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• W2027424250 abstract "Dendritic cells (DCs) are critical for the initiation of immune responses including activation of CD8 T cells. Intracellular reactive oxygen species (ROS) levels influence DC maturation and function. Intracellular heme, a product of catabolism of heme-containing metalloproteins, is a key inducer of ROS. Intracellular heme levels are regulated by heme oxygenase-1 (HO-1), which catalyzes the degradation of heme. Heme oxygenase-1 has been implicated in regulating DC maturation; however, its role in other DC functions is unclear. Furthermore, the signaling pathways modulated by HO-1 in DCs are unknown. In this study, we demonstrate that inhibition of HO-1 activity in murine bone marrow-derived immature DCs (iDCs) resulted in DCs with raised intracellular ROS levels, a mature phenotype, impaired phagocytic and endocytic function, and increased capacity to stimulate antigen-specific CD8 T cells. Interestingly, our results reveal that the increased ROS levels following HO-1 inhibition did not underlie the changes in phenotype and functions observed in these iDCs. Importantly, we show that the p38 mitogen-activated protein kinase (p38 MAPK), cAMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1) pathway is involved in the mediation of the phenotypic and functional changes arising from HO-1 inhibition. Furthermore, up-regulation of HO-1 activity rendered iDCs refractory to lipopolysaccharide-induced activation of p38 MAPK-CREB/ATF1 pathway and DC maturation. Finally, we demonstrate that treatment of iDC with the HO-1 substrate, heme, recapitulates the effects that result from HO-1 inhibition. Based on these results, we conclude that HO-1 regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signaling axis. Dendritic cells (DCs) are critical for the initiation of immune responses including activation of CD8 T cells. Intracellular reactive oxygen species (ROS) levels influence DC maturation and function. Intracellular heme, a product of catabolism of heme-containing metalloproteins, is a key inducer of ROS. Intracellular heme levels are regulated by heme oxygenase-1 (HO-1), which catalyzes the degradation of heme. Heme oxygenase-1 has been implicated in regulating DC maturation; however, its role in other DC functions is unclear. Furthermore, the signaling pathways modulated by HO-1 in DCs are unknown. In this study, we demonstrate that inhibition of HO-1 activity in murine bone marrow-derived immature DCs (iDCs) resulted in DCs with raised intracellular ROS levels, a mature phenotype, impaired phagocytic and endocytic function, and increased capacity to stimulate antigen-specific CD8 T cells. Interestingly, our results reveal that the increased ROS levels following HO-1 inhibition did not underlie the changes in phenotype and functions observed in these iDCs. Importantly, we show that the p38 mitogen-activated protein kinase (p38 MAPK), cAMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1) pathway is involved in the mediation of the phenotypic and functional changes arising from HO-1 inhibition. Furthermore, up-regulation of HO-1 activity rendered iDCs refractory to lipopolysaccharide-induced activation of p38 MAPK-CREB/ATF1 pathway and DC maturation. Finally, we demonstrate that treatment of iDC with the HO-1 substrate, heme, recapitulates the effects that result from HO-1 inhibition. Based on these results, we conclude that HO-1 regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signaling axis. Dendritic cells (DCs) 4The abbreviations used are:DCdendritic celliDCimmature dendritic cellHO-1heme oxygenase-1Nrf2nuclear factor-erythroid 