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.

Prevalence of Selected Polymorphisms of Il7R, CD226, CAPSL, and CLEC16A Genes in Children and Adolescents with Autoimmune Thyroid Diseases.

Hashimoto's thyroiditis (HT) and Graves' disease (GD) are common autoimmune endocrine disorders in children. Studies indicate that apart from environmental factors, genetic background significantly contributes to the development of these diseases. This study aimed to assess the prevalence of selected single-nucleotide polymorphisms (SNPs) of Il7R, CD226, CAPSL, and CLEC16A genes in children with autoimmune thyroid diseases. We analyzed SNPs at the locus rs3194051, rs6897932 of IL7R, rs763361 of CD226, rs1010601 of CAPSL, and rs725613 of CLEC16A gene in 56 HT patients, 124 GD patients, and 156 healthy children. We observed significant differences in alleles IL7R (rs6897932) between HT males and the control group (C > T, p = 0.028) and between all GD patients and healthy children (C > T, p = 0.035) as well as GD females and controls (C > T, p = 0.018). Moreover, the C/T genotype was less frequent in GD patients at rs6897932 locus and in HT males at rs1010601 locus. The presence of the T allele in the IL7R (rs6897932) locus appears to have a protective effect against HT in males and GD in all children. Similarly, the presence of the T allele in the CAPSL locus (rs1010601) seems to reduce the risk of HT development in all patients.

Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB.

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany 'Neem' (Azadirachta indica), was identified as glycoprotein, thus being named as 'Neem Leaf Glycoprotein' (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (Tc)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in Tc cells. METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of ß-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs. RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand's peripheral ß-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1. CONCLUSION: NLGP's binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including Tc cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.

Endothelial CLEC-1b plays a protective role against cancer hematogenous metastasis.

Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.

Interaction of human dendritic cell receptor DEC205/CD205 with keratins.

DEC205 (CD205) is one of the major endocytic receptors on dendritic cells and has been widely used as a receptor target in immune therapies. It has been shown that DEC205 can recognize dead cells through keratins in a pH-dependent manner. However, the mechanism underlying the interaction between DEC205 and keratins remains unclear. Here we determine the crystal structures of an N-terminal fragment of human DEC205 (CysR∼CTLD3). The structural data show that DEC205 shares similar overall features with the other mannose receptor family members such as the mannose receptor and Endo180, but the individual domains of DEC205 in the crystal structure exhibit distinct structural features that may lead to specific ligand binding properties of the molecule. Among them, CTLD3 of DEC205 adopts a unique fold of CTLD, which may correlate with the binding of keratins. Furthermore, we examine the interaction of DEC205 with keratins by mutagenesis and biochemical assays based on the structural information and identify an XGGGX motif on keratins that can be recognized by DEC205, thereby providing insights into the interaction between DEC205 and keratins. Overall, these findings not only improve the understanding of the diverse ligand specificities of the mannose receptor family members at the molecular level but may also give clues for the interactions of keratins with their binding partners in the corresponding pathways.

IL-23 induces CLEC5A+ IL-17A+ neutrophils and elicit skin inflammation associated with psoriatic arthritis.

IL-23-activation of IL-17 producing T cells is involved in many rheumatic diseases. Herein, we investigate the role of IL-23 in the activation of myeloid cell subsets that contribute to skin inflammation in mice and man. IL-23 gene transfer in WT, IL-23RGFP reporter mice and subsequent analysis with spectral cytometry show that IL-23 regulates early innate immune events by inducing the expansion of a myeloid MDL1+CD11b+Ly6G+ population that dictates epidermal hyperplasia, acanthosis, and parakeratosis; hallmark pathologic features of psoriasis. Genetic ablation of MDL-1, a major PU.1 transcriptional target during myeloid differentiation exclusively expressed in myeloid cells, completely prevents IL-23-pathology. Moreover, we show that IL-23-induced myeloid subsets are also capable of producing IL-17A and IL-23R+MDL1+ cells are present in the involved skin of psoriasis patients and gene expression correlations between IL-23 and MDL-1 have been validated in multiple patient cohorts. Collectively, our data demonstrate a novel role of IL-23 in MDL-1-myelopoiesis that is responsible for skin inflammation and related pathologies. Our data open a new avenue of investigations regarding the role of IL-23 in the activation of myeloid immunoreceptors and their role in autoimmunity.

[Durable remission of T-cell prolymphocytic leukemia with CLEC16A::IL2 after allogeneic hematopoietic stem cell transplantation].

