Mincle Signaling Research Articles

Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity

Mincle, expressed in activated phagocytes, recognizes the lipid ligand to activate the innate immune system. We have synthesized glycerol derivatives possessing simple alkyl chains or aromatic rings and elucidated their structure-activity relationships using a Mincle-mediated signaling assay. The activity depends on the length of the simple acyl chains of the glycerol derivatives.

Microglial Mincle receptor in the PVN contributes to sympathetic hyperactivity in acute myocardial infarction rat

Malignant ventricular arrhythmias (VAs) following myocardial infarction (MI) is a lethal complication resulting from sympathetic nerve hyperactivity. Numerous evidence have shown that inflammation within the paraventricular nucleus (PVN) participates in sympathetic hyperactivity. Our aim was to explore the role of Macrophage‐inducible C‐type lectin (Mincle) within the PVN in augmenting sympathetic activity following MI,and whether NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome/IL‐1β axis is involved in this activity. MI was induced by coronary artery ligation. Mincle expression localized in microglia within the PVN was markedly increased at 24 hours post‐MI together with sympathetic hyperactivity, as indicated by measurement of the renal sympathetic nerve activity (RSNA) and norepinephrine (NE) concentration. Mincle‐specific siRNA was administrated locally to the PVN, which consequently decreased microglial activation and sympathetic nerve activity. The MI rats exhibited a higher arrhythmia score after programmed electric stimulation than that treated with Mincle siRNA, suggesting that the inhibition of Mincle attenuated foetal ventricular arrhythmias post‐MI. The underlying mechanism of Mincle in sympathetic hyperactivity was investigated in lipopolysaccharide (LPS)‐primed naïve rats. Recombinant Sin3A‐associated protein 130kD (rSAP130), an endogenous ligand for Mincle, induced high levels of NLRP3 and mature IL‐1β protein. PVN‐targeted injection of NLRP3 siRNA or IL‐1β antagonist gevokizumab attenuated sympathetic hyperactivity. Together, the data indicated that the knockdown of Mincle in microglia within the PVN prevents VAs by attenuating sympathetic hyperactivity and ventricular susceptibility, in part by inhibiting its downstream NLRP3/IL‐1β axis following MI. Therapeutic interventions targeting Mincle signalling pathway could constitute a novel approach for preventing infarction injury.

Integrin CD11b negatively regulates Mincle-induced signaling via the Lyn\u2013SIRP\u03b1\u2013SHP1 complex

During mycobacteria infection, anti-inflammatory responses allow the host to avoid tissue damage caused by overactivation of the immune system; however, little is known about the negative modulators that specifically control mycobacteria-induced immune responses. Here we demonstrate that integrin CD11b is a critical negative regulator of mycobacteria cord factor-induced macrophage-inducible C-type lectin (Mincle) signaling. CD11b deficiency resulted in hyperinflammation following mycobacterial infection. Activation of Mincle by mycobacterial components turns on not only the Syk signaling pathway but also CD11b signaling and induces formation of a Mincle–CD11b signaling complex. The activated CD11b recruits Lyn, SIRPα and SHP1, which dephosphorylate Syk to inhibit Mincle-mediated inflammation. Furthermore, the Lyn activator MLR1023 effectively suppressed Mincle signaling, indicating the possibility of Lyn-mediated control of inflammatory responses. These results describe a new role for CD11b in fine-tuning the immune response against mycobacterium infection.

Trehalose dimycolate interferes with FcγR-mediated phagosome maturation through Mincle, SHP-1 and FcγRIIB signalling.

The causative agent of tuberculosis, Mycobacterium tuberculosis (M. tuberculosis), contains an abundant cell wall glycolipid and a crucial virulence factor, trehalose-6,6’-dimycolate (TDM). TDM causes delay of phagosome maturation and thus promotes survival of mycobacteria inside host macrophages by a not fully understood mechanism. TDM signals through the Monocyte-INducible C-type LEctin (Mincle), a recently identified pattern recognition receptor. Here we show that recruitment of Mincle by TDM coupled to immunoglobulin (Ig)G-opsonised beads during Fcγ receptor (FcγR)-mediated phagocytosis interferes with phagosome maturation. In addition, modulation of phagosome maturation by TDM requires SH2-domain-containing inositol polyphosphate 5’ phosphatase (SHP-1) and the FcγRIIB, which strongly suggests inhibitory downstream signalling of Mincle during phagosome formation. Overall, our study reveals important mechanisms contributing to the virulence of TDM.

