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• W2001947746 abstract "Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response–altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)–mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40–producing cells from the draining lymph nodes of treated Leishmania major–infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar–treated and control groups was not observed in draining lymph node cells from TLR2−/− mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections. Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response–altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)–mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40–producing cells from the draining lymph nodes of treated Leishmania major–infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar–treated and control groups was not observed in draining lymph node cells from TLR2−/− mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections. Pathogens that cause chronic infectious diseases often inhabit host phagocytic cells. Detection of pathogens and initiation of the innate immune response involve interactions between conserved motifs in the ligands as pathogen-associated molecular patterns and in pattern recognition receptors, including toll-like receptors (TLRs) and C-type lectin receptors expressed on host phagocytic cells.1Akira S. Pathogen recognition by innate immunity and its signaling.Proc Jpn Acad Ser B Phys Biol Sci. 2009; 85: 143-156Crossref PubMed Scopus (181) Google Scholar, 2Geijtenbeek T.B. van Vliet S.J. Engering A. ‘t Hart B.A. van Kooyk Y. Self- and nonself-recognition by C-type lectins on dendritic cells.Annu Rev Immunol. 2004; 22: 33-54Crossref PubMed Scopus (415) Google Scholar, 3Janeway Jr, C.A. Medzhitov R. Innate immune recognition.Annu Rev Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6150) Google Scholar Macrophages serve as a predominant phagocytic cell type specialized for identification, phagocytosis, and destruction of invading pathogens. Binding of microbial ligands to pattern recognition receptor triggers various crucial immune effector functions, including the production of proinflammatory cytokines. Glycolipids are major ligands whose components have been shown to induce immune modulation.4Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. Mycobacteria target DC-SIGN to suppress dendritic cell function.J Exp Med. 2003; 197: 7-17Crossref PubMed Scopus (895) Google Scholar, 5Sacks D. Sher A. Evasion of innate immunity by parasitic protozoa.Nat Immunol. 2002; 3: 1041-1047Crossref PubMed Scopus (298) Google Scholar, 6Sacks D.L. Modi G. Rowton E. Spath G. Epstein L. Turco S.J. Beverley S.M. The role of phosphoglycans in Leishmania-sand fly interactions.Proc Natl Acad Sci U S A. 2000; 97: 406-411Crossref PubMed Scopus (174) Google Scholar Carbohydrate chains are abundantly expressed on outer surfaces of most bacterial, protozoan, viral, and fungal pathogens. These chains are without peer in structural diversity. Leishmania parasites are covered by a complex glycocalyx whose glycoconjugate components are thought to be important factors in promoting virulence.7Beverley S.M. Turco S.J. Lipophosphoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania.Trends Microbiol. 1998; 6: 35-40Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 8Dermine J.F. Goyette G. Houde M. Turco S.J. Desjardins M. Leishmania donovani lipophosphoglycan disrupts phagosome microdomains in J774 macrophages.Cell Microbiol. 2005; 7: 1263-1270Crossref PubMed Scopus (56) Google Scholar, 9Descoteaux A. Turco S.J. Glycoconjugates in Leishmania infectivity.Biochim Biophys Acta. 1999; 1455: 341-352Crossref PubMed Scopus (173) Google Scholar, 10Gaur U. Showalter M. Hickerson S. Dalvi R. Turco S.J. Wilson M.E. Beverley S.M. Leishmania donovani lacking the Golgi GDP-Man transporter LPG2 exhibit attenuated virulence in mammalian hosts.Exp Parasitol. 2009; 122: 182-191Crossref PubMed Scopus (44) Google Scholar Leishmania metacyclic promastigote glycocalyx contains several phosphoglycans composed of polymeric disaccharide phosphate repeating units.11Thomas J.R. McConville M.J. Thomas-Oates J.E. Homans S.W. Ferguson M.A. Gorin P.A. Greis K.D. Turco S.J. Refined structure of the lipophosphoglycan of Leishmania donovani.J Biol Chem. 1992; 267: 6829-6833Abstract Full Text PDF PubMed Google Scholar One of the most abundant promastigote surface glycolipids on Leishmania spp. is lipophosphoglycan (LPG), containing 15 to 30 repeating units, which distally bears an external cap oligosaccharide and proximally is anchored to the parasite membrane by glycosyl phosphatidyl inositol.12Ilg T. Proteophosphoglycans of Leishmania.Parasitol Today. 