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• W2045821670 abstract "The induction of heme oxygenase-1 (HO-1; Hmox1) by inflammation, for instance in sepsis, is associated both with an anti-inflammatory response and with mitochondrial biogenesis. Here, we tested the idea that HO-1, acting through the Nfe2l2 (Nrf2) transcription factor, links anti-inflammatory cytokine expression to activation of mitochondrial biogenesis. HO-1 induction after LPS stimulated anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-1Ra) expression in mouse liver, human HepG2 cells, and mouse J774.1 macrophages but blunted tumor necrosis factor-α expression. This was accompanied by nuclear Nfe2l2 accumulation and led us to identify abundant Nfe2l2 and other mitochondrial biogenesis transcription factor binding sites in the promoter regions of IL10 and IL1Ra compared with pro-inflammatory genes regulated by NF-κΒ. Mechanistically, HO-1, through its CO product, enabled these transcription factors to bind the core IL10 and IL1Ra promoters, which for IL10 included Nfe2l2, nuclear respiratory factor (NRF)-2 (Gabpa), and MEF2, and for IL1Ra, included NRF-1 and MEF2. In cells, Hmox1 or Nfe2l2 RNA silencing prevented IL-10 and IL-1Ra up-regulation, and HO-1 induction failed post-LPS in Nfe2l2-silenced cells and post-sepsis in Nfe2l2−/− mice. Nfe2l2−/− mice compared with WT mice, showed more liver damage, higher mortality, and ineffective CO rescue in sepsis. Nfe2l2−/− mice in sepsis also generated higher hepatic TNF-α mRNA levels, lower NRF-1 and PGC-1α mRNA levels, and no enhancement of anti-inflammatory Il10, Socs3, or bcl-xL gene expression. These findings disclose a highly structured transcriptional network that couples mitochondrial biogenesis to counter-inflammation with major implications for immune suppression in sepsis. The induction of heme oxygenase-1 (HO-1; Hmox1) by inflammation, for instance in sepsis, is associated both with an anti-inflammatory response and with mitochondrial biogenesis. Here, we tested the idea that HO-1, acting through the Nfe2l2 (Nrf2) transcription factor, links anti-inflammatory cytokine expression to activation of mitochondrial biogenesis. HO-1 induction after LPS stimulated anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-1Ra) expression in mouse liver, human HepG2 cells, and mouse J774.1 macrophages but blunted tumor necrosis factor-α expression. This was accompanied by nuclear Nfe2l2 accumulation and led us to identify abundant Nfe2l2 and other mitochondrial biogenesis transcription factor binding sites in the promoter regions of IL10 and IL1Ra compared with pro-inflammatory genes regulated by NF-κΒ. Mechanistically, HO-1, through its CO product, enabled these transcription factors to bind the core IL10 and IL1Ra promoters, which for IL10 included Nfe2l2, nuclear respiratory factor (NRF)-2 (Gabpa), and MEF2, and for IL1Ra, included NRF-1 and MEF2. In cells, Hmox1 or Nfe2l2 RNA silencing prevented IL-10 and IL-1Ra up-regulation, and HO-1 induction failed post-LPS in Nfe2l2-silenced cells and post-sepsis in Nfe2l2−/− mice. Nfe2l2−/− mice compared with WT mice, showed more liver damage, higher mortality, and ineffective CO rescue in sepsis. Nfe2l2−/− mice in sepsis also generated higher hepatic TNF-α mRNA levels, lower NRF-1 and PGC-1α mRNA levels, and no enhancement of anti-inflammatory Il10, Socs3, or bcl-xL gene expression. These findings disclose a highly structured transcriptional network that couples mitochondrial biogenesis to counter-inflammation with major implications for immune suppression in sepsis. Early survivors of severe sepsis often develop immune suppression (1Rittirsch D. Flierl M.A. Ward P.A. Nat. Rev. Immunol. 