris-ciliary Body Explants. Each value represents the mean ± SEM. ****p < 0.0001, significantly different from stimulated PGE₂ release by LPS 50 ng/ml (n = 8).

Effect of SAC on LPS-induced PGE₂ Production in the presence of a CBS/CSE inhibitor in Porcine Iris-ciliary Body Explants

To assess the effect of SAC on LPS-induced PGE₂ production in the presence of AOAA (1 μM), porcine ICB explants were incubated with a submaximal concentration of SAC (100 nM). SAC elicited a significant (p < 0.0001) reversal of the AOAA-induced elevation in the LPS-induced PGE₂ release (Fig. 9).

Effects of COX Inhibitors on Lipopolysaccharide-induced PGE₂ Production in Porcine Iris-ciliary Body Explants

To assess the contribution of endogenous prostaglandins in the release of PGE₂ induced by LPS (50 ng/ml) in the porcine ICB explants, tissues were incubated with the non-selective COX inhibitors, indomethacin (10 μM) and flurbiprofen (30 μM). In the absence of LPS, both indomethacin and flurbiprofen had no significant (p > 0.05) action on basal PGE₂ concentrations (Table 3). In the presence of LPS-induced inflammation, both indomethacin and flurbiprofen significantly decreased PGE₂ release by 56.3% ± 2.8% (n = 9) and 61.6% ± 6.4% (n = 9), respectively (Table 2).

Table 2.

Effects of COX Inhibitors on Lipopolysaccharide (LPS)-induced PGE₂ Production in Iris-ciliary Body Explants.

TREATMENT	PGE2 CONCENTRATION (pg/ml/mg protein)
Basal	155.0 ± 14.6
LPS (50 ng/ml)	364.5 ± 3.8
Indomethacin (10 μM)	125.9 ± 11.5
LPS + Indomethacin (10 μM)	158.2 ± 2.8
Flurbiprofen (30 μM)	136.7 ± 6.5
LPS + Flurbiprofen (30 μM)	140.6 ± 6.4

Each value represents the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, sig

Each value represents the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, significantly different from stimulated PGE₂ release by LPS 50 ng/ml (n = 9).

Discussion

In 2004, Brito and colleagues developed a human ICB explant for studying acute anterior uveal inflammation using bacterial LPS as the insult and reported the release of pro-inflammatory mediators such as TNF-α and IL-6 into the culture media [47]. In the present study, we modified the protocol used by Brito et al. [47] for use with isolated porcine iris-ciliary bodies in culture. It is pertinent to note the use of cultured explants to study the inflammatory response triggered by LPS has been reported in human gestational tissues [48], lung explants [49], human abdominal aortic aneurysm explants [50], and adipose tissue [51]. Clearly, the approach used in the present study to investigate the pharmacological actions of H₂S-releasing compounds in the porcine explants exposed to LPS supports previous work reported in the literature [48–51].

We found that treatment with different concentrations of LPS produced an increase in the release of TNF-α, IL-6, and PGE₂ into the media, indicating that these mediators were associated with the acute inflammatory process. The use of LPS to induce inflammation in the anterior uvea has been reported in various experimental models of uveitis [48–54]. Indeed, LPS stimulated the production of pro-inflammatory cytokines such as TNF-α, IL-6, and PGE_2, which are commonly found in aqueous humor from patients with anterior uveitis and in experimental animals with EIU [55, 56]. It is pertinent to note that we were able to replicate the observation made by Brito et al. [47] on human ICB explants with the porcine tissue [47]. Taken together, data from the present study support the use of porcine tissues in studying inflammatory responses induced by LPS in the anterior uvea. The observation that the release of three distinct inflammatory mediators, TNF-α, IL-6, and PGE₂, can be measured in the media containing the ICB explants, validates the use of these inflammatory markers for assessing anterior uveal inflammation.

Interestingly, in the present study, we observed that LPS caused a concentration-dependent decrease in basal levels of IL-10, an anti-inflammatory cytokine in the porcine ICB explants, suggesting that only processes that favor the onset and maintenance of inflammation prevail with the insult triggered by the bacterial LPS. Other investigators have also reported a decrease in the concentration of IL-10 during an inflammatory process [57, 58]. IL-10 is an essential anti-inflammatory multifunctional cytokine produced primarily from T cells and activated macrophages [59]. There is evidence that IL-10 can also inhibit the production of pro-inflammatory cytokines in tissues/organs [60, 61]. When compared to our earlier observations, it appears that as LPS stimulates the release of pro-inflammatory mediators (TNF-α, IL-6, and PGE₂), it could be simultaneously suppressing the release of anti-inflammatory mediators such as IL-10. These findings are consistent with the studies reported by Vucevic et al., and other investigators which showed an inverse correlation between IL-6 and IL-10 levels in abdominal aortic aneurysm explant tissue cultures [50, 62]. In summary, data from the present study support the view that the bacterial LPS can initiate an inflammatory response in the porcine anterior uvea, ex vivo, a process that also inhibits the release of an anti-inflammatory cytokine such as IL-10.

