Minocycline Down-regulates MHC II Expression in Microglia and Macrophages through Inhibition of IRF-1 and Protein Kinase C (PKC)α/βII

Experimental allergic encephalomyelitis, an autoimmune disorder mediated by T cells, results in demyelination, inflammation, and axonal loss in the central nervous system (CNS). Microglia play a critical role in major histocompatibility complex class II (MHC II)-dependent antigen presentation and in reactivation of CNS-infiltrated encephalitogenic T cells. Minocycline, a tetracycline anti-biotic, has profound anti-inflammatory properties and is experimentally used for treatment of many CNS disorders; however, the mechanisms involved in minocycline effects remain unknown. We show that administration of minocycline for 2 weeks ameliorated clinical severity of experimental allergic encephalomyelitis, an effect that partially involves the down-regulation of MHC II proteins in the spinal cord. Therefore, we sought to elucidate the molecular mechanisms of minocycline inhibitory effects on MHC II expression in microglia. Although complex, the co-activator class II transactivator (CIITA) is a key regulator of MHC II expression. Here we show that minocycline inhibited interferonγ (IFNγ)-induced CIITA and MHC II mRNA. Interestingly, however, it was without effect on STAT1 phosphorylation or IRF-1 expression, transcription factors that are activated by IFNγ and necessary for CIITA expression. Further experiments revealed that MHC II expression is down-regulated in the presence of the PKCα inhibitor Goö6976. Minocycline inhibited IFNγ-induced PKCα/βII phosphorylation and the nuclear translocation of both PKCα/βII and IRF-1 that subsequently inhibits CIITA expression. Our present data delineate a molecular pathway of minocycline action that includes inhibitory effects on PKCα/βII and transcription factors that regulate the expression of critical inflammatory genes such as MHC II. Such a fundamental mechanism may underlie the pleiotropic effects of minocycline in CNS inflammatory disorders. Experimental allergic encephalomyelitis, an autoimmune disorder mediated by T cells, results in demyelination, inflammation, and axonal loss in the central nervous system (CNS). Microglia play a critical role in major histocompatibility complex class II (MHC II)-dependent antigen presentation and in reactivation of CNS-infiltrated encephalitogenic T cells. Minocycline, a tetracycline anti-biotic, has profound anti-inflammatory properties and is experimentally used for treatment of many CNS disorders; however, the mechanisms involved in minocycline effects remain unknown. We show that administration of minocycline for 2 weeks ameliorated clinical severity of experimental allergic encephalomyelitis, an effect that partially involves the down-regulation of MHC II proteins in the spinal cord. Therefore, we sought to elucidate the molecular mechanisms of minocycline inhibitory effects on MHC II expression in microglia. Although complex, the co-activator class II transactivator (CIITA) is a key regulator of MHC II expression. Here we show that minocycline inhibited interferonγ (IFNγ)-induced CIITA and MHC II mRNA. Interestingly, however, it was without effect on STAT1 phosphorylation or IRF-1 expression, transcription factors that are activated by IFNγ and necessary for CIITA expression. Further experiments revealed that MHC II expression is down-regulated in the presence of the PKCα inhibitor Goö6976. Minocycline inhibited IFNγ-induced PKCα/βII phosphorylation and the nuclear translocation of both PKCα/βII and IRF-1 that subsequently inhibits CIITA expression. Our present data delineate a molecular pathway of minocycline action that includes inhibitory effects on PKCα/βII and transcription factors that regulate the expression of critical inflammatory genes such as MHC II. Such a fundamental mechanism may underlie the pleiotropic effects of minocycline in CNS inflammatory disorders. Experimental allergic encephalomyelitis (EAE), 2The abbreviations used are: EAE, experimental allergic encephalomyelitis; CNS, central nervous system; MHC, major histocompatibility complex; CIITA, class II transactivator; IFN, interferon; IRF-1, IFNγ regulatory factor; PKC, protein kinase C; MS, multiple sclerosis; MOG, myelin oligodendrocyte glycoprotein; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription; JAK, Janus kinase; STAT, signal transducers and activators of transcription; MAP, mitogen-activated protein. an animal model of multiple sclerosis (MS), is an autoimmune disorder characterized by T cell-mediated inflammation, demyelination, and axonal loss in the CNS. Susceptibility to EAE and MS is associated with the major histocompatibility complex class II (MHC II) genes (1Gunther E. Walter L. Immunogenetics. 