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• W2033577622 abstract "•Fungal engagement of Mincle opposes dectin-1-induced antifungal TH1 immune responses•Mincle activates PKB and E3 ligase Mdm2 to target nuclear IRF1 for degradation•IRF1 is crucial to IL12A transcription via nucleosome remodeling•Reduced levels of IL-12, a TH1-polarizing cytokine, decreases protective TH1 responses Recognition of fungal pathogens by C-type lectin receptor (CLR) dectin-1 on human dendritic cells is essential for triggering protective antifungal TH1 and TH17 immune responses. We show that Fonsecaea monophora, a causative agent of chromoblastomycosis, a chronic fungal skin infection, evades these antifungal responses by engaging CLR mincle and suppressing IL-12, which drives TH1 differentiation. Dectin-1 triggering by F. monophora activates transcription factor IRF1, which is crucial for IL12A transcription via nucleosome remodeling. However, simultaneous F. monophora binding to mincle induces an E3 ubiquitin ligase Mdm2-dependent degradation pathway, via Syk-CARD9-mediated PKB signaling, that leads to loss of nuclear IRF1 activity, hence blocking IL12A transcription. The absence of IL-12 leads to impaired TH1 responses and promotes TH2 polarization. Notably, mincle is similarly exploited by other chromoblastomycosis-associated fungi to redirect TH responses. Thus, mincle is a fungal receptor that can suppress antifungal immunity and, as such, is a potential therapeutic target. Recognition of fungal pathogens by C-type lectin receptor (CLR) dectin-1 on human dendritic cells is essential for triggering protective antifungal TH1 and TH17 immune responses. We show that Fonsecaea monophora, a causative agent of chromoblastomycosis, a chronic fungal skin infection, evades these antifungal responses by engaging CLR mincle and suppressing IL-12, which drives TH1 differentiation. Dectin-1 triggering by F. monophora activates transcription factor IRF1, which is crucial for IL12A transcription via nucleosome remodeling. However, simultaneous F. monophora binding to mincle induces an E3 ubiquitin ligase Mdm2-dependent degradation pathway, via Syk-CARD9-mediated PKB signaling, that leads to loss of nuclear IRF1 activity, hence blocking IL12A transcription. The absence of IL-12 leads to impaired TH1 responses and promotes TH2 polarization. Notably, mincle is similarly exploited by other chromoblastomycosis-associated fungi to redirect TH responses. Thus, mincle is a fungal receptor that can suppress antifungal immunity and, as such, is a potential therapeutic target. Fungal species are ubiquitously present and pose considerable risk to human health. While opportunistic fungi cause disease in vulnerable patient groups, particular fungal strains are virulent regardless of host immunocompetence. Dendritic cells (DCs) have a key role in the generation of protective antifungal immunity by orchestrating activation and expansion of CD4+ effector T cell populations that restrict fungal growth and enable phagocytic clearance (Roy and Klein, 2012Roy R.M. Klein B.S. Dendritic cells in antifungal immunity and vaccine design.Cell Host Microbe. 2012; 11: 436-446Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Efficient host protection to fungi requires a coordinated immune response consisting of T helper cell (TH) type 1 and TH17 cells (Romani, 2011Romani L. Immunity to fungal infections.Nat. Rev. Immunol. 2011; 11: 275-288Crossref PubMed Scopus (877) Google Scholar, Wüthrich et al., 2012Wüthrich M. Deepe Jr., G.S. Klein B. Adaptive immunity to fungi.Annu. Rev. Immunol. 