There is abundant evidence of the anti-inflammatory role of H₂S in several tissues and organs [63, 64]. Due to the difficulties associated with handling this gas for experimental purposes, H₂S-releasing compounds serve as exogenous sources of this gas [65, 66]. In the present study, we examined the effects of fast- and slow-H₂S-releasing compounds, as well as garlic-derived H₂S-releasing compounds, on the production of pro- and anti-inflammatory mediators in the porcine ICB explants. Amongst the gas-releasing compounds, inorganic sulfide salts are the most widely used sources of exogenous H₂S, with NaHS considered as the prototypical fast-releasing H₂S compounds [39, 67]. The phosphorodithioate derivative, GYY4137 (morpholin-4-ium 4-methoxyphenyl-morpholino-phosphinodithioate) has been reported to demonstrate a slow-release H₂S kinetics in biological media [68]. In a series of experiments, we observed that both NaHS and GYY4137 elicited concentration-dependent attenuation of LPS-induced increases in the levels of TNF-α, IL-6, and PGE₂ in the culture media of porcine ICB explants, suggesting an anti-inflammatory role for both the fast- and slow-releasing H₂S compounds. Our data also shows that at an equi-effective concentration of LPS (25 ng/ml), GYY4137 was more potent than NaHS in reducing the release of pro-inflammatory mediators in cultured porcine ICB explants. Our observation is consistent with results reported by Karaman et al. [67], where GYY4137 was shown to be up to 10 times more potent than NaHS in reducing LPS-stimulated TNF-α and IL-6 levels in an in vitro model of airway inflammation in mice.

Naturally occurring H₂S-releasing compounds from garlic (such as S-allyl cysteine, DADS and DATTS) have been shown to release H₂S in biological media at a relatively slow rate over time [69, 70, 71]. In a series of experiments, we investigated the effects of DADS, SAC, DATTS on LPS-induced PGE₂ production in the porcine ICB explants. DADS, SAC, and DATTS elicited concentration-dependent decreases in the LPS-induced PGE₂ production in the explants. We found that DATTS was more potent than SAC and DADS in decreasing PGE₂ production at an equi-effective concentration of LPS (50 ng/ml). The observed discrepancy could be due to the ability of DATTS to release more H₂S than DADS over time, since the reactivity of these organosulfur H₂S-releasing compounds is directly related to the number of sulfur atoms present in the molecule [71, 72]. Attenuation of LPS-induced PGE₂ production has also been reported for SAC and other diallyl compounds from garlic by other investigators [73–78]. The observed ability of SAC to prevent inflammation may be due to its action in releasing H₂S since cysteine analogues such as S-propargyl-cysteine (SPRC) can increase levels of the gas in biological systems via mechanisms including direct H₂S donation and upregulation of the expression of enzymes responsible for the biosynthesis of H₂S [79, 80]. In the present study, the garlic-derived organosulfur compounds exhibited higher potencies in inhibiting the release of PGE₂ stimulated by LPS when compared to fast- and slow-H₂S-releasing compounds yielding the following rank order of potency: DATTS > > SAC > DADS > GYY4137 > NaHS.

We next examined the effect of DATTS on the production of IL-10, an anti-inflammatory cytokine, in the presence of LPS. We observed that DATTS significantly reversed the LPS-induced decrease in IL-10 levels in a concentration-dependent manner, indicating that DATTS can prevent the deleterious action of LPS on IL-10 production at the site of inflammation. Indeed, Flannigan et al. [80] demonstrated that a relationship exists between IL-10 and the pathway leading to H₂S production since the administration of IL-10 to IL-10-deficient mice restored colonic H₂S synthesis and reduced homocysteine levels. Be that as it may, the finding that DATTS released enough H₂S to prevent the inhibitory action of LPS on the production of IL-10, supports the anti-inflammatory role of H₂S in the porcine ICB explants.