Mincle activation enhances neutrophil migration and resistance to polymicrobial septic peritonitis

Sepsis is a systemic inflammatory response to bacterial infection. The therapeutic options for treating sepsis are limited. Impaired neutrophil recruitment into the infection site is directly associated with severe sepsis, but the precise mechanism is unclear. Here, we show that Mincle plays a key role in neutrophil migration and resistance during polymicrobial sepsis. Mincle-deficient mice exhibited lower survival rates in experimental sepsis from cecal ligation and puncture and Escherichia coli–induced peritonitis. Mincle deficiency led to higher serum inflammatory cytokine levels and reduced bacterial clearance and neutrophil recruitment. Transcriptome analyses revealed that trehalose dimycolate, a Mincle ligand, reduced the expression of G protein–coupled receptor kinase 2 (GRK2) in neutrophils. Indeed, GRK2 expression was upregulated, but surface expression of the chemokine receptor CXCR2 was downregulated in blood neutrophils from Mincle-deficient mice with septic injury. Moreover, CXCL2-mediated adhesion, chemotactic responses, and F-actin polymerization were reduced in Mincle-deficient neutrophils. Finally, we found that fewer Mincle-deficient neutrophils infiltrated from the blood circulation into the peritoneal fluid in bacterial septic peritonitis compared with wild-type cells. Thus, our results indicate that Mincle plays an important role in neutrophil infiltration and suggest that Mincle signaling may provide a therapeutic target for treating sepsis.

Abstract B20: The necrosome promotes pancreatic Oncogenesis via CXCL1 and mincle-induced immune Suppression

Abstract Neoplastic pancreatic epithelial cells are widely believed to die via Caspase 8-dependantapoptotic cell death and chemotherapy is thought to further promote tumor apoptosis. Conversely, disruption of apoptosis is a basic modality cancer cells exploit for survival. However, the role of necroptosis, or programmed necrosis, in pancreatic ductal adenocarcinoma (PDA) is uncertain. There are a multitude of potential inducers of necroptosis in PDA including ligation of TNFR1, CD95, TRAIL receptors, Toll-like receptors, ROS, and Chemotherapeutics. We doscovered that the principal components of the necrosome, RIP1 and RIP3, are highly expressed in PDA and are further upregulated by chemotherapy. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. However, belying these findings, in vivo RIP3 deletion or RIP1 inhibition was protective against oncogenic progression and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumor microenvironment (TME) associated with intact RIP1/RIP3 signaling was in-part contingent on necroptosis-induced CXCL1 expression whereas CXCL1 blockade was protective against PDA. Moreover, we found that cytoplasmic SAP130 was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle – its cognate receptor – was upregulated in tumor-infiltrating myeloid cells. Mincle ligation by SAP130 promoted oncogenesis whereas Mincle deletion was protective and phenocopied the immunogenic reprogramming of the TME characteristic of RIP3 deletion. Cellular depletion experiments suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects in the context of RIP3 or Mincle deletion. As such, T cells which are dispensable to PDA progression in hosts with intact RIP3 or Mincle signaling become reprogrammed into indispensable mediators of anti-tumor immunity in absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signaling which critically promote macrophage-induced adaptive immune suppression enabling PDA progression. Citation Format: Lena Seifert, Gregor Werba, Shaun Tiwari, Ly Nancy Ngoc Giao, Dalia Alqunaibit, Sara Alothman, Donnele Daley, Mautin Hundeyin, Vishnu R. Mani, Rocky Barilla, George Miller.{Authors}. The necrosome promotes pancreatic Oncogenesis via CXCL1 and mincle-induced immune Suppression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B20.

Mincle Signaling Promotes Con A Hepatitis.

