C-type Lectin Domain Family Research Articles

ASGR1 and Its Enigmatic Relative, CLEC10A.

The large family of C-type lectin (CLEC) receptors comprises carbohydrate-binding proteins that require Ca2+ to bind a ligand. The prototypic receptor is the asialoglycoprotein receptor-1 (ASGR1, CLEC4H1) that is expressed primarily by hepatocytes. The early work on ASGR1, which is highly specific for N-acetylgalactosamine (GalNAc), established the foundation for understanding the overall function of CLEC receptors. Cells of the immune system generally express more than one CLEC receptor that serve diverse functions such as pathogen-recognition, initiation of cellular signaling, cellular adhesion, glycoprotein turnover, inflammation and immune responses. The receptor CLEC10A (C-type lectin domain family 10 member A, CD301; also called the macrophage galactose-type lectin, MGL) contains a carbohydrate-recognition domain (CRD) that is homologous to the CRD of ASGR1, and thus, is also specific for GalNAc. CLEC10A is most highly expressed on immature DCs, monocyte-derived DCs, and alternatively activated macrophages (subtype M2a) as well as oocytes and progenitor cells at several stages of embryonic development. This receptor is involved in initiation of TH1, TH2, and TH17 immune responses and induction of tolerance in naïve T cells. Ligand-mediated endocytosis of CLEC receptors initiates a Ca2+ signal that interestingly has different outcomes depending on ligand properties, concentration, and frequency of administration. This review summarizes studies that have been carried out on these receptors.

The Superior Ability of Human BDCA3+ (CD141+) Dendritic Cells (DCs) to Cross-Present Antigens Derived From Necrotic Lung Cancer Cells.

Dendritic cells (DCs) play a key role in initiating and regulating the immune responses to pathogens, self-antigens, and cancers. Human blood DCs comprise a family of different subsets: plasmacytoid DCs (pDCs) and CD16+, CD1c/BDCA1+, and BDCA3+ (CD141+) myeloid DCs and possess different phenotypes and functional characteristics. Lung cancer is the most common cancer, with the highest morbidity and mortality in the world. However, which DC subset plays a leading role in the lung cancer immune responses is unclear. We reanalyzed C-type lectin domain family 9 member A (CLEC9A) and CD141 (THBD) gene expression profiles from the Cancer Genome Atlas (TCGA) database and performed the Kaplan-Meier survival analysis of overall survival for several cancers according to their expression levels. Next, we investigated the capacities of five human blood DC subsets to stimulate T cell proliferation and capture, process and (cross-) present tumor antigen. Human BDCA3+ (CD141+) DCs have a superior capacity to stimulate allogeneic CD4+T cells proliferation and induce superior Th1 response compared with other DC subsets. Interestingly, toll-like receptor (TLR) agonists have little effect on DCs to induce the proliferation of naïve CD4+ T cells, but contribute to their differentiation. Importantly, BDCA3+ (CD141+) DCs possess the most potent ability to cross-present human tumor antigen after their uptake of necrotic lung cancer cells despite their lower antigen uptake. These findings suggest that human BDCA3+ (CD141+) DCs are critical mediators of cytotoxic T lymphocyte responses against EGFR-positive lung cancer. Therefore, our findings may provide theoretical basis for the development of DC-based antitumor vaccines.

Histidine-Rich Glycoprotein Inhibits High-Mobility Group Box-1-Mediated Pathways in Vascular Endothelial Cells through CLEC-1A.

SummaryHigh-mobility group box-1 (HMGB1) protein has been postulated to play a pathogenic role in severe sepsis. Histidine-rich glycoprotein (HRG), a 75 kDa plasma protein, was demonstrated to improve the survival rate of septic mice through the regulation of neutrophils and endothelium barrier function. As the relationship of HRG and HMGB1 remains poorly understood, we investigated the effects of HRG on HMGB1-mediated pathway in endothelial cells, focusing on the involvement of specific receptors for HRG. HRG potently inhibited the HMGB1 mobilization and effectively suppressed rHMGB1-induced inflammatory responses and expression of all three HMGB1 receptors in endothelial cells. Moreover, we first clarified that these protective effects of HRG on endothelial cells were mediated through C-type lectin domain family 1 member A (CLEC-1A) receptor. Thus, current study elucidates protective effects of HRG on vascular endothelial cells through inhibition of HMGB1-mediated pathways may contribute to the therapeutic effects of HRG on severe sepsis.

