The use of mesenchymal stromal cells (MSCs) for treatment of bacterial infections, including systemic processes like sepsis, is an evolving field of investigation. The present study was designed to investigate the potential use of MSCs, harvested from compact bone, and their interactions with the innate immune system, during polymicrobial sepsis induced by cecal ligation and puncture (CLP). We also wanted to elucidate the role of endogenous HO-1 in MSCs during a systemic bacterial infection. MSCs harvested from the bones of heme oxygenase (HO)-1 deficient (−/−) and wild-type (+/+) mice improved the survival of HO-1−/− and HO-1+/+ recipient mice when administered after the onset of polymicrobial sepsis induced by cecal ligation and puncture (CLP), compared with the administration of fibroblast control cells. The MSCs, originating from compact bone in mice, enhanced the ability of neutrophils to phagocytize bacteria in vitro and in vivo, and to promote bacterial clearance in the peritoneum and blood after CLP. Moreover, after depleting neutrophils in recipient mice, the beneficial effects of MSCs were entirely lost, demonstrating the importance of neutrophils for this MSC response. MSCs also decreased multiple organ injury in susceptible HO-1−/− mice, when administered after the onset of sepsis. Taken together, these data demonstrate that the beneficial effects of treatment with MSCs after the onset of polymicrobial sepsis is not dependent on endogenous HO-1 expression, and that neutrophils are crucial for this therapeutic response.

We examined our hypothesis that heme-oxygenase-1 (HO-1)-derived carbon monoxide (CO) inhibits the release of highmobility group box 1 (HMGB1) in RAW264.7 cells activated with lipopolysaccharide (LPS) in vitro and in LPS-or cecal ligation and puncture (CLP)-induced septic mice in vivo, so that HO-1 induction or CO improves survival of sepsis in rodents. We found that pretreatment with HO-1 inducers (hemin, cobalt protoporphyrin IX) or transfection of HO-1 significantly inhibited HMGB1 release, which was blocked by HO-1 small interfering RNA, in cells activated by LPS. Carbon monoxide-releasing molecule 2 (CORM-2) but not bilirubin or deferoxamine inhibited HMGB1 release in LPS-activated macrophages. Oxyhemoglobin reversed the effect of HO-1 inducers on HMGB1 release. Translocation of HMGB1 from nucleus to cytosol was significantly inhibited by HO-1 inducers, CORM-2, or HO-1 transfection. Neutralizing antibodies to tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤, interferon-␤, and N -nitro-L-arginine methyl ester hydrochloride but not N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) significantly inhibited HMGB1 release in LPS-activated cells. Production of TNF-␣, IL-1␤, and IFN-␤ was significantly reduced by pretreatment of HO-1 inducers, CORM-2, or HO-1 transfection in LPS-activated cells. Plasma levels of HMGB1 in mice challenged with LPS or CLP were significantly reduced by the administration of HO-1 inducers or CORM-2, which was accompanied by either reduction (pretreatment) or no change (delayed administration) of serum TNF-␣ and IL-1␤ levels. Regardless of pretreatment or delayed administration, CORM-2 and hemin rescued mice from lethal endotoxemia and sepsis induced by LPS or CLP. Taken together, we concluded that HO-1-derived CO reduces HMGB1 release in LPS-activated cells and LPS-or CLP-induced animal model of sepsis.Sepsis is defined as a systemic inflammatory response syndrome from a microbial infection that results from excessive stimulation of the host immune system by pathogen components to produce various proinflammatory cytokines, and their overproduction causes systemic inflammation that can lead to the lethal multiple organ damage (Oberholzer et al., 2001). High-mobility group box 1 (HMGB1) is a chromatin-binding protein that participates in maintaining nucleosome structure and regulation of gene transcription (Landsman and Bustin, 1993). Various evidence indicated that HMGB1 is a necessary and sufficient late mediator of severe sepsis (Wang et al., 1999;Yang et al., 2004). Once released, HMGB1 can bind to cell-surface receptors, such as the receptor for advanced glycation end products and Toll-like receptors 2 and 4, and mediate various cellular responses, chemotactic cell movement, and release of proinflammatory Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.055137.ABBREVIATIONS: HMGB1, high-mobility group box 1; HO-1, heme-oxygenase-1; CORM-2, carbon monoxide-releasing molecule II; LPS, lip...

