SAP130 mediates crosstalk between hepatocyte ferroptosis and M1 macrophage polarization in PFOS-induced hepatotoxicity.

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant widely utilized in industrial manufacturing and daily life, leading to significant environmental accumulation and various public health issues. This study aims to characterize spliceosome-associated protein 130 (SAP130) as a key mediator of crosstalk between hepatocytes and macrophages, elucidating its role in PFOS-induced liver inflammation. The data demonstrate that PFOS exposure induces ferroptosis in mouse liver and AML12 cells. During ferroptosis, SAP130 is released from injured hepatocytes into the microenvironment, binding to macrophage-inducible C-type lectin (Mincle) and activating the Mincle/Syk signaling pathway in macrophages, ultimately promoting M1 polarization and exacerbating liver injury. Treatment with the ferroptosis inhibitor Ferrostatin-1 reduces SAP130 release, inhibits Mincle/Syk signaling activation, and mitigates inflammatory response. Furthermore, siSAP130 suppresses the activation of the Mincle signaling pathway and M1 polarization in BMDM cells. Conversely, treatment with the ferroptosis agonist Erastin enhances paracrine secretion of SAP130 and exacerbates inflammation. These findings emphasize the significance of hepatocyte-macrophage crosstalk as a critical pathway for PFOS-induced liver injury in mice while highlighting SAP130 as a pivotal regulator of ferroptosis and inflammation, thereby elucidating the potential mechanism of PFOS-induced liver injury.

Ectopic Expression of C-Type Lectin Mincle Renders Mice Susceptible to Staphylococcal Pneumonia.

Staphylococcus aureus is a prevalent pathogen in pneumonia and harbors glycolipids, which may serve as molecular patterns in Mincle (macrophage-inducible C-type lectin)-dependent pathogen recognition. We examined the role of Mincle in lung defense against S aureus in wild-type (WT), Mincle knockout (KO), and Mincle transgenic (tg) mice. Two glycolipids, glucosyl-diacylglycerol (Glc-DAG) and diglucosyl-diacylglycerol (Glc2-DAG), were purified, of which only Glc-DAG triggered Mincle reporter cell activation and professional phagocyte responses. Proteomic profiling revealed that Glc2-DAG blocked Glc-DAG-induced cytokine responses, thereby acting as inhibitor of Glc-DAG/Mincle signaling. WT mice responded to S aureus with a similar lung pathology as Mincle KO mice, most likely due to Glc2-DAG-dependent inhibition of Glc-DAG/Mincle signaling. In contrast, ectopic Mincle expression caused severe lung pathology in S aureus-infected mice, characterized by bacterial outgrowth and fatal pneumonia. Collectively, Glc2-DAG inhibits Glc-DAG/Mincle-dependent responses in WT mice, whereas sustained Mincle expression overrides Glc2-DAG-mediated inhibitory effects, conferring increased host susceptibility to S aureus.

Hydrogel activation of Mincle receptors for tumor cell processing: A novel approach in cancer immunotherapy.

An obstacle in current tumor immunotherapies lies in the challenge of achieving sustained and tumor-targeting T cell immunity, impeded by the limited antigen processing and cross-presentation of tumor antigens. Here, we propose a hydrogel-based multicellular immune factory within the body that autonomously converts tumor cells into an antitumor vaccine. Within the body, the scaffold, formed by a calcium-containing chitosan hydrogel complex (ChitoCa) entraps tumor cells and attracts immune cells to establish a durable and multicellular microenvironment. Within this context, tumor cells are completely eliminated by antigen-presenting cells (APCs) and processed for cross-antigen presentation. The regulatory mechanism relies on the Mincle receptor, a cell-phagocytosis-inducing C-type lectin receptor specifically activated on ChitoCa-recruited APCs, which serves as a recognition synapse, facilitating a tenfold increase in tumor cell engulfment and subsequent elimination. The ChitoCa-induced tumor cell processing further promotes the cross-presentation of tumor antigens to prime protective CD8+ T cell responses. Therefore, the ChitoCa treatment establishes an immune niche within the tumor microenvironment, resulting in effective tumor regression either used alone or in combination with other immunotherapies. This hydrogel-induced immune factory establishes a functional organ-like multicellular colony for tumor-specific immunotherapy, paving the way for innovative strategies in cancer treatment.

