Endogenous carbon monoxide (CO) exerts anti-inflammatory effects. Tristetraprolin (TTP) is known to destabilize pro-inflammatory transcripts. Here we found that exogenous CO enhanced the decay of TNF-α mRNA and suppressed TNF-α expression in LPS-activated macrophages from wild-type (WT) mice. However, TTP deficiency abrogated the effects of exogenous CO. While CO treatment prior to DSS administration in WT mice significantly reduced inflammatory cytokine levels and colitis, it failed to reduce the pro-inflammatory cytokine levels and colitis in TTP knockout (KO) mice. Our results demonstrate that TTP is a key factor mediating the anti-inflammatory action of CO in DSS-induced colitis.

Carbon monoxide (CO) can act as an anti-inflammatory effector in mouse models of lung injury and disease, through the downregulation of pro-inflammatory cytokines production, though the underlying mechanisms remain unclear. The nucleotide-binding oligomerization domain-, leucine-rich region-, and pyrin domain-containing-3 (NLRP3) inflammasome is a protein complex that regulates the maturation and secretion of pro-inflammatory cytokines, including interleukin-1β (IL-1β). In this report, we show that the CO-releasing molecule (CORM-2) can stimulate the expression of pyrin, a negative regulator of the NLRP3 inflammasome. CORM-2 increased the transcription of pyrin in the human leukemic cell line (THP-1) in the absence and presence of lipopolysaccharide (LPS). In THP-1 cells, CORM-2 treatment dose-dependently reduced the activation of caspase-1 and the secretion of IL-1β, and increased the levels of IL-10, in response to LPS and adenosine 5'-triphosphate (ATP), an NLRP3 inflammasome activation model. Genetic interference of IL-10 by small interfering RNA (siRNA) reduced the effectiveness of CORM-2 in inhibiting IL-1β production and in inducing pyrin expression. Genetic interference of pyrin by siRNA increased IL-1β production in response to LPS and ATP, and reversed CORM-2-dependent inhibition of caspase-1 activation. CO inhalation (250 ppm) in vivo increased the expression of pyrin and IL-10 in lung and spleen, and decreased the levels of IL-1β induced by LPS. Consistent with the induction of pyrin and IL-10, and the downregulation of lung IL-1β production, CO provided protection in a model of acute lung injury induced by intranasal LPS administration. These results provide a novel mechanism underlying the anti-inflammatory effects of CO, involving the IL-10-dependent upregulation of pyrin expression.

Endogenous carbon monoxide (CO) is produced by heme oxygenase-1 (HO)-1 which mediates the degradation of heme into CO, iron, and biliverdin. Also, CO ameliorates the human inflammatory bowel diseases and ulcerative colitis. However, the mechanism for the effect of CO on the inflammatory bowel disease has not yet been known. In this study, we showed that CO significantly increases survival percentage, body weight, colon length as well as histologic parameters in DSS-treated mice. In addition, CO inhalation significantly decreased DSS induced pro-inflammatory cytokines by inhibition of GSK-3β in mice model. To support the in vivo observation, TNF-α, iNOS and IL-10 after CO and LiCl treatment were measured in mesenteric lymph node cells (MLNs) and bone marrow-derived macrophages (BMMs) from DSS treated mice. In addition, we determined that CO potentially inhibited GSK-3β activation and decreased TNF-α and iNOS expression by inhibition of NF-κB activation in LPS-stimulated U937 and MLN cells pretreated with CO. Together, our findings indicate that CO attenuates DSS-induced colitis via inhibition of GSK-3β signaling in vitro and in vivo. Importantly, this is the first report that investigated the molecular mechanisms mediated the novel effects of CO via inhibition GSK-3β in DSS-induced colitis model.