2 (NF-E2) p45-related factor-2ROSreactive oxygen speciesNP68nucleoprotein 68NACN-acetylcysteineCREBcAMP-response element-binding proteinATF1activating transcription factor 1BVbiliverdinCFSEcarboxyfluorescein succinimidyl esterTLRToll-like receptorTCRT cell receptorCoPPcobalt(III) protoporphyrin IX chlorideSnPP-IXtin protoporphyrin IX dichlorideANOVAanalysis of variance. are potent antigen-presenting cells that play a major role in the initiation and regulation of the immune response (1Steinman R.M. Some interfaces of dendritic cell biology.APMIS. 2003; 111: 675-697Crossref PubMed Scopus (243) Google Scholar). Immature DCs (iDCs) are efficient at capturing extracellular antigens through several endocytic and phagocytic mechanisms (2Sallusto F. Cella M. Danieli C. Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.J. Exp. Med. 1995; 182: 389-400Crossref PubMed Scopus (2178) Google Scholar, 3Fanger N.A. Wardwell K. Shen L. Tedder T.F. Guyre P.M. Type I (CD64) and type II (CD32) Fc γ receptor-mediated phagocytosis by human blood dendritic cells.J. Immunol. 1996; 157: 541-548PubMed Google Scholar). However, iDCs are poorly immunogenic as they express low levels of MHC class II molecules and co-stimulatory receptors including CD86 and CD40 at the cell surface. Dendritic cell maturation is triggered through engagement of pattern recognition receptors such as the Toll-like receptors (TLRs) by pathogen-associated molecular patterns, e.g. bacterial lipopolysaccharide (LPS). Maturation is associated with morphological, phenotypic, and functional changes including up-regulation of cell surface MHC II, co-stimulatory molecules, loss of phagocytic capacity, and enhanced antigen-presenting capabilities, which are required for inducing competent T cell activation (4Chow A. Toomre D. Garrett W. Mellman I. Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane.Nature. 2002; 418: 988-994Crossref PubMed Scopus (353) Google Scholar, 5Casals C. Barrachina M. Serra M. Lloberas J. Celada A. Lipopolysaccharide up-regulates MHC class II expression on dendritic cells through an AP-1 enhancer without affecting the levels of CIITA.J. Immunol. 2007; 178: 6307-6315Crossref PubMed Scopus (33) Google Scholar, 6Angelini G. Gardella S. Ardy M. Ciriolo M.R. Filomeni G. Di Trapani G. Clarke F. Sitia R. Rubartelli A. Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1491-1496Crossref PubMed Scopus (303) Google Scholar). A key intracellular signaling pathway that governs DC maturation is the p38 MAPK pathway (7Dong C. Davis R.J. Flavell R.A. MAP kinases in the immune response.Annu. Rev. Immunol. 2002; 20: 55-72Crossref PubMed Scopus (1375) Google Scholar). The p38 MAPK signaling pathway positively regulates DC phenotype and cytokine production by driving the expression of multiple genes involved in DC maturation (8Nakahara T. Moroi Y. Uchi H. Furue M. Differential role of MAPK signaling in human dendritic cell maturation and Th1/Th2 engagement.J. Dermatol. Sci. 2006; 42: 1-11Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The major transcription factors that lie downstream of the p38 MAPK pathway are the cAMP-response element-binding protein (CREB) and activation transcription factor 1 (ATF1) (9Mellett M. Atzei P. Jackson R. O'Neill L.A. Moynagh P.N. Mal mediates TLR-induced activation of CREB and expression of IL-10.J. Immunol. 