The influence of CLEC5A on early macrophage-mediated inflammation in COPD progression.

Chronic obstructive pulmonary disease (COPD) is a complex syndrome with poorly understood mechanisms driving its early progression (GOLD stages 1-2). Elucidating the genetic factors that influence early-stage COPD, particularly those related to airway inflammation and remodeling, is crucial. This study analyzed lung tissue sequencing data from patients with early-stage COPD (GSE47460) and smoke-exposed mice. We employed Weighted Gene Co-Expression Network Analysis (WGCNA) and machine learning to identify potentially pathogenic genes. Further analyses included single-cell sequencing from both mice and COPD patients to pinpoint gene expression in specific cell types. Cell-cell communication and pseudotemporal analyses were conducted, with findings validated in smoke-exposed mice. Additionally, Mendelian randomization (MR) was used to confirm the association between candidate genes and lung function/COPD. Finally, functional validation was performed in vitro using cell cultures. Machine learning analysis of 30 differentially expressed genes identified 8 key genes, with CLEC5A emerging as a potential pathogenic factor in early-stage COPD. Bioinformatics analyses suggested a role for CLEC5A in macrophage-mediated inflammation during COPD. Two-sample Mendelian randomization linked CLEC5A single nucleotide polymorphisms (SNPs) with Forced Expiratory Volume in One Second (FEV1), FEV1/Forced Vital Capacity (FVC) and early/later on COPD. In vitro, the knockdown of CLEC5A led to a reduction in inflammatory markers within macrophages. Our study identifies CLEC5A as a critical gene in early-stage COPD, contributing to its pathogenesis through pro-inflammatory mechanisms. This discovery offers valuable insights for developing early diagnosis and treatment strategies for COPD and highlights CLEC5A as a promising target for further investigation.

REEP5 mediates the function of CLEC5A to alleviate myocardial infarction by inhibiting endoplasmic reticulum stress-induced apoptosis.

MI (myocardial infarction) often triggers severe heart failure and is one of the leading causes of death worldwide. Receptor expression-enhancing protein 5 (REEP5), a member of REEPs, acts as regulators of endoplasmic reticulum (ER) affecting cardiac functions. Based on GSE114695 profile data, REEP5 was decreased in the left ventricle of MI mice. However, its role and potential mechanism in MI remain to be investigated. In the present study, the mouse MI model was established by ligation of the left anterior descending artery. REEP5 expression was downregulated in the infarct penumbra area of MI mice. Next, its role during MI was explored by gain-of-function. Interestingly, REEP5 overexpression improved left ventricular function of mice with MI, accompanied with reduced infarct size. In cardiomyocytes, REEP5 overexpression inhibited ER stress, accompanied with repressive phosphorylation of PERK and IRE1α, and the decreased nuclear translocation of ATF6. Subsequently, REEP5 overexpression downregulated the levels of Chop and cleaved caspase-12, further alleviating ER stress-induced apoptosis, which was consistent with the in vivo results. Moreover, REEP5 was found to bind to C-type lectin member 5 A (CLEC5A), a protein that triggers cardiac dysfunction. CLEC5A, whose expression was elevated in hypoxia-induced cell models, led to cardiomyocyte apoptosis. Noteworthily, REEP5 overexpression markedly abolished the effects of CLEC5A on ER stress-induced apoptosis. Taken together, REEP5 mediated the function of CLEC5A to relieve MI via inhibiting ER stress-induced apoptosis in vivo and in vitro. REEP5 may be a promising target for treating MI.

Polymorphisms in CLEC5A and CLEC7A genes modify risk for inflammatory bowel disease.