2008; 8: 776-787Crossref PubMed Scopus (900) Google Scholar, 2Hotchkiss R.S. Opal S. N. Engl. J. Med. 2010; 363: 87-89Crossref PubMed Scopus (289) Google Scholar) and may later die with the multiple organ dysfunction syndrome (3Winters B.D. Eberlein M. Leung J. Needham D.M. Pronovost P.J. Sevransky J.E. Crit. Care Med. 2010; 38: 1276-1283Crossref PubMed Scopus (439) Google Scholar). A key effector of multiple organ dysfunction syndrome is the liver, which is integral to the host response, especially in infections that activate Toll-like receptor 4 and NF-κΒ-dependent cytokine synthesis (4Knolle P. Schlaak J. Uhrig A. Kempf P. Meyer zum Büschenfelde K.H. Gerken G. J. Hepatol. 1995; 22: 226-229Abstract Full Text PDF PubMed Scopus (297) Google Scholar). The persistence of inflammatory cytokines such as TNF-α and IL-1β perpetuates immune activation, causing tissue damage and remodeling (5Dinarello C.A. Annu. Rev. Immunol. 2009; 27: 519-550Crossref PubMed Scopus (2551) Google Scholar) and leads to sustained production of anti-inflammatory modulators and suppressors of adaptive immunity (6Madan R. Demircik F. Surianarayanan S. Allen J.L. Divanovic S. Trompette A. Yogev N. Gu Y. Khodoun M. Hildeman D. Boespflug N. Fogolin M.B. Gröbe L. Greweling M. Finkelman F.D. Cardin R. Mohrs M. Müller W. Waisman A. Roers A. Karp C.L. J. Immunol. 2009; 183: 2312-2320Crossref PubMed Scopus (239) Google Scholar, 7Lin C.Y. Tsai I.F. Ho Y.P. Huang C.T. Lin Y.C. Lin C.J. Tseng S.C. Lin W.P. Chen W.T. Sheen I.S. J. Hepatol. 2007; 46: 816-826Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). These anti-inflammatory modulators include the type II cytokine IL-10, the soluble IL-1 receptor antagonist (sIL-1Ra) 2The abbreviations used are: sIL-1Ra, soluble IL-1 receptor antagonist; HO-1, heme oxygenase-1; IL-1Ra, IL-1 receptor antagonist; NRF, nuclear respiratory factor; DCM/CO, CO-producing molecule dichloromethane; CREB, cAMP-response element-binding protein. (5Dinarello C.A. Annu. Rev. Immunol. 2009; 27: 519-550Crossref PubMed Scopus (2551) Google Scholar), and SOCS (suppressor of cytokine signaling) proteins (8Palmer D.C. Restifo N.P. Trends Immunol. 2009; 30: 592-602Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). IL-10 is widely expressed in the liver (9Saraiva M. O'Garra A. Nat. Rev. Immunol. 2010; 10: 170-181Crossref PubMed Scopus (2075) Google Scholar, 10Mosser D.M. Zhang X. Immunol. Rev. 2008; 226: 205-218Crossref PubMed Scopus (815) Google Scholar) by Kupffer cells (11Rai R.M. Loffreda S. Karp C.L. Yang S.Q. Lin H.Z. Diehl A.M. Hepatology. 1997; 25: 889-895Crossref PubMed Scopus (111) Google Scholar), stellate cells (12Wang S.C. Ohata M. Schrum L. Rippe R.A. Tsukamoto H. J. Biol. Chem. 1998; 273: 302-308Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and hepatocytes (13Bourdi M. Masubuchi Y. Reilly T.P. Amouzadeh H.R. Martin J.L. George J.W. Shah A.G. Pohl L.R. Hepatology. 2002; 35: 289-298Crossref PubMed Scopus (271) Google Scholar), where it contributes to LPS tolerance (14Menezes G.B. Lee W.Y. Zhou H. Waterhouse C.C. Cara D.C. Kubes P. J. Immunol. 2009; 183: 7557-7568Crossref PubMed Scopus (61) Google Scholar). The IL-10 receptor activates JAK/STAT (Janus kinase/signal transducer and activator of transcription) to block the production of TNF-α and other NF-κΒ-dependent mediators (15Lai C.F. Ripperger J. Morella K.K. Jurlander J. Hawley T.S. Carson W.E. Kordula T. Caligiuri M.A. Hawley R.G. Fey G.H. Baumann H. J. Biol. Chem. 1996; 271: 13968-13975Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), the basis for its anti-inflammatory effects (16Lee T.S. Chau L.Y. Nat. Med. 2002; 8: 240-246Crossref PubMed Scopus (914) Google Scholar). IL-10 also suppresses mononuclear cell function (17Mosser D.M. J. Leukoc. Biol. 2003; 73: 209-212Crossref PubMed Scopus (1448) Google Scholar), and IL-10 secretion by macrophages and neutrophils negatively regulates the response to LPS (18Siewe L. Bollati-Fogolin M. Wickenhauser C. Krieg T. Müller W. Roers A. Eur. J. Immunol. 