2011; 186: 4925-4935Crossref PubMed Scopus (55) Google Scholar, 10Wiggin G.R. Soloaga A. Foster J.M. Murray-Tait V. Cohen P. Arthur J.S. MSK1 and MSK2 are required for the mitogen- and stress-induced phosphorylation of CREB and ATF1 in fibroblasts.Mol. Cell. Biol. 2002; 22: 2871-2881Crossref PubMed Scopus (361) Google Scholar). Dendritic cell maturation and function are influenced by changes in cellular levels of reactive oxygen species (ROS) (11Rutault K. Alderman C. Chain B.M. Katz D.R. Reactive oxygen species activate human peripheral blood dendritic cells.Free Radic. Biol. Med. 1999; 26: 232-238Crossref PubMed Scopus (165) Google Scholar). Alterations in intracellular ROS can also impact on the activity of the p38 MAPK pathway (12Matos T.J. Duarte C.B. Gonçalo M. Lopes M.C. Role of oxidative stress in ERK and p38 MAPK activation induced by the chemical sensitizer DNFB in a fetal skin dendritic cell line.Immunol. Cell Biol. 2005; 83: 607-614Crossref PubMed Scopus (50) Google Scholar). Heme is a product of catabolism of heme-containing metalloproteins that has the potential to induce the generation of ROS (13Jeney V. Balla J. Yachie A. Varga Z. Vercellotti G.M. Eaton J.W. Balla G. Pro-oxidant and cytotoxic effects of circulating heme.Blood. 2002; 100: 879-887Crossref PubMed Scopus (503) Google Scholar). Accumulation of intracellular heme is prevented by the enzymatic activity of heme oxygenases (HO), in particular, the HO-1 isoform (14Abraham N.G. Kappas A. Pharmacological and clinical aspects of heme oxygenase.Pharmacol. Rev. 2008; 60: 79-127Crossref PubMed Scopus (919) Google Scholar). Heme oxygenase-1 catalyzes the degradation of heme into biliverdin (BV), free iron, and carbon monoxide (CO) (15Siow R.C. Sato H. Mann G.E. Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide?.Cardiovasc. Res. 1999; 41: 385-394Crossref PubMed Scopus (240) Google Scholar). We and others have identified a potential role for HO-1 in regulating DC maturation (16Al-Huseini L.M. Aw Yeang H.X. Sethu S. Alhumeed N. Hamdam J.M. Tingle Y. Djouhri L. Kitteringham N. Park B.K. Goldring C.E. Sathish J.G. Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38 MAPK-cAMP-responsive element binding protein/activating transcription factor 1 signaling.J. Biol. Chem. 2013; 288: 22281-22288Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Figueiredo R.T. Fernandez P.L. Mourao-Sa D.S. Porto B.N. Dutra F.F. Alves L.S. Oliveira M.F. Oliveira P.L. Graça-Souza A.V. Bozza M.T. Characterization of heme as activator of Toll-like receptor 4.J. Biol. Chem. 2007; 282: 20221-20229Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). However, it is unclear whether HO-1 utilizes the p38 MAPK pathway to mediate regulation of DC maturation. Furthermore, it is not known whether the primary effect of HO-1 in regulating DC maturation is by preventing elevation of intracellular ROS levels. Finally, it is unknown whether the HO-1 substrate, heme, is directly involved in regulating DC maturation and function through activation of the p38 MAPK-CREB/ATF1 pathway. In this study, we show that inhibition of HO-1 activity in mouse bone marrow-derived iDCs results in a mature phenotype associated with an impaired phagocytic and endocytic capacity and an enhanced ability to stimulate antigen-specific T cell proliferation. We also demonstrate that although HO-1 inhibition was accompanied by elevated ROS, the increased ROS was not required for inducing DC maturation. Importantly, we show that the DC maturation and functional changes brought about by HO-1 inhibition are mediated through the p38 MAPK-CREB/ATF1 pathway. Finally, we provide evidence for the induction of p38 MAPK-CREB/ATF1 activation and DC maturation by the HO-1 substrate, heme. We conclude that HO-1 regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signaling axis. dendritic cell immature dendritic cell heme oxygenase-1 nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 reactive oxygen species nucleoprotein 68 