Promotion of Th17 Polarized Immunity via Co-Delivery of Mincle Agonist and Tuberculosis Antigen Using Silica Nanoparticles.

Adjuvants and immunomodulators that effectively drive a Th17-skewed immune response are not part of the standard vaccine toolkit. Vaccine adjuvants and delivery technologies that can induce Th17 or Th1/17 immunity and protection against bacterial pathogens, such as tuberculosis (TB), are urgently needed. Th17-polarized immune response can be induced using agonists that bind and activate C-type lectin receptors (CLRs) such as macrophage inducible C-type lectin (Mincle). A simple but effective strategy was developed for codelivering Mincle agonists with the recombinant Mycobacterium tuberculosis fusion antigen, M72, using tunable silica nanoparticles (SNP). Anionic bare SNP, hydrophobic phenyl-functionalized SNP (P-SNP), and cationic amine-functionalized SNP (A-SNP) of different sizes were coated with three synthetic Mincle agonists, UM-1024, UM-1052, and UM-1098, and evaluated for adjuvant activity in vitro and in vivo. The antigen and adjuvant were coadsorbed onto SNP via electrostatic and hydrophobic interactions, facilitating multivalent display and delivery to antigen presenting cells. The cationic A-SNP showed the highest coloading efficiency for the antigen and adjuvant. In addition, the UM-1098-adsorbed A-SNP formulation demonstrated slow-release kinetics in vitro, excellent stability over 12 months of storage, and strong IL-6 induction from human peripheral blood mononuclear cells. Co-adsorption of UM-1098 and M72 on A-SNP significantly improved antigen-specific humoral and Th17-polarized immune responses in vivo in BALB/c mice relative to the controls. Taken together, A-SNP is a promising platform for codelivery and proper presentation of adjuvants and antigens and provides the basis for their further development as a vaccine delivery platform for immunization against TB or other diseases for which Th17 immunity contributes to protection.

Mincle receptor in macrophage and neutrophil contributes to the unresolved inflammation during the transition from acute kidney injury to chronic kidney disease.

Background: Recent studies have demonstrated a strong association between acute kidney injury (AKI) and chronic kidney disease (CKD), while the unresolved inflammation is believed to be a driving force for this chronic transition process. As a transmembrane pattern recognition receptor, Mincle (macrophage-inducible C-type lectin, Clec4e) was identified to participate in the early immune response after AKI. However, the impact of Mincle on the chronic transition of AKI remains largely unclear. Methods: We performed single-cell RNA sequencing (scRNA-seq) with the unilateral ischemia-reperfusion (UIR) murine model of AKI at days 1, 3, 14 and 28 after injury. Potential effects and mechanism of Mincle on renal inflammation and fibrosis were further validated in vivo utilizing Mincle knockout mice. Results: The dynamic expression of Mincle in macrophages and neutrophils throughout the transition from AKI to CKD was observed. For both cell types, Mincle expression was significantly up-regulated on day 1 following AKI, with a second rise observed on day 14. Notably, we identified distinct subclusters of Minclehigh neutrophils and Minclehigh macrophages that exhibited time-dependent influx with dual peaks characterized with remarkable pro-inflammatory and pro-fibrotic functions. Moreover, we identified that Minclehigh neutrophils represented an "aged" mature neutrophil subset derived from the "fresh" mature neutrophil cluster in kidney. Additionally, we observed a synergistic mechanism whereby Mincle-expressing macrophages and neutrophils sustained renal inflammation by tumor necrosis factor (TNF) production. Mincle-deficient mice exhibited reduced renal injury and fibrosis following AKI. Conclusion: The present findings have unveiled combined persistence of Minclehigh neutrophils and macrophages during AKI-to-CKD transition, contributing to unresolved inflammation followed by fibrosis via TNF-α as a central pro-inflammatory cytokine. Targeting Mincle may offer a novel therapeutic strategy for preventing the transition from AKI to CKD.