2006; 36: 3248-3255Crossref PubMed Scopus (101) Google Scholar). Absence of IL-10 increases cytokine production (19Standiford T.J. Strieter R.M. Lukacs N.W. Kunkel S.L. J. Immunol. 1995; 155: 2222-2229PubMed Google Scholar, 20Berg D.J. Kühn R. Rajewsky K. Müller W. Menon S. Davidson N. Grünig G. Rennick D. J. Clin. Invest. 1995; 96: 2339-2347Crossref PubMed Scopus (483) Google Scholar) and inflammation-induced mortality (21van der Poll T. Marchant A. Buurman W.A. Berman L. Keogh C.V. Lazarus D.D. Nguyen L. Goldman M. Moldawer L.L. Lowry S.F. J. Immunol. 1995; 155: 5397-5401PubMed Google Scholar), which is reversed by restoration of IL-10 levels (22Gérard C. Bruyns C. Marchant A. Abramowicz D. Vandenabeele P. Delvaux A. Fiers W. Goldman M. Velu T. J. Exp. Med. 1993; 177: 547-550Crossref PubMed Scopus (722) Google Scholar, 23Howard M. Muchamuel T. Andrade S. Menon S. J. Exp. Med. 1993; 177: 1205-1208Crossref PubMed Scopus (720) Google Scholar). Inflammation also induces HO-1, which metabolizes heme and releases CO, iron, and biliverdin (24Maines M.D. Antioxid. Redox Signal. 2005; 7: 1761-1766Crossref PubMed Scopus (130) Google Scholar), and promotes cell protection (25Otterbein L.E. Soares M.P. Yamashita K. Bach F.H. Trends Immunol. 2003; 24: 449-455Abstract Full Text Full Text PDF PubMed Scopus (1020) Google Scholar, 26Yao P. Hao L. Nussler N. Lehmann A. Song F. Zhao J. Neuhaus P. Liu L. Nussler A. Am. J. Physiol. Gastrointest. Liver Physiol. 2009; 296: G1318-G1323Crossref PubMed Scopus (53) Google Scholar, 27Chhikara M. Wang S. Kern S.J. Ferreyra G.A. Barb J.J. Munson P.J. Danner R.L. PLoS One. 2009; 4: e8139Crossref PubMed Scopus (48) Google Scholar). HO-1 is involved in sepsis in macrophage IL-10 induction and suppression of TNF-α and nitric oxide synthase-2 (16Lee T.S. Chau L.Y. Nat. Med. 2002; 8: 240-246Crossref PubMed Scopus (914) Google Scholar, 28Drechsler Y. Dolganiuc A. Norkina O. Romics L. Li W. Kodys K. Bach F.H. Mandrekar P. Szabo G. J. Immunol. 2006; 177: 2592-2600Crossref PubMed Scopus (105) Google Scholar) but also mediates the anti-inflammatory effects of adiponectin in Kupffer cells (29Mandal P. Park P.H. McMullen M.R. Pratt B.T. Nagy L.E. Hepatology. 2010; 51: 1420-1429Crossref PubMed Scopus (118) Google Scholar) and activates mitochondrial biogenesis (30Piantadosi C.A. Carraway M.S. Babiker A. Suliman H.B. Circ. Res. 2008; 103: 1232-1240Crossref PubMed Scopus (443) Google Scholar). Mitochondrial damage without ischemia or hypoxia is experimentally and clinically well recognized in sepsis (31Kantrow S.P. Taylor D.E. Carraway M.S. Piantadosi C.A. Arch Biochem. Biophys. 1997; 345: 278-288Crossref PubMed Scopus (143) Google Scholar, 32Brealey D. Brand M. Hargreaves I. Heales S. Land J. Smolenski R. Davies N.A. Cooper C.E. Singer M. Lancet. 2002; 360: 219-223Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar, 33Crouser E.D. Julian M.W. Huff J.E. Struck J. Cook C.H. Crit. Care Med. 2006; 34: 2439-2446Crossref PubMed Scopus (91) Google Scholar, 34Singer M. Crit. Care Med. 2007; 35: S441-S448Crossref PubMed Scopus (180) Google Scholar). During the resolution phase, energy homeostasis is restored by mitochondrial biogenesis (35Suliman H.B. Carraway M.S. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2003; 167: 570-579Crossref PubMed Scopus (125) Google Scholar, 36Suliman H.B. Carraway M.S. Welty-Wolf K.E. Whorton A.R. Piantadosi C.A. J. Biol. Chem. 2003; 278: 41510-41518Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 37Suliman H.B. Welty-Wolf K.E. Carraway M.S. Schwartz D.A. Hollingsworth J.W. Piantadosi C.A. Faseb J. 2005; 19: 1531-1533Crossref PubMed Scopus (91) Google Scholar, 38Haden D.W. Suliman H.B. Carraway M.S. Welty-Wolf K.E. Ali A.S. Shitara H. Yonekawa H. Piantadosi C.A. Am. J. Respir. Crit. Care Med. 2007; 176: 768-777Crossref PubMed Scopus (144) Google Scholar, 39Suliman H.B. Welty-Wolf K.E. Carraway M. Tatro L. Piantadosi C.A. Cardiovasc. Res. 2004; 64: 279-288Crossref PubMed Scopus (215) Google Scholar) activated by innate immunity (37Suliman H.B. Welty-Wolf K.E. Carraway M.S. Schwartz D.A. Hollingsworth J.W. Piantadosi C.A. Faseb J. 2005; 19: 1531-1533Crossref PubMed Scopus (91) Google Scholar) as well as by electrophiles or oxidants that activate the basic leucine zipper transcription factor, Nfe2l2 (Nrf2) (40Kensler T.W. Wakabayashi N. Biswal S. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 89-116Crossref PubMed Scopus (2813) Google Scholar). Electrophiles free Nfe2l2 from its Keap1 docking protein, allowing it to enter the nucleus and induce xenobiotic and antioxidant genes (40Kensler T.W. Wakabayashi N. Biswal S. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 89-116Crossref PubMed Scopus (2813) Google Scholar), including Hmox1 (41Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar). Endogenous CO also stimulates mitochondrial H2O2 production (42Suliman H.B. Carraway M.S. Ali A.S. Reynolds C.M. Welty-Wolf K.E. Piantadosi C.A. J. Clin. Invest. 2007; 117: 3730-3741PubMed Google Scholar), which acts as a retrograde signal for mitochondrial biogenesis in part through Nfe2l2 nuclear accumulation (30Piantadosi C.A. Carraway M.S. Babiker A. Suliman H.B. Circ. Res. 2008; 103: 1232-1240Crossref PubMed Scopus (443) Google Scholar). These findings suggest the hypothesis that HO-1/CO, acting through Nfe2l2, links anti-inflammatory gene expression to the transcriptional program for mitochondrial biogenesis. To test this hypothesis, we evaluated HO-1 and Nfe2l2 regulation and causal activation of mitochondrial biogenesis and anti-inflammation in cells challenged with LPS and in mice challenged with Escherichia coli sepsis. We also specifically investigated IL-10 and sIL-1Ra gene regulation by Nfe2l2 and other transcription factors of mitochondrial biogenesis, and in mice in sepsis, we also measured hepatic anti-inflammatory Socs3 and bcl-xL genes to check for their association with the mitochondrial biogenesis program. Evidence of an expanded network would signify the coupling of mitochondrial biogenesis to immune counter-regulation and implicate the genetic response to mitochondrial damage/repair as a factor in the apparent immune paralysis encountered in sepsis. Primary antibodies were from Santa Cruz Biotechnology unless otherwise specified; secondary antibodies, including antibodies for fluorescence microscopy, were from Invitrogen. siRNA oligonucleotides were from Ambion. Mouse recombinant TNF-α, IL-10, E. coli LPS, and dichloromethane (DCM) were from Sigma. Human HepG2 (hepatocellular carcinoma) cells were purchased from ATCC (Manassas, VA) and cultured in 5% CO2 in RMPI 1640 (Hyclone) containing 10% FCS, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Studies were conducted in murine J774.1 macrophages (ATCC) at 85% confluence in RPMI 1640 supplemented with FCS. Both cell lines were exposed to LPS at 15 ng/ml and exposed to CO using the CO-producing molecule dichloromethane (DCM/CO; 50–100 μm), which generates CO through the cytochrome P450 system. Cells were transfected with scrambled (negative control) or targeted siRNA using FuGENE HD (Roche Applied Science) to achieve efficiencies of >70%. The studies were approved by the Duke Institutional Animal Care and Use Committee. WT male C57BL/6 (The