P14 Comparing the differential effects of novel, slow-release H2S donors on inflammatory mediators and cell proliferation in human joint and immune cells

Abstract

Acute inflammation is the host response to infection and tissue damage, and serves to limit harm to the body. However, dysregulated or chronic inflammation underlies the pathology of many diseases, such as autoimmune disease. Elevated H2S levels are present in the joints of rheumatoid arthritis patients, and increased levels of H2S and the H2S synthesising enzyme CSE are observed in vitro and in vivo following exposure to an inflammatory stimulus. However, the role of H2S in inflammation is unclear, with pro-and anti-inflammatory effects observed. The method of H2S delivery may be, at least partially, responsible for these discrepancies. For instance, fast release donors that deliver an instant bolus of H2S (e.g. NaSH/Na2S) invariably have pro-inflammatory effects in vivo, whereas slow-release donors that deliver H2S in a manner comparable to endogenous production are predominantly anti-inflammatory. Recently, attention has focused on the slow-release H2S donor GYY4137, with down-regulation of pro-inflammatory mediators and pro-inflammatory enzyme activity, as well as reduced joint swelling observed, supporting an anti-inflammatory role of H2S in vitro and in vivo. Here the effects of GYY4137 and two novel slow-release H2S donors, AP105 and AP106, which release H2S at different rates (AP105 > AP106 > GYY4137), were compared by examining their effects on pro-inflammatory mediators, pro-inflammatory enzyme activity and NF-κB signalling. Also, the effect on the proliferation of primary human synoviocytes (HFLS) and Jurkat T-cells, whose hyper-proliferation contributes to joint inflammation, were investigated. Following stimulation of HFLS with a cocktail of pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ) in the presence or absence of H2S donors, pro-inflammatory mediator levels were measured in the culture media. Significantly decreased IL-6 levels (p < 0.05; ANOVA) were observed following exposure to AP105 or AP106 (250 μM or 500 μM), and significantly decreased PGE2 levels were observed following exposure to GYY4137 (250 μM) or AP105 (250 μM or 500 μM). In-vitro recombinant enzyme activity assays showed that TNF-α converting enzyme (TACE) and cycloxygenase-2 (COX-2) activity were significantly inhibited by all three slow-release H2S donors (200–1000 μM). However, whereas small, significant decreases in matrix metalloprotease-2 (MMP-2) activity were observed with AP105 and AP106 (250 μM or 500 μM), significantly increased MMP-2 activity was observed with GYY4137 (250 μM). Jurkat T-cell numbers were significantly decreased following 72 h culture in the presence of AP105 (62.5 μM or 250 μM) or AP106 (250 μM), whereas significantly increased Jurkat T-cell numbers were observed with 62.5 μM AP106. GYY4137 had no effect on Jurkat proliferation, and there was no significant effect on cell viability with any of the donors, except a small decrease with AP105 or AP106 at 250 μM (3.43% and 2.93% c.f. control, respectively). A significant decrease in HFLS numbers was observed with AP105 (250 μM or 500 μM) after 72 h, either in standard culture or following stimulation with TNF-α, with no significant effect of AP106 or GYY4137 under the same conditions. Following cytokine stimulation of HFLS there was no effect of GYY4137, AP105 or AP106 on I-κB phosphorylation at early time-points. With exceptions, the down-regulation of pro-inflammatory mediators, pro-inflammatory enzyme activity and reduced T-cell and HFLS cell proliferation observed supports an anti-inflammatory role for H2S from slow-release H2S donors. This work was supported by the Wellcome Trust.