Mincle as a potential intervention target for the prevention of inflammation and fibrosis (Review).

Macrophage­inducible C­type lectin receptor (Mincle) is predominantly found on antigen­presenting cells. It can recognize specific ligands when stimulated by certain pathogens such as fungi and Mycobacterium tuberculosis. This recognition triggers the activation of the nuclear factor­κB pathway, leading to the production of inflammatory factors and contributing to the innate immune response of the host. Moreover, Mincle identifies lipid damage­related molecules discharged by injured cells, such as Sin3­associated protein 130, which triggers aseptic inflammation and ultimately hastens the advancement of renal damage, autoimmune disorders and malignancies by fostering tissue inflammation. Presently, research on the functioning of the Mincle receptor in different inflammatory and fibrosis­associated conditions has emerged as a popular topic. Nevertheless, there remains a lack of research on the impact of Mincle in promoting long­lasting inflammatory reactions and fibrosis. Additional investigation is required into the function of Mincle receptors in chronological inflammatory reactions and fibrosis of organ systems, including the progression from inflammation to fibrosis. Hence, the present study showed an overview of the primary roles and potential mechanism of Mincle in inflammation, fibrosis, as well as the progression of inflammation to fibrosis. The aim of the present study was to clarify the potential mechanism of Mincle in inflammation and fibrosis and to offer perspectives for the development of drugs that target Mincle.

Punicalagin promotes mincle-mediated phagocytosis of macrophages via the NF-κB and MAPK signaling pathways.

Punicalagin (PUN) is a polyphenol derived from the pomegranate peel. It has been reported to have many beneficial effects, including anti-inflammatory, anti-oxidant, and anti-proliferation. However, the role of PUN in macrophage phagocytosis is currently unknown. In this study, we found that pre-treatment with PUN significantly enhanced phagocytosis by macrophages in a time- and dose-dependent manner in vitro. Moreover, KEGG enrichment analysis by RNA-sequencing showed that differentially expressed genes following PUN treatment were significantly enriched in phagocyte-related receptors, such as the C-type lectin receptor signaling pathway. Among the C-type lectin receptor family, Mincle (Clec4e) significantly increased at the mRNA and protein level after PUN treatment, as shown by qRT-PCR and western blotting. Small interfering RNA (siRNA) mediated knockdown of Mincle in macrophages resulted in down regulation of phagocytosis. Furthermore, western blotting showed that PUN treatment enhanced the phosphorylation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) in macrophages at the early stage. Mincle-mediated phagocytosis by PUN was inhibited by PDTC (a NF-κB inhibitor) and SB203580 (a p38 MAPK inhibitor). In addition, PUN pre-treatment enhanced phagocytosis by peritoneal and alveolar macrophages in vivo. After intraperitoneal injection of Escherichia coli (E.coli), the bacterial load of peritoneal lavage fluid and peripheral blood in PUN pre-treated mice decreased significantly. Similarly, the number of bacteria in the lung tissue significantly reduced after intranasal administration of Pseudomonas aeruginosa (PAO1). Taken together, our results reveal that PUN enhances bacterial clearance in mice by activating the NF-κB and MAPK pathways and upregulating C-type lectin receptor expression to enhance phagocytosis by macrophages.

Isorhamnetin ameliorates cisplatin-induced acute kidney injury in mice by activating SLPI-mediated anti-inflammatory effect in macrophage.