A 64-year-old woman presented with fine motor impairment in both hands. MRI revealed a contrast-enhanced lesion in the medulla oblongata. Lymphoid cells with abnormal blebs were observed and a CD4+/CD8+ double positive (DP) T cell population was detected by flow cytometry (FCM) in the bone marrow (BM) and the peripheral blood (PB). CLEC16A::IL2 fusion gene was identified by whole exome sequencing with DNA prepared from DP T cells. Clonal rearrangement of the T-cell receptor gene and expression of TCL1A protein were detected. This led to a diagnosis of T-cell prolymphocytic leukemia (T-PLL) with central nervous system (CNS) infiltration. Abnormal cells in BM and PB became undetectable on microscopy and FCM, and the CNS lesion disappeared on MRI after second-line therapy with alemtuzumab. Meanwhile, the CLEC16A::IL2 fusion mRNA remained detectable in PB. Allogeneic hematopoietic stem-cell transplantation was performed, and the fusion mRNA has now been undetectable for more than 5 years since transplantation. This is the first report of a T-PLL case with a CLEC16A::IL2 fusion gene.

Cirrhosis-downregulated LSECtin can be retrieved by cytokines, shifts the TLR-induced LSECs secretome and correlates with the hepatic Th response.

BACKGROUND AND AIMS: We evaluated tolerogenic C-type lectin LSECtin loss in cirrhosis and its potential regulation by cytokines. METHODS: Liver tissue from patients with cirrhosis and healthy controls, immortalised and generated LSECtin-CRISPR immortalised LSECs, and murine primary LSECs from the CCl4 model were handled. RESULTS: LSECtin expression was reduced in liver tissue from cirrhotic patients, and it decreased from compensated to decompensated disease. Increased phosphorylation of MAPK, Akt and NFkB was observed upon LSECtin stimulation in LSEC murine cell line, showing a pattern of inflammatory and chemotactic cytokines either restrained (IL-10, CCL4) or unrestrained (TNF-α, IL-1ß, IL-6, CCL2). CD44 attenuated whereas LAG-3 increased all substrates phosphorylation in combination with TLR4 and TLR2 ligands except for NFkB. TNF-α, IL-1 ß, IL-6 and CCL2 were restrained by LSECtin crosslinking on TLRs studied. Conversely, IL-10 and CCL4 were upregulated, suggesting a LSECtin-TLRs synergistic effect. Also, LSECtin was significantly induced after IL-13 stimulation or combined with anti-inflammatory cytokines in cirrhotic and immortalised LSECs. Th17 and regulatory T cells were progressively increased in the hepatic tissue from compensated to decompensated patients. A significant inverse correlation was present between gene expression levels of CLEC4G/LSECtin and RORγT and FOXP3 in liver tissues. CONCLUSION: LSECtin restrains TLR proinflammatory secretome induced on LSECs by interfering immune response control, survival and MAPKs signalling pathways. The cytokine-dependent induction of LSECtin and the association between LSECtin loss and Th17 cell subset expansion in the liver, provides a solid background for exploring LSECtin retrieval as a mechanism to reprogram LSEC homeostatic function hampered during cirrhosis.

CD301 and LSECtin glycan-binding receptors of innate immune cells serve as prognostic markers and potential predictors of immune response in breast cancer subtypes.

Glycosylation is a prominent posttranslational modification, and alterations in glycosylation are a hallmark of cancer. Glycan-binding receptors, primarily expressed on immune cells, play a central role in glycan recognition and immune response. Here, we used the recombinant C-type glycan-binding receptors CD301, Langerin, SRCL, LSECtin, and DC-SIGNR to recognize their ligands on tissue microarrays (TMA) of a large cohort (n = 1859) of invasive breast cancer of different histopathological types to systematically determine the relevance of altered glycosylation in breast cancer. Staining frequencies of cancer cells were quantified in an unbiased manner by a computer-based algorithm. CD301 showed the highest overall staining frequency (40%), followed by LSECtin (16%), Langerin (4%) and DC-SIGNR (0.5%). By Kaplan-Meier analyses, we identified LSECtin and CD301 as prognostic markers in different breast cancer subtypes. Positivity for LSECtin was associated with inferior disease-free survival in all cases, particularly in estrogen receptor positive (ER+) breast cancer of higher histological grade. In triple negative breast cancer, positivity for CD301 correlated with a worse prognosis. Based on public RNA single-cell sequencing data of human breast cancer infiltrating immune cells, we found CLEC10A (CD301) and CLEC4G (LSECtin) exclusively expressed in distinct subpopulations, particularly in dendritic cells and macrophages, indicating that specific changes in glycosylation may play a significant role in breast cancer immune response and progression.

PvMR1, a novel C-type lectin plays a crucial role in the antibacterial immune response of Pacific white shrimp, Penaeus vannamei.