Jackson Laboratory) and Nfe2l2−/− mice (RIKEN) on a C57BL/6 background were used at 12–16 weeks of age. They were inoculated with 107 or 108 CFU live E. coli (serotype 086a:K61, ATCC, Rockville, MD) by peritoneal fibrin clot implantation followed by 1.5 ml of sterile 0.9% NaCl. This model activates hepatic mitochondrial biogenesis (37Suliman H.B. Welty-Wolf K.E. Carraway M.S. Schwartz D.A. Hollingsworth J.W. Piantadosi C.A. Faseb J. 2005; 19: 1531-1533Crossref PubMed Scopus (91) Google Scholar, 43Suliman H.B. Babiker A. Withers C.M. Sweeney T.E. Carraway M.S. Tatro L.G. Bartz R.R. Welty-Wolf K.E. Piantadosi C.A. Free Radic. Biol. Med. 2010; 48: 736-746Crossref PubMed Scopus (21) Google Scholar). For CO exposures, mice breathed 250 or 500 ppm CO in air for 1 h (carboxyhemoglobin levels 10–20%) to induce mitochondrial biogenesis (42Suliman H.B. Carraway M.S. Ali A.S. Reynolds C.M. Welty-Wolf K.E. Piantadosi C.A. J. Clin. Invest. 2007; 117: 3730-3741PubMed Google Scholar). At the appropriate times, mice were killed by isofluorane, and the livers were harvested and frozen at −80 °C. Fresh livers were flushed with 0.9% cold NaCl, perfusion-fixed with 10% formalin, and divided into lobes. After 24 h, the livers were transferred to 70% ethanol and stored at 4 °C. The lobes were block-cut and embedded in paraffin, and 4-μm sections were randomly cut and stained with H&E and photographed at 200×. Mouse and human IL10 promoter loci were aligned, and DNA sequence homology was computed with the web-based Regulatory Visualization Tools for Alignment (rVISTA; www.gsd.lbl.gov/vista) (44Loots G.G. Ovcharenko I. Nucleic Acids Res. 2004; 32: W217-W221Crossref PubMed Scopus (349) Google Scholar, 45Loots G.G. Ovcharenko I. Pachter L. Dubchak I. Rubin E.M. Genome Res. 2002; 12: 832-839Crossref PubMed Scopus (366) Google Scholar). Promoter analysis was performed with consensus transcription factor binding sequences located with DNASIS (Hitachi Software; Alameda, CA) and confirmed with MatInspector (Genomatix Software; München, Germany). Predicted binding sites for Nfe2l2/Nrf2 (accession no. NM_006164 (human); accession no. NM_010902 (mouse)), MEF2A (myocyte enhancer factor 2A, accession no. NM_001130926 (human); accession no. NM_001033713 (mouse)), NRF-1 (accession no. NM_005011 (human) and accession no. NM_001164226 (mouse)), and NRF-2α/Gabpa (accession no. NM_001197297 (human); accession no. NM_008065 (mouse)) of at least 85% homology were identified in human and mouse promoters. Nuclear liver and cell extracts were prepared as described (36Suliman H.B. Carraway M.S. Welty-Wolf K.E. Whorton A.R. Piantadosi C.A. J. Biol. Chem. 2003; 278: 41510-41518Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 37Suliman H.B. Welty-Wolf K.E. Carraway M.S. Schwartz D.A. Hollingsworth J.W. Piantadosi C.A. Faseb J. 2005; 19: 1531-1533Crossref PubMed Scopus (91) Google Scholar). Transcriptional activation of Nfe2l2 and MEF2A was measured using the TRANS-AM kit (Active Motif, Carlsbad, CA). ChIP assays were performed with ChIP-IT and Re-ChIP-IT kits (Active Motif) following the manufacturer's protocol (42Suliman H.B. Carraway M.S. Ali A.S. Reynolds C.M. Welty-Wolf K.E. Piantadosi C.A. J. Clin. Invest. 