OBJECTIVE: Isorhamnetin (IH) has been reported to have significant anti-inflammatory effects in various diseases, but its role and mechanism in AKI remain unclear. This study aimed to explore the potential role and mechanism of isorhamnetin in inhibiting macrophage related inflammation and improving AKI injury. METHODS: We established an AKI mouse model by intraperitoneal injection of cisplatin in vivo, and constructed an inflammatory cell model by stimulating RAW264.7 cells with LPS. Creatinine and urea nitrogen were measured to evaluate the changes of renal function in AKI mice. The changes of renal pathological structure were observed by H&E staining. The inflammatory factor-related proteins and RNA expression levels were detected by Western blot and real time PCR. RESULTS: Isorhamnetin protected the kidney from cisplatin induced AKI and significantly inhibited the mRNA and protein levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) both in AKI kidney and LPS-stimulated RAW264.7 cells. Interestingly, the data also demonstrated that isorhamnetin significantly upregulated the expression of secretory leukocyte peptidase inhibitor (SLPI), an anti-inflammatory factor, in AKI kidney and LPS-stimulated macrophages, as well as inhibited the M1 macrophage and activated M2 macrophage in vitro. Blocking of SLPI by siRNA activated Mincle-associated inflammatory signaling in macrophages, and the inhibitory effect of isorhamnetin on inflammation was significantly attenuated. CONCLUSION: Isorhamnetin inhibits macrophage inflammation and protects kidney in AKI may be related to downregulating Mincle/Syk/NF-κB-maintained macrophage phenotype by activating SLPI.

Stereoselective Synthesis of the Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc Trisaccharide: a new Ligand for DCAR and Mincle C-Type Lectin Receptors.

In this work, we have discovered that the Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc trisaccharide, a fragment of the B antigen Type-1, is a new ligand of two C-type lectin receptors (CLRs) i. e. DCAR and Mincle which are key players in different types of autoimmune diseases. Accordingly, we report here on a straightforward methodology to access pure Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc trisaccharide. A spacer with a terminal primary amine group was included at the reducing end of the GlcNAc residue thus ensuring the further functionalization of the trisaccharide Gal-α-(1→3)-Gal-ß-(1→3)-GlcNAc.

SARS-CoV-2 N protein induced acute kidney injury in diabetic db/db mice is associated with a Mincle-dependent M1 macrophage activation.

"Cytokine storm" is common in critically ill COVID-19 patients, however, mechanisms remain largely unknown. Here, we reported that overexpression of SARS-CoV-2 N protein in diabetic db/db mice significantly increased tubular death and the release of HMGB1, one of the damage-associated molecular patterns (DAMPs), to trigger M1 proinflammatory macrophage activation and production of IL-6, TNF-α, and MCP-1 via a Mincle-Syk/NF-κB-dependent mechanism. This was further confirmed in vitro that overexpression of SARS-CoV-2 N protein caused the release of HMGB1 from injured tubular cells under high AGE conditions, which resulted in M1 macrophage activation and production of proinflammatory cytokines via a Mincle-Syk/NF-κB-dependent mechanism. This was further evidenced by specifically silencing macrophage Mincle to block HMGB1-induced M1 macrophage activation and production of IL-6, TNF-α, and MCP-1 in vitro. Importantly, we also uncovered that treatment with quercetin largely improved SARS-CoV-2 N protein-induced AKI in db/db mice. Mechanistically, we found that quercetin treatment significantly inhibited the release of a DAMP molecule HMGB1 and inactivated M1 pro-inflammatory macrophage while promoting reparative M2 macrophage responses by suppressing Mincle-Syk/NF-κB signaling in vivo and in vitro. In conclusion, SARS-CoV-2 N protein-induced AKI in db/db mice is associated with Mincle-dependent M1 macrophage activation. Inhibition of this pathway may be a mechanism through which quercetin inhibits COVID-19-associated AKI.

Infiltrating myeloid cell-derived properdin markedly promotes microglia-mediated neuroinflammation after ischemic stroke.