2001; 53: 520-542Crossref PubMed Scopus (99) Google Scholar, 2de Graaf K.L. Barth S. Herrmann M.M. Storch M.K. Otto C. Olsson T. Melms A. Jung G. Wiesmuller K.H. Weissert R. J. Immunol. 2004; 173: 2792-2802Crossref PubMed Scopus (9) Google Scholar), suggesting that presentation of antigens on MHC II plays an important role in CD4+ T cell activation and disease initiation. After priming of encephalitogenic T cells in the periphery, they migrate into the CNS, where they trigger inflammatory responses including microglial activation. To sustain their activation, the self-reactive T cells have to be reactivated in the CNS by encountering their cognate antigen bound to MHC II. Microglia, the resident innate immune cells of the CNS, have been shown to be the main antigen-presenting cells of the CNS (3Aloisi F. Glia. 2001; 36: 165-179Crossref PubMed Scopus (1046) Google Scholar, 4O'Keefe G.M. Nguyen V.T. Benveniste E.N. J. Neurovirol. 2002; 8: 496-512Crossref PubMed Scopus (77) Google Scholar). They normally express low levels of MHC II proteins; however, in inflammatory or neurodegenerative conditions, activated microglia highly up-regulate MHC II and co-stimulatory molecules (5Kreutzberg G.W. Trends Neurosci. 1996; 19: 312-318Abstract Full Text Full Text PDF PubMed Scopus (3753) Google Scholar). It was recently shown that microglial paralysis inhibited the development and maintenance of inflammatory CNS lesions, providing the first direct evidence of microglial significance in EAE pathology (6Heppner F.L. Greter M. Marino D. Falsig J. Raivich G. Hovelmeyer N. Waisman A. Rulicke T. Prinz M. Priller J. Becher B. Aguzzi A. Nat. Med. 2005; 11: 146-152Crossref PubMed Scopus (610) Google Scholar). Microglial activation precedes the clinical onset of EAE and infiltration of peripheral myeloid cells into the CNS, suggesting that they play a critical role in the induction and progression of EAE (7Ponomarev E.D. Shriver L.P. Maresz K. Dittel B.N. J. Neurosci. Res. 2005; 81: 374-389Crossref PubMed Scopus (330) Google Scholar). Therefore, the inhibition of microglial activation and MHC II expression may be therapeutically significant in MS. In recent years, minocycline, a semisynthetic antibiotic of the tetracycline family, has emerged as a potent anti-inflammatory drug that has been shown to be beneficial in animal models of many CNS disorders including ischemia (8Yrjanheikki J. Keinanen R. Pellikka M. Hokfelt T. Koistinaho J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15769-15774Crossref PubMed Scopus (871) Google Scholar), Huntington disease (9Chen M. Ona V.O. Li M. Ferrante R.J. Fink K.B. Zhu S. Bian J. Guo L. Farrell L.A. Hersch S.M. Hobbs W. Vonsattel J.P. Cha J.H. Friedlander R.M. Nat. Med. 2000; 6: 797-801Crossref PubMed Scopus (108) Google Scholar), Parkinson disease (10Du Y. Ma Z. Lin S. Dodel R.C. Gao F. Bales K.R. Triarhou L.C. Chernet E. Perry K.W. Nelson D.L. Luecke S. Phebus L.A. Bymaster F.P. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14669-14674Crossref PubMed Scopus (694) Google Scholar), amyotrophic lateral sclerosis (11Zhu S. Stavrovskaya I.G. Drozda M. Kim B.Y. Ona V. Li M. Sarang S. Liu A.S. Hartley D.M. Wu du C. Gullans S. Ferrante R.J. Przed-borski S. Kristal B.S. Friedlander R.M. Nature. 2002; 417: 74-78Crossref PubMed Scopus (977) Google Scholar), and MS (12Metz L.M. Zhang Y. Yeung M. Patry D.G. Bell R.B. Stoian C.A. Yong V.W. Patten S.B. Duquette P. Antel J.P. Mitchell J.R. Ann. Neurol. 2004; 55: 756Crossref PubMed Scopus (156) Google Scholar). We have previously shown that minocycline ameliorated the clinical course of EAE and decreased MHC II expression in the spinal cord (13Popovic N. Schubart A. Goetz B.D. Zhang S.C. Linington C. Duncan I.D. Ann. Neurol. 