Background: Inflammatory bowel disease (IBD) seems to arise from an interplay between genetic and environmental factors. CLEC5A and CLEC7A genes code for 2 members of the C-type lectin receptor superfamily, which participate in the immune response against various pathogens, mediating inflammatory signaling. CLEC5A polymorphisms have been linked to the risk of Crohn's disease (CD), whereas CLEC7A has been implicated in fungal dysbiosis, chemically induced colitis in mice and undertreated ulcerative colitis (UC) in humans. This study aimed to explore how specific CLEC5A and CLEC7A polymorphisms contribute to the development of CD and UC. Methods: One hundred twelve CD patients, 94 UC patients and 164 sex- and age- matched healthy individuals were genotyped for the single nucleotide polymorphisms rs2078178 and rs16910631 of the CLEC7A gene, and rs1285933 of the CLEC5A gene. Results: The CLEC7A rs2078178 AA genotype was more frequent in UC patients compared to healthy individuals, The CLEC7A rs16910631 CT genotype was significantly associated with UC risk compared to healthy individuals, while there was no statistical correlation with CD. The CLEC5A rs1285933 GA genotype was found to be protective against UC and CD, and the AA genotype against CD. Carriers of the rs1285933 A allele appeared to have reduced susceptibility to CD, implying that the presence of the A allele could be protective against CD development. Conclusions: This is the first study to correlate the CLEC5A rs1285933 polymorphism with the risk for UC. The rs2078178 AA genotype and the CLEC7A rs16910631 CT could be promising biomarkers for UC susceptibility.

A Macrophage-Related Gene Signature for Identifying COPD Based on Bioinformatics and ex vivo Experiments.

Background: This study aims to investigate the association between immune cells and the development of COPD, while providing a new method for the diagnosis of COPD according to the changes in immune microenvironment. Methods: In this study, the "CIBERSORT" algorithm was used to estimate the tissue infiltration of 22 types of immune cells in GSE20257 and GSE10006. The "limma" package was used for differentially expressed analysis. The key modules associated with vital immune cells were identified using WGCNA. GO and KEGG enrichment analysis revealed the biological functions of the candidate genes. Ultimately, a novel diagnostic prediction model was constructed via machine learning methods and multivariate logistic regression analysis based on GSE20257. Furthermore, we examined the stability of the model on one internal test set (GSE10006), three external test sets (GSE8545, GSE57148 and GSE76925), one single-cell transcriptome dataset (GSE167295), macrophages (THP-M cells) and lung tissue from COPD patients. Results: M0 macrophages (AUC > 0.7 in GSE20257 and GSE10006) were considered as the most important immune cells through exploring the immune microenvironment landscapes in COPD patients and healthy controls. The differentially expressed genes from GSE20257 and GSE10006 were divided into six and five modules via WGCNA, respectively. The green module in GSE20257 (cor = 0.41, P < 0.001) and the brown module in GSE10006 (cor = 0.67, P < 0.001) were highly correlated with M0 macrophages and were selected as key modules. Forty-one intersected genes obtained from two modules were primarily involved in regulation of cytokine production, regulation of innate immune response, specific granule, phagosome, lysosome, ferroptosis, and other biological processes. On the basis of the candidate genetic markers further characterized via the "Boruta" and "LASSO" algorithm for COPD, a diagnostic model comprising CLEC5A, FTL and SLC2A3 was constructed, which could accurately distinguish COPD patients from healthy controls in multiple datasets. GSE20257 as the training set has an AUC of 0.916. The AUCs of the internal test set and three external test sets were 0.873, 0.932, 0.675 and 0.688, respectively. Single-cell sequencing analysis suggested that CLEC5A, FTL and SLC2A3 were expressed in macrophages from COPD patients. The expressions of CLEC5A, FTL and SLC2A3 were up-regulated in THP-M cells and lung tissue from COPD patients. Conclusion: According to the variations of immune microenvironment in COPD patients, we constructed and validated a novel macrophage M0-associated diagnostic model with satisfactory predictive value. CLEC5A, FTL and SLC2A3 are expected to be promising targets of immunotherapy in COPD.

Phellopterin attenuates ovarian cancer proliferation and chemoresistance by inhibiting the PU.1/CLEC5A/PI3K-AKT feedback loop.

Ovarian cancer is known as the second leading cause of gynecologic cancer-associated deaths in women worldwide. Developing new and effective compounds to alleviate chemoresistance is an urgent priority in ovarian cancer. Here, we aimed to reveal the biological function and underlying mechanisms of phellopterin, a naturally sourced ingredient of Angelica dahurica, in ovarian cancer progression as well as evaluate the therapeutic potential of phellopterin in ovarian cancer patients. In this investigation, we found that phellopterin mitigated DNA replication and induced cell cycle arrest, apoptosis, and DNA damage, attenuating cell proliferation and chemoresistance of ovarian cancer. Interestingly, bioinformatics analyses of data from our RNA sequencing and The Cancer Genome Atlas ovarian cancer dataset suggested that phellopterin presented anti-cancer activities in ovarian cancer cells by modulating signals affecting ovarian cancer progression and identified phellopterin as a potential compound in improving ovarian cancer patients' prognosis. In addition, the C-Type Lectin Domain Containing 5A (CLEC5A) was demonstrated as a downstream effector of phellopterin and involved in a positive PU.1/CLEC5A/PI3K-AKT feedback loop. Interestingly, phellopterin might inactivate the positive feedback circuit to suppress ovarian cancer progression. Collectively, our investigation revealed that phellopterin mitigated ovarian cancer proliferation and chemoresistance through suppressing the PU.1/CLEC5A/PI3K-AKT feedback loop, and predicted phellopterin as a new and effective cytotoxic drug and CLEC5A as a potential target for the treatment of ovarian cancer.