2012; 30: 115-148Crossref PubMed Scopus (157) Google Scholar). Both TH1 and TH17 cells are involved in chemotaxis and activation of phagocytes, particularly macrophages and neutrophils, via secretion of interferon-γ (IFN-γ) and IL-17, respectively (De Luca et al., 2010De Luca A. Zelante T. D’Angelo C. Zagarella S. Fallarino F. Spreca A. Iannitti R.G. Bonifazi P. Renauld J.C. Bistoni F. et al.IL-22 defines a novel immune pathway of antifungal resistance.Mucosal Immunol. 2010; 3: 361-373Crossref PubMed Scopus (209) Google Scholar, Korn et al., 2009Korn T. Bettelli E. Oukka M. Kuchroo V.K. IL-17 and Th17 Cells.Annu. Rev. Immunol. 2009; 27: 485-517Crossref PubMed Scopus (3780) Google Scholar). TH1-produced IFN-γ is essential for optimal activation of phagocytic effector cell functions (e.g., release of nitric oxide and production of reactive oxygen intermediates) to combat fungal persistence (Schroder et al., 2004Schroder K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-gamma: an overview of signals, mechanisms and functions.J. Leukoc. Biol. 2004; 75: 163-189Crossref PubMed Scopus (2910) Google Scholar). The critical role of TH1 cells is underscored by the susceptibility of IFN-γ knockout mice to fungal infections (Káposzta et al., 1998Káposzta R. Tree P. Maródi L. Gordon S. Characteristics of invasive candidiasis in gamma interferon- and interleukin-4-deficient mice: role of macrophages in host defense against Candida albicans.Infect. Immun. 1998; 66: 1708-1717PubMed Google Scholar) and the successful use of IFN-γ therapy in controlling human mycoses (Stevens et al., 2006Stevens D.A. Brummer E. Clemons K.V. Interferon- γ as an antifungal.J. Infect. Dis. 2006; 194: S33-S37Crossref PubMed Scopus (26) Google Scholar). Chromoblastomycosis is a chronic progressive fungal infection of skin and subcutaneous tissue that occurs worldwide and is caused by traumatic inoculation of a specific group of dematiaceous fungi, most commonly Fonsecaea, Cladophialophora, and Phialophora species (Queiroz-Telles et al., 2009Queiroz-Telles F. Esterre P. Perez-Blanco M. Vitale R.G. Salgado C.G. Bonifaz A. Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment.Med. Mycol. 2009; 47: 3-15Crossref PubMed Scopus (224) Google Scholar). Fungal strains that cause chromoblastomycosis are highly pathogenic and affect immunocompetent hosts (Sousa et al., 2011Sousa Mda. G. Reid D.M. Schweighoffer E. Tybulewicz V. Ruland J. Langhorne J. Yamasaki S. Taylor P.R. Almeida S.R. Brown G.D. Restoration of pattern recognition receptor costimulation to treat chromoblastomycosis, a chronic fungal infection of the skin.Cell Host Microbe. 2011; 9: 436-443Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Lesions of chromoblastomycosis patients that manifest a high fungal load are characterized by the presence of TH2 cells (d’Avila et al., 2003d’Avila S.C. Pagliari C. Duarte M.I. The cell-mediated immune reaction in the cutaneous lesion of chromoblastomycosis and their correlation with different clinical forms of the disease.Mycopathologia. 2003; 156: 51-60Crossref PubMed Scopus (53) Google Scholar). TH2 cells are detrimental for antifungal defense as they oppose fungal elimination (Romani, 2011Romani L. Immunity to fungal infections.Nat. Rev. Immunol. 2011; 11: 275-288Crossref PubMed Scopus (877) Google Scholar), suggesting that these fungi have evolved to escape or manipulate innate and adaptive immune responses. C-type lectin receptors (CLRs), a prominent class of pathogen recognition receptors (PRRs), expressed on DCs couple innate recognition of fungal carbohydrates to expression of cytokines involved in TH polarization (Wevers et al., 2013Wevers B.A. Geijtenbeek T.B. Gringhuis S.I. C-type lectin receptors orchestrate antifungal immunity.Future Microbiol. 