2007; 117: 3730-3741PubMed Google Scholar). For liver, ∼200 mg was diced on ice in PBS and fixed in 1% (v/v) formaldehyde at room temperature to cross-link protein to DNA. Reactions were stopped in 0.125 m glycine, the samples were centrifuged twice, and the final pellet was suspended in ChIP buffer. Genomic DNA was sheared by sonication, and lysates were tumbled overnight at 4 °C with salmon sperm DNA/protein A-agarose and anti-RNA polymerase II (Re-ChIP-IT), anti-MEF2, anti-Nfe2l2, and anti-NRF-1 (Cell Signaling) or normal rabbit IgG. Complexes were precipitated, washed, and eluted with 1% SDS and 100 mm NaHCO3. After reversal of protein/DNA cross-linking, DNA was purified and amplified across the IL10 and IL1Ra promoter regions of interest. ChIP samples were normalized to input chromatin (ΔCt) and enrichment defined as change in Ct in treated versus untreated samples (ΔCt), relative to IgG controls. Liver homogenate, cell lysate, and nuclear extract proteins were separated by SDS-PAGE, and Western blots were performed. After the transfer, primary and secondary antibodies were applied, and the signals were developed using ECL. Blots were quantified in the mid-dynamic range, and the protein density was expressed relative to stable reference proteins. For immunochemistry, cells were grown in one-well chamber slides to ∼70% confluence. After reagent additions, the cells were washed in PBS, fixed in 2% paraformaldehyde, and washed with 1% Triton X-100 for 15 min at room temperature. Cells were labeled with primary antibodies to HO-1 (1:1000) and MEF2 (catalog no. sc-55500; 1:800). Fluorescence microscopy was performed on a Nikon H550S microscope (46Suliman H.B. Sweeney T.E. Withers C.M. Piantadosi C.A. J. Cell Sci. 2010; 123: 2565-2575Crossref PubMed Scopus (60) Google Scholar). We performed qRT-PCR on an ABI PRISM 7000 system with TaqMan gene expression and premix assays (Applied Biosystems). 18 S rRNA served as an endogenous control. Quantification of gene expression was determined using the comparative threshold cycle CT and RQ method. The mt DNA copy number was determined by quantitative PCR using SYBR Green and the ABI system (42Suliman H.B. Carraway M.S. Ali A.S. Reynolds C.M. Welty-Wolf K.E. Piantadosi C.A. J. Clin. Invest. 2007; 117: 3730-3741PubMed Google Scholar, 47Suliman H.B. Carraway M.S. Tatro L.G. Piantadosi C.A. J. Cell Sci. 2007; 120: 299-308Crossref PubMed Scopus (178) Google Scholar). Grouped data are expressed as means ± S.E. for replicates of four to six. Significance was tested by an unpaired t test or with two-way analysis of variance using commercial software unless indicated otherwise. Statistical significance required p < 0.05. HO-1 induction does not invariably stimulate IL10, so we confirmed that its CO product increases IL-10 mRNA levels in mouse liver, HepG2 cells, and J774.1 macrophages. In C57/BL/6 mice, 1 h of CO breathing (250 or 500 ppm) increased hepatic IL-10 mRNA levels by ∼60% at 24 h (Fig. 1A). In HepG2 cells, DCM/CO at low μm levels doubled IL-10 mRNA content by 24 h (Fig. 1B). Comparably, LPS stimulated IL-10 production, and LPS + DCM/CO produced an additive effect. In J774.1 cells, IL-10 mRNA levels followed the same patterns, including after LPS + DCM/CO (data not shown). Dose- and time-dependent increases in hepatic IL-10 and HO-1 protein levels after CO administration were documented by Western blot (Fig. 1, C and D). In an E. coli peritonitis model in mice, hepatic HO-1 protein levels were also significantly up-regulated at 24 h (Fig. 1E). IL-10 was constitutive and strongly up-regulated in sepsis but well after TNF-α production. In HepG2 cells, DCM/CO or LPS challenge up-regulated HO-1 and IL-10 protein expression by 1–6 h. Peak responses for both occurred 24 h after these exposures (Fig. 2A). LPS + DCM/CO significantly enhanced HO-1 and IL-10 induction compared with either alone, whereas IL-10 up-regulation was blocked by Hmox1 RNA silencing (Fig. 2A). HO-1 was thus required for increased IL-10 expression by DCM/CO and LPS. IL-10 (10 ng/ml) also induced cellular HO-1 expression within 6 h (Fig. 2A). To show that LPS and CO up-regulate the transcriptional program of mitochondrial biogenesis in HepG2 cells, we measured protein for NRF-1 and the mitochondrial transcription factor