SAC has been reported to act as a substrate for endogenous H₂S biosynthesis in tissues and organs [79, 82]. We next investigated the role of an enzyme of the H₂S trans-sulfuration pathway, CBS/CSE in the pharmacological effects produced by SAC on PGE₂ production. There are several reports of the use of inhibitors of CBS/CSE such as AOAA and PAG (propargylglycine) to assess the pharmacological role of H₂S in various cells, tissues, and organs [83, 84]. In the present study, we utilized AOAA to assess the role of CBS/CSE in the endogenous production of H₂S during LPS-induced inflammation. We found that in the presence of AOAA, there was an enhancement of PGE₂ release in LPS-treated porcine ICB explants, suggesting that inhibition of gas production further exacerbated the inflammation caused by LPS. Interestingly, treatment with SAC which releases H₂S was able to reverse this response. These observations imply that endogenously produced H₂S via a CBS/CSE-mediated pathway serves an anti-inflammatory role during the inflammation process. The replacement of depleted endogenous H₂S content elicited by SAC was able to attenuate the inflammatory process and restore the anti-inflammatory action of the gas. A similar observation was made by Zhao et al. and other investigators who found that the inhibition of CBS activity by AOAA suppressed the protective effects of S-adenosyl-l-methionine (SAM) and H₂S-releasing compounds on the secretion of pro-inflammatory cytokines due to astrocyte activation induced by LPS [85–88].

In another series of experiments, we sought to determine the source of PGE₂ in the LPS-induced inflammatory response in porcine ICB explants. We investigated the effect of two COX inhibitors (indomethacin and flurbiprofen) on PGE₂ levels in the LPS-induced inflammation model. In the presence of an equi-effective concentration of LPS (50 ng/ml), both indomethacin and flurbiprofen caused significant (p < 0.01) decreases in LPS-induced PGE₂ release from the porcine ICB explants. The observed ability of COX inhibitors to attenuate inflammation-induced release of a mediator such as PGE₂ confirms that active biosynthesis of endogenous prostaglandins contributes to this complex immune response.

A major significance of data obtained from the present study is that it supports the view that H₂S-releasing compounds can inhibit the release of some mediators of inflammation. Data from the present study showing that SAC reversed the increased inflammation caused by the inhibition of an enzyme of the H₂S trans-sulfuration pathway indicate that CBS/CSE activities could serve as a marker of inflammation in the pre-clinical and clinical settings [89]. A low expression or activity of these H₂S trans-sulfuration enzymes demonstrated by low concentrations of the gas could indicate the presence of an inflammatory process in the anterior uvea. In support this assertion, evidence from our laboratory demonstrated that in porcine ICB explants, increasing concentrations of LPS caused a concentration-related decrease in the H₂S levels indicating inflammation is associated with a decreased concentration of the gas [44]. Observations from the present study showing the ability of DATTS to prevent the decrease in IL-10 production induced by LPS, suggest that these gas-releasing compounds may have a potential therapeutic utility in enhancing the production of anti-inflammatory cytokines in uveitis.

Conclusions

We conclude that the porcine ICB explants can serve as a model for studying inflammation induced by bacterial LPS. Furthermore, this model can be used to assess the effect of LPS on the release of both pro- and anti-inflammatory mediators in the anterior uvea. Both fast- and slow-releasing H₂S compounds, as well as garlic-derived H₂S-releasing compounds, were effective in reducing LPS-induced inflammation in porcine anterior uveal explants, ex vivo. Inhibition of the biosynthesis of H₂S via the CBE/CSE and 3-MST pathways exacerbated the LPS-induced inflammation, a response that was reversed in the presence of a H₂S-releasing compound. H₂S-releasing compounds can also prevent the deleterious action of the bacterial LPS on the production of an anti-inflammatory cytokine in this model. The ability of LPS to increase the release of PGE₂ in the explants was due, at least in part, to the endogenous biosynthesis of prostaglandins in the anterior uvea.

Figure 8.

Effect of SAC on LPS-Induced PGE₂ Production in Cultured Porcine Iris-ciliary Body Explants in the Presence of AOAA. Each value represents the mean ± SEM. ****p < 0.0001, significantly different from stimulated PGE₂ release by LPS (50 ng/ml) in the presence of AOAA (n = 8).

Funding Statement

This project was supported by Grant Number 1R15EY032274-01 from the National Institute of Health, National Eye Institute.

Data Availability

Datasets supporting the conclusions of this article are included within the article and its additional files.