BACKGROUND: Emerging evidence has shown that myeloid cells that infiltrate into the peri-infarct region may influence the progression of ischemic stroke by interacting with microglia. Properdin, which is typically secreted by immune cells such as neutrophils, monocytes, and T cells, has been found to possess damage-associated molecular patterns (DAMPs) properties and can perform functions unrelated to the complement pathway. However, the role of properdin in modulating microglia-mediated post-stroke neuroinflammation remains unclear. METHODS: Global and conditional (myeloid-specific) properdin-knockout mice were subjected to transient middle cerebral artery occlusion (tMCAO). Histopathological and behavioral tests were performed to assess ischemic brain injury in mice. Single-cell RNA sequencing and immunofluorescence staining were applied to explore the source and the expression level of properdin. The transcriptomic profile of properdin-activated primary microglia was depicted by transcriptome sequencing. Lentivirus was used for macrophage-inducible C-type lectin (Mincle) silencing in microglia. Conditioned medium from primary microglia was administered to primary cortex neurons to determine the neurotoxicity of microglia. A series of cellular and molecular biological techniques were used to evaluate the proinflammatory response, neuronal death, protein-protein interactions, and related signaling pathways, etc. RESULTS: The level of properdin was significantly increased, and brain-infiltrating neutrophils and macrophages were the main sources of properdin in the ischemic brain. Global and conditional myeloid knockout of properdin attenuated microglial overactivation and inflammatory responses at the acute stage of tMCAO in mice. Accordingly, treatment with recombinant properdin enhanced the production of proinflammatory cytokines and augmented microglia-potentiated neuronal death in primary culture. Mechanistically, recombinant properdin served as a novel ligand that activated Mincle receptors on microglia and downstream pathways to drive primary microglia-induced inflammatory responses. Intriguingly, properdin can directly bind to the microglial Mincle receptor to exert the above effects, while Mincle knockdown limits properdin-mediated microglial inflammation. CONCLUSION: Properdin is a new medium by which infiltrating peripheral myeloid cells communicate with microglia, further activate microglia, and exacerbate brain injury in the ischemic brain, suggesting that targeted disruption of the interaction between properdin and Mincle on microglia or inhibition of their downstream signaling may improve the prognosis of ischemic stroke.

Characterisation of the bovine C-type lectin receptor Mincle and potential evidence for an endogenous ligand.

Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6'-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macrophages. However, boMincle mRNA transcripts were also detected in granulocytes, B cells, and T cells. Finally, we show that boMincle binds to isolated bovine CD4+ T cells in a specific manner, indicating the potential to recognise endogenous ligands. This suggests that the receptor might also play a role in homeostasis in cattle.

Aryl-functionalised α,α'-Trehalose 6,6'-Glycolipid Induces Mincle-independent Pyroptotic Cell Death.

α,α'-Trehalose 6,6'-glycolipids have long been known for their immunostimulatory properties. The adjuvanticity of α,α'-trehalose 6,6'-glycolipids is mediated by signalling through the macrophage inducible C-type lectin (Mincle) and the induction of an inflammatory response. Herein, we present an aryl-functionalised trehalose glycolipid, AF-2, that leads to the release of cytokines and chemokines, including IL-6, MIP-2 and TNF-α, in a Mincle-dependent manner. Furthermore, plate-coated AF-2 also leads to the Mincle-independent production of IL-1ß, which is unprecedented for this class of glycolipid. Upon investigation into the mode of action of plate-coated AF-2, it was observed that the treatment of WT and Mincle-/- bone marrow derived macrophages (BMDM), murine RAW264.7 cells, and human monocytes with AF-2 led to lytic cell death, as evidenced using Sytox Green and lactate dehydrogenase assays, and confocal and scanning electron microscopy. The requirement for functional Gasdermin D and Caspase-1 for IL-1ß production and cell death by AF-2 confirmed pyroptosis as the mode of action of AF-2. The inhibition of NLRP3 and K+ efflux reduced AF-2 mediated IL-1ß production and cell death, and allowed us to conclude that AF-2 leads to Capase-1 dependent NLRP3 inflammasome-mediated cell death. The unique mode of action of plate-coated AF-2 was surprising and highlights how the physical presentation of Mincle ligands can lead to dramatically different immunological outcomes.