N-acetylcysteine cAMP-response element-binding protein activating transcription factor 1 biliverdin carboxyfluorescein succinimidyl ester Toll-like receptor T cell receptor cobalt(III) protoporphyrin IX chloride tin protoporphyrin IX dichloride analysis of variance. All reagents were from Sigma-Aldrich unless otherwise stated. FCS, DextranFITC (40,000 Mr), and carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen); tin protoporphyrin IX dichloride (SnPP-IX) and cobalt(III) protoporphyrin IX chloride (CoPP) (Tocris Bioscience, Bristol, UK); and SB203580 (Cell Signaling Technology, Danvers, MA) were also purchased for the study. Mice transgenic for the H-2Db-restricted T cell receptor (TCR)-αβ transgene, F5, were a kind gift from Dr. James Matthews (Cardiff, Wales, UK). Mice were maintained at the Biomedical Services Unit, University of Liverpool. Protocols described herein were undertaken in accordance with criteria outlined in the license granted under the Animals (Scientific Procedures) Act 1986 (PPL 40/3379). Mouse bone marrow-derived iDCs were generated according to published protocol (18Lutz M.B. Kukutsch N. Ogilvie A.L. Rössner S. Koch F. Romani N. Schuler G. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.J. Immunol. Methods. 1999; 223: 77-92Crossref PubMed Scopus (2502) Google Scholar). DCs were stained with fluorescent αCD11cTC (Invitrogen) and αCD86FITC or αMHC IIPE (BD Biosciences) antibodies for 30 min on ice, washed, acquired on a BD FACSCanto II flow cytometer (BD Biosciences), and analyzed using Cyflogic software (version 1.2.1 CyFlo Ltd.). Apoptotic thymocytes were generated from mouse thymi treated with 1 μm dexamethasone for 18 h. Apoptotic thymocytes were labeled with the intracellular fluorescent dye, CFSE, and co-cultured with plate-adherent DCs for 2 h at 37 or 4 °C. Cells were stained with αCD11cTC prior to analysis by flow cytometry. Jurkat T cells were fluorescently labeled with CFSE, and necrosis was induced by snap-freezing in liquid nitrogen. Necrotic cells were co-cultured with DCs as above and analyzed by flow cytometry. DCs were incubated with 0.5 μg/ml DextranFITC (40,000 Mr) for different time points at 37 °C. Cells were stained for surface expression of CD11c as described above and analyzed by flow cytometry. Untreated or treated iDCs were stained using the fluorescent ROS indicator, dihydroethidium, according to Ref. 19Burnaugh L. Sabeur K. Ball B.A. Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium.Theriogenology. 2007; 67: 580-589Crossref PubMed Scopus (78) Google Scholar and analyzed by flow cytometry. F5 CD8 T cell proliferation was quantified as described previously (20Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1.J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar). Briefly, iDCs were pulsed with a concentration range of antigenic peptide (NP68), washed, and co-cultured with F5 CD8 T cells for 72 h. [3H]Thymidine was added for the last 16 h. Cells were harvested onto glass fiber filter mats and read on a scintillation counter (MicroBeta TriLux; PerkinElmer Life Sciences, Buckinghamshire, UK). DCs were lysed, and 5 μg of lysate protein was resolved by SDS-PAGE, transferred to PVDF membranes (Bio-Rad; Hertfordshire, UK), blocked, and probed for proteins of interest using the appropriate primary antibodies (phospho-p38 MAPK and phospho-CREB (Cell Signaling Technology, Danvers, MA) and α-tubulin (Santa Cruz Biotechnologies)) followed by horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and visualized using the ECL system (PerkinElmer Life Sciences). Raw data obtained were analyzed using unpaired t test, one-way ANOVA and the Mann-Whitney U