2000; 16: 489-497Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar Many studies have shown that purified LPG has a significant effect on parasite survival, attributed to suppression of host signaling and evasion of activated complement.7Beverley S.M. Turco S.J. Lipophosphoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania.Trends Microbiol. 1998; 6: 35-40Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 8Dermine J.F. Goyette G. Houde M. Turco S.J. Desjardins M. Leishmania donovani lipophosphoglycan disrupts phagosome microdomains in J774 macrophages.Cell Microbiol. 2005; 7: 1263-1270Crossref PubMed Scopus (56) Google Scholar, 9Descoteaux A. Turco S.J. Glycoconjugates in Leishmania infectivity.Biochim Biophys Acta. 1999; 1455: 341-352Crossref PubMed Scopus (173) Google Scholar Multiple elegant genetic studies altering Leishmania glycolipid expression identified that these molecules are important for parasite virulence and persistence, although there are critical species differences regarding the complex multiple enzymes required to assemble a virulent parasite glycocalyx.13Turco S.J. Spath G.F. Beverley S.M. Is lipophosphoglycan a virulence factor? a surprising diversity between Leishmania species.Trends Parasitol. 2001; 17: 223-226Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar Pathogenic species of Mycobacterium evolved similar strategies to establish long-term infection. The unique composition of the mycobacterial multiglycosylated envelope complex lipoglycans mannosylated lipoarabinomannan (ManLAM) and lipomannan, strategically located at the host-pathogen interface, contributes to immune alteration.14Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan.J Biol Chem. 2003; 278: 5513-5516Crossref PubMed Scopus (222) Google Scholar, 15Naderer T. Vince J.E. McConville M.J. Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host.Curr Mol Med. 2004; 4: 649-665Crossref PubMed Scopus (116) Google Scholar, 16Guenin-Mace L. Simeone R. Demangel C. Lipids of pathogenic Mycobacteria: contributions to virulence and host immune suppression.Transbound Emerg Dis. 2009; 56: 255-268Crossref PubMed Scopus (84) Google Scholar The three distal glycosylated portions of ManLAM were shown to promote mycobacterial binding to the lectin binding receptor DC-SIGN and to promote mycobacterial virulence.14Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan.J Biol Chem. 2003; 278: 5513-5516Crossref PubMed Scopus (222) Google Scholar LPG and ManLAM, implicated in modulating inflammatory responses and supporting continuation of chronic disease,17Vercellone A. Nigou J. Puzo G. Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis.Front Biosci. 1998; 3: e149-e163PubMed Google Scholar provide the cap sugars of focus for this study. In this study, we use unit oligosaccharide carbohydrates coating inert latex beads as a novel model system to determine the immunologic activity of subunit sugars. We used five cap sugars found on Mycobacterium tuberculosis and Mycobacterium bovis ManLAM, Leishmania mexicana and Leishmania major promastigote LPG, in vitro noninfective mutant Leishmania donovani LPG, and inert control sugar lactose to identify the ability of different oligosaccharides to alter the innate immune response. We found that trimannose, found on mycobacterial ManLAM and Leishmania spp. LPG, alters the production of IL-12p40 in macrophages in vitro and in vivo by engaging the TLR2 pathway. When introduced into mice, the trimannose-coated bead had increased numbers of IL-12p40–producing cells and production of proinflammatory cytokines in wild-type but not TLR2−/− mice. Understanding how subunit oligosaccharides, singularly or combined, produce immune alteration aids in the production of responsive targeted immunostimulants against complex pathogens, including M. tuberculosis and Leishmania. Murine macrophage cell line J774.A1 (American Type Culture Collection, Rockville, MD) was cultured in macrophage media (Dulbecco's modified Eagle's medium, 20 mmol/L HEPES, pH 7.3 to 7.4, supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL of penicillin, and 100 μL/mL of streptomycin) at 37°C in 5% CO2. Bone marrow–derived macrophages were obtained as previously as described.18Mukbel R. Petersen C.A. Jones D.E. Soluble factors from Leishmania major-specific CD4(+)T cells and B cells limit L. amazonensis amastigote survival within infected macrophages.Microbes Infect. 