Con A hepatitis is regarded as a T cell-mediated model of acute liver injury. Mincle is a C-type lectin receptor that is critical in the immune response to mycobacteria and fungi but does not have a well-defined role in preclinical models of non-pathogen-mediated inflammation. Because Mincle can ligate the cell death ligand SAP130, we postulated that Mincle signaling drives intrahepatic inflammation and liver injury in Con A hepatitis. Acute liver injury was assessed in the murine Con A hepatitis model using C57BL/6, Mincle(-/-), and Dectin-1(-/-) mice. The role of C/EBPβ and hypoxia-inducible factor-1α (HIF-1α) signaling was assessed using selective inhibitors. We found that Mincle was highly expressed in hepatic innate inflammatory cells and endothelial cells in both mice and humans. Furthermore, sterile Mincle ligands and Mincle signaling intermediates were increased in the murine liver in Con A hepatitis. Most significantly, Mincle deletion or blockade protected against Con A hepatitis, whereas Mincle ligation exacerbated disease. Bone marrow chimeric and adoptive transfer experiments suggested that Mincle signaling in infiltrating myeloid cells dictates disease phenotype. Conversely, signaling via other C-type lectin receptors did not alter disease course. Mechanistically, we found that Mincle blockade decreased the NF-κβ-related signaling intermediates C/EBPβ and HIF-1α, both of which are necessary in macrophage-mediated inflammatory responses. Accordingly, Mincle deletion lowered production of nitrites in Con A hepatitis and inhibition of both C/EBPβ and HIF-1α reduced the severity of liver disease. Our work implicates a novel innate immune driver of Con A hepatitis and, more broadly, suggests a potential role for Mincle in diseases governed by sterile inflammation.

The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincle-induced immune suppression.

Neoplastic pancreatic epithelial cells are widely believed to die via Caspase 8-dependant apoptotic cell death and chemotherapy is thought to further promote tumor apoptosis1. Conversely, disruption of apoptosis is a basic modality cancer cells exploit for survival2,3. However, the role of necroptosis, or programmed necrosis, in pancreatic ductal adenocarcinoma (PDA) is uncertain. There are a multitude of potential inducers of necroptosis in PDA including ligation of TNFR1, CD95, TRAIL receptors, Toll-like receptors, ROS, and Chemotherapeutics4,5. Here we report that the principal components of the necrosome, RIP1 and RIP3, are highly expressed in PDA and are further upregulated by chemotherapy. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo RIP3 deletion or RIP1 inhibition was protective against oncogenic progression and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumor microenvironment (TME) associated with intact RIP1/RIP3 signaling was in-part contingent on necroptosis-induced CXCL1 expression whereas CXCL1 blockade was protective against PDA. Moreover, we found that cytoplasmic SAP130 was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle – its cognate receptor – was upregulated in tumor-infiltrating myeloid cells. Mincle ligation by SAP130 promoted oncogenesis whereas Mincle deletion was protective and phenocopied the immunogenic reprogramming of the TME characteristic of RIP3 deletion. Cellular depletion experiments suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects in the context of RIP3 or Mincle deletion. As such, T cells which are dispensable to PDA progression in hosts with intact RIP3 or Mincle signaling become reprogrammed into indispensable mediators of anti-tumor immunity in absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signaling which critically promote macrophage-induced adaptive immune suppression enabling PDA progression.

Fonsecaea pedrosoi-induced Th17-cell differentiation in mice is fostered by Dectin-2 and suppressed by Mincle recognition.

Chromoblastomycosis is a chronic skin infection caused by the pigmented saprophytic mould Fonsecaea pedrosoi. Chronicity of infection can be broken by a coordinated innate recognition of the spores by pattern recognition receptors. While Mincle signaling via the Syk/Card9 pathway is required for fungal recognition by host cells, it is not sufficient for host control. Exogenously applied TLR agonists are necessary to promote the induction of proinflammatory cytokines and clearance of infection in vivo. Here, we investigated whether costimulation by TLR agonists fosters the development of adaptive immune responses, by examining the development of fungus‐specific T cells. Subcutaneous infection of mice with F. pedrosoi spores induced the activation, expansion, and differentiation of Ag‐specific CD4+ T cells but TLR costimulation did not further augment these T‐cell responses. The Dectin‐2/FcRγ/Card9 signaling pathway promoted the differentiation of fungus‐specific CD4+ T cells into Th17 cells, whereas Mincle inhibited the development of this T‐helper subset in infected mice. These results indicate differential roles for Dectin‐2 and Mincle in the generation of adaptive immune responses to F. pedrosoi infection.

C-Type Lectin Receptor MCL Facilitates Mincle Expression and Signaling through Complex Formation.