2013; 8: 839-854Crossref PubMed Scopus (20) Google Scholar). Interleukin (IL)-12p70 is crucial for TH1 differentiation (Moser and Murphy, 2000Moser M. Murphy K.M. Dendritic cell regulation of TH1-TH2 development.Nat. Immunol. 2000; 1: 199-205Crossref PubMed Scopus (981) Google Scholar), while activation and maintenance of TH17 cells requires secretion of IL-1β, IL-6, and IL-23 by DCs (Korn et al., 2009Korn T. Bettelli E. Oukka M. Kuchroo V.K. IL-17 and Th17 Cells.Annu. Rev. Immunol. 2009; 27: 485-517Crossref PubMed Scopus (3780) Google Scholar, Zielinski et al., 2012Zielinski C.E. Mele F. Aschenbrenner D. Jarrossay D. Ronchi F. Gattorno M. Monticelli S. Lanzavecchia A. Sallusto F. Pathogen-induced human TH17 cells produce IFN-γ or IL-10 and are regulated by IL-1β.Nature. 2012; 484: 514-518Crossref PubMed Scopus (696) Google Scholar). CLR-induced signaling pathways that induce human antifungal TH polarization programs are now being defined. Dectin-1 is an important fungal sensor, and dectin-1 triggering activates Syk, which induces assembly of a signaling complex consisting of CARD9, Bcl-10, and MALT1 (Gross et al., 2006Gross O. Gewies A. Finger K. Schäfer M. Sparwasser T. Peschel C. Förster I. Ruland J. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity.Nature. 2006; 442: 651-656Crossref PubMed Scopus (669) Google Scholar), resulting in activation of both classical and noncanonical NF-κB pathways to induce expression of TH1- and TH17-polarizing cytokines (Gringhuis et al., 2009Gringhuis S.I. den Dunnen J. Litjens M. van der Vlist M. Wevers B.A. Bruijns S.C. Geijtenbeek T.B. Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-kappaB activation through Raf-1 and Syk.Nat. Immunol. 2009; 10: 203-213Crossref PubMed Scopus (348) Google Scholar). CLR mincle is also involved in host responses against several fungi, including Malassezia, some Candida species, and Fonsecaea pedrosoi (Sousa et al., 2011Sousa Mda. G. Reid D.M. Schweighoffer E. Tybulewicz V. Ruland J. Langhorne J. Yamasaki S. Taylor P.R. Almeida S.R. Brown G.D. Restoration of pattern recognition receptor costimulation to treat chromoblastomycosis, a chronic fungal infection of the skin.Cell Host Microbe. 2011; 9: 436-443Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, Wells et al., 2008Wells C.A. Salvage-Jones J.A. Li X. Hitchens K. Butcher S. Murray R.Z. Beckhouse A.G. Lo Y.L. Manzanero S. Cobbold C. et al.The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans.J. Immunol. 2008; 180: 7404-7413Crossref PubMed Scopus (332) Google Scholar, Yamasaki et al., 2009Yamasaki S. Matsumoto M. Takeuchi O. Matsuzawa T. Ishikawa E. Sakuma M. Tateno H. Uno J. Hirabayashi J. Mikami Y. et al.C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia.Proc. Natl. Acad. Sci. USA. 2009; 106: 1897-1902Crossref PubMed Scopus (329) Google Scholar); however, its contribution to antifungal immunity remains unclear. Mincle transduces Syk-CARD9 signaling via the paired Fc receptor common γ-chain (FcRγ) adaptor (Ishikawa et al., 2009Ishikawa E. Ishikawa T. Morita Y.S. Toyonaga K. Yamada H. Takeuchi O. Kinoshita T. Akira S. Yoshikai Y. Yamasaki S. Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle.J. Exp. Med. 2009; 206: 2879-2888Crossref PubMed Scopus (560) Google Scholar, Strasser et al., 2012Strasser D. Neumann K. Bergmann H. Marakalala M.J. Guler R. Rojowska A. Hopfner K.P. Brombacher F. Urlaub H. Baier G. et al.Syk kinase-coupled C-type lectin receptors engage protein kinase C-σ to elicit Card9 adaptor-mediated innate immunity.Immunity. 2012; 36: 32-42Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, Yamasaki et al., 2008Yamasaki S. Ishikawa E. Sakuma M. Hara H. Ogata K. Saito T. Mincle is an ITAM-coupled activating receptor that senses damaged cells.Nat. Immunol. 