test. p values < 0.05 were considered to be statistically significant. To investigate the role of HO-1 in DC maturation and function, we treated iDCs with the inhibitor of HO-1 activity, SnPP-IX. Inhibition of HO-1 by SnPP-IX in iDCs resulted in increased levels of MHC II and CD86 cell surface expression in comparison with untreated DCs (Fig. 1A, panel i, MHC II 58.2 ± 3.7% versus 18.5 ± 0.8%, p < 0.05; Fig. 1A, panel ii, CD86 64.3 ± 4.5% versus 12.0 ± 1.6%, p < 0.05). Furthermore, MHC II and CD86 levels on SnPP-IX-treated iDCs were comparable with LPS-treated iDCs (MHC II 61.9 ± 4.1% and CD86 67.4 ± 3.2%). Following maturation, DCs have reduced phagocytic and endocytic capabilities (21Rescigno M. Granucci F. Citterio S. Foti M. Ricciardi-Castagnoli P. Coordinated events during bacteria-induced DC maturation.Immunol. Today. 1999; 20: 200-203Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 22Banchereau J. Briere F. Caux C. Davoust J. Lebecque S. Liu Y.J. Pulendran B. Palucka K. Immunobiology of dendritic cells.Annu. Rev. Immunol. 2000; 18: 767-811Crossref PubMed Scopus (5617) Google Scholar). As HO-1 inhibition resulted in DC maturation, we examined the antigen acquisition capacity of SnPP-IX-treated iDCs. Our results revealed that SnPP-IX-treated iDCs had a reduced capacity to phagocytose both necrotic cells (Fig. 1B, panel i, 4.9 ± 0.7 versus 3.4 ± 0.6-fold increase over baseline, p < 0.05) and apoptotic cells (Fig. 1B, panel ii, 6.9 ± 1.7 versus 4.0 ± 0.5-fold increase over baseline, p < 0.05) when compared with their untreated control. Results also demonstrated that SnPP-IX-treated iDCs had a diminished capacity to endocytose dextran in comparison with their untreated controls (Fig. 1C, 46.0 ± 1.0% versus 75.0 ± 2.6% at 15 min, 65.7 ± 1.5% versus 84.0 ± 2.0% at 30 min, and 70.7 ± 2.1% versus 88.7 ± 1.2% at 60 min, p < 0.05). Low levels of co-stimulatory molecule expression in iDCs render them unable to stimulate a fully competent antigen-specific CD8 T cell response (23Bachmann M.F. Speiser D.E. Mak T.W. Ohashi P.S. Absence of co-stimulation and not the intensity of TCR signaling is critical for the induction of T cell unresponsiveness in vivo.Eur. J. Immunol. 1999; 29: 2156-2166Crossref PubMed Scopus (17) Google Scholar). However, up-regulation of these surface molecules in mature DCs enhances their ability to induce T cell activation (24Lanzavecchia A. Sallusto F. Regulation of T cell immunity by dendritic cells.Cell. 2001; 106: 263-266Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar). As SnPP-IX-treated iDCs exhibit a mature phenotype, we expected that this would be associated with an enhanced capacity to induce DC-mediated antigen-specific CD8 T cell activation. To test this, we utilized a TCR transgenic mouse model, F5, wherein the CD8 T cells exclusively express the F5 T cell receptor (F5 TCR) that specifically recognizes the MHC-I (H2-Db)-restricted antigenic peptide, NP68, when presented by DCs (25Mamalaki C. Norton T. Tanaka Y. Townsend A.R. Chandler P. Simpson E. Kioussis D. Thymic depletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 11342-11346Crossref PubMed Scopus (143) Google Scholar). Functional consequences of altered DC co-stimulatory receptor expression were assessed by the ability of NP68-bearing DCs to stimulate antigen-specific F5 CD8 T cell proliferation. We observed that SnPP-IX-treated DCs elicited enhanced DC-mediated antigen-specific F5 CD8 T cell proliferation in relation to the untreated control at all NP68 concentrations as shown in Fig. 1D (2.5-fold at 1 and 10 nm to 1.4-fold at 100 nm, p < 0.05). Taken together, these findings indicate that inhibition of HO-1 activity