C-type lectin receptors expressed in APCs are recently defined pattern recognition receptors that play a crucial role in immune responses against pathogen-associated molecular patterns. Among pathogen-associated molecular patterns, cord factor (trehalose-6,6'-dimycolate [TDM]) is the most potent immunostimulatory component of the mycobacterial cell wall. Two C-type lectin receptors, macrophage-inducible C-type lectin (Mincle) and macrophage C-type lectin (MCL), are required for immune responses against TDM. Previous studies indicate that MCL is required for TDM-induced Mincle expression. However, the mechanism by which MCL induces Mincle expression has not been fully understood. In this study, we demonstrate that MCL interacts with Mincle to promote its surface expression. After LPS or zymosan stimulation, MCL-deficient bone marrow-derived dendritic cells (BMDCs) had a lower level of Mincle protein expression, although mRNA expression was comparable with wild-type BMDCs. Meanwhile, BMDCs from MCL transgenic mice showed an enhanced level of Mincle expression on the cell surface. MCL was associated with Mincle through the stalk region and this region was necessary and sufficient for the enhancement of Mincle expression. This interaction appeared to be mediated by the hydrophobic repeat of MCL, as substitution of four hydrophobic residues within the stalk region with serine (MCL(4S)) abolished the function to enhance the surface expression of Mincle. MCL(4S) mutant failed to restore the defective TDM responses in MCL-deficient BMDCs. These results suggest that MCL positively regulates Mincle expression through protein-protein interaction via its stalk region, thereby magnifying Mincle-mediated signaling.

Mincle signaling in the innate immune response after traumatic brain injury.

The innate immune response contributes to the inflammatory activity after traumatic brain injury (TBI). In the present study we identify macrophage-inducible C-type lectin (mincle) as a pattern recognition receptor that contributes to innate immunity in neurons after TBI. Here we report that mincle is activated by SAP130 in cortical neurons in culture, resulting in production of the inflammatory cytokine TNF. In addition, mincle and SAP130 are elevated in the brain and cerebrospinal fluid of humans after TBI and the brain of rodents after fluid percussion brain injury. Thus, these findings suggest the involvement of mincle to the pathology of TBI. Importantly, blocking mincle with a neutralizing antibody against mincle in cortical neurons in culture treated with SAP130 resulted in inhibition of mincle signaling and decreased TNF production. Therefore, our findings identify mincle as a contributor to the inflammatory response after TBI.

When PRRs Collide: Mincle Meddles with Dectin and Toll

Pattern recognition receptors (PRR) promote pathogen eradication and shape adaptive immunity. Although evidence suggests PRRs can antagonize each other, few detailed mechanisms are known. Wevers etal. (2014) uncover a mechanism of PRR antagonism where fungal-induced Mincle signaling suppresses IL-12 transcription induced by other PRRs and thereby abates antifungal immunity.

Mincle signaling contributes to the inflammatory response after traumatic brain injury (P1297)

Abstract The inflammatory response after traumatic brain injury (TBI) contributes to neuronal death and progressive axonal loss to areas undamaged by the initial trauma. Moreover, anti-inflammatory therapies offer great hope to improve outcomes after TBI. Thus, it is important to find therapeutic targets to better control the inflammatory response after injury. In this study we used cells in culture, an animal model of TBI as well as brain and CSF samples obtained from patients with TBI to investigate, after injury, the role of the CLR family member, macrophage-inducible C-type lectin (mincle) in the production of the inflammatory cytokine TNF. Here we report that mincle is activated after TBI in both, humans and rodents. Our data show that moderate fluid percussion brain injury (using male Sprague-Dawley rats (250-350 g) subjected to moderate fluid-percussion brain injury (1.8-2.2 atm)) is associated with increase expression of SAP130, which activates mincle signaling resulting in phosphorylation of the enzyme syk. Moreover, we identified increased expression of SAP130 and mincle in cortical neurons after TBI. In addition, we show that SAP130 stimulates mincle signaling in primary neuronal cultures, resulting in increase expression of the inflammatory cytokine TNF. Acute TNF expression after TBI is associated with deleterious outcomes. In conclusion, these findings suggest mincle as a novel therapeutic target that can be used to lower the levels of TNF after CNS injury.

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.