2008; 9: 1179-1188Crossref PubMed Scopus (535) Google Scholar). Mincle can heterodimerize with another CLR, macrophage C-type lectin (MCL), forming a functional trimeric receptor complex with FcRγ (Lobato-Pascual et al., 2013Lobato-Pascual A. Saether P.C. Fossum S. Dissen E. Daws M.R. Mincle, the receptor for mycobacterial cord factor, forms a functional receptor complex with MCL and FcεRI-γ.Eur. J. Immunol. 2013; 43: 3167-3174Crossref PubMed Scopus (86) Google Scholar). In mice, mincle agonists promote induction of TH1 and TH17 responses (Schoenen et al., 2010Schoenen H. Bodendorfer B. Hitchens K. Manzanero S. Werninghaus K. Nimmerjahn F. Agger E.M. Stenger S. Andersen P. Ruland J. et al.Cutting edge: mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate.J. Immunol. 2010; 184: 2756-2760Crossref PubMed Scopus (380) Google Scholar). However, how human mincle affects cytokine transcription and subsequently TH differentiation remains unclear. Here, we identify mincle as a suppressor of antifungal defenses by suppressing IL-12. We show that interferon regulatory factor 1 (IRF1) is crucial for IL-12 production by inducing nucleosome remodeling of IL12A. Dectin-1 triggering by F. monophora induced IRF1-dependent IL-12p35 mRNA expression, but our study demonstrates that F. monophora simultaneously triggered mincle, which led to specific degradation of IRF1, thereby suppressing IL12A transcription. Mincle signaling led to E3 ubiquitin ligase Mdm2-dependent proteasomal degradation of IRF1 in the nucleus. IL-12p70 suppression redirected TH differentiation from TH1 to TH2 responses, thereby adversely affecting antifungal defense mechanisms. Thus, human mincle is a prominent modulator of antifungal immunity and immune suppressor and might be targeted to treat chromoblastomycosis as well as disorders characterized by aberrant IL-12-driven inflammation, such as autoimmune diabetes. Stimulation of primary human DCs with heat-killed conidia from chromoblastomycosis isolate F. monophora induced expression of maturation markers CD80, CD86, CD83, and HLA-DR (Figure S1 available online) as well as expression of IL-6, IL-1β, IL-23, and IL-12p40, but not IL-12p70 (Figure 1A). We next examined the lack of IL-12p70 expression. Whereas TLR4 triggering by lipopolysaccharide (LPS) induced strong IL-12p70 secretion, simultaneous stimulation with F. monophora severely suppressed LPS-mediated IL-12p70 release (Figure 1B). IL-12p70 comprises of IL-12p35 and IL-12p40 subunits (Hunter, 2005Hunter C.A. New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions.Nat. Rev. Immunol. 2005; 5: 521-531Crossref PubMed Scopus (685) Google Scholar). F. monophora-stimulated DCs produced IL-12p40 as well as IL-23, which consists of IL-12p40 and IL-23p19 subunits (Figure 1A), indicating that IL-12p70 expression is restricted at the level of IL-12p35. F. monophora alone failed to trigger IL-12p35 expression, while LPS-induced IL-12p35 mRNA levels were significantly blocked after coexposure to F. monophora (Figure 1C). F. monophora-mediated suppression of LPS-induced IL-12p35 mRNA expression was even more evident with live fungi (Figure 1C). In contrast, Candida albicans strongly induced IL-12p35 and enhanced LPS-induced IL-12p35 mRNA expression (Figure 1D). These data strongly suggest that F. monophora actively suppresses IL-12p35 production. We next set out to identify the innate receptor(s) involved in F. monophora responses and IL-12p35 suppression. Recombinant human dectin-1 interacted with F. monophora as well as C. albicans (Figure 1E). We found that blocking antibodies against dectin-1 abrogated F.