C-type lectins (CTLs) are important immune molecules in innate immune, which participate in non-self recognition and clearance of pathogens. Here, a new CTL with two distinct C-type lectin domains (CTLDs) from Pacific white shrimp Penaeus vannamei, designated as PvMR1 was identified. The obtained PvMR1 coding sequence (CDS) was 1044 bp long encoding a protein with 347 amino acids. PvMR1 had two CTLD, a conserved mannose-specific EPN motif and a galactose-specific QPD motif, clustering into the same branch as the crustacean CTLs. PvMR1 was widely distributed in shrimp tissues with the highest transcription level in the hepatopancreas, with significantly induced mRNA expression on the hepatopancreas and intestines after immune challenge with Vibrio anguillarum. In vitro assays with recombinant PvMR1 (rPvMR1) protein revealed that it exhibited a wide range of antimicrobial activity, bacterial binding ability, and bacterial agglutination activity in a Ca2+-independent manner. Moreover, PvMR1 promoted bacterial phagocytosis in hemocytes. Furthermore, rPvMR1 treatment could significantly enhance the bacterial clearance in hemolymph and greatly improved the survival of shrimp under V. anguillarum infection in vivo. These results collectively suggest that PvMR1 plays an important role in antibacterial immune response of P. vannamei.

LDLa containing C-type lectin mediates phagocytosis of V.anguillarum and regulates immune effector genes in shrimp.

C-type lectins (CTLs) function as pattern recognition receptors (PRRs) by recognizing invading microorganisms, thereby triggering downstream immune events against infected pathogens. In this study, a novel CTL containing a low-density lipoprotein receptor class A (LDLa) domain was obtained from Litopenaeus vannamei, designed as LvLDLalec. Stimulation by the bacterial pathogen Vibrio anguillarum (V. anguillarum) resulted in remarkable up-regulation of LvLDLalec, as well as release of LvLDLalec into hemolymph. The rLvLDLalec protein possessed broad-spectrum bacterial binding and agglutinating activities, as well as hemocyte attachment ability. Importantly, LvLDLalec facilitated the bacterial clearance in shrimp hemolymph and protected shrimp from bacterial infection. Further studies revealed that LvLDLalec promoted hemocytes phagocytosis against V. anguillarum and lysosomes were involved in the process. Meanwhile, LvLDLalec participated in humoral immunity through activating and inducing nuclear translocation of Dorsal to regulate phagocytosis-related genes and antimicrobial peptides (AMPs) genes, thereby accelerated the removal of invading pathogens in vivo and improved the survival rate of L. vannamei. These results unveil that LvLDLalec serves as a PRR participate in cellular and humoral immunity exerting opsonin activity to play vital roles in the immune regulatory system of L. vannamei.

CLEC19A overexpression inhibits tumor cell proliferation/migration and promotes apoptosis concomitant suppression of PI3K/AKT/NF-κB signaling pathway in glioblastoma multiforme.

BACKGROUND: GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. METHODS: To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. RESULTS: Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. CONCLUSIONS: To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

Fungal chitin-binding glycoprotein induces Dectin-2-mediated allergic airway inflammation synergistically with chitin.

Although chitin in fungal cell walls is associated with allergic airway inflammation, the precise mechanism underlying this association has yet to be elucidated. Here, we investigated the involvement of fungal chitin-binding protein and chitin in allergic airway inflammation. Recombinant Aspergillus fumigatus LdpA (rLdpA) expressed in Pichia pastoris was shown to be an O-linked glycoprotein containing terminal α-mannose residues recognized by the host C-type lectin receptor, Dectin-2. Chitin particles were shown to induce acute neutrophilic airway inflammation mediated release of interleukin-1α (IL-1α) associated with cell death. Furthermore, rLdpA-Dectin-2 interaction was shown to promote phagocytosis of rLdpA-chitin complex and activation of mouse bone marrow-derived dendritic cells (BMDCs). Moreover, we showed that rLdpA potently induced T helper 2 (Th2)-driven allergic airway inflammation synergistically with chitin, and Dectin-2 deficiency attenuated the rLdpA-chitin complex-induced immune response in vivo. In addition, we showed that serum LdpA-specific immunoglobulin levels were elevated in patients with pulmonary aspergillosis.

A convenient assay for soluble Dectin-1 lectin domain binding to insoluble ß-glucans.

