Hydrogen sulfide modulates chromatin remodeling and inflammatory mediator production in response to endotoxin, but does not play a role in the development of endotoxin tolerance

Abstract\ud \ud Background\ud Pretreatment with low doses of LPS (lipopolysaccharide, bacterial endotoxin) reduces the pro-inflammatory response to a subsequent higher LPS dose, a phenomenon known as endotoxin tolerance. Moreover, hydrogen sulfide (H2S), an endogenous gaseous mediator (gasotransmitter) can exert anti-inflammatory effects. Here we investigated the potential role of H2S in the development of LPS tolerance. THP1 differentiated macrophages were pretreated with the H2S donor NaHS (1 mM) or the H2S biosynthesis inhibitor aminooxyacetic acid (AOAA, 1 mM).\ud \ud \ud Methods\ud To induce tolerance, cells were treated with a low concentration of LPS (0.5 μg/ml) for 4 or 24 h, and then treated with a high concentration of LPS (1 μg/ml) for 4 h or 24 h. In in vivo studies, male wild-type and CSE-/- mice were randomized to the following groups: Control (vehicle); Endotoxemic saline for 3 days before the induction of endotoxemia with 10 mg/kg LPS) mg/kg; Tolerant (LPS at 1 mg/kg for 3 days, followed LPS at 10 mg/kg). Animals were sacrificed after 4 or 12 h; plasma IL-6 and TNF-α levels were measured. Changes in histone H3 and H4 acetylation were analyzed by Western blotting.\ud \ud \ud Results\ud LPS tolerance decreased pro-inflammatory cytokine production. AOAA did not affect the effect of tolerance on reducing cytokine production. Treatment of the cells with the H2S donor reduced cytokine production. Induction of the tolerance increased the acetylation of H3; AOAA reduced histone acetylation. H2S donation increased histone acetylation. Tolerance did not affect the responses to H2S with respect to histone acetylation.\ud \ud \ud Conclusions\ud In conclusion, both LPS tolerance and H2S donation decrease LPS-induced cytokine production in vitro and modulate histone acetylation. However, endogenous, CSE-derived H2S does not appear to play a significant role in the development of LPS tolerance.This work was supported by a grant from the National Institutes of Health\ud (R01 GM107846) to C.S

Identification of Pharmacological Modulators of HMGB1-Induced Inflammatory Response by Cell-Based Screening

High mobility group box 1 (HMGB1), a highly conserved, ubiquitous protein, is released into the circulation during sterile inflammation (e.g. arthritis, trauma) and circulatory shock. It participates in the pathogenesis of delayed inflammatory responses and organ dysfunction. While several molecules have been identified that modulate the release of HMGB1, less attention has been paid to identify pharmacological inhibitors of the downstream inflammatory processes elicited by HMGB1 (C23-C45 disulfide C106 thiol form). In the current study, a cell-based medium-throughput screening of a 5000+ compound focused library of clinical drugs and drug-like compounds was performed in murine RAW264.7 macrophages, in order to identify modulators of HMGB1-induced tumor-necrosis factor alpha (TNFα) production. Clinically used drugs that suppressed HMGB1-induced TNFα production included glucocorticoids, beta agonists, and the anti-HIV compound indinavir. A re-screen of the NIH clinical compound library identified beta-agonists and various intracellular cAMP enhancers as compounds that potentiate the inhibitory effect of glucocorticoids on HMGB1-induced TNFα production. The molecular pathways involved in this synergistic anti-inflammatory effect are related, at least in part, to inhibition of TNFα mRNA synthesis via a synergistic suppression of ERK/IκB activation. Inhibition of TNFα production by prednisolone+salbutamol pretreatment was also confirmed in vivo in mice subjected to HMGB1 injection; this effect was more pronounced than the effect of either of the agents administered separately. The current study unveils several drug-like modulators of HMGB1-mediated inflammatory responses and offers pharmacological directions for the therapeutic suppression of inflammatory responses in HMGB1-dependent diseases. © 2013 Gerö et al