-monophora-induced IL-6, IL-1β, IL-23p19, and IL-12p40 mRNA expression, as were the responses induced by dectin-1 agonist curdlan (Figure 1F). Moreover, DCs derived from a donor carrying a homozygous Y238X dectin-1 mutation, hence lacking functional dectin-1 expression (Ferwerda et al., 2009Ferwerda B. Ferwerda G. Plantinga T.S. Willment J.A. van Spriel A.B. Venselaar H. Elbers C.C. Johnson M.D. Cambi A. Huysamen C. et al.Human dectin-1 deficiency and mucocutaneous fungal infections.N. Engl. J. Med. 2009; 361: 1760-1767Crossref PubMed Scopus (601) Google Scholar) (Figure S2), did not produce IL-6, IL-1β, IL-23, and IL-12p70 after F. monophora stimulation (Figures 1G and S2). In contrast, both LPS and C. albicans (strain CBS2712, which is not strictly dependent on dectin-1 for triggering cytokine responses) (Gringhuis et al., 2011Gringhuis S.I. Wevers B.A. Kaptein T.M. van Capel T.M. Theelen B. Boekhout T. de Jong E.C. Geijtenbeek T.B. Selective C-Rel activation via Malt1 controls anti-fungal T(H)-17 immunity by dectin-1 and dectin-2.PLoS Pathog. 2011; 7: e1001259Crossref PubMed Scopus (132) Google Scholar) induced cytokines in the absence of functional dectin-1 (Figure 1G). Furthermore, blocking dectin-1 signaling did not interfere with F. monophora-mediated suppression of LPS-induced IL-12p35 mRNA expression (Figure 1H). These data indicate that dectin-1 recognition of F. monophora is crucial for induction of cytokine gene transcription, whereas another innate receptor is responsible for suppression of IL-12p35. We found that F. monophora, but not C. albicans, interacted with recombinant human mincle (Figure 1E). As mincle is constitutively expressed on human immature DCs (Figure S2), we investigated whether mincle is involved in IL-12p35 suppression by F. monophora by silencing mincle expression by RNAi (Figure S3). Strikingly, mincle silencing restored IL-12p35 mRNA and IL-12p70 protein expression in response to F. monophora, without affecting curdlan-induced IL-12p35 synthesis (Figures 1I and S4). We next determined whether mincle-mediated IL-12p35 suppression required the presence of MCL, which can form functional complexes with mincle and FcRγ (Lobato-Pascual et al., 2013Lobato-Pascual A. Saether P.C. Fossum S. Dissen E. Daws M.R. Mincle, the receptor for mycobacterial cord factor, forms a functional receptor complex with MCL and FcεRI-γ.Eur. J. Immunol. 2013; 43: 3167-3174Crossref PubMed Scopus (86) Google Scholar). MCL silencing did not interfere with mincle surface expression, while, vice versa, mincle silencing only slightly affected cell surface expression of MCL (Figure S3). Importantly, MCL silencing did not interfere with IL-12p35 suppression by F. monophora (Figure 1I), indicating that the suppression is independent of MCL. To further examine the involvement of mincle in IL-12p35 suppression, we used trehalose-6,6-dibehenate (TDB), a known mincle and MCL agonist (Ishikawa et al., 2009Ishikawa E. Ishikawa T. Morita Y.S. Toyonaga K. Yamada H. Takeuchi O. Kinoshita T. Akira S. Yoshikai Y. Yamasaki S. Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle.J. Exp. Med. 2009; 206: 2879-2888Crossref PubMed Scopus (560) Google Scholar, Miyake et al., 2013Miyake Y. Toyonaga K. Mori D. Kakuta S. Hoshino Y. Oyamada A. Yamada H. Ono K.i. Suyama M. Iwakura Y. Yoshikai Y. Yamasaki S. C-type lectin MCL is an FcRgamma-coupled receptor that mediates the adjuvanticity of mycobacterial cord factor.Immunity. 