Hepatic ischemia/reperfusion (I/R) injury can arise as a complication of liver surgery and transplantation. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, modulates inflammation and apoptosis in response to oxidative stress. SIRT1, which is regulated by p53 and microRNA-34a (miR-34a), can modulate non-alcoholic fatty liver disease, fibrosis and cirrhosis. Since carbon monoxide (CO) inhalation can protect against hepatic I/R, we hypothesized that CO could ameliorate hepatic I/R injury by regulating the miR-34a/SIRT1 pathway. Livers from mice pretreated with CO, or PFT, a p53 inhibitor, displayed reduced production of pro-inflammatory mediators, including TNF-α, iNOS, interleukin (IL)-6, and IL-1β after hepatic I/R injury. SIRT1 expression was increased by CO or PFT in the liver after I/R, whereas acetylated p65, p53 levels, and miR-34a expression were decreased. CO increased SIRT1 expression by inhibiting miR-34a. Both CO and PFT diminished pro-inflammatory cytokines production in vitro. Knockdown of SIRT1 in LPS-stimulated macrophages increased NF-κB acetylation, and increased pro-inflammatory cytokines. CO treatment reduced miR-34a expression and increased SIRT1 expression in oxidant-challenged hepatocytes; and rescued SIRT1 expression in p53-expressing or miR-34a transfected cells. In response to CO, enhanced SIRT1 expression mediated by miR-34a inhibition protects against liver damage through p65/p53 deacetylation, which may mediate inflammatory responses and hepatocellular apoptosis. The miR-34a/SIRT1 pathway may represent a therapeutic target for hepatic injury.

Carbon monoxide (CO) can confer protection against cellular stress, whereas the potential involvement of autophagy and lysosomal biogenesis remains incompletely understood. We demonstrate here that the activation of protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) with CO increased the nuclear translocation of transcription factor EB (TFEB). PERK activation by CO increased intracellular Ca2+ concentration and the phosphatase activity of calcineurin against TFEB. Moreover, we found that in the deficiency of TFEB, CO not only failed to recruit Parkin to the mitochondria but also failed to increase expression of lysosomal genes such as Lamp1, CathB, and TPP1. Therefore, we suggest that CO increases mitophagy through TFEB nuclear translocation by PERK-calcinuerin activation. In addition, the inhibition of TFEB with siRNA against TFEB abrogated the increase of mtDNA with CO, markers of mitochondrial biogenesis such as PGC1α, NRF1, and TFAM, and the mitochondrial proteins COX II, COX IV, and cytochrome c. To investigate the effects of CO on mitochondrial homeostasis in vivo, mice were treated with lipopolysaccharide (LPS)/D-galactosamine (D-GalN). CO inhalation reduced liver injury after challenge with LPS/GalN. Furthermore, CO inhalation increased TFEB activation, mitophagy and mitochondrial biogenesis in mice treated with LPS/GalN. Our findings describe novel mechanisms underlying CO-dependent cytoprotection in hepatocytes and liver tissue via activation of TFEB-dependent mitophagy and associated induction of both lysosomal and mitochondrial biogenesis.

Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, has emerged as one of the most common causes of chronic liver disease in developed countries over the last decade. NAFLD comprises a spectrum of pathological hepatic changes, including steatosis, steatohepatitis, advanced fibrosis, and cirrhosis. Autophagy, a homeostatic process for protein and organelle turnover, is decreased in the liver during the development of NAFLD. Previously, we have shown that carbon monoxide (CO), a reaction product of heme oxygenase (HO) activity, can confer protection in NAFLD, though the molecular mechanisms remain unclear. We therefore investigated the mechanisms underlying the protective effect of CO on methionine/choline-deficient (MCD) diet-induced hepatic steatosis. We found that CO induced sestrin-2 (SESN2) expression through enhanced mitochondrial ROS production and protected against MCD-induced NAFLD progression through activation of autophagy. SESN2 expression was increased by CO or CO-releasing molecule (CORM2), in a manner dependent on signaling through the protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor-2 alpha (eIF2α)/ activating transcription factor-4 (ATF4)-dependent pathway. CO-induced SESN2 upregulation in hepatocytes contributed to autophagy induction through activation of 5'-AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR) complex I (mTORC1). Furthermore, we demonstrate that CO significantly induced the expression of SESN2 and enhanced autophagy in the livers of MCD-fed mice or in MCD-media treated hepatocytes. Conversely, knockdown of SESN2 abrogated autophagy activation and mTOR inhibition in response to CO. We conclude that CO ameliorates hepatic steatosis through the autophagy pathway induced by SESN2 upregulation.