Mesenchymal stem cells transplantation combined with IronQ attenuates ICH-induced inflammation response via Mincle/syk signaling pathway.

BACKGROUND: Intracerebral hemorrhage (ICH) is a severe brain-injured disease accompanied by cerebral edema, inflammation, and subsequent neurological deficits. Mesenchymal stem cells (MSCs) transplantation has been used as a neuroprotective therapy in nervous system diseases because of its anti-inflammatory effect. Nevertheless, the biological characteristics of transplanted MSCs, including the survival rate, viability, and effectiveness, are restricted because of the severe inflammatory response after ICH. Therefore, improving the survival and viability of MSCs will provide a hopeful therapeutic efficacy for ICH. Notably, the biomedical applications of coordination chemistry-mediated metal-quercetin complex have been verified positively and studied extensively, including growth-promoting and imaging probes. Previous studies have shown that the iron-quercetin complex (IronQ) possesses extraordinary dual capabilities with a stimulating agent for cell growth and an imaging probe by magnetic resonance imaging (MRI). Therefore, we hypothesized that IronQ could improve the survival and viability of MSCs, displaying the anti-inflammation function in the treatment of ICH while also labeling MSCs for their tracking by MRI. This study aimed to explore the effects of MSCs with IronQ in regulating inflammation and further clarify their potential mechanisms. METHODS: C57BL/6 male mice were utilized in this research. A collagenase I-induced ICH mice model was established and randomly separated into the model group (Model), quercetin gavage group (Quercetin), MSCs transplantation group (MSCs), and MSCs transplantation combined with IronQ group (MSCs + IronQ) after 24 h. Then, the neurological deficits score, brain water content (BWC), and protein expression, such as TNF-α, IL-6, NeuN, MBP, as well as GFAP, were investigated. We further measured the protein expression of Mincle and its downstream targets. Furthermore, the lipopolysaccharide (LPS)-induced BV2 cells were utilized to investigate the neuroprotection of conditioned medium of MSCs co-cultured with IronQ in vitro. RESULTS: We found that the combined treatment of MSCs with IronQ improved the inflammation-induced neurological deficits and BWC in vivo by inhibiting the Mincle/syk signaling pathway. Conditioned medium derived from MSCs co-cultured with IronQ decreased inflammation, Mincle, and its downstream targets in the LPS-induced BV2 cell line. CONCLUSIONS: These data suggested that the combined treatment exerts a collaborative effect in alleviating ICH-induced inflammatory response through the downregulation of the Mincle/syk signaling pathway following ICH, further improving the neurologic deficits and brain edema.

Peroxiredoxin 1 aggravates acute kidney injury by promoting inflammation through Mincle/Syk/NF-κB signaling.

Damage-associated molecular patterns (DAMPs) are a cause of acute kidney injury (AKI). Our knowledge of these DAMPs remains incomplete. Here, we report serum peroxiredoxin 1 (Prdx1) as a novel DAMP for AKI. Lipopolysaccharide (LPS) and kidney ischemia/reperfusion injury instigated AKI with concurrent increases in serum Prdx1 and reductions of Prdx1 expression in kidney tubular epithelial cells. Genetic knockout of Prdx1 or use of a Prdx1-neutralizing antibody protected mice from AKI and this protection was impaired by introduction of recombinant Prdx1 (rPrdx1). Mechanistically, lipopolysaccharide increased serum and kidney proinflammatory cytokines, macrophage infiltration, and the content of M1 macrophages. All these events were suppressed in Prdx1-/- mice and renewed upon introduction of rPrdx1. In primary peritoneal macrophages, rPrdx1 induced M1 polarization, activated macrophage-inducible C-type lectin (Mincle) signaling, and enhanced proinflammatory cytokine production. Prdx1 interacted with Mincle to initiate acute kidney inflammation. Of note, rPrdx1 upregulated Mincle and the spleen tyrosine kinase Syk system in the primary peritoneal macrophages, while knockdown of Mincle abolished the increase in activated Syk. Additionally, rPrdx1 treatment enhanced the downstream events of Syk, including transcription factor NF-κB signaling pathways. Furthermore, serum Prdx1 was found to be increased in patients with AKI; the increase of which was associated with kidney function decline and inflammatory biomarkers in patient serum. Thus, kidney-derived serum Prdx1 contributes to AKI at least in part by activating Mincle signaling and downstream pathways.