2002; 51: 215-223Crossref PubMed Scopus (259) Google Scholar); however, the exact molecular mechanisms underlying these anti-inflammatory effects of minocycline remain unknown. Therefore, in this study, we investigated the molecular mechanisms of minocycline effects on MHC II expression both in vivo in an EAE model and in vitro using primary rat microglial and macrophage cultures. Since MHC II expression in microglia is requisite for T cell reactivation, understanding the mechanism whereby minocycline represses MHC II expression may elucidate critical pathways that can be targeted by other therapies to treat EAE or MS. The regulation of MHC II protein expression is under the control of a complex cytokine network that requires the coordinated action of multiple transcription factors and co-activators. Among the latter, class II transactivator (CIITA), a non-DNA-binding protein, was identified as a key regulator of both constitutive and inducible MHC II expression (14Harton J.A. Ting J.P. Mol. Cell. Biol. 2000; 20: 6185-6194Crossref PubMed Scopus (185) Google Scholar) (see Fig. 9). Our data reveal that minocycline inhibits MHC II expression in microglia via the inhibition of CIITA expression by a mechanism involving the inhibition of IFNγ regulatory factor (IRF-1) translocation to the nucleus in a PKCα/β-dependent manner. Animals—Female Dark Agouti rats and Sprague-Dawley rats purchased from Harlan Sprague Dawley, Inc. (Madison, WI) were housed under standard conditions. All experiments were conducted with approval of Research Animal Resources Center of the University of Wisconsin. MOG Purification—DH5α cells transfected with pQE12 vector with rat myelin oligodendrocyte glycoprotein (MOG) insert (amino acids 1-125) with a His6 sequence at the 3′ end was a generous gift of N. Ruddle (Yale University School of Medicine). After isopropyl-1-thio-β-d-galactopyranoside induction (4 h) of protein expression, bacterial extract was prepared using BugBuster reagent (Novagen, Madison, WI). MOG protein was purified from cell extract using His-Bind columns (Novagen) under denaturating conditions following the manufacturer’s protocol. Isolated proteins were then dialyzed against acetate buffer for 24 h using Slide-A-Lyzer dialysis cassette (Pierce). To determine the purity of isolated MOG, proteins were run on 10% SDS-PAGE gel and stained with GelCode blue stain reagent (Pierce). Induction of EAE and Minocycline Treatment—EAE was induced in 150-175 g of Dark Agouti female rats by immunization with 10 μg of MOG in complete Freund’s adjuvant. MOG protein in phosphate-buffered saline was emulsified with an equal volume of complete Freund’s adjuvant, to a final concentration of 10 μg of MOG and g of in of were by of a at the of the were with complete Freund’s were and for clinical of EAE, on the following loss of complete The first clinical 10 and Minocycline was a at the first of clinical and then a for at the of with phosphate-buffered saline by were and into and in were in and then for 24 h by into and at were from the with a and used for and and The of was using were at a and as the of the of the Microglial rat microglial were prepared by the from Sprague-Dawley rat as we previously M. Duncan I.D. J. PubMed Scopus Google Scholar). were and with for at The was by the of and the was with a and and cell were in with 10% and and in After 10 the were for and was and for 10 at g to microglial cells. The microglia were in the and in at a of or at a of of were isolated from Sprague-Dawley rats by the with of After the cells were in with 10% 10 MOPS, and on a at a of After a at the was to cells. cell extract was prepared by cell in buffer 2 2 were by 10% SDS-PAGE and to were in for h at by with primary and were used at a at II) and IRF-1 were used at a at After the were with or for h at by with system was used to and data from the After were with buffer for at and with or for h at Although the PKCα is for the PKCα it has with the microglial cells or were as were with for 10 by a in 10% to were stained with and (MHC II) at or for h at by a with with or were stained with microglial cells were for 