2006; 8: 2547-2555Crossref PubMed Scopus (16) Google Scholar Briefly, femur and tibia bone marrow cells were extracted and plated with 30 mL of macrophage media at 37°C in 5% CO2. On day 7, nonadherent cells were removed and adherent cells were harvested. Live cells were counted via Trypan blue exclusion and were resuspended in complete tissue culture medium for further experimentation. Cap sugar–coated beads (CSCBs) were synthesized as described previously.19Song E.H. Osanya A.O. Petersen C.A. Pohl N.L. Synthesis of multivalent tuberculosis and Leishmania-associated capping carbohydrates reveals structure-dependent responses allowing immune evasion.J Am Chem Soc. 2010; 132: 11428-11430Crossref PubMed Scopus (12) Google Scholar Briefly, 1-μm fluorescein isothiocyanate–labeled latex beads (Sigma-Aldrich, St Louis, MO) were linked to each oligosaccharide via amide linkage. Pathogen-derived oligosaccharides were selected for bead attachment and further investigation based on previous studies describing distal cap carbohydrate moieties from Leishmania and Mycobacterium spp.11Thomas J.R. McConville M.J. Thomas-Oates J.E. Homans S.W. Ferguson M.A. Gorin P.A. Greis K.D. Turco S.J. Refined structure of the lipophosphoglycan of Leishmania donovani.J Biol Chem. 1992; 267: 6829-6833Abstract Full Text PDF PubMed Google Scholar, 17Vercellone A. Nigou J. Puzo G. Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis.Front Biosci. 1998; 3: e149-e163PubMed Google Scholar To N link oligosaccharides to commercially produced beads, approximately 50 Eq of each synthesized cap sugar was used for each coupling reaction. A total of 3.5 × 10−4 mmol of cap sugar was required to attain 100% coverage of a single 1-μm latex bead. Carbohydrate linkage was tested via Kaiser colorimetric assay,20Kaiser E. Colescott R.L. Bossinger C.D. Cook P.I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides.Anal Biochem. 1970; 34: 595-598Crossref PubMed Scopus (3505) Google Scholar and a phenol–sulfuric acid assay was used to confirm the sugar density on each bead.21Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Colorimetric method for determination of sugars and related substances.Analytic Chem. 1956; 28: 350-356Crossref Scopus (40620) Google Scholar Beads were normalized to 2.65 × 1010 beads/mL in sterile water at 4°C. Bone marrow–derived macrophages and J774.A1 cells were plated in 24-well plates with glass coverslips at a density of 5 × 105 cells/mL. After 24 hours, macrophages were activated with 100 U/mL of interferon-γ (IFN-γ; BD Pharmingen, San Diego, CA) and 100 ng/mL of lipopolysaccharide (LPS) (Escherichia coli J5; Sigma-Aldrich), followed by the addition of CSCBs at multiplicity of infection of 5:1 beads per cell. Uptake and internalization were assessed via microscopy, and bead entry into cells was not statistically different between bead types (data not shown). Blockade of TLR2 and mannose receptor (MR) on the surface of J774.A1 cells was mediated through the addition of extracellular domain blocking antibodies [anti-human/mouse CD282 (TLR2), clone T2.5 (eBioscience, San Diego, CA), 10 μg/mL, and anti-mouse CD206 (MR), clone MRSD3 (BioLegend, San Diego, CA), 20 μg/mL] for 2 hours, followed by the addition of CSCBs for 16 hours. Control nonspecific antibody responses were analyzed via the addition of mouse IgG1 isotype for TLR2 (20 μg/mL) and rat anti-mouse IgG2a (eBioscience) for CD206/MR (20 μg/mL). Supernatants from bone marrow–derived macrophages and J774.A1 cultures were harvested at various time points after bead treatment. IL-12p40 enzyme-linked immunosorbent assay (ELISA) was performed using commercially available purified IL-12p40 and biotinylated anti–IL-12p40 antibodies (BD Pharmingen), peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), and ABTS microwell peroxidase substrate (Roche, Indianapolis, IN). For in vitro Luminex analysis, cells