Thermophobic Trehalose Glycopolymers as Smart C-Type Lectin Receptor Vaccine Adjuvants.

Herein, this work reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1-2 °C changes in temperature about their lower critical solution temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia is created. Thermophobic adjuvants are synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly-N-isoporpylacrylamide (NIPAM) via reversible addition fragmentation chain transfer (RAFT) polymerization. The resulting thermophobic adjuvants exhibit LCSTs near 37 °C, and self-assembled into nanoparticles with temperature-dependent sizes (90-270 nm). Thermophobic adjuvants activate HEK-mMINCLE and other innate immune cell lines as well as primary mouse bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs). Inflammatory cytokine production is attenuated under conditions mimicking pyrexia (above the LCST) relative to homeostasis (37 °C) or below the LCST. This thermophobic behavior correlated with decreased adjuvant Rg is observed by DLS, as well as glycolipid-NIPAM shielding interactions are observed by NOESY-NMR. In vivo, thermophobic adjuvants enhance efficacy of a whole inactivated influenza A/California/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4+ /44+ /62L+ lung and lymph node central memory T cells, as well as providing better protection from morbidity after viral challenge relative to unadjuvanted control vaccine. Together, these results demonstrate the first adjuvants with potency regulated by temperature. This work envisions that with further investigation, this approach can enhance vaccine efficacy while maintaining safety.

Benzyl isothiocyanate improves the prognosis of Aspergillus fumigatus keratitis by reducing fungal load and inhibiting Mincle signal pathway.

Aspergillus fumigatus keratitis is a potential blinding disease associated with A. fumigatus invasion and excessive inflammatory response. Benzyl isothiocyanate (BITC) is a secondary metabolite with broad antibacterial and anti-inflammatory activity extracted from cruciferous species. However, the role of BITC in A. fumigatus keratitis has not been discovered yet. This study aims to explore the antifungal and anti-inflammatory effects and mechanisms of BITC in A. fumigatus keratitis. Our results provided evidences that BITC exerted antifungal effects against A. fumigatus by damaging cell membranes, mitochondria, adhesion, and biofilms in a concentration-dependent manner. In vivo, fungal load and inflammatory response including inflammatory cell infiltration and pro-inflammatory cytokine expression were reduced in BITC-treated A. fumigatus keratitis. Additionally, BITC significantly decreased Mincle, IL-1ß, TNF-α, and IL-6 expression in RAW264.7 cells that stimulated by A. fumigatus or Mincle ligand trehalose-6,6-dibehenate. In summary, BITC possessed fungicidal activities and could improve the prognosis of A. fumigatus keratitis by reducing fungal load and inhibiting the inflammatory response mediated by Mincle.

Synthesis and Biological Evaluation of Trehalose-based Bi-aryl Derivatives as C-type Lectin Ligands.