Glucocorticoids Suppress Mitochondrial Oxidant Production via Upregulation of Uncoupling Protein 2 in Hyperglycemic Endothelial Cells

<div><p>Diabetic complications are the leading cause of morbidity and mortality in diabetic patients. Elevated blood glucose contributes to the development of endothelial and vascular dysfunction, and, consequently, to diabetic micro- and macrovascular complications, because it increases the mitochondrial proton gradient and mitochondrial oxidant production. Therapeutic approaches designed to counteract glucose-induced mitochondrial reactive oxygen species (ROS) production in the vasculature are expected to show efficacy against all diabetic complications, but direct pharmacological targeting (scavenging) of mitochondrial oxidants remains challenging due to the high reactivity of some of these oxidant species. In a recent study, we have conducted a medium-throughput cell-based screening of a focused library of well-annotated pharmacologically active compounds and identified glucocorticoids as inhibitors of mitochondrial superoxide production in microvascular endothelial cells exposed to elevated extracellular glucose. The goal of the current study was to investigate the mechanism of glucocorticoids' action. Our findings show that glucocorticoids induce the expression of the mitochondrial UCP2 protein and decrease the mitochondrial potential. UCP2 silencing prevents the protective effect of the glucocorticoids on ROS production. UCP2 induction also increases the oxygen consumption and the “proton leak” in microvascular endothelial cells. Furthermore, glutamine supplementation augments the effect of glucocorticoids via further enhancing the expression of UCP2 at the translational level. We conclude that UCP2 induction represents a novel experimental therapeutic intervention in diabetic vascular complications. While direct repurposing of glucocorticoids may not be possible for the therapy of diabetic complications due to their significant side effects that develop during chronic administration, the UCP2 pathway may be therapeutically targetable by other, glucocorticoid-independent pharmacological means.</p></div

Glucocorticoids decrease the glucose-induced mitochondrial ROS production in microvascular endothelial cells.

<p><b>A, B:</b> b.End3 microvascular endothelial cells were exposed to high glucose for 7 days and were treated with dexamethasone at the indicated concentrations for 3 days. <b>A:</b> ROS production was measured with the mitochondrial superoxide specific MitoSOX Red and <b>(B)</b> the viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>C, D:</b> b.End3 cells were exposed to high glucose for 7 days and were treated with mifepristone at the indicated concentrations for 3 days. <b>C:</b> ROS production was measured with the mitochondrial superoxide specific MitoSOX Red and <b>(D)</b> viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>E, F</b>: EA.hy926 human venous endothelial cells were exposed to high glucose for 7 days and treated with dexamethasone at the indicated concentrations for 3 days. <b>E</b>: The mitochondrial superoxide production was determined by MitoSOX Red stain and <b>F</b>: the viability by Hoechst 33342 DNA dye uptake. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 glucocorticoid treatment significantly reduced the ROS production.)</p

High glucose exposure induces mitochondrial hyperpolarization and oxidant production in b.End3 microvascular endothelial cells.

<p><b>A-F:</b> Confluent b.End3 endothelial cells were maintained in low or high-glucose containing medium for 3–12 days and metabolic indices and ROS production were determined. <b>A:</b> The mitochondrial citric acid cycle activity was determined by measuring the MTT converting capacity of the cells. <b>B:</b> The anaerobic metabolic capacity of the cells was determined by measuring the LDH activity of the cells. <b>C:</b> The mitochondrial membrane potential was determined by JC-1 dye. <b>D:</b> The cellular ATP content was measured. <b>E, F:</b> The cellular ROS production was determined by (<b>E</b>) measuring the mitochondrial superoxide generation and (<b>F</b>) the cellular H₂O₂ production. <b>G:</b> Respective levels of the mitochondrial respiratory complexes were determined by MitoProfile total OXPHOS antibody cocktail. Representative blot image and densitometric analysis results are shown. (*p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium.)</p

Time course of steroid induced UCP2 expression.