BACKGROUND AND PURPOSE Recently, metformin, a well‐known anti‐diabetic drug, has been shown to possess anti‐inflammatory activities. This study investigated the effect of metformin on the expression of pro‐inflammatory cytokines including high mobility group box 1 (HMGB1) in lipopolysaccharide (LPS)‐treated animals and cells. EXPERIMENTAL APPROACH We investigated whether metformin inhibits the release of HMGB1 in LPS‐treated RAW 264.7 cells and increases survival rate in endotoxaemic mice (lethal endotoxaemia was induced by an i.p. injection of LPS). This was achieved by a range of techniques including Western blotting, enzyme‐linked immunosorbent assay, specific pharmacological inhibitors, knock out of α1‐subunit of AMP‐activated protein kinase (AMPK) and recombinant HMGB1. KEY RESULTS Both pre‐ and post‐treatment with metformin significantly improved survival of animals during lethal endotoxaemia (survival rate was monitored up to 2 weeks), decreased serum levels of tumour necrosis factor‐alpha (TNF‐α), interleukin‐1β, HMGB1 expression and myeloperoxidase activity in lungs. However, metformin failed to improve survival in endotoxaemic animals that had additionally been treated with recombinant HMGB1. In an in vitro study, metformin dose‐dependently inhibited production of pro‐inflammatory cytokines and HMGB1 release. Metformin activated AMPK by its phosphorylation. Compound C (pharmacological inhibitor of AMPK) and siAMPKα1 reversed the anti‐inflammatory effect of metformin in LPS‐treated cells. CONCLUSIONS AND IMPLICATIONS Our data indicate that metformin significantly attenuates the pro‐inflammatory response induced by LPS both in vivo and in vitro. Metformin improved survival in a mouse model of lethal endotoxaemia by inhibiting HMGB1 release. AMPK activation was implicated as one of the mechanisms contributing to this inhibition of HMGB1 secretion.

Ultraviolet (UV) radiation can cause inflammatory changes and may further contribute to skin carcinogenesis. Anthocyanins are known to be powerful antioxidants that help protect plants from UV damage. Recently, we isolated anthocyanins from black soybean [Glycine max (L.) Merr] seed coats. Thus, we investigated the protective effect of anthocyanins from black soybean seed coats on UVB radiation-induced inflammatory responses and the molecular mechanism responsible for regulation of apoptosis and inflammatory responses. Anthocyanins inhibited UVB-induced cylooxygenase-2 (COX-2) and PGE 2 production through a nuclear factor-kappaB-dependent pathway and regulation of the PI3 kinase/Akt pathway activated by UVB in a human keratinocyte cell line, HaCaT. Topical application of anthocyanins prior to UVB irradiation of hairless mice also inhibited induction of COX-2 and PGE 2. In conclusion, it is suggested that anthocyanins from the seed coat of black soybeans can be used as a useful drug to modulate oxidative disorders including UVB-induced inflammation.

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Activation of nicotinic acetylcholine receptor alpha7 subunit (α7nAChR) by nicotine leads to the improved survival rate in experimental model of sepsis. Previously, we demonstrated that heme oxygenase (HO)-1 inducers or carbon monoxide significantly increased survival of lipopolysaccharide (LPS)-induced and cecal ligation and puncture-induced septic mice by reduction of high mobility group box 1 release, a late mediator of sepsis. However, that activation of α7nAChR by nicotine provides anti-inflammatory action through HO-1 upregulation has not been elucidated. Here we show that HO-1-inducible effect by nicotine was mediated through sequential event-Ca(2+) influx, classical protein kinase C activation, and reactive oxygen species production-which activates phosphoinositol-3-kinase/Akt/Nrf-2 pathway. In addition, HO-1 is required for nicotine-mediated suppression of tumor necrosis factor-α, inducible nitric oxide synthase, and high mobility group box 1 expression induced by LPS in macrophages, as evidenced by the fact that nicotine failed to inhibit production of these mediators when HO-1 was suppressed. Importantly, nicotine-induced survival rate was reduced by inhibition of HO-1 in LPS- and cecal ligation and puncture-treated septic mice. Collectively, these data suggest that activation of α7nAChR by nicotine is critical in the regulation of anti-inflammatory process, which could be mediated through HO-1 expression. Thus, we conclude that activation of α7nAChR by nicotine provides anti-inflammatory action through HO-1 upregulation.