The identification of Mincle as the C-type lectin receptor on innate immune cells responsible for binding TDM and the realization that this receptor could be key to productive vaccines for mycobacterial infection has raised interest in the development of synthetic Mincle ligands as novel adjuvants. We recently reported on the synthesis and evaluation of Brartemicin analog UM-1024 that demonstrated Mincle agonist activity, exhibiting potent Th1/Th17 adjuvant activity that was greater than that of trehalose dibehenate (TDB). Our pursuit to understand Mincle/ligand relationships and improve the pharmacologic properties of the ligands has expanded and continues to reveal new and exciting structure activity relationships. Herein we report the synthesis of novel bi-aryl trehalose derivatives in good to excellent yields. These compounds were evaluated for their ability to engage the human Mincle receptor and tested for the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel bi-aryl derivatives revealed that bi-aryl trehalose ligand 3D showed relatively high potency in cytokine production in comparison to trehalose glycolipid adjuvant TDB and the natural ligand TDM and induced dose-dependent, Mincle selective stimulation in hMincle HEK reporter cells. Also, through computational studies, we provide an insight into the potential mode of binding of 6,6'-Biaryl trehalose compounds on human Mincle receptor.

TNF promotes DECTIN2 family C-type lectin receptor expression in human macrophages.

TNF blockade constitutes an effective therapy for inflammatory bowel disease, yet increases the risk for infection, including active tuberculosis. The DECTIN2 family C-type lectin receptors MINCLE, MCL, and DECTIN2 sense mycobacterial ligands and activate myeloid cells. In mice, upregulation of DECTIN2 family C-type lectin receptor after stimulation with Mycobacterium bovis Bacille Calmette-Guérin requires TNF. Here, we investigated whether TNF controls inducible C-type lectin receptor expression in human myeloid cells. Monocyte-derived macrophages were stimulated with Bacille Calmette-Guérin and the TLR4 ligand lipopolysaccharide, and expression of C-type lectin receptor was analyzed. Bacille Calmette-Guérin and lipopolysaccharide strongly upregulated messenger RNA expression of DECTIN2 family C-type lectin receptor but not of DECTIN1. Bacille Calmette-Guérin and lipopolysaccharide also induced robust production of TNF. Recombinant TNF was sufficient to upregulate expression of DECTIN2 family C-type lectin receptor. Blocking TNF with the TNFR2-Fc fusion protein etanercept abrogated, as expected, the effect of recombinant TNF and impaired induction of DECTIN2 family C-type lectin receptor by Bacille Calmette-Guérin and lipopolysaccharide. Flow cytometry confirmed upregulation of MCL at the protein level by recombinant TNF and showed inhibition of Bacille Calmette-Guérin-induced MCL by etanercept. To investigate the impact of TNF on C-type lectin receptor expression in vivo, we analyzed peripheral blood mononuclear cells of patients with inflammatory bowel disease and observed downregulation of MINCLE and MCL expression after therapeutic TNF blockade. Together, TNF is sufficient to upregulate DECTIN2 family C-type lectin receptor in human myeloid cells and contributes to this process after encounter with Bacille Calmette-Guérin or lipopolysaccharide. Impaired C-type lectin receptor expression in patients receiving TNF blockade may dampen the sensing of microbes and defense against infection.

Direct activation of microglia by ß-glucosylceramide causes phagocytosis of neurons that exacerbates Gaucher disease.

Gaucher disease (GD) is the most common lysosomal storage disease caused by recessive mutations in the degrading enzyme of ß-glucosylceramide (ß-GlcCer). However, it remains unclear how ß-GlcCer causes severe neuronopathic symptoms, which are not fully treated by current therapies. We herein found that ß-GlcCer accumulating in GD activated microglia through macrophage-inducible C-type lectin (Mincle) to induce phagocytosis of living neurons, which exacerbated Gaucher symptoms. This process was augmented by tumor necrosis factor (TNF) secreted from activated microglia that sensitized neurons for phagocytosis. This characteristic pathology was also observed in human neuronopathic GD. Blockade of these pathways in mice with a combination of FDA-approved drugs, minocycline (microglia activation inhibitor) and etanercept (TNF blocker), effectively protected neurons and ameliorated neuronopathic symptoms. In this study, we propose that limiting unrestrained microglia activation using drug repurposing provides a quickly applicable therapeutic option for fatal neuronopathic GD.