affects DC phenotypic maturation, antigen acquisition ability, and antigen-specific CD8 T cell stimulatory capacity. Dendritic cell maturation and function are influenced by intracellular ROS levels (26Kantengwa S. Jornot L. Devenoges C. Nicod L.P. Superoxide anions induce the maturation of human dendritic cells.Am. J. Respir. Crit. Care Med. 2003; 167: 431-437Crossref PubMed Scopus (103) Google Scholar). To test whether HO-1 activity is required for regulation of ROS levels in DCs, we treated iDCs with SnPP-IX and measured the ROS levels using the fluorescent redox-sensitive probe, dihydroethidium. Dihydroethidium reports on superoxide levels, and superoxide is a key ROS that has been shown to induce the maturation of DCs (26Kantengwa S. Jornot L. Devenoges C. Nicod L.P. Superoxide anions induce the maturation of human dendritic cells.Am. J. Respir. Crit. Care Med. 2003; 167: 431-437Crossref PubMed Scopus (103) Google Scholar). We found that HO-1 inhibition resulted in a significant increase in intracellular ROS levels in iDCs as shown in Fig. 2 (46.6 ± 8.7% versus 14.0 ± 0.7%, p < 0.05), an increase similar to that observed in the LPS-treated iDCs (47.6 ± 14.2%). Intracellular ROS can be lowered by ROS scavengers such as vitamins C and E (16Al-Huseini L.M. Aw Yeang H.X. Sethu S. Alhumeed N. Hamdam J.M. Tingle Y. Djouhri L. Kitteringham N. Park B.K. Goldring C.E. Sathish J.G. Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38 MAPK-cAMP-responsive element binding protein/activating transcription factor 1 signaling.J. Biol. Chem. 2013; 288: 22281-22288Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and here we observed that treatment with vitamins C and E resulted in significant reduction in ROS levels induced by SnPP-IX treatment in iDCs (Fig. 2, 18.8 ± 9.4% versus 46.6 ± 8.7%, p < 0.05). Similar effects were also observed in LPS- and vitamin-treated iDCs (Fig. 2). These findings implicate HO-1 activity in preventing elevation of intracellular ROS levels in iDCs. To test whether DC maturation induced by HO-1 inhibition was a result of increased intracellular ROS levels, we treated HO-1-inhibited iDCs with vitamins C and E and examined MHC II and CD86 expression. Our results revealed that there were no significant differences between SnPP-IX treatment alone or in combination with vitamins C and E in iDC expression of MHC II (Fig. 3A, panel i, 57.2 ± 9.0% versus 51.8 ± 5.6%, p > 0.05) and CD86 (Fig. 3A, panel ii, 61.6 ± 9.1% versus 54.2 ± 4.9%, p > 0.05). In addition, no significant differences in the expression of MHC II and CD86 was observed in iDCs treated either with LPS alone or with LPS and vitamins C and E (Fig. 3A, panel i, 64.2 ± 9.0% versus 49.9 ± 9.1%, p > 0.05 and Fig. 3A, panel ii, 64.4 ± 6.0% versus 58.3 ± 3.6%, p > 0.05, respectively). Similarly, there were no differences observed in iDC-mediated antigen-specific F5 CD8 T cell proliferation upon SnPP-IX treatment alone or in combination with vitamins C and E (Fig. 3B). These observations suggest that the altered iDC phenotype and function induced by HO-1 inhibition are not a result of elevated ROS in these iDCs. Activation of the p38 MAPK-CREB/ATF1 signaling pathway has been shown to be involved in DC maturation (27Arrighi J.F. Rebsamen M. Rousset F. Kindler V. Hauser C. A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-α, and contact sensitizers.J. Immunol. 