Carbon monoxide (CO) may exert important roles in physiological and pathophysiological states through the regulation of cellular signaling pathways. CO can protect organ tissues from ischemia/reperfusion (I/R) injury by modulating intracellular redox status and by inhibiting inflammatory, apoptotic, and proliferative responses. However, the cellular mechanisms underlying the protective effects of CO in organ I/R injury remain incompletely understood. In this study, a murine model of hepatic warm I/R injury was employed to assess the role of glycogen synthase kinase-3 (GSK3) and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways in the protective effects of CO against inflammation and injury. Inhibition of GSK3 through the PI3K/Akt pathway played a crucial role in CO-mediated protection. CO treatment increased the phosphorylation of Akt and GSK3-beta (GSK3β) in the liver after I/R injury. Furthermore, administration of LY294002, an inhibitor of PI3K, compromised the protective effect of CO and decreased the level of phospho-GSK3β after I/R injury. These results suggest that CO protects against liver damage by maintaining GSK3β phosphorylation, which may be mediated by the PI3K/Akt signaling pathway. Our study provides additional support for the therapeutic potential of CO in organ injury and identifies GSK3β as a therapeutic target for CO in the amelioration of hepatic injury.

The resolution phase of acute inflammation is essential for tissue homeostasis, yet the underlying mechanisms remain unclear. We demonstrate that resolution of inflammation involves interactions between CD38 and tristetraprolin (TTP). During the onset of acute inflammation, CD38 levels are increased, leading to the production of Ca2+-signaling messengers, nicotinic acid adenine dinucleotide phosphate (NAADP), ADP ribose (ADPR), and cyclic ADPR (cADPR) from NAD(P)+. To initiate the onset of resolution, TTP expression is increased by the second messengers, NAADP and cADPR, which downregulate CD38 expression. The activation of TTP by Sirt1-dependent deacetylation, in response to increased NAD+ levels, suppresses the acute inflammatory response and decreases Rheb expression, inhibits mTORC1, and induces autophagolysosomes for bacterial clearance. TTP may represent a mechanistic target of anti-inflammatory agents, such as carbon monoxide. TTP mediates crosstalk between acute inflammation and autophagic clearance of bacteria from damaged tissue in the resolution of inflammation during sepsis.

Heme oxygenase-1 (HO-1) can exert anti-inflammatory and antioxidant effects. Acute lung injury (ALI) is associated with increased inflammation and influx of proinflammatory cells and mediators in the airspaces and lung parenchyma. In this study, we demonstrate that pterostilbene 4'-β-glucoside (4-PG), the glycosylated form of the antioxidant pterostilbene (PTER), can protect against lipopolysaccharide- (LPS-) or Pseudomonas aeruginosa- (P. aeruginosa-) induced ALI when applied as a pretreatment or therapeutic post-treatment, via the induction of HO-1. To determine whether HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG, we subjected mice genetically deficient in Hmox-1 to LPS-induced ALI and evaluated histological changes, HO-1 expression, and proinflammatory cytokine levels in bronchoalveolar lavage (BAL) fluid. 4-PG exhibited protective effects on LPS- or P. aeruginosa-induced ALI by ameliorating pathological changes in lung tissue and decreasing proinflammatory cytokines. In addition, HO-1 expression was significantly increased by 4-PG in cells and in mouse lung tissues. The glycosylated form of pterostilbene (4-PG) was more effective than PTER in inducing HO-1 expression. Genetic deletion of Hmox-1 abolished the protective effects of 4-PG against LPS-induced inflammatory responses. Furthermore, we found that 4-PG decreased both intracellular ROS levels and mitochondrial (mt) ROS production in a manner dependent on HO-1. Pharmacological application of the HO-1 reaction product carbon monoxide (CO), but not biliverdin or iron, conferred protection in Hmox-1-deficient macrophages. Taken together, these results demonstrate that 4-PG can increase HO-1 expression, which plays a critical role in ameliorating intracellular and mitochondrial ROS production, as well as in downregulating inflammatory responses induced by LPS. Therefore, these findings strongly suggest that HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG.