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• W2110432110 abstract "Galectin-3 is a β-galactoside–binding animal lectin with diverse functions, including regulation of T helper (Th) 1 and Th2 responses. Current data indicate that galectin-3 expressed in dendritic cells (DCs) may be contributory. Th17 cells have emerged as critical inducers of tissue inflammation in autoimmune disease and important mediators of host defense against fungal pathogens, although little is known about galectin-3 involvement in Th17 development. We investigated the role of galectin-3 in the induction of Th17 immunity in galectin-3–deficient (gal3−/−) and gal3+/+ mouse bone marrow–derived DCs. We demonstrate that intracellular galectin-3 negatively regulates Th17 polarization in response to the dectin-1 agonist curdlan (a β-glucan present on the cell wall of fungal species) and lipopolysaccharide, agents that prime DCs for Th17 differentiation. On activation of dectin-1, gal3−/− DCs secreted higher levels of the Th17-axis cytokine IL-23 compared with gal3+/+ DCs and contained higher levels of activated c-Rel, an NF-κB subunit that promotes IL-23 expression. Levels of active Raf-1, a kinase that participates in downstream inhibition of c-Rel binding to the IL23A promoter, were impaired in gal3−/− DCs. Modulation of Th17 by galectin-3 in DCs also occurred in vivo because adoptive transfer of gal3−/− DCs exposed to Candida albicans conferred higher Th17 responses and protection against fungal infection. We conclude that galectin-3 suppresses Th17 responses by regulating DC cytokine production. Galectin-3 is a β-galactoside–binding animal lectin with diverse functions, including regulation of T helper (Th) 1 and Th2 responses. Current data indicate that galectin-3 expressed in dendritic cells (DCs) may be contributory. Th17 cells have emerged as critical inducers of tissue inflammation in autoimmune disease and important mediators of host defense against fungal pathogens, although little is known about galectin-3 involvement in Th17 development. We investigated the role of galectin-3 in the induction of Th17 immunity in galectin-3–deficient (gal3−/−) and gal3+/+ mouse bone marrow–derived DCs. We demonstrate that intracellular galectin-3 negatively regulates Th17 polarization in response to the dectin-1 agonist curdlan (a β-glucan present on the cell wall of fungal species) and lipopolysaccharide, agents that prime DCs for Th17 differentiation. On activation of dectin-1, gal3−/− DCs secreted higher levels of the Th17-axis cytokine IL-23 compared with gal3+/+ DCs and contained higher levels of activated c-Rel, an NF-κB subunit that promotes IL-23 expression. Levels of active Raf-1, a kinase that participates in downstream inhibition of c-Rel binding to the IL23A promoter, were impaired in gal3−/− DCs. Modulation of Th17 by galectin-3 in DCs also occurred in vivo because adoptive transfer of gal3−/− DCs exposed to Candida albicans conferred higher Th17 responses and protection against fungal infection. We conclude that galectin-3 suppresses Th17 responses by regulating DC cytokine production. IL-17–producing CD4+ T helper 17 (Th17) cells play an essential role in the clearance of extracellular bacterial and fungal pathogens1Huang W. Na L. Fidel P.L. Schwarzenberger P. Requirement of interleukin-17A for systemic anti-Candida albicans host defense in mice.J Infect Dis. 2004; 190: 624-631Crossref PubMed Scopus (696) Google Scholar, 2Conti H.R. Shen F. Nayyar N. Stocum E. Sun J.N. Lindemann M.J. Ho A.W. Hai J.H. Yu J.J. Jung J.W. Filler S.G. Masso-Welch P. Edgerton M. Gaffen S.L. Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis.J Exp Med. 2009; 206: 299-311Crossref PubMed Scopus (741) Google Scholar and promote inflammatory responses involved in autoimmune disease.3Cua D.J. Sherlock J. Chen Y. Murphy C.A. Joyce B. Seymour B. Lucian L. To W. Kwan S. Churakova T. Zurawski S. Wiekowski M. Lira S.A. Gorman D. Kastelein R.A. Sedgwick J.D. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.Nature. 2003; 421: 744-748Crossref PubMed Scopus (2319) Google Scholar, 4Langrish C.L. Chen Y. Blumenschein W.M. Mattson J. Basham B. Sedgwick J.D. McClanahan T. Kastelein R.A. Cua D.J. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3174) Google Scholar The factors associated with Th17 cell development have been well characterized and include cytokines such as transforming growth factor-β (TGF-β) and IL-6, which promote Th17 differentiation in mice,5Bettelli E. Carrier Y. Gao W. Korn T. Strom T.B. Oukka M. Weiner H.L. Kuchroo V.K. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.Nature. 2006; 441: 235-238Crossref PubMed Scopus (5434) Google Scholar, 6Veldhoen M. Hocking R.J. Atkins C.J. Locksley R.M. Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.Immunity. 2006; 24: 179-189Abstract Full Text Full Text PDF PubMed Scopus (2955) Google Scholar and IL-23, which is required for Th17 cell expansion and effector functions.4Langrish C.L. Chen Y. Blumenschein W.M. Mattson J. Basham B. Sedgwick J.D. McClanahan T. Kastelein R.A. Cua D.J. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3174) Google Scholar Dendritic cells (DCs) express germline-encoded pattern recognition receptors (PRRs) that recognize conserved molecular components expressed on microbial pathogens7Clark G.J. Angel N. Kato M. Lopez J.A. MacDonald K. Vuckovic S. Hart D.N.J. The role of dendritic cells in the innate immune system.Microbes Infect. 2000; 2: 257-272Crossref PubMed Scopus (96) Google Scholar and are capable of generating the appropriate co-stimulatory molecules and cytokines that support Th17 development. Among the receptors known to induce Th17-promoting factors is dectin-1, a C-type lectin receptor that recognizes β-glucans expressed on cell walls of fungi.8Ariizumi K. Shen G.-L. Shikano S. Xu S. Ritter R. Kumamoto T. Edelbaum D. Morita A. Bergstresser P.R. Takashima A. Identification of a novel dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning.J Biol Chem. 2000; 275: 20157-20167Crossref PubMed Scopus (375) Google Scholar, 9Brown G.D. Gordon S. Immune recognition: a new receptor for β-glucans.Nature. 2001; 413: 36-37Crossref PubMed Scopus (1265) Google Scholar Engagement of dectin-1 with the β-glucan curdlan or Candida albicans has been shown to stimulate IL-23 production in DCs and promote Th17 cell responses in vitro and in vivo.10LeibundGut-Landmann S. Grosz O. Robinson M.J. Osorio F. Slack E.C. Tsoni S.V. Schweighoffer E. Tybulewicz V. Brown G.D. Ruland J. Reis e Sousa C. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17.Nat Immunol. 2007; 8: 630-638Crossref PubMed Scopus (916) Google Scholar, 11Gringhuis S.I. den Dunnen J. Litjens M. van der Vlist M. Wevers B. Bruijns S.C.M. Geijtenbeek T.B.H. Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-κB activation through Raf-1 and Syk.Nat Immunol. 2009; 10: 203-213Crossref PubMed Scopus (336) Google Scholar Members of the Toll-like receptor (TLR) family, specifically TLR2, TLR3, TLR4, and TLR9, have also been reported to elicit Th17 responses through the induction of Th17-promoting cytokines.6Veldhoen M. Hocking R.J. Atkins C.J. Locksley R.M. Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.Immunity. 2006; 24: 179-189Abstract Full Text Full Text PDF PubMed Scopus (2955) Google Scholar, 12Acosta-Rodriguez E.V. Napolitani G. Lanzavecchia A. Sallusto F. Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17-producing human T helper cells.Nat Immunol. 2007; 8: 942-949Crossref PubMed Scopus (1499) Google Scholar Galectin-3 is a β-galactoside–binding animal lectin, a pleiotropic protein capable of participating in a variety of cellular processes through intracellular and extracellular mechanisms.13Liu F.-T. Patterson R.J. Wang J.L. Intracellular functions of galectins.Biochim Biophys Acta. 2002; 1572: 263-273Crossref PubMed Scopus (538) Google Scholar, 14Ochieng J. Furtak V. Lukyanov P. Extracellular functions of galectin-3.Glycoconj J. 2004; 19: 527-535Crossref PubMed Scopus (279) Google Scholar Extracellular galectin-3 has been shown to modulate cell adhesion, cell activation, and cell migration,15Dumic J. Dabelic S. Flogel M. Galectin-3: an open-ended story.Biochim Biophys Acta. 2006; 1760: 616-635Crossref PubMed Scopus (808) Google Scholar whereas intracellular galectin-3 has been implicated in the regulation of cell survival,16Yang R.-Y. Hsu D.K. Liu F.-T. Expression of galectin-3 modulates T-cell growth and apoptosis.Proc Natl Acad Sci U S A. 1996; 93: 6737-6742Crossref PubMed Scopus (660) Google Scholar pre-mRNA splicing,17Dagher S.F. Wang J.L. Patterson R.J. Identification of galectin-3 as a factor in pre-mRNA splicing.Proc Natl Acad Sci U S A. 1995; 92: 1213-1217Crossref PubMed Scopus (354) Google Scholar and phagocytosis.18Sano H. Hsu D.K. Apgar J.R. Yu L. Sharma B.B. Kuwabara I. Izui S. Liu F.-T. Critical role of galectin-3 in phagocytosis by macrophages.J Clin Invest. 2003; 112: 389-397Crossref PubMed Scopus (320) Google Scholar We previously demonstrated that intracellular galectin-3 translocates to lipid raft microdomains in mouse bone marrow–derived DCs (BMDCs) on chemokine receptor activation and positively regulates DC migration.19Hsu D.K. Chernyavsky A.I. Chen H.-Y. Yu L. Grando S.A. Liu F.-T. Endogenous galectin-3 is localized in membrane lipid rafts and regulates migration of dendritic cells.J Invest Dermatol. 2008; 129: 573-583Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Increasing evidence suggests that galectin-3 may also play a role in the regulation of Th1/Th2 differentiation by affecting IL-12 production in DCs.20Zuberi R.I. Hsu D.K. Kalayci O. Chen H.-Y. Sheldon H.K. Yu L. Apgar J.R. Kawakami T. Lilly C.M. Liu F.-T. Critical role for galectin-3 in airway inflammation and bronchial hyperresponsiveness in a murine model of asthma.Am J Pathol. 2004; 165: 2045-2053Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 21Bernardes E.S. Silva N.M. Ruas L.P. Mineo J.R. Loyola A.M. Hsu D.K. Liu F.-T. Chammas R. Roque-Barreira M.C. Toxoplasma gondii infection reveals a novel regulatory role for galectin-3 in the interface of innate and adaptive immunity.Am J Pathol. 2006; 168: 1910-1920Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 22Saegusa J. Hsu D.K. Chen H.-Y. Yu L. Fermin A. Fung M.A. Liu F.-T. Galectin-3 is critical for the development of the allergic inflammatory response in a mouse model of atopic dermatitis.Am J Pathol. 2009; 174: 922-931Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar However, the role of galectin-3 in Th17 development remains largely undefined. Regarding innate immunity, galectin-3 has been shown to be involved in the recognition of several microbial species and innate defense.23Rabinovich G.A. Gruppi A. Galectins as immunoregulators during infectious processes: from microbial invasion to the resolution of the disease.Parasite Immunol. 2005; 27: 103-114Crossref PubMed Scopus (103) Google Scholar Its expression is essential for the recognition of C. albicans by macrophages,24Jouault T. El Abed-El Behi M. Martínez-Esparza M. Breuilh L. Trinel P.-A. Chamaillard M. Trottein F. Poulain D. Specific recognition of Candida albicans by macrophages requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling.J Immunol. 2006; 177: 4679-4687Crossref PubMed Scopus (190) Google Scholar, 25Esteban A. Popp M.W. Vyas V.K. Strijbis K. Ploegh H.L. Fink G.R. Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages.Proc Natl Acad Sci U S A. 2011; 108: 14270-14275Crossref PubMed Scopus (107) Google Scholar and it has fungicidal activity against Candida species,26Kohatsu L. Hsu D.K. Jegalian A.G. Liu F.-T. Baum L.G. Galectin-3 induces death of Candida species expressing specific beta-1,2-linked mannans.J Immunol. 2006; 177: 4718-4726Crossref PubMed Scopus (177) Google Scholar indicating a direct role for the lectin in antifungal immunity. Whether galectin-3 plays a role in antifungal defense through the Th17 response has not been investigated. Using an ovalbumin (OVA)-specific T-cell activation model, we compared antigen-specific Th17 polarization induced by gal3+/+ and gal3−/− BMDCs. Gal3−/− DCs primed with curdlan or high-dose lipopolysaccharide (LPS) produced higher levels of IL-17–axis cytokines and induced higher Th17 responses compared with gal3+/+ DCs. These findings were also observed in the presence of lactose in culture media, suggesting that galectin-3 affects DC functions through an intracellular mechanism. In addition to differences in cytokine secretion, gal3−/− DCs treated with either curdlan or high-dose LPS exhibited impaired mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, suggesting that galectin-3 may regulate DC cytokine expression in response to dectin-1 and TLR4 stimulation through a common mechanism. Furthermore, curdlan-stimulated gal3−/− DCs expressed higher levels of c-Rel, a critical transcription factor involved in IL-23 production. On closer examination of dectin-1 signaling pathways, we found that curdlan-stimulated gal3−/− DCs expressed lower levels of phosphorylated Raf-1, a serine-threonine kinase that negatively regulates IL-23. In agreement with our in vitro studies, the adoptive transfer of C. albicans–exposed gal3−/− DCs into mice induced higher Th17 responses and promoted greater fungal clearance than gal3+/+ DCs, indicating that galectin-3 in DCs plays a pivotal role in the induction of antifungal immunity in vivo. Taken together, these findings reveal a novel role for endogenous galectin-3 in the regulation of DC cytokine expression involved in Th17 induction. Thus, targeting galectin-3 in DCs may be a means to modulate adaptive immune responses to fungal infections and in autoimmune disease. Gal3−/− mice were developed as previously described27Hsu D.K. Yang R.-Y. Pan Z. Yu L. Salomon D.R. Fung-Leung W.-P. Liu F.-T. Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses.Am J Pathol. 2000; 156: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar and were backcrossed to C57BL/6 mice for nine generations. Gal3+/+ and gal3−/− littermates obtained from heterozygous breeders were used to prepare BMDCs. OVA-TCR transgenic mice (OT-II) on the C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice used in adoptive transfer experiments were bred in-house or were purchased from The Jackson Laboratory. All the experiments were approved by the Institutional Animal Care and Use Committee of the University of California, Davis (Sacramento, CA) or the National Taiwan University College of Medicine (Taipei, Taiwan). LPS from Escherichia coli O111:B4 was from List Biological Laboratories (Campbell, CA). Heat-killed C. albicans (HKCA) was from InvivoGen (San Diego, CA). β-Lactose, sucrose, and curdlan were from Sigma-Aldrich (St. Louis, MO). Recombinant granulocyte-macrophage colony-stimulating factor, IL-1β, IL-6, IL-10, IL-12, IL-17A, and interferon γ (IFN-γ) were from PeproTech (Rocky Hill, NJ) and recombinant IL-23 was from eBioscience Inc. (San Diego, CA). All capture and detection antibodies for enzyme-linked immunosorbent assays (ELISAs) and neutralizing antibodies against IL-6 (MP5-20F3), IL-23 p19 (G23-8), and IL-12/23 p40 (C17.8) were from eBioscience Inc. Anti–phospho-p44/p42 (ERK1/2) mitogen-activated protein kinase, total p44/p42, and anti–phospho–Raf-1 (Ser 338) (56A6) were from Cell Signaling Technology Inc. (Danvers, MA), and anti–phospho–Raf-1 (Tyr340/341) (44-506G) was from Invitrogen (Carlsbad, CA). Total Raf-1 antibody was from BD Transduction Laboratories (San Jose, CA). Egg whites from organic eggs were extracted under sterile conditions and were lyophilized using a SpeedVac concentrator (Thermo Fisher Scientific Inc., Rockford, IL). Lyophilized egg whites were reconstituted in sterile PBS, and the protein concentrations were determined by bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL). Endotoxin levels were below the level of detection, as determined by Pyrogent gel clot limulus amebocyte lysate assay (Lonza Inc., Walkersville, MD). BMDCs were generated from gal3+/+ and gal3−/− mice by culturing bone marrow cells for 8 to 10 days as described previously28Inaba K. Inaba M. Romani N. Aya H. Deguchi M. Ikehara S. Muramatsu S. Steinman R.M. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.J Exp Med. 1992; 176: 1693-1702Crossref PubMed Scopus (3275) Google Scholar in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Thermo Fisher Scientific Inc.), penicillin/streptomycin (Invitrogen), 2ME (Gibco, Carlsbad, CA), HEPES (Mediatech Inc., a Corning subsidiary, Manassas, VA), minimal essential medium nonessential amino acids (Invitrogen), and 20 ng/mL of granulocyte-macrophage colony-stimulating factor (PeproTech). For measuring cytokine production, 2 × 105 DCs per well were stimulated with 0.1 or 100 ng/mL of LPS; 1, 10, or 100 μg/mL of curdlan; or 105, 106, 107 cells/mL of HKCA in 96-well plates at 200 μL per well for 24 hours. CD4+ cells were purified from OT-II splenocytes using biotinylated anti-mouse CD4+ and a biotin selection kit (STEMCELL Technologies Inc., Vancouver, BC, Canada) according to the manufacturer's instructions. For OT-II activation, DCs were pulsed overnight (approximately 16 hours) with 100 μg/mL of endotoxin-free OVA alone or in the presence of 0.1 ng/mL of LPS, 100 ng/mL of LPS, or 10 μg/mL of curdlan. DCs were washed and co-cultured with CD4+ OT-II cells (100,000 CD4+ cells per well and 20,000 DCs per well) in 96-well round-bottomed plates. In some conditions, neutralizing antibodies were added at 10 μg/mL on day 0. Cell culture supernatants were harvested after 3 days, and cytokines were measured by ELISA. DCs were plated in media without fetal bovine serum at 2 × 106 cells per well in 12-well plates for 2 hours and then were left untreated or stimulated with curdlan or LPS for 5, 10, 30, or 60 minutes. Cell lysates were prepared by resuspending cell pellets in lysis buffer [20 mmol/L Tris (pH 7.4), 137 mmol/L NaCl, 2 mmol/L EDTA (pH 7.4), 1% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany), 25 mmol/L β-glycerophosphate, 2 mmol/L sodium pyrophosphate, 10% glycerol, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 5 mmol/L NaF, and 1× protease inhibitor cocktail] before centrifugation at 12,000 rpm, 4°C for 20 minutes. Twenty micrograms of protein were loaded per lane onto a 10% SDS-PAGE gel, and the separated proteins were transferred onto polyvinylidene difluoride membranes. Membranes were incubated with the indicated antibodies, and bands were detected by using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific Inc.). For cell surface staining of co-stimulatory molecules, DCs were incubated with anti–CD80-PE, anti–CD86-PE, and anti–major histocompatibility complex II–fluorescein isothiocyanate (eBiosciences Inc.) in PBS containing 1% fetal bovine serum and 0.09% sodium azide for 30 minutes and were analyzed by flow cytometry (BD FACScan; BD Biosciences, San Jose, CA). To compare cell viability in DCs after activation, cells were first stained with anti–CD11c–fluorescein isothiocyanate (eBiosciences Inc.) and were subsequently stained with 7-aminoactinomycin D (Sigma-Aldrich) in PBS on ice for 30 minutes. After washing, cells were fixed in 1% paraformaldehyde supplemented with 50 μg/mL of actinomycin D before flow cytometry analysis. To compare regulatory T cell (Treg) induction by curdlan-OVA–stimulated DCs, T-cell–DC co-cultures were harvested after 3 days and were stained using anti–CD4-PE-Cy5 (eBioscience Inc.) and anti–CD25-PE (BD Pharmingen, San Diego, CA). Cells were fixed and permeabilized using a BD Cytofix/Cytoperm kit before intracellular staining with anti–Foxp3–fluorescein isothiocyanate (BD Pharmingen). DCs were stimulated with curdlan for 15 minutes and then were lysed with buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% Nonidet P-40 (Caledon Laboratories Ltd., Georgetown, ON, Canada), 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 5 mmol/L NaF, and protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were immunoprecipitated with mouse anti–Raf-1 and recombinant Protein G-Sepharose 4B (Zymed-Invitrogen, Carlsbad, CA). Phosphorylated Raf-1 was detected by immunoblotting with rabbit anti–phospho–Raf-1 (Ser338) and rabbit anti–phospho–Raf-1 (Tyr340/341). 1 × 106 DCs per milliliter were stimulated with curdlan for 2 hours. Nuclear extracts from DCs were prepared using an NE-PER nuclear and cytoplasmic extraction reagents kit (Thermo Fisher Scientific Inc.). NF-κB DNA binding was performed by using a TransAM NF-κB family kit (Active Motif, Carlsbad, CA). The protocol for immunization with antigen-pulsed DCs was adapted from that of d’Ostiani et al29d’Ostiani C.F. Del Sero G. Bacci A. Montagnoli C. Spreca A. Mencacci A. Ricciardi-Castagnoli P. Romani L. Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans.J Exp Med. 2000; 191: 1661-1674Crossref PubMed Scopus (439) Google Scholar with slight modifications. DCs were stimulated with 5 × 105 HKCA cells/mL overnight. Cells were washed in sterile PBS and were s.c. injected twice, a week apart, into C57BL/6 mice at a concentration of 5 × 105 cells per mouse in 50 μL of PBS. Seven days after the last injection of DCs, splenocytes were harvested and restimulated with HKCA at 1 × 105 cells/mL. CD4+ cells were also purified from splenocytes and were restimulated with HKCA-treated antigen-presenting cells. After 72 hours, supernatants were harvested for cytokines. The following protocol for adoptive transfer of DCs was followed: 1 × 106 DCs/mL were co-cultured with 1 × 106 viable C. albicans cells/mL in RPMI 1640 medium at room temperature for 1 hour. The mixture was subsequently i.v. injected into wild-type (gal3+/+) mice (500 μL per mouse). Brain and kidney were harvested 3 and 6 days after infection. To determine the cytokine levels, brain and kidney tissues were homogenized in lysis buffer (RayBiotech Inc., Norcross, GA). Homogenate supernatants were collected after centrifugation, and cytokine concentrations were measured by ELISA. For fungal burden determination, tissues were homogenized in RPMI 1640 medium using a tissue grinder. Homogenates were serially diluted and plated on YPD agar. Yeast colonies were counted 3 days after incubation at 30°C. CD4+ T cells from the spleen were purified using an EasySep mouse CD4+ selection kit (STEMCELL Technologies Inc.). Total RNA was extracted from purified CD4+ T cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with Moloney Murine Leukemia Virus High-Performance Reverse Transcriptase (Epicentre Biotechnologies, Madison, WI) according to the manufacturer's instructions. Real-time quantitative PCR (qPCR) was performed by using the iCycler real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). The primers (forward and reverse) used for qPCR are as follows: mIl17a: 5′-AGGCAGCAGCGATCATCC-3′ and 5′-TGGAACGGTTGAGGTAGTCTG-3′; mIl-17f: 5′-CTGGAGGATAACACTGTGAGAGT-3′ and 5′-TGCTGAATGGCGACGGAGTTC-3′; RORγt: 5′-CGCCTCACCTGACCTACC-3′ and 5′-TTGCCTCGTTCTGGACTATAC-3′; RORα: 5′-TCTCCCTGCGCTCTCCGCAC-3′ and 5′-TCCACAGATCTTGCATGGA-3′; and mβ-actin: 5′-TGTATGAAGGCTTTGGTCTCCCT-3′ and 5′-AGGTGTGCACTTTTATTGGTCTCAA-3′. Each sample was analyzed in triplicate. The relative cytokine mRNA expression level of each sample was normalized against β-actin expression. H&E and PAS staining of tissue sections was performed in the Pathology Core Laboratory at the Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. Enumeration of infiltrated neutrophils in H&E slides was performed by a certified veterinary pathologist at the Institute of Biomedical Sciences Pathology Core Laboratory based on the presence of polymorphic nuclei and appropriate cell size. Statistical analysis of experimental groups was performed by unpaired Student’s t-tests using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA). P < 0.05 was considered significant. β-Glucans present on the cell wall of fungal species activate dectin-1 and induce the production of Th1- and Th17-polarizing cytokines in DCs.10LeibundGut-Landmann S. Grosz O. Robinson M.J. Osorio F. Slack E.C. Tsoni S.V. Schweighoffer E. Tybulewicz V. Brown G.D. Ruland J. Reis e Sousa C. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17.Nat Immunol. 