Genetic association analysis of CLEC5A and CLEC7A gene single-nucleotide polymorphisms and Crohn's disease.

BACKGROUNDCrohn’s disease (CD) is characterized by a multifactorial etiology and a significant impact of genetic traits. While NOD2 mutations represent well established risk factors of CD, the role of other genes is incompletely understood.AIMTo challenge the hypothesis that single nucleotide polymorphisms (SNPs) in the genes CLEC5A and CLEC7A, two members of the C-type lectin domain family of pattern recognition receptors, may be associated with CD.METHODSSNPs in CLEC5A, CLEC7A and the known CD risk gene NOD2 were studied using real time PCR-based SNP assays. Therefore, DNA samples from 175 patients and 157 healthy donors were employed. Genotyping data were correlated with clinical characteristics of the patients and the results of gene expression data analyses.RESULTSIn accordance with previous studies, rs2066844 and rs2066847 in NOD2 were found to be significantly associated with CD (allelic P values = 0.0368 and 0.0474, respectively). Intriguingly, for genotype AA of rs1285933 in CLEC5A, a potential association with CD (recessive P = 0.0523; odds ratio = 1.90) was observed. There were no associations between CD and SNPs rs2078178 and rs16910631 in CLEC7A. Variants of rs1285933 had no impact on CLEC5A gene expression. In contrast, genotype-dependent differences of CXCL5 expression in peripheral blood mononuclear cells were observed. There is no statistical interaction between the tested SNPs of NOD2 and CLEC5A, suggesting of a novel pathway contributing to the disease.CONCLUSIONOur data encourage enlarged follow-up studies to further address an association of SNP rs1285933 in CLEC5A with CD. The C-type lectin domain family member also deserves attention regarding a potential role in the pathophysiology of CD.

Elevated Expression of C-Type Lectin Domain Family 5-Member A (CLEC5A) and Its Relation to Inflammatory Parameters and Disease Course in Adult-Onset Still's Disease.

C-type lectin domain family 5-member A (CLEC5A) associates with adaptor DAP12 (DNAX activation protein 12) to form receptor complexes involved in inflammatory responses. We postulated a potential role of CLEC5A in the pathogenesis of adult-onset Still's disease (AOSD) and aimed to investigate CLEC5A expression and its association with activity parameters and disease course. In 34 AOSD patients and 12 healthy controls (HC), circulating levels of CLEC5A-expressing monocytes or granulocytes were determined by flow cytometry analysis, the mRNA expression of CLEC5A and DAP12 on PBMCs by quantitative PCR, and plasma levels of proinflammatory cytokines by ELISA. AOSD patients had significantly higher percentages and mean fluorescence intensity (MFI) of CLEC5A-expressing monocytes (median 62.1% and 3.20, respectively) or granulocytes (72.6% and 3.22, respectively) compared with HC (in monocytes: 17.0% and 0.65, both p < 0.001; in granulocytes: 67.3%, p < 0.05 and 0.90, p < 0.001; respectively). Patients also had significantly higher levels of CLEC5A mRNA expression on PBMCs compared with HC (median 1.77 vs. 0.68, p < 0.05). The levels of CLEC5A-expressing monocytes or granulocytes were positively associated with activity scores and levels of IL-1β and IL-18 in AOSD patients. The patients with a systemic pattern had significantly higher levels of CLEC5A-expressing granulocytes and IL-18 compared to those with a chronic articular pattern of disease course. After 6 months of therapy, levels of CLEC5A-expressing monocytes and granulocytes significantly declined, paralleling the decrease of AOSD activity. Elevated CLEC5A levels and their positive association with activity parameters suggest that CLEC5A is involved in the pathogenesis and may serve as an activity indicator of AOSD.