2001; 166: 3837-3845Crossref PubMed Scopus (375) Google Scholar). Activation of this pathway is accompanied by an increase in the serine phosphorylation status of p38 MAPK (28Shanware N.P. Zhan L. Hutchinson J.A. Kim S.H. Williams L.M. Tibbetts R.S. Conserved and distinct modes of CREB/ATF transcription factor regulation by PP2A/B56γ and genotoxic stress.PLoS One. 2010; 5: e12173Crossref PubMed Scopus (18) Google Scholar, 29Ardeshna K.M. Pizzey A.R. Devereux S. Khwaja A. The PI3 kinase, p38 SAP kinase, and NF-κB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells.Blood. 2000; 96: 1039-1046Crossref PubMed Google Scholar). To investigate whether p38 MAPK is involved in the HO-1-mediated regulation of DC function, we first examined the phosphorylation status of p38 MAPK upon SnPP-IX treatment. As demonstrated in Fig. 4A, panel i, HO-1 inhibition resulted in a marked increase in p38 MAPK phosphorylation, and this was independent of ROS status, as we found that the increased p38 MAPK phosphorylation following HO-1 inhibition remained unaffected when iDCs were co-treated with vitamins and SnPP-IX (Fig. 4A, panel i). We then examined the phosphorylation status of proteins that are downstream of p38 MAPK, i.e. CREB/ATF-1 (Fig. 4A, panel ii). We observed a marked induction in phospho-CREB/ATF-1in iDCs treated with SnPP-IX, and no difference was noted in this phosphorylation status when the cells were treated with vitamins along with SnPP-IX. A similar pattern was observed with LPS stimulation. We then assessed the requirement of the p38 MAPK pathway for the induction of DC maturation elicited by HO-1 inhibition using a pharmacological inhibitor of p38 MAPK (SB203580) and examined its effects on SnPP-IX-treated iDC phenotype and function. As shown in Fig. 4B, inhibition of p38 MAPK activity prevented the increase in co-stimulatory molecule expression induced by SnPP-IX treatment of iDCs (MHC II 43.5 ± 1.0 versus 15.9 ± 0.6%, p < 0.05, panel i; CD86 40.4 ± 0.9% versus 14.8 ± 2.3%, p < 0.05, panel ii). Furthermore, p38 MAPK inhibition also prevented the enhanced antigen-specific F5 CD8 T cell proliferation mediated by SnPP-IX-treated iDCs (Fig. 4C, reduction of 2.5-fold at 1 nm NP68, p < 0.05, 2-fold at 10 nm NP68, p < 0.05, and 1.4-fold at 100 nm, p < 0.05, which are comparable with iDCs). To test whether inhibition of p38 MAPK activity in SnPP-IX-treated DCs, which resulted in reduced DC maturation phenotype, is manifested through altered CREB/ATF1 signaling, DCs were treated with SnPP-IX and SB203580. Western immunoblotting revealed that SnPP-IX treatment resulted in increased CREB/ATF1 phosphorylation (Fig. 4D). Furthermore, the enhanced CREB/ATF1 phosphorylation was markedly reduced by p38 MAPK inhibition. Collectively, these results suggest that HO-1 regulates DC phenotype and function through modulation of the p38 MAPK-CREB/ATF1 signaling axis. To test whether the effects of HO-1 inhibition in DCs can be reversed by antioxidants other than vitamins, we used NAC as an additional ROS scavenger. NAC has been shown to be a potent ROS scavenger in DCs (30Sheng K.C. Pietersz G.A. Tang C.K. Ramsland P.A. Apostolopoulos V. Reactive oxygen species level defines two functionally distinctive stages of inflammatory dendritic cell development from mouse bone marrow.J. Immunol. 2010; 184: 2863-2872Crossref PubMed Scopus (48) Google Scholar) that can elevate or replenish intracellular glutathione levels (31Zafarullah M. Li W.Q. Sylvester J. Ahmad M. Molecular mechanisms of N-acetylcysteine actions.Cell. Mol. Life Sci. 2003; 60: 6-20Crossref PubMed Scopus (1064) Google Scholar). We observed that, as with vitamins C and E, NAC was effective in lowering the increased intracellular ROS levels induced by SnPP-IX in iDCs (Fig. 5A, SnPP-IX versus SnPP-IX + NAC 46.6 ± 8.7% versus 20.2 ± 9.8%, p < 0.05). To further confirm the effects that ROS reduction by NAC have on DC maturation, we treated iDCs with NAC alone