2007; 8: 630-638Crossref PubMed Scopus (916) Google Scholar To study the effect of galectin-3 on DC cytokine production in response to dectin-1 activation, we compared cytokine profiles in gal3−/− and gal3+/+ DCs after stimulation with the β-glucan curdlan. Curdlan-stimulated gal3−/− DCs secreted higher levels of IL-23 compared with gal3+/+ DCs. In contrast, levels of IL-1β, IL-6, IL-10, and IL-12 were comparable (Figure 1), and levels of TGF-β were undetectable (data not shown). Differences in cytokine levels were not due to differences in cell death after stimulation (Supplemental Figure S1A). Furthermore, curdlan induced surface expression of co-stimulatory molecules on gal3−/− and gal3+/+ DCs to a similar extent (Supplemental Figure S1, B and C). To investigate the ability of galectin-3 to influence DC-mediated Th17 polarization in response to β-glucan, we pulsed gal3−/− or gal3+/+ DCs with endotoxin-free OVA in the presence of curdlan, co-cultured the cells with OT-II CD4+ cells, and measured T-cell cytokines in co-culture supernatants. Gal3−/− DCs induced more antigen-specific IL-17 production relative to gal3+/+ DCs after curdlan treatment (Figure 2A). Apart from Th17 cells, curdlan-stimulated DCs also promote the induction of Th1 cells.10LeibundGut-Landmann S. Grosz O. Robinson M.J. Osorio F. Slack E.C. Tsoni S.V. Schweighoffer E. Tybulewicz V. Brown G.D. Ruland J. Reis e Sousa C. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17.Nat Immunol. 2007; 8: 630-638Crossref PubMed Scopus (916) Google Scholar Curdlan-primed gal3−/− DCs induced higher IFN-γ secretion in T cells relative to gal3+/+ DCs; however, the differences were not statistically significant. As expected, DCs pulsed with endotoxin-free OVA alone failed to activate OT-II cells, presumably owing to relatively low levels of co-stimulatory molecule expression.30Peters M. Dudziak K. Stiehm M. Bufe A. T-cell polarization depends on concentration of the danger signal used to activate dendritic cells.Immunol Cell Biol. 2010; 88: 537-544Crossref PubMed Scopus (36) Google Scholar Using a different approach, we cultured gal3−/− and gal3+/+ DCs with curdlan for 16 hours and tested Th17-polarizing activity in the presence of the superantigen Staphylococcus Enterotoxin B. In agreement with the findings from the OVA-specific T-cell activation model described in Figure 2A, curdlan-primed gal3−/− DCs induced higher IL-17 production in CD4+ cells compared with gal3+/+ DCs (Figure 2B). Tregs have been reported to act as a source of TGF-β to drive Th17 differentiation.6Veldhoen M. Hocking R.J. Atkins C.J. Locksley R.M. Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.Immunity. 2006; 24: 179-189Abstract Full Text Full Text PDF PubMed Scopus (2955) Google Scholar To determine whether galectin-3 in DCs indirectly affected Th17 responses by altering Treg development, we compared the number of CD4+CD25+Foxp3+ cells induced by curdlan-primed gal3−/− and gal3+/+ DCs. The frequencies of Foxp3+ Treg cells induced by both DC genotypes were comparable (Supplemental Figure S2), suggesting that the higher Th17-polarized responses by gal3−/− DCs were independent of Treg induction. Curdlan-induced Th17 cell differentiation was shown to depend on IL-23,10LeibundGut-Landmann S. Grosz O. Robinson M.J. Osorio F. Slack E.C. Tsoni S.V. Schweighoffer E. Tybulewicz V. Brown G.D. Ruland J. Reis e Sousa C. Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17.Nat Immunol. 2007; 8: 630-638Crossref PubMed Scopus (916) Google Scholar suggesting that the enhanced Th17 induction by gal3−/− DCs in this model may be due to differential IL-23 secretion by gal3−/− and gal3+/+ DCs (Figure 1). To confirm the importance of IL-23 in Th17 induction by curdlan-primed DCs, we performed the OVA-specific T-cell activation experiment in the presence of IL-23–neutralizing antibodies. Neutralization of IL-23 p19 decreased Th17 differentiation induced by gal3−/− and gal3+/+ DCs. Unexpectedly, neutralization of IL-23 p40 did not have a significant effect (Figure 3). On the other hand, it reduced Th1 development induced by gal3−/− and gal3+/+ DCs, likely due to the targeting of IL-12 by antibody, which shares the p40 chain with IL-23. As expected, blocking p19 had no effect on Th1-polarized resp" @default.
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