CLEC3B as a potential diagnostic and prognostic biomarker in lung cancer and association with the immune microenvironment

BackgroundLung cancer is the leading cause of cancer-related mortality globally. Discovering effective biomarkers for early diagnosis and prognosis is important to reduce the mortality rate and ensure efficient therapy for lung cancer patients. C-type lectin domain family 3 member B (CLEC3B) has been reported in various cancers, but its correlation with lung cancer remains elusive.MethodsThe GEO, TCGA and Oncomine databases were analyzed to examine the expression of CLEC3B in lung cancer. The CLEC3B mRNA levels in 15 patient tissue samples were detected by real-time PCR and the CLEC3B protein levels in 34 patient tissue samples were detected by immunohistochemistry. A Chi-square test was performed to analyze the correlation of CLEC3B expression and clinicopathological factors. The diagnostic value of CLEC3B was revealed by receiver operating characteristic (ROC) curves. Univariate and multivariate Cox proportional hazards regression models and Kaplan–Meier plots were used to evaluate the prognostic value of CLEC3B in lung cancer. The TIMER database was used to evaluate the correlation of CLEC3B and immune infiltration. Gene set enrichment analysis revealed tumor‐associated biological processes related to CLEC3B.ResultsCLEC3B is significantly downregulated in lung cancer patients compared with nontumor controls according to database analysis and patient tissue sample detection (p < 0.001). Specifically, CLEC3B is significantly downregulated in stage IA lung cancer patients (p < 0.001) and has a high diagnostic accuracy (area under the receiver operating characteristic curve > 0.9). Moreover, low expression of CLEC3B is related to poor progression-free survival (HR = 0.60, 95% CI 0.49–0.74, p = 8.3e−07) and overall survival (HR = 0.66, 95% CI 0.58–0.75, p = 2.1e−10), indicating it as a risk factor for lung cancer. Multivariate analysis value showed that low expression of CLEC3B may be an independent risk factor for disease‐free survival in lung cancer patients (HR = 0.655, 95% CI 0.430–0.996, Cox p = 0.048). In addition, we also investigated the potential role of CLEC3B in tumor-immune interactions and found that CLEC3B might be associated with the immune infiltration and immune activation of lung cancer, especially in squamous cell carcinoma.ConclusionsOur findings indicate that CLEC3B expression is downregulated in lung cancer and reveal the diagnostic and prognostic potential of CLEC3B in lung cancer and its potential as an immune-related therapeutic target in lung cancer.

Expression of CLEC4G in liver disease and its correlation with hepatocellular carcinoma

Objective To investigate the expression level of C-type lectin domain family 4 member G (CLEC4G) in liver disease tissues and its correlation with the clinicopathological characteristics of hepatocellular carcinoma (HCC) patients. Methods The cancer tissue and the corresponding adjacent tissues (at least 2 cm from the edge of the cancer tissue), cut in surgeries from January to December in 2019, of 40 HCC patients in Zhuzhou Central Hospital, as well as 10 normal liver tissue samples (seen as far away as possible from the edge of the cancer tissue with naked eyes) and 10 liver cirrhosis samples were analyzed retrospectively. The tumor genome atlas (TCGA) database was used to screen the HCC transcriptome data sets, and bioinformatics methods were used to make expression heat maps and box maps which can help analyze the difference of CLEC4G in cancer and adjacent tissues. The mRNA expression level of CLEC4G was detected by conducting real-time fluorescence quantitative PCR (qRT-PCR), and the protein expression level of CLEC4G was detected by immunohistochemistry (IHC). The measurement data were expressed as mean±standard deviation (Mean±SD). Group t test was used for inter-group comparison. The counting information was expressed as a percentage (%). The χ2 test was adopted to analyze the correlation between CLEC4G expression level and the clinicopathological features of patients. Results The expression level of CLEC4G in cancer tissues was significantly decreased in heat map compared with that in adjacent tissues. In the box figure, the relative expression of CLEC4G mRNA in the cancer tissues was (82.5±18.9) and (3 354.4±296.2) in paracancer tissues, with statistically significant difference (P<0.001). Respectively, qRT-PCR and IHC showed that mRNA of CLEC4G were abundant in normal liver tissues (3 301.3±286.4), while they were very little in liver cancer tissues (63.6±32.9), significantly decreasing in liver cirrhosis (1 742.6±208.7) and paracancer tissues (1 553.2±249.9), with statistically significant difference (P<0.001). Moreover, low CLEC4G expression level was associated with tumor vascular metastasis in HCC patients. Conclusions CLEC4G is highly expressed in normal liver tissue, but with the progression of malignant liver disease, it is significantly decreased with little expression in HCC tissue. It can be expected to be a good marker for the pathological diagnosis of HCC. Key words: Carcinoma, hepatocellular; Liver cirrhosis; Neoplasms, vascular tissue; Liver tissue

Unraveling the transcriptional determinants of liver sinusoidal endothelial cell specialization.

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.

Molecular structure, expression, and functional role of Clec11a in skeletal biology and cancers.

C-type lectin domain family 11 member A (Clec11a), also known as stem cell growth factor (SCGF), C-type lectin superfamily member 3 (CLECSF3), or osteolectin was initially identified as a growth factor for hematopoietic progenitor cells. The human Clec11a gene encodes a polypeptide of 323 amino acids with characteristics of a secreted glycoprotein encompassing two integrin-binding motifs, RGD (Arg-Gly-Asp) and LDT (Leu-Asp-Thr), a putative leucine zipperdomain, and a functional C-type lectin domain. It regulates hematopoietic differentiation and homeostasisand exhibits a protective effect against severe malarial anemia and lipotoxicity. Furthermore, Clec11a promotes the differentiation of mesenchymal progenitors into mature osteoblasts in vitro and plays an important role in the maintenance of adult skeleton age-related bone loss and fracture repair. Receptor ligand binding results in activation of downstream signaling cascades including glycogen synthase kinase 3 (GSK3), β-catenin, and Wnt, resulting in the expression of osteoblast-related gene transcripts including Alp, Runx2, Lef1, and Axin2. In addition, Clec11a is also associated with the development of several cancers, including leukemia, multiple myeloma, and gastrointestinal tract tumors. To date, however, the mechanisms governing transcription regulation of the Clec11a gene arenot known and remainto be uncovered. Understanding the function and mechanism of action of Clec11a will pave the way for the development of Clec11a as a novel therapeutic target for conditions such as cancer, anemia, and skeletal diseases.

High expression levels of CLEC4M indicate poor prognosis in patients with hepatocellular carcinoma.

The identification of novel and accurate biomarkers is important to improve the prognosis of patients with hepatocellular carcinoma (HCC). C-Type lectin domain family 4 member M (CLEC4M) is involved in the progression of numerous cancer types. However, the clinical significance of CLEC4M in HCC is yet to be elucidated. The aim of the present study is to evaluate the involvement of CLEC4M in HCC progression. The expression level of CLEC4M was determined in tumor, and their corresponding adjacent non-tumor tissues derived from 88 patients with HCC, using immunohistochemistry, western blot and reverse transcription-quantitative PCR. The correlation between CLEC4M expression and certain clinicopathological characteristics was retrospectively analyzed. The results suggested that CLEC4M was specifically labeled in sinusoidal endothelial cells, in both HCC and non-tumor tissues. Moreover, the expression of CLEC4M in tumor tissues was significantly lower than that in non-tumor tissues (P<0.0001), which indicated its potential as a biomarker of the development of HCC. Subsequently, correlation analysis suggested that the relatively higher CLEC4M expression in HCC tissues was significantly associated with increased microvascular invasion (P=0.008), larger tumor size (P=0.018), absence of tumor encapsulation (P<0.0001) and lower tumor differentiation (P=0.019). Notably, patients with high CLEC4M expression levels in their tumor tissues experienced more frequent recurrence and shorter overall survival (OS) times compared with the low-expression group. Furthermore, CLEC4M expression in tumor tissues was identified as an independent and significant risk factor for recurrence-free survival and OS. The results of the present study suggest that CLEC4M may be a valuable biomarker for the prognosis of the patients with HCC, postoperatively.

CLEC3B protects H9c2 cardiomyocytes from apoptosis caused by hypoxia via the PI3K/Akt pathway.

Ischemic heart disease (IHD) is one of the leading causes of death worldwide. C-type lectin domain family 3 member B (CLEC3B) is a C-type lectin superfamily member and is reported to promote tissue remodeling. The serum levels of CLEC3B are downregulated in patients with cardiovascular disease. However, the molecular mechanisms of CLEC3B in IHD is not well-characterized. Therefore, we overexpressed CLEC3B and silenced CLEC3B in H9c2 rat cardiomyocytes for the first time. We then constructed a model of IHD in vitro through culturing H9c2 cardiomyocytes in serum-free medium under oxygen-deficit conditions. Then, Cell Counting Kit-8 (CCK-8), flow cytometry, qRT-PCR, and western blot assays were performed to investigate cell viability, apoptosis, and expression levels of CLEC3B, phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), and cleaved-caspase 3. We observed that the mRNA expression of CLEC3B was decreased in hypoxic H9c2 cardiomyocytes (P<0.05). Overexpression of CLEC3B increased cell viability (P<0.01), inhibited cell apoptosis (P<0.05), upregulated the levels of p-PI3K/PI3K and p-Akt/Akt (P<0.01 or P<0.05), and downregulated expression of cleaved-caspase 3 (P<0.001) in hypoxic H9c2 cardiomyocytes while silencing of CLEC3B caused the opposite results. Inhibition of the PI3K/Akt pathway reversed the protective effect of CLEC3B on hypoxic H9c2 cardiomyocytes. Our study demonstrated that CLEC3B alleviated the injury of hypoxic H9c2 cardiomyocytes via the PI3K/Akt pathway.

Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism.

Osteoporosis and osteoporotic fractures severely compromise quality of life in elderly people and lead to early death. Human umbilical cord mesenchymal stromal cell (MSC)-derived extracellular vesicles (hucMSC-EVs) possess considerable therapeutic effects in tissue repair and regeneration. Thus, in the present study, we investigated the effects of hucMSC-EVs on primary and secondary osteoporosis and explored the underlying mechanisms.Methods: hucMSCs were isolated and cultured. EVs were obtained from the conditioned medium of hucMSCs and determined by using transmission electron microscopy, dynamic light scattering and Western Blot analyses. The effects of hucMSC-EVs on ovariectomy-induced postmenopausal osteoporosis and tail suspension-induced hindlimb disuse osteoporosis in mouse models were assessed by using microcomputed tomography, biomechanical, histochemical and immunohistochemical, as well as histomorphometric analyses. Proteomic analysis was applied between hucMSC-EVs and hucMSCs to screen the candidate proteins that mediate hucMSC-EVs function. The effects of hucMSC-EVs on osteogenic and adipogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), and osteoclastogenesis of the macrophage cell line RAW264.7 in vitro were determined by using cytochemical staining and quantitative real-time PCR analysis. Subsequently, the roles of the key protein in hucMSC-EVs-induced regulation on BMSCs and RAW264.7 cells were evaluated.Results: hucMSCs were able to differentiate into osteoblasts, adipocytes or chondrocytes and positively expressed CD29, CD44, CD73 and CD90, but negatively expressed CD34 and CD45. The morphological assessment revealed the typical cup- or sphere-shaped morphology of hucMSC-EVs with diameters predominantly ranging from 60 nm to 150 nm and expressed CD9, CD63, CD81 and TSG101. The systemic administration of hucMSC-EVs prevented bone loss and maintained bone strength in osteoporotic mice by enhancing bone formation, reducing marrow fat accumulation and decreasing bone resorption. Proteomic analysis showed that the potently pro-osteogenic protein, CLEC11A (C-type lectin domain family 11, member A) was very highly enriched in hucMSC-EVs. In addition, hucMSC-EVs enhanced the shift from adipogenic to osteogenic differentiation of BMSCs via delivering CLEC11A in vitro. Moreover, CLEC11A was required for the inhibitory effects of hucMSC-EVs on osteoclast formation.Conclusion: Our results suggest that hucMSC-EVs serve as a critical regulator of bone metabolism by transferring CLEC11A and may represent a potential agent for prevention and treatment of osteoporosis.

Histidine-Rich Glycoprotein Inhibits High Mobility Group Box 1-Mediated Pathways in Vascular Endothelial Cells