<p><b>A-D:</b> Confluent b.End3 endothelial cells were exposed to <b>A, C, D</b>: dexamethasone (1 μM) or <b>B:</b> mifepristone (3 μM) for the indicated time period. <b>A, B</b>: UCP2 mRNA expression was measured by UCP2 Taqman assay using rRNA normalization. <b>C, D</b>: UCP2 protein expression was measured by Western blotting. Representative blot image (<b>C</b>) and densitometry results (<b>D</b>) are shown. <b>E, F:</b> HepG2 human liver cells were treated with dexamethasone (1 μM, <b>E</b>) or mifepristone (3 μM, <b>F</b>) for the indicated time periods and UCP2 expression was determined by Taqman assay. (*p<0.05 glucocorticoid treatment induced significant changes in the UCP2 expression.)</p

Glutamine potentiates the dexamethasone-mediated UCP2 induction and inhibition of ROS production.

<p><b>A-D</b>: b.End3 cells were exposed to hyperglycemia for 7 days and were treated subsequently with dexamethasone (1 μM) and the indicated amount of glutamine for 6 hours (<b>A</b>) or 3 days (<b>B-D</b>). <b>C</b>: The mitochondrial superoxide production was measured by MitoSOX Red. <b>C-D</b>: UCP2 protein expression was measured by Western blotting. Representative blot image (<b>C</b>) and densitometric analysis results (<b>D</b>) are shown. (High-glucose exposure induced significant increase in the mitochondrial ROS production. #p<0.05 dexamethasone significantly decreased the ROS production compared to the high glucose group, *p<0.05 glutamine treatment resulted in a significant decrease in ROS production.)</p

Mifepristone inhibits the glucose-induced mitochondrial ROS production in microvascular endothelial cells.

<p><b>A-D:</b> b.End3 microvascular endothelial cells were exposed to high glucose for 7 days and were treated with dexamethasone and mifepristone (<b>A, B</b>) or with mifepristone alone (<b>C, D</b>) at the indicated concentrations for 3 days. <b>A, C:</b> The ROS production was measured with the mitochondrial superoxide specific MitoSOX Red <b>B, D:</b> The viability was determined by measuring the Hoechst 33342 DNA dye uptake. <b>E, F</b>: bEnd.3 BALB/c murine microvascular endothelial cells were exposed to high glucose for 7 days and were treated with mifepristone for 3 days at the indicate concentrations. <b>E</b>: The mitochondrial ROS production was measured by MitoSOX Red and <b>F</b>: the viability was determined by the Hoechst 33342 uptake. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 glucocorticoid treatment significantly reduced the ROS production, §p<0.05 mifepristone induced significant reduction in the ROS generation compared to dexamethasone alone.)</p

Glucocorticoid steroids block the glucose-induced mitochondrial hyperpolarization and induce UCP2 expression.

<p><b>A, B, D, E:</b> b.End3 endothelial cells were exposed to high glucose for 7 days and treated with the dexamethasone (1 μM) or mifepristone (3 μM) for 3 days. <b>A, B:</b> Changes in the mitochondrial potential were determined by measuring the MitoTracker Green uptake (<b>A</b>) or the ratio of the mitochondrial J-aggregate and the free cytoplasmic form of JC-1 (<b>B</b>). <b>C, F</b>: EA.hy926 human venous cells were exposed to high glucose for 7 days and treated with dexamethasone (1 μM) for 3 days. <b>C</b>: The mitochondrial membrane potential was measured by JC-1 dye. <b>D, E, F</b>: UCP2 expression was determined by realtime PCR using Taqman assays. (#p<0.05 high-glucose exposure induced significant changes compared to cells maintained in low glucose containing medium,*p<0.05 glucocorticoid treatment induced significant changes.)</p