24 h as were prepared using a protein (Novagen) to the manufacturer’s protocol. proteins were then by 10% SDS-PAGE and by and microglial cells were on at the of After a with minocycline, cells were with IFNγ with or without minocycline for 24 were in of reagent and was isolated to the manufacturer’s protocol. was with for at and μg of was then used for reverse transcription using and reverse RT, of was for of using The of and for and for MHC and for The were on a gel and by were by of and using All in vitro experiments were at and data the experimental were significant at The clinical of and were by a of Minocycline the of of Dark Agouti rats with 10 μg of MOG in a disease course with the onset The clinical course of disease was in with the and revealed a significant infiltration of into the of both the and the of the spinal cord in EAE, an effect that was ameliorated by minocycline the and the of in the spinal cord were decreased by minocycline treatment the and of in the spinal cord. of spinal cord from control and EAE or with minocycline were used for and infiltration of in both and of spinal cord was in EAE Minocycline MHC II in EAE in Dark Agouti rats is characterized by inflammatory in the spinal cord by a microglial In the of cells Fig. and is 10 the of MHC cells and suggesting that microglia express MHC II in the a cells with a can be in the of the spinal cord the onset of the we in the of however, the of cells was in to and we a in of microglial cells from to suggesting was a significant in the of cells at the onset of the disease that with the of cells. 2 weeks the clinical onset of EAE, the of and MHC cells was at the of with minocycline for 2 weeks decreased the of cells by suggesting decreased microglial decreased of from the into the CNS The of MHC cells was by in the minocycline MHC II levels were disease onset minocycline treatment was Minocycline MHC II and CIITA in was in primary microglial cells are they express MHC II under conditions. of cells with IFNγ for 24 h induced MHC II expression in of the cells, and this was decreased by treatment with minocycline with the of MHC II proteins by revealed an in MHC II proteins minocycline treatment Minocycline decreased MHC II suggesting that the inhibitory effects of minocycline at the the co-activator CIITA, an inducible non-DNA-binding protein, is for MHC II expression (see Fig. we investigated the of minocycline to CIITA expression as a mechanism of minocycline inhibitory effects on MHC II mRNA. Minocycline decreased IFNγ-induced CIITA suggesting that this drug involved in CIITA MHC II and CIITA mRNA. microglial cells were with IFNγ in the presence or of minocycline for 24 was isolated and used for to levels of MHC II and microglia express MHC II or for both proteins was induced by IFNγ and was in the presence of Minocycline STAT1 or determine the mechanism of minocycline inhibitory effects on MHC II expression, we investigated effects on the involved in IFNγ-induced CIITA and MHC II expression. The pathway is central to IFNγ-induced we first the effect of minocycline on the activation of STAT1 of microglia with IFNγ for in STAT1 activation, was by minocycline phosphorylation of that inhibitory effect on MHC II expression of this protein. We investigated minocycline effects on IRF-1 expression, a transcription factor for CIITA expression, and for MHC II In the levels of IRF-1 protein were with cells, and this was by minocycline PKCα/βII in MHC II on has been shown to IFNγ-induced expression of MHC II in the macrophage cell M. M. A. J. Immunol. PubMed Scopus Google Scholar). In with an effect of minocycline on inhibition of in microglial cells M. Duncan I.D. J. PubMed Scopus Google Scholar), suggesting that minocycline may MHC II via inhibition of To this we first the role of in MHC II regulation in rat primary microglial cells. MHC II expression induced by IFNγ was in the presence of the inhibitor a highly inhibitor of PKCα and and revealed that MHC II expression in microglia was in the presence of the Minocycline and of PKCα/βII and phosphorylation of suggesting that minocycline may Interestingly, the in IFNγ-induced IRF-1 levels was by suggesting that that are targeted by minocycline be