were cultured in triplicate, and draining lymph node (DLN) cells from in vivo experiments were cultured in duplicate; cell-free supernatants were harvested after 72 hours of culture and then were analyzed for the concentration of tumor necrosis factor-α (TNF-α), IFN-γ, IL-6, IL-4, IL-10, and IL-12p40 (in vitro analysis) and TNF-α, IFN-γ, IL-6, IL-4, IL-10, IL-17, leukemia inhibitory factor, IL-5, and IL-13 (ex vivo analysis) using multiplexed flow cytometric assay (Luminex, Austin, TX). For in vitro analysis, 50 μL of bead suspension was added to each well and was washed. Fifty microliters of sample and standard were added to each well, plate sealed, and shaken for 30 seconds at 1100 rpm and then were incubated for 1 hour at 300 rpm at room temperature and washed; 25 μL of prediluted detection antibody was added and mixed. The plate was incubated for 30 minutes at 300 rpm in the dark. After washing, 50 μL of 1× streptavidin-phosphatidylethanolamine was added to each well and incubated for 10 minutes. Plate was washed and resuspended in 125 μL of the assay buffer, sealed, mixed, and immediately read. Ex vivo samples were processed using a Milliplex map mouse cytokine/chemokine kit (Millipore, Billerica, MA) as described in the package insert. Samples were incubated with the beads for 2 hours at room temperature followed by O/N incubation at 4°C, and samples were acquired using the Luminex default settings. Median fluorescent intensity data were analyzed using the five-parameter logistic curve fitting method for calculating cytokine and chemokine concentrations. IL-12 ELIspots were performed on DLN cells. Immulon 2 plates (Fisher, Fair Lawn, NJ) were coated with 5 μg/mL of IL-12 (R&D Systems, Minneapolis, MN) overnight at 4°C. After washing with PBS, commercially available biotinylated anti–IL-12p40 antibodies (BD Pharmingen) were added at a 1:10,000 dilution in 5% fetal bovine serum overnight at 4°C. ELIspots were developed using 2-amino-2-methyl-1-propanol (ICN Biomedicals Inc., Aurora, OH) and 5-bromo-4-chloro-3-indoly-phosphate (Fisher) and were counted within 24 hours. Five mice per experimental group were injected with CSCBs and infected with L. major in the footpad. Mice were humanely euthanized at time 0 (2 hours), 24 hours, and 48 hours; footpads were processed for histopathologic analysis, and DLNs were processed for IL-12p40 ELIspot and Luminex analysis. All experiments involving animals were performed as approved by the Iowa State University Institutional Animal Care and Use Committee. From each experimental group (n = 5), after humane euthanasia, infected footpads from three mice were processed for immunohistochemical analysis to classify bead-containing cells as C11b+, CD11c+, non-CD11b+, or non-CD11c+ for histologic characterization of the inflammatory response. Entire cross sections of each footpad from three mice per treatment group were evaluated for degree of cellular inflammation. Frozen sections of each footpad were sectioned at 5 μm, stained with H&E for histopathologic evaluation and immunofluorescent labeling for CD11b+ and CD11c+ cells using biotin-labeled anti-mouse CD11b and CD11c antibodies (eBioscience), followed by Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc.). All the slides were examined, counted, and described by a board-certified veterinary pathologist. At least 20 fields (40×) were evaluated per treatment group. The degree of cellular inflammation was assessed via a rising scale from 0 to 3, with a score of 0 representing no inflammation; 1, small numbers of inflammatory cells; 2, moderate numbers of inflammatory cells; and 3, large numbers of inflammatory cells. Percentage of bead-positive CD11b/c was determined by counting the number of fluorescent beads that co-localized with a CD11b- or CD11c-positive cell relative to the total number of beads counted. Statistical analysis was assessed by one-way analysis of variance and between two values was determined via a post–analysis of variance Tukey's multiple comparison test. P < 0.05 was considered statistically significant. This difference was confirmed via Student's t-test. Previous studies have demonstrated that the highly glycosylated distal third of LPG and ManLAM confer and exhibit immunomodulatory activity.10Gaur U. Showalter M. Hickerson S. Dalvi R. Turco S.J. Wilson M.E. Beverley S.M. Leishmania donovani lacking the Golgi GDP-Man transporter LPG2 exhibit attenuated virulence in mammalian hosts.Exp Parasitol. 