[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis H37Ra produces a range of immunogenic β-gentiobiosyl diacylglycerides. We report the total synthesis of several candidate structures and show that these compounds signal weakly through mouse, but not human, Mincle. Structure-activity relationships reveal a striking dependence upon acyl chain length for gentiobiosyl diacylglyceride signalling through Mincle. Significantly, a truncated β-glucosyl diglyceride was shown to provide potent signalling through both human and mouse Mincle and could activate murine bone marrow derived dendritic cells.
Chemical Communications 08/2015; DOI:10.1039/c5cc04773k · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An enantioselective synthesis of (+)-corynomycolic acid, and its elaboration to esters of trehalose, glucose and glycerol, is described. Trehalose dicorynomycolate and trehalose monocorynomycolate activate human and mouse Mincle as effectively as trehalose dimycolate (cord factor). Glucose monomycolate is revealed to be a potent activator of both mouse and human Mincle. Glycerol monomycolate signals through human Mincle, with the activity predominantly residing in the 2'S-isomer.
Chemical Communications 02/2015; 51(24). DOI:10.1039/C5CC00085H · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation.
[Show abstract][Hide abstract] ABSTRACT: Mycobacteria possess various immunomodulatory molecules on the cell wall. Mannose-capped lipoarabinomannan (Man-LAM), a major lipoglycan of Mycobacterium tuberculosis, has long been known to have both inhibitory and stimulatory effects on host immunity. However, the direct Man-LAM receptor that explains its pleiotropic activities has not been clearly identified. Here, we report that a C-type lectin receptor Dectin-2 (gene symbol Clec4n) is a direct receptor for Man-LAM. Man-LAM activated bone-marrow-derived dendritic cells (BMDCs) to produce pro- and anti-inflammatory cytokines, whereas it was completely abrogated in Clec4n–/– BMDCs. Man-LAM promoted antigen-specific T cell responses through Dectin-2 on DCs. Furthermore, Man-LAM induced experimental autoimmune encephalitis (EAE) as an adjuvant in mice, whereas Clec4n–/– mice were resistant. Upon mycobacterial infection, Clec4n–/– mice showed augmented lung pathology. These results demonstrate that Dectin-2 contributes to host immunity against mycobacterial infection through the recognition of Man-LAM
[Show abstract][Hide abstract] ABSTRACT: Mycobacteria possess various immunomodulatory molecules on the cell wall. Mannose-capped lipoarabinomannan (Man-LAM), a major lipoglycan of Mycobacterium tuberculosis, has long been known to have both inhibitory and stimulatory effects on host immunity. However, the direct Man-LAM receptor that explains its pleiotropic activities has not been clearly identified. Here, we report that a C-type lectin receptor Dectin-2 (gene symbol Clec4n) is a direct receptor for Man-LAM. Man-LAM activated bone-marrow-derived dendritic cells (BMDCs) to produce pro- and anti-inflammatory cytokines, whereas it was completely abrogated in Clec4n–/– BMDCs. Man-LAM promoted antigen-specific T cell responses through Dectin-2 on DCs. Furthermore, Man-LAM induced experimental autoimmune encephalitis (EAE) as an adjuvant in mice, whereas Clec4n–/– mice were resistant. Upon mycobacterial infection, Clec4n–/– mice showed augmented lung pathology. These results demonstrate that Dectin-2 contributes to host immunity against mycobacterial infection through the recognition of Man-LAM.
[Show abstract][Hide abstract] ABSTRACT: Axonal Guillain-Barré syndrome (GBS) is an autoimmune neuropathy characterized by limb weakness and/or paralysis due to the presence of autoantibodies against brain glycolipids. The immune receptors that recognize these autoimmune targets have not been described. In this study, 12 C-type lectin and 10 immunoglobulin-like receptors were screened for their potential ligands from the brain glycolipids, which are the binding targets for GBS autoantibodies. These glycolipids were GM1, GM2, GD1a, GD1b, GQ1b, crude gangliosides, and 3-O-sulfo-β-d-galactosylceramide C24:1 (designated as C24:1). A direct interaction between ligand and receptor was examined using an ELISA-based binding assay. C-type lectin (CLEC5a, SIGNR3) and immunoglobulin-like receptors (TREM2, TREM3, LMIR2, LMIR5, LMIR7, LMIR8) interacted with C24:1. In addition, TREM3 did bind to GQ1b. LMIR5 interacted with GD1a, GQ1b, and crude gangliosides. Binding with highest affinity was observed for the LMIR5-C24:1 interaction, which was selected for further verification. C24:1 was found to induce MCP-1 production, but not proinflammatory cytokines, in basophils. C24:1-induced MCP-1 production was significantly reduced in DAP12(-/-) basophils. Importantly, LMIR5 ligation by C24:1 resulted in NFAT activation through DAP12 in LMIR5-expressing reporter cells. Structural analysis showed that LMIR5 recognized the 3-O-sulfo-β-d-galactose moiety of C24:1. The findings indicated that C24:1 is a potential ligand for DAP12-coupled LMIR5.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.