A (Fig. 2B). Both proteins responded to both stimuli in a pattern similar to that of IL-10. IL-10 administration in HepG2 cells inhibited the LPS induction of TNF-α and nitric oxide synthase-2, but IL-10 suppression of TNF-α and nitric oxide synthase-2 was reduced markedly by Hmox1 RNA silencing (Fig. 2C). The same result was obtained in J774.1 cells (Fig. 2D), indicating that HO-1/CO is required for IL-10 to inhibit the LPS response. In macrophages, IL-10 is associated with the anti-inflammatory molecule sIL-1Ra (15Lai C.F. Ripperger J. Morella K.K. Jurlander J. Hawley T.S. Carson W.E. Kordula T. Caligiuri M.A. Hawley R.G. Fey G.H. Baumann H. J. Biol. Chem. 1996; 271: 13968-13975Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), which could also connect counter-inflammation to mitochondrial biogenesis. We assessed hepatic sIL-1Ra mRNA levels in mice 24 h after CO exposure (250 or 500 ppm for 1 h) and demonstrated a pattern of induction similar to that of IL-10 (Fig. 3A). In the liver (90), sIL-1Ra protein levels after CO exposure corroborated the mRNA data; the protein increased significantly compared with controls (Fig. 3A). In HepG2 and in J774.1 cells, sIL-1Ra protein increased in response to LPS and to DCM/CO with a greater effect of LPS in J774.1 cells and DCM/CO in HepG2 cells (Fig. 3, B and C). The use of both agents had additive effects in both cell lines. Hmox1 or Nfe2l2 RNA silencing diminished the sIL-1Ra responses to the stimuli equally (Fig. 3, B and C). These results imply that CO and LPS elicit anti-inflammatory cytokine expression through multiple and overlapping but not identical transcriptional elements. A common induction pattern for IL-10 transcription and mitochondrial biogenesis by CO and LPS raised the prospect of a common regulatory pathway, for instance through Nfe2l2 (Nrf2) signaling (42Suliman H.B. Carraway M.S. Ali A.S. Reynolds C.M. Welty-Wolf K.E. Piantadosi C.A. J. Clin. Invest. 2007; 117: 3730-3741PubMed Google Scholar), which increases both HO-1 (48Piantadosi C.A. Carraway M.S. Suliman H.B. Free Radic. Biol. Med. 2006; 40: 1332-1339Crossref PubMed Scopus (59) Google Scholar) and IL-10 expression. We used multiple approaches to examine the role of Nfe2l2 as well as other transcriptional regulators of mitochondrial biogenesis in IL10 and IL1Ra gene regulation. A computer analysis of the human and mouse promoters using MatInspector and the TRANSFAC database (Genomatix Software) detected putative binding sites for Nfe2l2 and at least three other relevant transcription factors: NRF-1, nuclear respiratory factor-2 (NRF-2), and MEF2. We aligned the IL10 promoter sequences with DNA Block Aligner (DNAsis software) to identify species-conserved regions that are most likely to represent functional elements (supplemental Fig. S1A). Putative binding sites for Nfe2l2, NRF-2 (Gabpa), and MEF2 were identified in the first 500 bp after the TATA box with >90% canonical consensus and >85% conservation scores. Using the same stringency, we identified putative binding sites on the IL1Ra promoter for NRF-1 and MEF2 (supplemental Fig. S1B). MEF2 has been associated with IL-10 expression, but HO-1/CO is not known to activate MEF2 genes. In the liver, MEF2A and -2D regulate cell activity and growth, e.g. in stellate cells (49Wang X. Tang X. Gong X. Albanis E. Friedman S.L. Mao Z. Gastroenterology. 2004; 127: 1174-1188Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and MEF2A is a target of NRF-1, which is activated by HO-1/CO (50Czubryt M.P. McAnally J. Fishman G.I. Olson E.N. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1711-1716Crossref PubMed Scopus (344) Google Scholar). Although HO-1/CO activates Nfe2l2 (51Lee B.S. Heo J. Kim Y.M. Shim S.M. Pae H.O. Kim Y.M. Chung H.T. Biochem. Biophys. Res. Commun. 