2009; 122: 182-191Crossref PubMed Scopus (44) Google Scholar, 12Ilg T. Proteophosphoglycans of Leishmania.Parasitol Today. 2000; 16: 489-497Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 14Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan.J Biol Chem. 2003; 278: 5513-5516Crossref PubMed Scopus (222) Google Scholar, 15Naderer T. Vince J.E. McConville M.J. Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host.Curr Mol Med. 2004; 4: 649-665Crossref PubMed Scopus (116) Google Scholar, 16Guenin-Mace L. Simeone R. Demangel C. Lipids of pathogenic Mycobacteria: contributions to virulence and host immune suppression.Transbound Emerg Dis. 2009; 56: 255-268Crossref PubMed Scopus (84) Google Scholar More recently, distal cap sugars were shown to lead to differential virulence17Vercellone A. Nigou J. Puzo G. Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis.Front Biosci. 1998; 3: e149-e163PubMed Google Scholar and alterations in T-cell responses,22Adams E.W. Ratner D.M. Seeberger P.H. Hacohen N. Carbohydrate-mediated targeting of antigen to dendritic cells leads to enhanced presentation of antigen to T cells.Chembiochem. 2008; 9: 294-303Crossref PubMed Scopus (67) Google Scholar but activity could not be attributed to oligosaccharide alone either because purification of the cap sugars from a heterogeneous population of small saccharides present at the end of larger glycoconjugates leads to a muddle of many different sugars or could be demonstrated only after conjugation of oligosaccharide(s) to peptide antigens. We overcame these problems by developing novel automated synthesis processes to build pathogen-derived oligosaccharides and amide link them to fluorescein isothiocyanate–conjugated latex beads.19Song E.H. Osanya A.O. Petersen C.A. Pohl N.L. Synthesis of multivalent tuberculosis and Leishmania-associated capping carbohydrates reveals structure-dependent responses allowing immune evasion.J Am Chem Soc. 2010; 132: 11428-11430Crossref PubMed Scopus (12) Google Scholar Four different pathogen CSCBs, with uncoated beads and inert lactose-coated beads as negative controls, provided a starting point from which to define specific biologic responses to these pathogen-derived oligosaccharides (Figure 1). These pathogen-derived and virulence-related oligosaccharides included α-linked trimannose (Figure 1D), previously shown to cap ManLAM of M. tuberculosis17Vercellone A. Nigou J. Puzo G. Relationships between the structure and the roles of lipoarabinomannans and related glycoconjugates in tuberculosis pathogenesis.Front Biosci. 1998; 3: e149-e163PubMed Google Scholar and LPG of L. donovani.11Thomas J.R. McConville M.J. Thomas-Oates J.E. Homans S.W. Ferguson M.A. Gorin P.A. Greis K.D. Turco S.J. Refined structure of the lipophosphoglycan of Leishmania donovani.J Biol Chem. 1992; 267: 6829-6833Abstract Full Text PDF PubMed Google Scholar Loss of this oligosaccharide cap from M. bovis bacillus Calmette-Guerin and M. tuberculosis ManLAM was shown to reverse inhibition of LPS-induced IL-12 production by human dendritic cells.23Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor.J Immunol. 2001; 166: 7477-7485PubMed Google Scholar We demonstrated the chemical composition and surface availability of these latex bead–bound cap sugars.19Song E.H. Osanya A.O. Petersen C.A. Pohl N.L. Synthesis of multivalent tuberculosis and Leishmania-associated capping carbohydrates reveals structure-dependent responses allowing immune evasion.J Am Chem Soc. 2010; 132: 11428-11430Crossref PubMed Scopus (12) Google Scholar Given previous results regarding the effect of the larger pathogen glycoconjugates ManLAM and LPG on critical cytokine production by macrophages and dendritic cells, we used novel bead-linked oligosaccharides to determine the role of these five pathogen-derived cap sugars alone in altering in vitro macrophage cytokine production. IL-12 is the primary cytokine for initiation of a Th1 immune response.24Heinzel F.P. Schoenhaut D.S. Rerko R.M. Rosser L.E. Gately M.K. Recombinant interleukin 12 cures mice infected with Leishmania major.J Exp Med. 1993; 177: 1505-1509Crossref PubMed Scopus (749) Google Scholar, 25Reiner S.L. Zheng S. Wang Z.E. Stowring L. Locksley R.M. Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection.J Exp Med. 1994; 179: 447-456Crossref PubMed Scopus (341) Google Scholar, 26Sacks D. Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice.Nat Rev Immunol. 2002; 2: 845-858Crossref PubMed Scopus (931) Google Scholar A Th1 response is necessary for in vitro and in vivo removal of intracellular Leishmania and Mycobacterium spp.23Nigou J. Zelle-Rieser C. Gilleron M. Thurnher M. Puzo G. Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor.J Immunol. 2001; 166: 7477-7485PubMed Google Scholar, 27Zaph C. Scott P. Interleukin-12 regulates chemokine gene expression during the early immune response to Leishmania major.Infect Immun. 2003; 71: 1587-1589Crossref PubMed Scopus (32) Google Scholar, 28Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity.Nat Rev Immunol. 2003; 3: 133-146Crossref PubMed Scopus (2958) Google Scholar, 29Jiao X. Lo-Man R. Guermonprez P. Fiette L. Deriaud E. Burgaud S. Gicquel B. Winter N. Leclerc C. Dendritic cells are host cells for mycobacteria in vivo that trigger innate and acquired immunity.J Immunol. 2002; 168: 1294-1301PubMed Google Scholar, 30Aguilar Torrentera F. Laman J.D. Van Meurs M. Adorini L. Muraille E. Carlier Y. Endogenous interleukin-12 is critical for controlling the late but not the early stage of Leishmania mexicana infection in C57BL/6 mice.Infect Immun. 2002; 70: 5075-5080Crossref PubMed Scopus (20) Google Scholar Based on the importance of IL-12 in the in vitro removal of Leishmania and Mycobacterium spp., we tested the ability of CSCBs to drive differential cytokine responses through measurement via ELISA of IL-12p40 production in bead-treated J774 cells. LPS/IFN-γ–stimulated J774 cells exposed to each CSCB induced three alternative responses (Figure 2, A and B). Treatment of J774 cells with trimannose-coated beads led to significantly decreased IL-12p40 production after activation with IFN-γ and LPS (Figure 2A) compared with control IFN-γ and LPS-stimulated cells. Dimannose- and galactose-coated beads increased IL-12p40 production above that observed in cells stimulated with IFN-γ and LPS (Figure 2B). Uncoated beads and beads coated with lactose, a relatively inert sugar, did not significantly change IL-12p40 production from that of control IFN-γ and LPS-stimulated J774 cells (Figure 2, A and B). A similar profile of IL-12p40 production was found using bone marrow–derived macrophages. Rate of bead entry was determined via microscopy and was found to be similar for all bead treatments. To identify whether the differential cytokine production pattern observed after bead treatment was specific to IL-12p40, we analyzed the production of multiple cytokines via Luminex assay. We observed altered proinflammatory cytokine production due to bead treatment in J774 cells, most significantly TNF-α and IL-6 (Figure 2, C and D). There were no significant differences in the production of cytokines not expected to be produced by in vitro macrophages: IL-4, IL-10, and IFN-γ (data not shown). Alteration of macrophage cytokine production by these simple bead-linked oligosaccharides demonstrates that alteration of macrophage cytokine responses by more complex glycolipids, including ManLAM and LPG, is likely to be partially due to alteration by subunit distal cap oligosaccharides. Based on the observed different responses from these cap oligosaccharides, we predicted that these differences may be mediated through varied C-type lectin receptors. Production of IL-12p40 and proinflammatory cytokines by macrophages is initiated by engagement of lectin-binding receptors by one or multiple carbohydrate-bearing ligands found on complex pathogen surfaces.1Akira S. Pathogen recognition by innate immunity and its signaling.Proc Jpn Acad Ser B Phys Biol Sci. 2009; 85: 143-156Crossref PubMed Scopus (181) Google Scholar, 31Bhatt K. Salgame P. Host innate immune response to Mycobacterium tuberculosis.J Clin Immunol. 2007; 27: 347-362Crossref PubMed Scopus (162) Google Scholar" @default.
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• W2001947746 date "2011-09-01" @default.
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• W2001947746 title "Pathogen-Derived Oligosaccharides Improve Innate Immune Response to Intracellular Parasite Infection" @default.
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