Background: High mobility group box-1 (HMGB1) protein has been postulated to play a pathogenic role in severe sepsis. Histidine-rich glycoprotein (HRG) was proposed as a new biomarker to predict the outcome of sepsis patients and demonstrated to improve survival rate and relieve symptom of septic mice. However, its effect on HMGB1 signaling has never been investigated. Our research focuses on the effects of HRG on HMGB1 mobilization and HMGB1-mediated pathway in endothelial cells and the involvement of specific receptors for HRG. Methods: A variety of biochemical assays were applied to explore the effects of HRG on HMGB1 release and HMGB1-mediated signal pathway in EA.hy 926 endothelial cells. The receptor for HRG was identified using co-immunoprecipitation experiments and surface plasmon resonance analysis. Findings: HRG potently inhibited the LPS-induced translocation and release of HMGB1 and effectively suppressed rHMGB1-induced ICAM-1 and VCAM-1 expression, inflammatory cytokines secretion and NF-κB activation in EA.hy 926 endothelial cells. HRG also inhibited HMGB1 release and HMGB1-mediated inflammatory responses in LPS-injected mice. Moreover, we first clarified that these protective effects of HRG on endothelial cells were mediated through C-type lectin domain family 1 member A (CLEC-1A) receptor. Interpretation: The present findings strongly support the supplementary therapy with HRG in sepsis and will provide the new insights into the understanding of the functional role of CLEC1A in the maintenance of vascular endothelial cells. Funding Statement: This research was supported by grants from AMED (no. 18im0210109h0002) and from the Secom Science and Technology Foundation to M.N., a Grant-in-Aid for Scientific Research (no.15H04686 to M.N.), a Grant-in-Aid for Young Scientists (no. 17K15580 to H.W.), and a Grant-in-Aid for Scientific Research (no. 16K08232 to K.T.) from the Japan Society for the Promotion of Science (JSPS). The author S.G. was supported by the scholarship from China Scholarship Council. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Human neutrophils were isolated from peripheral blood obtained from healthy volunteers in accordance with ethics approval and guidelines of Okayama University and the Declaration of Helsinki. All animal experiments were approved by the university's committee and performed according to the guidelines of Okayama University on animal experiments.

The Candida albicans exotoxin candidalysin promotes alcohol-associated liver disease

Alcohol-associated liver disease is a leading indication for liver transplantation and a leading cause of mortality. Alterations to the gut microbiota contribute to the pathogenesis of alcohol-associated liver disease. Patients with alcohol-associated liver disease have increased proportions of Candida spp. in the fecal mycobiome, yet little is known about the effect of intestinal Candida on the disease. Herein, we evaluated the contributions of Candida albicans and its exotoxin candidalysin in alcohol-associated liver disease. C. albicans and the extent of cell elongation 1 (ECE1) were analyzed in fecal samples from controls, patients with alcohol use disorder and those with alcoholic hepatitis. Mice colonized with different and genetically manipulated C. albicans strains were subjected to the chronic-plus-binge ethanol diet model. Primary hepatocytes were isolated and incubated with candidalysin. The percentages of individuals carrying ECE1 were 0%, 4.76% and 30.77% in non-alcoholic controls, patients with alcohol use disorder and patients with alcoholic hepatitis, respectively. Candidalysin exacerbates ethanol-induced liver disease and is associated with increased mortality in mice. Candidalysin enhances ethanol-induced liver disease independently of the β-glucan receptor C-type lectin domain family 7 member A (CLEC7A) on bone marrow-derived cells, and candidalysin does not alter gut barrier function. Candidalysin can damage primary hepatocytes in a dose-dependent manner in vitro and is associated with liver disease severity and mortality in patients with alcoholic hepatitis. Candidalysin is associated with the progression of ethanol-induced liver disease in preclinical models and worse clinical outcomes in patients with alcoholic hepatitis. Candidalysin is a peptide toxin secreted by the commensal gut fungus Candida albicans. Candidalysin enhances alcohol-associated liver disease independently of the β-glucan receptor CLEC7A on bone marrow-derived cells in mice without affecting intestinal permeability. Candidalysin is cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease.

Protective effects of Clec11a in islets against lipotoxicity via modulation of proliferation and lipid metabolism in mice

The lipotoxicity is considered as one of the risk for diabetes. Here we report C-type lectin domain family 11, member A (Clec11a) as a new regulator in islet playing a protective role in lipotoxicity induced dysfunction. Islet transcriptome sequencing was performed using the high-fat diet induced obesity (DIO) mice model. We found a significant decrease of Clec11a expression in islets of DIO mice compared to normal control mice, which was further confirmed by real-time PCR. Immunostaining demonstrated the localization of the Clec11a protein in mouse islets. Administration of recombinant human Clec11a (rClec11a) protein promoted the proliferation of islet cells and rescued the inhibition of fatty acid on cell proliferation, which involved the activation of Erk signaling pathway. We also found that the rClec11a altered the expression of genes involved in lipid metabolism.

Antimicrobial peptides derived from the cartilage.-specific C-type Lectin Domain Family 3 Member A (CLEC3A) – potential in the prevention and treatment of septic arthritis

To investigate the antimicrobial activity of peptides derived from C-type Lectin Domain Family 3 Member A (CLEC3A), shed light on the mechanism of antimicrobial activity and assess their potential application in prevention and treatment of septic arthritis. We performed immunoblot to detect CLEC3A peptides in human cartilage extracts. To investigate their antimicrobial activity, we designed peptides and recombinantly expressed CLEC3A domains and used them to perform viable count assays using E.coli, P.aeruginosa and S.aureus. We investigated the mechanism of their antimicrobial activity by fluorescence and scanning electron microscopy, performed ELISA-style immunoassays and transmission electron microscopy to test for lipopolysaccharide binding and surface plasmon resonance to test for lipoteichoic acid (LTA) binding. We coated CLEC3A peptides on titanium, a commonly used prosthetic material, and performed fluorescence microscopy to quantify bacterial adhesion. Moreover, we assessed the peptides' cytotoxicity against primary human chondrocytes using MTT cell viability assays. CLEC3A fragments were detected in human cartilage extracts. Moreover, bacterial supernatants lead to fragmentation of recombinant and cartilage-derived CLEC3A. CLEC3A-derived peptides killed E.coli, P.aeruginosa and S.aureus, permeabilized bacterial membranes and bound lipopolysaccharide and LTA. Coating CLEC3A antimicrobial peptides (AMPs) on titanium lead to significantly reduced bacterial adhesion to the material. In addition, microbicidal concentrations of CLEC3A peptides invitro displayed no direct cytotoxicity against primary human chondrocytes. We identify cartilage-specific AMPs originating from CLEC3A, resolve the mechanism of their antimicrobial activity and point to a novel approach in the prevention and treatment of septic arthritis using potent, non-toxic, AMPs.

Morphometric image analysis of placental C-type lectin domain family 2, member D (CLEC2D) immuno-expression in HIV associated pre-eclampsia.

ObjectiveC-type lectin domain family 2, member D (CLEC2D) is implicated in the immune response. Pre-eclampsia and HIV infection have opposing immune responses. In view of the high prevalence of HIV infection and pre-eclampsia in South Africa, this study assessed the placental immuno-expression of CLEC2D in HIV associated pre-eclampsia.MethodPlacental tissue was obtained from 60 pregnancies which were categorized according to pregnancy type (pre-eclamptic or normotensive) and HIV status (positive or negative). Immunohistochemistry and morphometric image analysis were used to evaluate placental CLEC2D immuno-expression.ResultsCLEC2D expression was significantly decreased in the conducting villi of pre-eclamptic vs normotensive placentae (p = 0.0418) but was increased in the exchange villi, albeit non-significant (p = 0.4948). HIV positive status intensified placental CLEC2D immuno-expression in conducting (p = 0.0312) and exchange (p = 0.0025) villi. CLEC2D expression was significantly different in exchange vs conducting villi (p < 0.0001) and across study groups (p = 0.0003). Normotensive; HIV negative placentae (control) had a non-significant difference in CLEC2D expression across villi types, however significant difference was noted within the remaining groups: normotensive; HIV positive (p < 0.05), pre-eclamptic; HIV positive (p < 0.01 and pre-eclamptic; HIV negative (p < 0.001).ConclusionThe contrasting expression of CLEC2D in HIV infection and pre-eclampsia is demonstrative of the immunosuppressive and pro-inflammatory roles of the respective pathologies. However, this implication may be confounded by highly active anti-retroviral treatment (HAART).

Platelet membrane lipid rafts protein composition varies following GPVI and CLEC-2 receptors activation

Lipid rafts are membrane microdomains that have been proposed to play an important role in several platelet-signalling cascades, including those mediated by the receptors Glycoprotein VI (GPVI), and C-type lectin domain family 1 member B (CLEC-2), both involved in thrombus formation. We have performed a LC-MS/MS proteomic analysis of lipid rafts isolated from platelets activated through GPVI and CLEC-2 as well as from resting platelets. Our aim was to determine the magnitude of changes in lipid rafts protein composition and to elucidate the relevance of these alterations in platelet function. A number of relevant signalling proteins were found enriched in lipid rafts following platelet activation (such as the tyrosine protein kinases Fyn, Lyn and Yes; the G proteins G(i) and G(z); and cAMP protein kinase). Interestingly, our results indicate that the relative enrichment of lipid rafts in these signalling proteins may not be a consequence of protein translocation to these domains upon platelet stimulation, but the result of a massive loss in cytoskeletal proteins after platelet activation. Thus, this study may help to better understand the effects of platelet activation in the reorganization of lipid rafts and set the basis for further proteomic studies of these membrane microdomains in platelets. SignificanceWe performed the first proteomic comparative analysis of lipid rafts- protein composition in platelets activated through GPVI and CLEC-2 receptors and in resting state. We identified a number of signalling proteins essential for platelet activation relatively enriched in platelets activated through both receptors, and we show that lipid rafts reorganization upon platelet activation leads to a loss in cytoskeletal proteins, highly associated to these domains in resting platelets.