involved in IRF-1 expression. IFNγ IRF-1 to the where it to the of CIITA, a transactivator critical for MHC II The recent by M. M. A. J. Immunol. PubMed Scopus Google Scholar) that in PKCα to the nucleus with IRF-1, where it IRF-1 IFNγ we sought to in microglia the effects of minocycline on the nuclear of PKCα/βII and IRF-1 the inhibitor translocation of both proteins IFNγ-induced translocation of both proteins into the nucleus was by minocycline treatment that is decreased IRF-1 in the presence of Minocycline MHC II data show a potent inhibitory effect of minocycline on MHC II expression in we were in minocycline MHC II expression. isolated from Sprague-Dawley rats express levels of MHC II proteins In the presence of the inhibitor MHC expression was suggesting that in to MHC II expression, PKCα is involved in MHC II in minocycline inhibited PKCα phosphorylation in suggesting that inhibitory effect on MHC II expression is mediated via with Minocycline has profound anti-inflammatory properties from antibiotic Since minocycline has the of the this drug is for treatment of CNS disorders. In EAE, minocycline at the onset of clinical decreased the severity of the clinical course of The significant in clinical the and was the first of minocycline this is important in MS minocycline treatment be at the of In study, of the spinal cord revealed that minocycline both the and the of and decreased expression of MHC II in microglia. Microglia are the primary antigen-presenting cells of the CNS, and they are the main immune cells this disease G.M. Nguyen V.T. Benveniste E.N. J. Neurovirol. 2002; 8: 496-512Crossref PubMed Scopus (77) Google Scholar). In EAE, activated microglia into and cells, and they up-regulate levels of MHC II and co-stimulatory proteins that are necessary for antigen presentation to the encephalitogenic T cells (7Ponomarev E.D. Shriver L.P. Maresz K. Dittel B.N. J. Neurosci. Res. 2005; 81: 374-389Crossref PubMed Scopus (330) Google Scholar). activated microglia can T cells from the periphery, the inflammatory in the CNS. of that the immune induced by immunization with MOG is mediated by CD4+ T cells, the of T cells and cells has been shown S. D. J. P. L. Immunol. 2005; PubMed Scopus Google Scholar, J. Neurol. Res. PubMed Scopus Google Scholar). The of microglia to CD4+ cells and to CD4+ cells in vitro in an and MHC is S. D. J. P. L. Immunol. 2005; PubMed Scopus Google Scholar, A. K. S. E. Immunol. PubMed Scopus Google Scholar, K. C. P. S. C. A. J. Immunol. PubMed Scopus Google Scholar). The significance of microglial cells in EAE as as in other CNS is and in recent years, these cells have an for development of for CNS disorders. Although the exact mechanisms of minocycline anti-inflammatory effects are they may the inhibition of the inhibition of inducible and and the of cytokine (9Chen M. Ona V.O. Li M. Ferrante R.J. Fink K.B. Zhu S. Bian J. Guo L. Farrell L.A. Hersch S.M. Hobbs W. Vonsattel J.P. Cha J.H. Friedlander R.M. Nat. Med. 2000; 6: 797-801Crossref PubMed Scopus (108) Google Scholar, M. Duncan I.D. J. PubMed Scopus Google Scholar, S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, Kim B.S. W. Res. 2004; PubMed Scopus Google Scholar, V. L.M. Yong V.W. 2002; PubMed Scopus Google Scholar). It has been shown that of these effects are at in by the inhibition of (10Du Y. Ma Z. Lin S. Dodel R.C. Gao F. Bales K.R. Triarhou L.C. Chernet E. Perry K.W. Nelson D.L. Luecke S. Phebus L.A. Bymaster F.P. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14669-14674Crossref PubMed Scopus (694) Google Scholar, M. Duncan I.D. J. PubMed Scopus Google Scholar, Koistinaho J. Immunol. 2001; PubMed Scopus Google Scholar), the exact and molecular mechanisms of minocycline on the expression of these genes remain Our results show that inhibition of MHC II expression by minocycline involves the inhibition of that subsequently nuclear translocation of the transcription factor IRF-1, MHC II expression. has such pleiotropic effects in cells, the of minocycline to such a of expression may the with levels of MHC II molecules are on antigen-presenting cells such as cells and in other cell including MHC II expression can be induced by In MS or EAE, IFNγ is by T cells into the CNS The regulation of MHC II proteins is complex and at the involving transcription factors on the MHC II 9). these are for activation of MHC II on their of CIITA, a co-activator that to the complex of transcription factors on the MHC II to is for both constitutive and MHC II expression (14Harton J.A. Ting J.P. Mol. Cell. Biol. 2000; 20: 6185-6194Crossref PubMed Scopus (185) Google Scholar, V. C.A. A. B. B. PubMed Scopus Google Scholar). inducible MHC II expression and have constitutive MHC II expression on of cells S. S.C. W. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of the CIITA is and is by the of on cell G.M. Nguyen V.T. Benveniste E.N. J. Neurovirol. 2002; 8: 496-512Crossref PubMed Scopus (77) Google Scholar, E. Ting J.P. Immunol. 2005; PubMed Scopus Google Scholar). the constitutive expression of CIITA, and MHC II in cells. is for constitutive expression in and is used to regulate the expression of CIITA in cells such as microglia G.M. Nguyen V.T. Benveniste E.N. J. Neurovirol. 2002; 8: 496-512Crossref PubMed Scopus (77) Google Scholar). After to IFNγ a to the activation of STAT1 activation is subsequently involved in the regulation of IRF-1 and CIITA expression. STAT1 and IRF-1 transcription factors are necessary for inducible CIITA expression from 9). In IFNγ induced MHC II expression in of microglial cells, was by in the presence of Since results that minocycline decreased IFNγ-induced CIITA we that minocycline may the activation of STAT1 IRF-1 in primary microglial cells. we that minocycline IFNγ-induced STAT1 phosphorylation or IRF-1 expression, suggesting that these transcription factors are direct for minocycline have that the activation of is for MHC II expression, and the PKCα has been identified to regulate IFNγ-induced expression of MHC II by the of IRF-1 on the CIITA in macrophage cells M. M. A. J. Immunol. PubMed Scopus Google Scholar). Our present data revealed that is involved in both the constitutive and the MHC II expression in and We have previously shown that minocycline inhibited activation of and suggesting that minocycline with however, we was by minocycline M. Duncan I.D. J. PubMed Scopus Google Scholar). Since it is that PKCα is involved in the regulation of MHC II expression in in this study, we investigated the minocycline effect on this in microglia. Minocycline decreased IFNγ-induced PKCα/βII phosphorylation and nuclear translocation of this It is to PKCα and PKCα have to with translocation of IRF-1 induced by IFNγ was inhibited in the presence of a PKCα suggesting that this is it PKCα IRF-1 translocation into the In we that minocycline inhibited nuclear translocation of IRF-1, decreased of IRF-1 in the with the inhibitory effect of minocycline on CIITA expression. Interestingly, we that PKCα/βII is involved in the regulation of both the and the MHC II however, are to elucidate the exact mechanism of PKCα the of minocycline to MHC II expression that this drug the antigen presentation of antigen-presenting cells, such as cells. is necessary for the activation of CD4+ T cells in the to their infiltration to the CNS. In study, the administration of minocycline from the of immunization in a EAE model in a significant in the onset and severity of the clinical course of disease (13Popovic N. Schubart A. Goetz B.D. Zhang S.C. Linington C. Duncan I.D. Ann. Neurol. 2002; 51: 215-223Crossref PubMed Scopus (259) Google Scholar), suggesting that the inhibition of MHC II expression on peripheral cells be of the mechanisms by minocycline anti-inflammatory effects in this EAE In data show that minocycline decreased the severity of the clinical course of EAE and that these effects may be at partially mediated by the inhibitory effect of minocycline on the MHC II expression in microglia. Our in vitro experiments on primary microglial cells revealed that the molecular mechanisms of minocycline action the inhibition of IFNγ-induced PKCα/βII phosphorylation and inhibition of IRF-1 translocation to the in decreased CIITA expression. expression of this co-activator is to the expression of MHC providing a the severity of EAE and decreased antigen presentation of CNS resident microglia. We are to of for critical and on the