2013; 38: 1050-1062Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Similar to F. monophora, triggering of DCs by TDB alone failed to induce IL-12p35 but suppressed LPS-induced IL-12p35 mRNA expression (Figure 1J). Mincle, but not MCL, silencing effectively prevented suppression of LPS-induced IL-12p35 mRNA expression by TDB (Figure 1J). Taken together, these data demonstrate that dectin-1 induces cytokine expression in response to F. monophora, whereas mincle signaling suppresses IL-12p35 transcription. We set out to elucidate the mechanism behind mincle-mediated IL-12p35 suppression. As both dectin-1 and mincle signal via Syk-CARD9 (Gross et al., 2006Gross O. Gewies A. Finger K. Schäfer M. Sparwasser T. Peschel C. Förster I. Ruland J. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity.Nature. 2006; 442: 651-656Crossref PubMed Scopus (669) Google Scholar, Yamasaki et al., 2008Yamasaki S. Ishikawa E. Sakuma M. Hara H. Ogata K. Saito T. Mincle is an ITAM-coupled activating receptor that senses damaged cells.Nat. Immunol. 2008; 9: 1179-1188Crossref PubMed Scopus (535) Google Scholar), we combined TLR4 triggering with selective mincle stimulation by TDB to distinguish between dectin-1- and mincle-mediated effects. Silencing of Syk, CARD9, Bcl-10, or MALT1 abrogated the suppressive effects of TDB on LPS-induced IL-12p35 expression (Figure 2A), demonstrating that mincle suppresses IL-12p35 via Syk-CARD9-Bcl-10-MALT1-mediated signaling. To further identify mincle signaling components, we utilized a panel of small molecule inhibitors. We noted in particular that pretreatment of DCs with wortmannin, an irreversible PI3K inhibitor, abrogated IL-12p35 suppression by TDB and restored expression to LPS-induced levels (Figure 2B). Similarly, inhibition of PKB (or Akt), a well-established effector of PI3K, by triciribine (Figure 2B) or PKB silencing (Figure 2C) blocked TDB-mediated suppression of IL-12p35. Thus, the PI3K-PKB cascade plays a primary role in mincle-mediated IL-12p35 suppression. Indeed, TDB, in contrast to curdlan or LPS, strongly induced PKB kinase activity (Figure 2D). PKB activation requires phosphorylation at Thr308 and Ser473, which is controlled by PI3K via kinases PDK1 and mTORC2, respectively (Sarbassov et al., 2005Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5223) Google Scholar). Mincle stimulation led to PKB phosphorylation at both Thr308 and Ser473 (Figure 2E), while LPS and curdlan had no effect (Figure S5), correlating with PKB activity (Figure 2D). PKB phosphorylation after TDB stimulation was dependent on PI3K activity (Figure 2E) as well as mincle and Syk-CARD9-Bcl-10-MALT1-mediated signaling as silencing of these proteins inhibited TDB-induced PKB phosphorylation (Figure 2F). Furthermore, we found that F. monophora induced PI3K-dependent PKB phosphorylation via mincle (Figures 2E and 2F), which relied on Syk and the CARD9-Bcl-10-MALT1 scaffold (Figure 2F). These results indicate that mincle suppresses dectin-1-induced IL-12p35 expression in response to F. monophora by activation of PI3K-PKB signaling, through a Syk-CARD9-Bcl-10-MALT1-dependent pathway. IL12A transcription is rigidly controlled; in resting cells, the IL12A promoter is assembled into stable nucleosomes, whereas upon stimulation, repositioning of nucleosome 2 (nuc-2) allows binding of, among others, NF-κB and subsequent transcriptional initiation by RNA polymerase II (RNAPII) (Goriely et al., 2003Goriely S. Demonté D. Nizet S. De Wit D. Willems F. Goldman M. Van Lint C. Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites.Blood. 2003; 101: 4894-4902Crossref PubMed Scopus (41) Google Scholar). We observed that, although F. monophora interfered with NF-κB p65 recruitment to the IL12A promoter (Figure 3A), fungal stimulation did not abrogate nuclear translocation of p65 (Figure 3B). Indeed, F. monophora alone induced nuclear translocation of p65, c-Rel, and RelB (Figure 3B), a hallmark of dectin-1 signaling (Gringhuis et al., 2009Gringhuis S.I. den Dunnen J. Litjens M. van der Vlist M. Wevers B.A. Bruijns S.C. Geijtenbeek T.B. Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-kappaB activation through Raf-1 and Syk.Nat. Immunol. 2009; 10: 203-213Crossref PubMed Scopus (348) Google Scholar). Moreover, RNAPII recruitment to the IL12A promoter was almost completely abolished (Figure 3A). Since these data suggest that mincle signaling interferes with IL12A transcription prior to RNAPII and NF-κB binding, we next examined whether F. monophora interferes with IL12A nucleosome remodeling. F. monophora did not induce IL12A nucleosome remodeling, whereas curdlan and LPS evoked complete remodeling (Figure 3C). Notably, LPS-induced IL12A nucleosome remodeling was attenuated after costimulation with F. monophora (Figure 3C), while mincle silencing restored LPS-induced IL12A nucleosome remodeling (Figure 3D). These results strongly suggest that F.-monophora-induced mincle signaling inhibits IL-12p35 expression by interfering with nucleosome remodeling events indispensable for transcriptional activation at the IL12A promoter. The nuc-2 promoter region of IL12A contains an IFN-stimulated response element (ISRE) that can be bound by IRF1 (Liu et al., 2003Liu J. Cao S. Herman L.M. Ma X. Differential regulation of interleukin (IL)-12 p35 and p40 gene expression and interferon (IFN)-gamma-primed IL-12 production by IFN regulatory factor 1.J. Exp. Med. 2003; 198: 1265-1276Crossref PubMed Scopus (159) Google Scholar). We found that IRF1 silencing abrogated IL-12p35 expression in response to curdlan or LPS stimulation (Figure 4A). In accordance, both dectin-1 and TLR4 triggering induced IRF1 recruitment to the ISRE site of the IL12A promoter (Figure 4B). Strikingly, IRF1 silencing completely abrogated IL12A nucleosome remodeling induced by dectin-1 and TLR4 signaling (Figure 4C), which coincided with impaired recruitment of RNAPII and p65 to the IL12A promoter (Figure 4D). These data show that IRF1 plays an essential role in IL12A transcription in response to both dectin-1 and TLR4 via nucleosome remodeling. We next investigated whether mincle triggering affects IL12A nucleosome remodeling by interfering with IRF1 activation. We found that both curdlan and LPS induced nuclear translocation of IRF1 (Figure 4E). Remarkably, simultaneous stimulation with F. monophora completely abrogated LPS-induced nuclear IRF1 accumulation (Figure 4F). F. monophora alone did not induce nuclear IRF1 accumulation (Figure 4F). Also, we observed a complete absence of nuclear IRF1 after TLR4 and mincle costimulation (Figures 4G and 4H). Silencing of either mincle or downstream effector PKB restored nuclear IRF1 accumulation after LPS and TDB costimulation, as well as F. monophora stimulation, without affecting LPS-induced nuclear IRF1 accumulation or cytoplasmic IRF1 expression (Figure 4I). These data show that PKB-mediated mincle signaling restricts nuclear accumulation of IRF1, suggesting that this mechanism underlies the block in IL12A nucleosomal remodeling and subsequent transcription in response to F. monophora. IRF1 steady-state levels are tightly controlled by a balance between synthesis and ubiquitin-mediated proteolysis (Landré et al., 2013Landré V. Pion E. Narayan V. Xirodimas D.P. Ball K.L. DNA-binding regulates site-specific ubiquitination of IRF-1.Biochem. J. 2013; 449: 707-717Crossref PubMed Scopus (20) Google Scholar, Nakagawa and Yokosawa, 2000Nakagawa K. Yokosawa H. Degradation of transcription factor IRF-1 by the ubiquitin-proteasome pathway. The C-terminal region governs the protein stability.Eur. J. Biochem. 