2006; 343: 965-972Crossref PubMed Scopus (86) Google Scholar), nothing addresses whether IL-10 expression depends on the other transcription factors. Preliminary studies indicated that hepatic nuclear Nfe2l2, MEF2, and NRF-2α (Gabpa) protein increase after CO breathing in mice; 1 h of CO increased all three nuclear proteins by 6 h (Fig. 4A). The peak effect was at 250 ppm, which we used subsequently. In HepG2 cells, DCM/CO also increased nuclear MEF2, Nfe2l2, and NRF-2 levels and was additive to the LPS effect (Fig. 4B). Hmox1 RNA silencing decreased nuclear Nfe2l2 and NRF-2 levels but only modestly decreased nuclear MEF2 levels (Fig. 4B). Nfe2l2 RNA silencing, however, sharply decreased nuclear MEF2 and slightly decreased NRF-2 levels (Fig. 4B). In J774.1 macrophages, Hmox1 silencing had a smaller effect than in HepG2 cells, whereas Nfe2l2 silencing had a similar effect in both cell lines (Fig. 4C). Replicate studies confirmed that Nfe2l2 silencing produced more attenuation of nuclear MEF and NRF-2 accumulation than did Hmox1 silencing. Transcription factor binding activities were measured in nuclear extracts of HepG2 cells for the Nfe2l2 and MEF2 sequences on the human IL10 promoter. These assays showed increased binding activity for both transcription factors after DCM/CO exposure (Fig. 5, A and B). The LPS-stimulated binding activity for both proteins was comparatively modest; by implication, LPS induction of IL-10 involves not only HO-1, but also other elements, e.g. NF-κΒ. Nfe2l2 and MEF2A nuclear translocation depends on phosphorylation, which is regulated, for instance, by Akt/PKB and p38 MAPK. NRF-1 translocation is also regulated by Akt/PKB. We evaluated the influence of p38 and Akt on the CO response in HepG2 cells with inhibition of p38 (SB203580; SB20) or PI-3K/Akt (LY294002; LY29). Activation of Nfe2l2 by DCM/CO was partly blocked by p38 inhibition, but fully abrogated by Akt inhibition. The effect of DCM/CO on MEF2 activation was blocked by inhibiting either kinase (Fig. 5, A and B). To determine how HO-1/CO promotes IL-10 transcription, we generated cross-linked nuclear lysates from HepG2 cells after a 60-min exposure to DCM/CO, LPS, or both. Sequential ChIP assay (ChIP-ReChIP) was performed on genomic DNA with anti-RNA polymerase II or with control IgG (data not shown) and probed for interactions with Nfe2l2, MEF2, and NRF-2 (Gabpa) at the human IL10 promoter. In control nuclei, almost no polymerase II signal was detected at the IL10 promoter, but the signal was increased by DCM/CO and LPS (Fig. 5C). The largest increases occurred after LPS + DCM/CO consistent with the changes in IL-10 mRNA expression. Chromatin IP assays were performed to evaluate IL10 promoter binding to MEF2, Nfe2l2, and NRF-2 using validated antibodies and primer sets for these sites. We also checked the IL1Ra promoter for NRF-1 and MEF2 binding at our predicted binding sites. IL10 promoter DNA did not co-precipitate with control IgG (Fig. 5D). ChIP assays for Nfe2l2 detected no transcription factor binding at the IL10 promoter in control or LPS-stimulated cells, but Nfe2l2 binding was detectable after DCM/CO (Fig. 5D). MEF2 and NRF-2 occupancy was minimal in control HepG2 nuclei, after DCM/CO and LPS, both factors were bound, and DCM +LPS produced stronger binding (Fig. 5D). IL1Ra promoter DNA did not precipitate with control IgG, and nonstimulated nuclei showed no MEF2 or NRF-1 binding, but both transcription factors bou" @default.
• W2045821670 created "2016-06-24" @default.
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• W2045821670 date "2011-05-01" @default.
• W2045821670 modified "2023-09-26" @default.
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