Impact of uterine macrophage phenotype on placental retention in dairy cows

Reproductive diseases affect 25% of dairy cows in the US and often develop from retention of the placenta. It is well established that expulsion of the placenta is a highly regulated inflammatory process, but the mechanisms by which dysregulation of uterine immune responses impair this process are poorly understood. In healthy non-ruminants, pro-inflammatory M1 macrophages are predominant in uterine tissue after parturition. However, macrophage phenotype in the postpartum bovine uterus is unknown. Our study compared macrophage phenotypes in the uterine caruncles of multiparous dairy cows that during the first day postpartum either retained (RET, n = 5) or had normal expulsion (NOR, n = 5) of placenta. Immune cells were sorted magnetically from the caruncular endometrial cell fraction using the CD172a marker and monocyte/macrophage population was characterized using flow cytometry. Transcriptional and protein expression studies were performed on uterine caruncles. Compared to NOR, RET samples showed a lower CD14+/CD16+ expression (P < 0.05) in caruncle monocyte/macrophage population. As opposed to NOR, RET further demonstrated greater expression of anti-inflammatory M2 macrophage associated genes CD206, C-type lectin domain family 7 member A (CLEC7A), and RNASE6. In addition, caruncles from RET showed decreased signal transducer and activator of transcription 3 (STAT3) activation, an important promoter of proteolytic activity, compared to NOR. Our studies demonstrate that there is an overall lower number of macrophage populations in the caruncle of cows with RET placenta and these are polarized towards M2 phenotype. Excessive accumulation of M2 macrophages may lead to reduced trafficking of immune cells into the caruncle thus impairing the inflammatory, phagocytic and proteolytic processes that lead to placental expulsion.

Probing the Virtual Proteome to Identify Novel Disease Biomarkers.

Proteomic approaches allow measurement of thousands of proteins in a single specimen, which can accelerate biomarker discovery. However, applying these technologies to massive biobanks is not currently feasible because of the practical barriers and costs of implementing such assays at scale. To overcome these challenges, we used a "virtual proteomic" approach, linking genetically predicted protein levels to clinical diagnoses in >40 000 individuals. We used genome-wide association data from the Framingham Heart Study (n=759) to construct genetic predictors for 1129 plasma protein levels. We validated the genetic predictors for 268 proteins and used them to compute predicted protein levels in 41 288 genotyped individuals in the Electronic Medical Records and Genomics (eMERGE) cohort. We tested associations for each predicted protein with 1128 clinical phenotypes. Lead associations were validated with directly measured protein levels and either low-density lipoprotein cholesterol or subclinical atherosclerosis in the MDCS (Malmö Diet and Cancer Study; n=651). In the virtual proteomic analysis in eMERGE, 55 proteins were associated with 89 distinct diagnoses at a false discovery rate q<0.1. Among these, 13 associations involved lipid (n=7) or atherosclerosis (n=6) phenotypes. We tested each association for validation in MDCS using directly measured protein levels. At Bonferroni-adjusted significance thresholds, levels of apolipoprotein E isoforms were associated with hyperlipidemia, and circulating C-type lectin domain family 1 member B and platelet-derived growth factor receptor-β predicted subclinical atherosclerosis. Odds ratios for carotid atherosclerosis were 1.31 (95% CI, 1.08-1.58; P=0.006) per 1-SD increment in C-type lectin domain family 1 member B and 0.79 (0.66-0.94; P=0.008) per 1-SD increment in platelet-derived growth factor receptor-β. We demonstrate a biomarker discovery paradigm to identify candidate biomarkers of cardiovascular and other diseases.