2000; 267: 1680-1686Crossref PubMed Scopus (73) Google Scholar). We next investigated whether mincle interferes with IRF1 by inducing its degradation. Treatment with proteasome inhibitor MG-132 reversed the block in nuclear IRF1 accumulation after LPS-TDB costimulation (Figure 5A), indicating that IRF1 is indeed targeted for proteasomal degradation by mincle. Since PKB can induce the E3 ubiquitin ligase activity of Mdm2, which recently was found to aid in IRF1 proteasomal degradation (Landré et al., 2013Landré V. Pion E. Narayan V. Xirodimas D.P. Ball K.L. DNA-binding regulates site-specific ubiquitination of IRF-1.Biochem. J. 2013; 449: 707-717Crossref PubMed Scopus (20) Google Scholar, Mayo and Donner, 2001Mayo L.D. Donner D.B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus.Proc. Natl. Acad. Sci. USA. 2001; 98: 11598-11603Crossref PubMed Scopus (952) Google Scholar, Zhou et al., 2001Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation.Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (785) Google Scholar), we explored a possible Mdm2 contribution. Remarkably, Mdm2 silencing completely abrogated mincle-mediated IL-12p35 suppression in response to TDB and F. monophora—without affecting LPS-induced IL-12p35 expression (Figure 5B). To regulate IRF1, Mdm2 must gain nuclear entry, a process which requires phosphorylation of Mdm2 at Ser166 by PKB (Mayo and Donner, 2001Mayo L.D. Donner D.B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus.Proc. Natl. Acad. Sci. USA. 2001; 98: 11598-11603Crossref PubMed Scopus (952) Google Scholar, Zhou et al., 2001Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation.Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (785) Google Scholar). We found that mincle triggering by both TDB and F. monophora, but not TLR4 triggering, induced nuclear translocation of Mdm2 (Figure 5C). Only Mdm2 present within the nucleus was phosphorylated at key residue Ser166 (Figure 5C). Mdm2 Ser166 phosphorylation following TDB and F. monophora stimulation was abrogated by silencing of mincle, Syk, CARD9, Bcl-10, and MALT1, or chemical inhibition of PI3K or PKB (Figures 5D and S5). Similarly, mincle and PKB silencing blocked Mdm2 nuclear translocation (Figure 5C). TDB-induced Mdm2 phosphorylation and nuclear translocation was not affected by LPS stimulation (Figures 5C and S5). Since Mdm2 ubiquitin ligase activity relies on association with its substrates (Haupt et al., 1997Haupt Y. Maya R. Kazaz A. Oren M. Mdm2 promotes the rapid degradation of p53.Nature. 1997; 387: 296-299Crossref PubMed Scopus (3705) Google Scholar, Yang et al., 2008Yang J.Y. Zong C.S. Xia W. Yamaguchi H. Ding Q. Xie X. Lang J.Y. Lai C.C. Chang C.J. Huang W.C. et al.ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation.Nat. Cell Biol. 2008; 10: 138-148Crossref PubMed Scopus (534) Google Scholar), we prepared nuclear extracts in the presence of MG-132 to block degradation, allowing us to determine whether Mdm2 interacts with IRF1 within the nucleus. We found that nuclear IRF1 immunoprecipitated together with Mdm2 in both TDB-stimulated and F. monophora-stimulated, but not LPS-stimulated, DCs (Figure 5E). The importance of Mdm2 in IRF1 regulation was" @default.
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• W2033577622 date "2014-04-01" @default.
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• W2033577622 title "Fungal Engagement of the C-Type Lectin Mincle Suppresses Dectin-1-Induced Antifungal Immunity" @default.
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• W2033577622 doi "https://doi.org/10.1016/j.chom.2014.03.008" @default.
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