and in combination with SnPP-IX. Our results showed that there were no significant differences between the expression of MHC II and CD86 in SnPP-IX-treated iDCs or SnPP-IX- and NAC-treated iDCs (Fig. 5B, panel i, 57.2 ± 9.0% versus 54.7 ± 5.8%, p > 0.05 and Fig. 5B, panel ii, 61.6 ± 9.1% versus 56.7 ± 4.7%, p > 0.05 respectively). We next examined whether NAC affects the SnPP-IX-mediated activation of p38 MAPK-CREB/ATF1 pathway in DCs. Results showed that NAC does not affect the phosphorylation of p38 MAPK and CREB/ATF1 induced by SnPP-IX (Fig. 5C). NAC had similar effects on LPS-induced changes in DC phenotype and signaling. These results further support the proposition that the effects of HO-1 inhibition on DCs are not dependent on ROS. To further test the involvement of HO-1 in the regulation of DC phenotype and function through p38 MAPK-CREB/ATF1, we investigated the effect of HO-1 up-regulation on LPS-triggered DC maturation and CREB/ATF1 phosphorylation. CoPP can be used to up-regulate the expression of HO-1 (32Rosa A.O. Egea J. Lorrio S. Rojo A.I. Cuadrado A. López M.G. Nrf2-mediated haeme oxygenase-1 up-regulation induced by cobalt protoporphyrin has antinociceptive effects against inflammatory pain in the formalin test in mice.Pain. 2008; 137: 332-339Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). We demonstrated that both basal and LPS-induced up-regulation of MHC II molecules expression was significantly reduced (Fig. 6A, panel i) when iDCs were treated with CoPP (at concentrations of 10 and 20 μm). HO-1 induction also causes significant reduction in LPS-induced up-regulation of CD86 (Fig. 6A, panel ii). Furthermore, CoPP treatment resulted in a significant reduction in LPS-treated DC-mediated antigen-specific F5 CD8 T cell proliferation (Fig. 6B.) To evaluate the influence of CoPP treatment on LPS-induced phosphorylation of CREB and ATF1, iDCs were treated with CoPP and stimulated with LPS. Cobalt protoporphyrin treatment markedly reduced CREB/ATF1 phosphorylation in LPS-treated DCs as shown in Fig. 6C. These results strengthen the evidence for the regulation of DC function by HO-1. Inhibition of HO-1 is thought to result in intracellular accumulation of the HO-1 substrate, heme (33Wagener F.A. Volk H.D. Willis D. Abraham N.G. Soares M.P. Adema G.J. Figdor C.G. Different faces of the heme-heme oxygenase system in inflammation.Pharmacol. Rev. 2003; 55: 551-571Crossref PubMed Scopus (466) Google Scholar). When heme (as hemin, the oxidized form of heme), is added to innate immune cells, it can accumulate within the cells (34Dang T.N. Robinson S.R. Dringen R. Bishop G.M. Uptake, metabolism and toxicity of hemin in cultured neurons.Neurochem. Int. 2011; 58: 804-811Crossref PubMed Scopus (33) Google Scholar, 35Hualin C. Wenli X. Dapeng L. Xijing L. Xiuhua P. Qingfeng P. The anti-inflammatory mechanism of heme oxygenase-1 induced by hemin in primary rat alveolar macrophages.Inflammation. 2012; 35: 1087-1093Crossref PubMed Scopus (44) Goo" @default.
• W2027424250 created "2016-06-24" @default.
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• W2027424250 creator A5018148199 @default.
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• W2027424250 creator A5056960672 @default.
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• W2027424250 creator A5070054598 @default.
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• W2027424250 date "2014-06-01" @default.
• W2027424250 modified "2023-10-14" @default.
• W2027424250 title "Heme Oxygenase-1 Regulates Dendritic Cell Function through Modulation of p38 MAPK-CREB/ATF1 Signaling" @default.
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