In-Silico CLEC5A mRNA expression analysis to predict Dengue susceptibility in cancer patients.

Dengue fever is the fastest-growing infectious disease in the world. It is the leading vector-borne viral neglected tropical disease. The most acute immune response to dengue virus infection is dengue shock syndrome and hemorrhagic fever, which is due to the activation of CLEC5A C-type lectin domain family 5, member A (CLEC5A). It is a cell surface receptor, and its well-known ligand is the dengue virus. It gets activated by the attachment of dengue virion, which, as a result, phosphorylates its adaptor protein DAP12 leading to the induction of various pro-inflammatory cytokines. Clinical data suggested that the kidneys and lungs are among the major hit organs in the case of severe dengue infection. Here we predict kidney and lung cancer patients are vulnerable to dengue virus infection as CLEC5A mRNA expression in tumor samples using publicly available software such as TIMER and GEPIA database. We also identified the immunomodulatory role CLEC5A gene therefore targeting it could be a vital tool to cure dengue.

CLEC5A regulates the proliferation and migration of colon cancer via the AKT/mTOR signaling pathway.

Background: The purpose of this study is to understand the CLEC5A mechanism in colon cancer's proliferation and migration. Methods: The CLEC5A expression levels in colon cancer tissues were analyzed using bioinformatics method based on Oncomine and The Cancer Genome Atlas (TCGA) databases, which were further tested by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). The CLEC5A expression levels in 4 types of colon cancer cell lines (HCT116, SW620, HT29, and SW480) were also examined by qRT-PCR. We constructed CLEC5A knockdown cell lines and used colony formation, Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assays for investigating the CLEC5A function in colon cancer's proliferation and migration. A CLEC5A silencing nude mice model was established to measure the scale, weight, and growth rate of tumor xenograft. In CLEC5A knockdown cell lines and xenograft tissues, the levels of cell cycle and epithelial-mesenchymal transition (EMT)-related proteins were detected using Western blot (WB), and the phosphorylation levels of AKT/mTOR pathway key proteins were also detected by WB. On the basis of gene expression data retrieved from TCGA database, a relevance between CLEC5A and AKT/mTOR pathway in colon cancer was examined by gene set enrichment analysis (GSEA), and correlation analysis of CLEC5A and COL1A1 was employed to confirm their interaction. Results: Bioinformatics analysis, IHC staining, and qRT-PCR assay results all showed the significant high levels of CLEC5A expression in colon cancer tissues and cells, and positive links between CLEC5A levels and lymph node metastasis, vascular metastasis, and tumor-node-metastasis (TNM) stages of colon cancer patients. The suppressive effects of CLEC5A knockdown on colon cancer's proliferation and migration were verified in cell function and nude mice tumorigenesis assays. WB analysis further indicated that CLEC5A knockdown could inhibit cell cycle, and EMT processes, as well as AKT/mTOR pathway phosphorylation in colon cancer. On the basis of TCGA data, CLEC5A's activation effect on AKT/mTOR pathway had been confirmed by GSEA analysis, and the interaction between CLEC5A and COL1A1 was also revealed through correlation analysis in colon cancer. Conclusions: CLEC5A may promote the development and migration of colon cancer by triggering the AKT/mTOR signaling pathway. Furthermore, COL1A1 could serve as the target gene of CLEC5A.

CLEC5A mediates Zika virus-induced testicular damage.

BACKGROUND: Zika virus (ZIKV) infection is clinically known to induce testicular swelling, termed orchitis, and potentially impact male sterility, but the underlying mechanisms remain unclear. Previous reports suggested that C-type lectins play important roles in mediating virus-induced inflammatory reactions and pathogenesis. We thus investigated whether C-type lectins modulate ZIKV-induced testicular damage. METHODS: C-type lectin domain family 5 member A (CLEC5A) knockout mice were generated in a STAT1-deficient immunocompromised background (denoted clec5a-/-stat1-/-) to enable testing of the role played by CLEC5A after ZIKV infection in a mosquito-to-mouse disease model. Following ZIKV infection, mice were subjected to an array of analyses to evaluate testicular damage, including ZIKV infectivity and neutrophil infiltration estimation via quantitative RT-PCR or histology and immunohistochemistry, inflammatory cytokine and testosterone detection, and spermatozoon counting. Furthermore, DNAX-activating proteins for 12 kDa (DAP12) knockout mice (dap12-/-stat1-/-) were generated and used to evaluate ZIKV infectivity, inflammation, and spermatozoa function in order to investigate the potential mechanisms engaged by CLEC5A. RESULTS: Compared to experiments conducted in ZIKV-infected stat1-/- mice, infected clec5a-/-stat1-/- mice showed reductions in testicular ZIKV titer, local inflammation and apoptosis in testis and epididymis, neutrophil invasion, and sperm count and motility. CLEC5A, a myeloid pattern recognition receptor, therefore appears involved in the pathogenesis of ZIKV-induced orchitis and oligospermia. Furthermore, DAP12 expression was found to be decreased in the testis and epididymis tissues of clec5a-/-stat1-/- mice. As for CLEC5A deficient mice, ZIKV-infected DAP12-deficient mice also showed reductions in testicular ZIKV titer and local inflammation, as well as improved spermatozoa function, as compared to controls. CLEC5A-associated DAP12 signaling appears to in part regulate ZIKV-induced testicular damage. CONCLUSIONS: Our analyses reveal a critical role for CLEC5A in ZIKV-induced proinflammatory responses, as CLEC5A enables leukocytes to infiltrate past the blood-testis barrier and induce testicular and epididymal tissue damage. CLEC5A is thus a potential therapeutic target for the prevention of injuries to male reproductive organs in ZIKV patients.

CLEC5A expression can be triggered by spike glycoprotein and may be a potential target for COVID-19 therapy.

The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins. Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo, and in silico assays. The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor-binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Finally, CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.

In Silico Design of siRNAs for Silencing CLEC5A Receptor as a Potential Therapeutic Approach Against Dengue and Japanese Encephalitis Virus Infection in Human.

Dengue and Japanese encephalitis virus (JEV) are mosquito-borne RNA viruses that can cause severe illness leading to death in the tropics and subtropics. Both of these viruses interact directly with the C-type lectin domain family 5, member A receptor (CLEC5A) on human macrophages which stimulates the release of proinflammatory cytokines. Since blockade of this interaction has been shown to suppress the secretion of cytokines, CLEC5A is considered a potential target for the development of new treatments to reduce virus-induced brain damage. Developing a vaccine against dengue is challenging because this virus can cause disease through 4 different serotypes. Therefore, the vaccine must immunize against all 4 serotypes to be effective, while unvaccinated people still contract JEV and suffer from its complications. Small interfering RNAs (siRNAs) play an important role in regulating gene expression by causing the degradation of target mRNAs. In this study, we attempted to rationally design potential siRNA molecules using various software, targeting the CLEC5A gene. In total, 3 siRNAs were found to be potential candidates for CLEC5A silencing. They showed good target accessibility, optimum guanine-cytosine (GC) content, the least chance of off-target effects, positive energy of folding, and strong interaction with Argonaute2 protein as denoted by a negative docking energy score. In addition, molecular dynamics simulation of the siRNA-Ago2-docked complexes showed the stability of the complexes over 1.5 nanoseconds. These predicted siRNAs might effectively downregulate the expression of the CLEC5A receptor and thus prove vital in the treatment of dengue and JEV infections.

A Pan-Cancer Analysis Reveals CLEC5A as a Biomarker for Cancer Immunity and Prognosis.

Background: CLEC5A is a member of the C-type lectin superfamily. It can activate macrophages and lead to a series of immune-inflammation reactions. Previous studies reveal the role of CLEC5A in infection and inflammation diseases. Method: We acquire and analyze data from The Cancer Genome Atlas (TCGA) database, Genotype-Tissue Expression (GTEx) database, and other comprehensive databases via GSCALite, cBioPortal, and TIMER 2.0 platforms or software. Single-cell sequencing analysis was performed for quantifying the tumor microenvironment of several types of cancers. Results: CLEC5A is differentially expressed in a few cancer types, of which overexpression accompanies low overall survival of patients. DNA methylation mainly negatively correlates with CLEC5A expression. Moreover, CLEC5A is positively related to immune infiltration, including macrophages, cancer-associated fibroblasts (CAFs), and regulatory T cells (Tregs). Immune checkpoint genes are significantly associated with CLEC5A expression in diverse cancers. In addition, CLEC5A expression correlates with mismatch repair (MMR) in several cancers. Tumor mutation burden (TMB), microsatellite instability (MSI), and neoantigens show a positive association with CLEC5A expression in several cancers. Furthermore, CLEC5A in cancer correlates with signal transduction, the immune system, EMT, and apoptosis process. The drug sensitivity analysis screens out potential therapeutic agents associated with CLEC5A expression, including FR-180204, Tivozanib, OSI-930, Linifanib, AC220, VNLG/124, Bexarotene, omacetaxine mepesuccinate, narciclasine, leptomycin B, PHA-793887, LRRK2-IN-1, and CR-1-31B. Conclusion: CLEC5A overexpresses in multiple cancers in contrast to normal tissues, and high CLEC5A expression predicts poor prognosis of patients and immune infiltration. CLEC5A is a potential prognostic biomarker of diverse cancers and a target for anti-tumor therapy.

CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation.

BACKGROUND: Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. RESULTS: We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a-/-/tlr2-/- mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a-/-/tlr2-/- mice. CONCLUSIONS: This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

CLEC5a-directed bispecific antibody for effective cellular phagocytosis.

While antibody-dependent cellular phagocytosis mediated by activating Fcγ receptor is a key mechanism underlying many antibody drugs, their full therapeutic activities can be restricted by the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we describe a bispecific antibody approach that harnesses phagocytic receptor CLEC5A (C-type Lectin Domain Containing 5A) to drive Fcγ receptor-independent phagocytosis, potentially circumventing the negative impact of FcγRIIB. First, we established the effectiveness of such an approach by constructing bispecific antibodies that simultaneously target CLEC5A and live B cells. Furthermore, we demonstrated its in vivo application for regulatory T cell depletion and subsequent tumor regression.

The Role of Plasma Cell-Free Mitochondrial DNA and Nuclear DNA in Systemic Lupus Erythematosus.

BACKGROUND: The roles of plasma cell-free (pcf) mitochondrial DNA (mtDNApcf) and nuclear DNA (nDNApcf) in the pathogenesis of systemic lupus erythematosus (SLE) remain unclear. We analyzed the relative copies of mtDNApcf and nDNApcf and investigated their association with the levels of plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG), plasma malondialdehyde (MDA) and mRNA of leukocyte C-type lectin domain family 5 member A (CLEC5A) in SLE patients. METHODS: A total of 80 SLE patients and 43 healthy controls (HCs) were enrolled. Their plasma samples were subjected to the measurements of mtDNApcf copies, nDNApcf copies, 8-OHdG and MDA, respectively. Their leukocytes were analyzed for CLEC5A mRNA expression. RESULTS: SLE patients had higher nDNApcf copies (2.84 ± 1.99 vs. 2.00 ± 0.88, p = 0.002), lower mtDNApcf copies (4.81 ± 6.33 vs. 9.83 ± 14.20, p = 0.032), higher plasma 8-OHdG (0.227 ± 0.085 vs. 0.199 ± 0.041 ng/mL, p = 0.016), lower plasma MDA (3.02 ± 2.20 vs. 4.37 ± 2.16 µM, p = 0.001) and similar leukocyte CLEC5A mRNA expression levels (1.21 ± 1.17 vs. 1.26 ± 1.05, p = 0.870), as compared with those of HCs. Among the HCs, SLE patients with SLE Disease Activity Index (SLEDAI) ≤8, and SLE patients with SLEDAI >8, their respective mtDNApcf copies decreased stepwisely (9.83 ± 14.20 vs. 6.28 ± 7.91 vs. 3.19 ± 3.35, p = 0.054). The nDNApcf copies of HCs, SLE patients without nephritis, and SLE patients with nephritis were increased stepwisely (2.00 ± 0.88 vs. 2.63 ± 1.74 vs. 3.16 ± 2.34, p = 0.043). Among SLE patients, higher nDNApcf copies were associated with higher levels of plasma 8-OHdG (p < 0.001) but lower plasma MDA (p = 0.019). Among HCs but not SLE patients, higher nDNApcf copies (p = 0.013) or lower mtDNApcf copies (p < 0.001) were related to higher levels of leukocyte CLEC5A mRNA expression. CONCLUSIONS: Higher nDNApcf, lower mtDNApcf, increased ROS-elicited oxidative DNA damage and dysregulated leukocyte CLEC5A expression might be implicated in the pathogenesis of SLE.

Identification of an Immune-Related Prognostic Gene CLEC5A Based on Immune Microenvironment and Risk Modeling of Ovarian Cancer.

Objective: To understand the immune characteristics of the ovarian cancer (OC) microenvironment and explore the differences of immune-related molecules and cells to establish an effective risk model and identify the molecules that significantly affected the immune response of OC, to help guide the diagnosis. Methods: First, we calculate the TMEscore which reflects the immune microenvironment, and then analyze the molecular differences between patients with different immune characteristics, and determine the prognostic genes. Then, the risk model was established by least absolute shrinkage and selection operator (LASSO) analysis and combined with clinical data into a nomogram for diagnosis and prediction. Subsequently, the potential gene CLEC5A influencing the immune response of OC was identified from the prognostic genes by integrative immune-stromal analysis. The genomic alteration was explored based on copy number variant (CNV) and somatic mutation data. Results: TMEscore was a prognostic indicator of OC. The prognosis of patients with high TMEscore was better. The risk model based on immune characteristics was a reliable index to predict the prognosis of patients, and the nomogram could comprehensively evaluate the prognosis of patients. Besides, CLEC5A was closely related to the abundance of immune cells, immune response, and the expression of immune checkpoints in the OC microenvironment. OC cells with high expression of CLEC5A increased the polarization of M2 macrophages. CLEC5A expression was significantly associated with TTN and CDK12 mutations and affected the copy number of tumor progression and immune-related genes. Conclusion: The study of immune characteristics in the OC microenvironment and the risk model can reveal the factors affecting the prognosis and guide the clinical hierarchical treatment. CLEC5A can be used as a potential key gene affecting the immune microenvironment remodeling of OC, which provides a new perspective for improving the effect of OC immunotherapy.

C-type lectins and extracellular vesicles in virus-induced NETosis.

Dysregulated formation of neutrophil extracellular traps (NETs) is observed in acute viral infections. Moreover, NETs contribute to the pathogenesis of acute viral infections, including those caused by the dengue virus (DV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Furthermore, excessive NET formation (NETosis) is associated with disease severity in patients suffering from SARS-CoV-2-induced multiple organ injuries. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and other members of C-type lectin family (L-SIGN, LSECtin, CLEC10A) have been reported to interact with viral glycans to facilitate virus spreading and exacerbates inflammatory reactions. Moreover, spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A (CLEC5A) has been shown as the pattern recognition receptor for members of flaviviruses, and is responsible for DV-induced cytokine storm and Japanese encephalomyelitis virus (JEV)-induced neuronal inflammation. Moreover, DV activates platelets via CLEC2 to release extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs). The DV-activated EXOs (DV-EXOs) and MVs (DV-MVs) stimulate CLEC5A and Toll-like receptor 2 (TLR2), respectively, to enhance NET formation and inflammatory reactions. Thus, EVs from virus-activated platelets (PLT-EVs) are potent endogenous danger signals, and blockade of C-type lectins is a promising strategy to attenuate virus-induced NETosis and intravascular coagulopathy.

CLEC5A knockdown protects against cardiac dysfunction after myocardial infarction by suppressing macrophage polarization, NLRP3 inflammasome activation, and pyroptosis.

Increasing evidence has shown that the NOD-like receptor protein 3 (NLRP3) inflammasome and pyroptotic cell death play vital roles in the pathophysiology of myocardial infarction (MI), a common cardiovascular disease characterized by cardiac dysfunction. C-type lectin member 5A (CLEC5A) has been reported to be strongly associated with activation of the NLRP3 inflammasome and pyroptosis. In this study, an in vivo MI model was established by ligation of the left anterior descending coronary artery in male C57BL/6 mice, and CLEC5A knockdown was further achieved by intra-myocardial injection of adenovirus delivering shRNA-CLEC5A. CLEC5A was found to be highly expressed in the left ventricle of MI mice, while CLEC5A knockdown alleviated cardiac dysfunction in MI mice. In addition, MI-induced classical activation of macrophages was significantly inhibited after CLEC5A silencing. Additionally, CLEC5A knockdown dramatically inhibited MI-triggered activation of NLRP3 inflammasome, pyroptosis, and NF-κB signaling in the left ventricle of mice. In vitro experiments further validated that CLEC5A knockdown suppressed M1 polarization in LPS/IFNγ-stimulated RAW264.7 cells and inhibited the polarized RAW264.7-induced activation of NLRP3 inflammasome/pyroptosis signaling in co-cultured cardiomyocytes. In conclusion, CLEC5A knockdown protects against MI-induced cardiac dysfunction by regulating macrophage polarization, NLRP3 inflammasome, and cell pyroptosis.

Activation of an Effective Immune Response after Yellow Fever Vaccination Is Associated with the Genetic Background and Early Response of IFN-γ and CLEC5A.

The yellow fever vaccine (YF17DD) is highly effective with a single injection conferring protection for at least 10 years. The YF17DD induces polyvalent responses, with a TH1/TH2 CD4+ profile, robust T CD8+ responses, and synthesis of interferon-gamma (IFN-γ), culminating in high titers of neutralizing antibodies. Furthermore, C-type lectin domain containing 5A (CLEC5A) has been implicated in innate outcomes in other flaviviral infections. Here, we conducted a follow-up study in volunteers immunized with YF17DD, investigating the humoral response, cellular phenotypes, gene expression, and single nucleotide polymorphisms (SNPs) of IFNG and CLEC5A, to clarify the role of these factors in early response after vaccination. Activation of CLEC5A+ monocytes occurred five days after vaccination (DAV). Following, seven DAV data showed activation of CD4+ and CD8+T cells together with early positive correlations between type II IFN and genes of innate antiviral response (STAT1, STAT2, IRF7, IRF9, OAS1, and RNASEL) as well as antibody levels. Furthermore, individuals with genotypes rs2430561 AT/AA, rs2069718 AG/AA (IFNG), and rs13237944 AC/AA (CLEC5A), exhibited higher expression of IFNG and CLEC5A, respectively. Together, we demonstrated that early IFN-γ and CLEC5A responses, associated with rs2430561, rs2069718, and rs13237944 genotypes, may be key mechanisms in the long-lasting immunity elicited by YF17DD.

Specific macrophage subsets accumulate in human subcutaneous and omental fat depots during obesity.

Obesity is a chronic inflammatory disease associated with adipose tissue macrophage (ATM) activation. ATMs from lean mice contribute to tissue homeostasis by their M2-oriented polarization, whereas obesity leads to an increase of M1 inflammatory ATMs that underlies obesity-related metabolic disorders. In humans, studies characterizing ATMs and their functional status are limited. Here we investigated ATM phenotype in visceral (VAT) and subcutaneous (SAT) adipose tissue from healthy lean and obese individuals using two molecules previously identified as markers of M1-like and M2-like/tissue-resident macrophages, the C-type lectin CLEC5A and the scavenger receptor CD163L1, respectively. CD163L1 was expressed by the majority of ATMs, and CD163L1+ ATM density was greater with respect to cells expressing the pan-macrophage markers CD68 or CD11b. ATM counts in SAT, but not in VAT, increased in obese compared to lean individuals, measured with the three markers. Accordingly, CD163L1, CD68 and ITGAM gene expression was significantly enhanced in obese with respect to control individuals only in SAT. CLEC5A+ ATMs had a proinflammatory profile and were abundant in the lean VAT, but their density diminished in obesity. The only ATM subset that increased its counts in the obese VAT had a mixed M1-like (CD11c+ CD163- CD209- ) and M2-like (CLEC5A- CD206+ ) phenotype. ATM expansion was dominated by a subset of M2-like macrophages (CD11c- CLEC5A- CD163+ CD206+ CD209+ ) in the obese SAT, with a minor contribution of a CD11c+ CLEC5A- ATM subpopulation. Thus, both SAT and VAT seems to limit inflammation during obesity by differentially altering their ATM subset composition.

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

BACKGROUND: Crohn'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. AIM: To 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. METHODS: SNPs 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. RESULTS: In 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. CONCLUSION: Our 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.