Proceedings of the National Academy of Sciences 07/2014; 111(30). DOI:10.1073/pnas.1406259111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: LMIR5/CD300b is an activating immunoglobulin-like receptor whose extracellular domain (LMIR5-Fc) is constitutively released from immune cells. The release of LMIR5-Fc is augmented upon stimulation with TLR agonists. LMIR5-Fc is reported to possess inflammatory activity and amplify LPS-induced lethal inflammation; however, its action mechanism has not been clarified. This study was aimed to identify receptors for LMIR5-Fc. Using NF-κB reporter cells in human monocytes THP1, LMIR5-Fc was solely found to trigger NF-κB activation among various signaling receptors examined. In addition, an injection of LMIR5-Fc into the mouse peritoneal resulted in a rapid production of inflammatory mediators and an amplification of LPS activity. Moreover, LMIR5-Fc-induced cytokine production was markedly reduced in TLR4-deficient mouse macrophages. Using TLR4 reporter cells, the LMIR5-Fc sample that contained a trace amount of endotoxin under the sensitivity of reporter cells triggered a potent NF-κB activation. Furthermore, the inflammatory activity of LMIR5-Fc was completely lost by heating but unchanged by polymyxin B pretreatment. Using TLR4 fusion protein, TLR4 was found to interact specifically with LMIR5-overexpressing cells. Therefore, LMIR5-Fc is new inflammatory mediator and endogenous ligand of TLR4. This study provides an insight into the positive feedback mechanism of inflammation through TLR4-LMIR5-Fc axis.
[Show abstract][Hide abstract] ABSTRACT: An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174-176 and 195-196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knockout mice (i.e. hMincle(+) mice) were established and compared with non-transgenic mice (i.e. mMincle(+) mice). We showed that macrophages derived from hMincle(+) mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle(+) mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle(+), but not mMincle(+) mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.
[Show abstract][Hide abstract] ABSTRACT: Immune activation as a result of the recognition of damage-associated molecular patterns needs to be controlled. In this issue of Immunity, Neumann et al. (2014) demonstrates that Clec12a is a receptor for dead cells through the recognition of uric acid crystals and contributes to the dampening of the responses.
[Show abstract][Hide abstract] ABSTRACT: The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.Immunology and Cell Biology advance online publication, 18 March 2014; doi:10.1038/icb.2014.18.
[Show abstract][Hide abstract] ABSTRACT: T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.
[Show abstract][Hide abstract] ABSTRACT: Guinea pig is a widely used animal for research and development of tuberculosis vaccines, since its pathological disease process is similar to that present in humans. We have previously reported that two C-type lectin receptors, Mincle (macrophage inducible C-type lectin, also called Clec4e) and MCL (macrophage C-type lectin, also called Clec4d), recognize the mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM). Here, we characterized the function of the guinea pig homologue of Mincle (gpMincle) and MCL (gpMCL). gpMincle directly bound to TDM and transduced an activating signal through ITAM-bearing adaptor molecule, FcRγ. Whereas, gpMCL lacked C-terminus and failed to bind to TDM. mRNA expression of gpMincle was detected in the spleen, lymph nodes and peritoneal macrophages and it was strongly up-regulated upon stimulation of zymosan and TDM. The surface expression of gpMincle was detected on activated macrophages by a newly established monoclonal antibody that also possesses a blocking activity. This antibody potently suppressed TNF production in BCG-infected macrophages. Collectively, gpMincle is the TDM receptor in the guinea pig and TDM-Mincle axis is involved in host immune responses against mycobacteria.
PLoS ONE 02/2014; 9(2):e88747. DOI:10.1371/journal.pone.0088747 · 3.23 Impact Factor