ß-Glucan is a homopolymer with a backbone of ß-1,3-linked glucose residues. The solubility and biological activity of ß-glucan can be influenced by the length of the backbone and the length/interval of the ß-1,6 branches. Dectin-1 is crucial in innate immunity through its binding to exogenous ß-glucans. However, there are few quantitative binding affinities available and there is no comprehensive comparative analysis of the binding of Dectin-1 to insoluble ß-glucans. Here, we have developed a simple binding assay for the interaction between Dectin-1 lectin domain (Dectin-1 CTLD) and insoluble ß-glucans. We utilized the paramylon particle as a model of insoluble ß-glucans. Dectin-1 CTLD bound to paramylon (particle size 3.1 µm) was separated from unbound Dectin-1 CTLD by centrifugation using a membrane filter (pore size 0.2 µm). The protein in the filtrate was quantified by SDS-PAGE and densitometry. The amount decreased in proportion to the amount of paramylon in the mixture. A control experiment using the Dectin-1 CTLD inactive mutant W221A showed that the mutant passes through the filter without binding paramylon. These results are evidence of site-specific binding of Dectin-1 CTLD to paramylon and demonstrate that the separation of paramylon-bound/unbound Dectin-1 CTLD is achievable through centrifugation using a filter. The assay was extended to other insoluble ß-glucans including curdlan. Additionally, it can be utilized in competitive inhibition experiments with soluble short-chain ß-glucans such as laminarin. The assay system allows for quantitative comparison of the affinities between insoluble and soluble ß-glucans and Dectin-1 CTLD, and should be useful because of its low-tech convenience.

CLEC11A expression as a prognostic biomarker in correlation to immune cells of gastric cancer.

Gastric cancer (GC) is a prevalent malignant cancer characterized by a poor survival rate. The C-type lectin domain family 11 member A (CLEC11A) is part of the C-type lectin superfamily, and its dysregulation has been implicated in the progression of several cancers. The specific role of CLEC11A and its association with immune infiltration in GC, however, remains unclear. In this study, we employed The Cancer Genome Atlas (TCGA) database, Gene Expression Omnibus (GEO) database, Tumor IMmune Estimation Resource (TIMER) database, Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, Kaplan-Meier plotter databases, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and the CIBERSORT algorithm to investigate CLEC11A expression, its prognostic significance, its association with tumor immune infiltration, and gene function enrichment in GC. We conducted western blotting, Cell Counting Kit-8 (CCK-8), wound healing, and transwell assays to validate CLEC11A's function. We found that CLEC11A expression was significantly elevated in GC when compared to adjacent non-tumor tissues. Elevated CLEC11A expression was strongly associated with poor survival outcomes and advanced clinicopathological stages.  Moreover, heightened CLEC11A expression positively correlated with immunomodulators, chemokines, and the infiltration of immune cells, especially M2 macrophages, in GC. Additionally, CLEC11A silencing suppressed GC cells proliferation, migration and invasion in vitro. Our results elucidate the functions of CLEC11A in GC, suggesting its potential as a valuable prognostic biomarker and therapeutic target for GC immunotherapy.

Identification and characterization of an L-type lectin from obscure puffer Takifugu obscurus in response to bacterial infection.

L-type lectins (LTLs) contain a carbohydrate recognition domain homologous to leguminous lectins, and have functions in selective protein trafficking, sorting and targeting in the secretory pathway of animals. In this study, a novel LTL, designated as ToERGIC-53, was cloned and identified from obscure puffer Takifugu obscurus. The open reading frame of ToERGIC-53 contained 1554 nucleotides encoding 517 amino acid residues. The deduced ToERGIC-53 protein consisted of a signal peptide, a leguminous lectin domain (LTLD), a coiled-coil region, and a transmembrane region. Quantitative real-time PCR showed that ToERGIC-53 was expressed in all examined tissues, with the highest expression level in the liver. The expression of ToERGIC-53 was significantly upregulated after infection with Vibrio harveyi and Staphylococcus aureus. Recombinant ToERGIC-53-LTLD (rToERGIC-53-LTLD) protein could not only agglutinate and bind to one Gram-positive bacterium (S. aureus) and three Gram-negative bacteria (V. harveyi, V. parahaemolyticus and Aeromonas hydrophila), but also bind to glycoconjugates on the surface of bacteria such as lipopolysaccharide, peptidoglycan, mannose and galactose. In addition, rToERGIC-53-LTLD inhibited the growth of bacteria in vitro. All these results suggested that ToERGIC-53 might be a pattern recognition receptor involved in antibacterial immune response of T. obscurus.

Comparison of skin mucus lectins among longtooth grouper Epinephelus bruneus and giant grouper E. lanceolatus as well as the hybrid, Kue-Tama, and their binding abilities to the skin fluke Benedeniaepinepheli.

Interspecific hybrids of farm-raised fish are becoming popular in aquaculture owing to their advantages over pure species, including improved growth and higher resistance to infectious diseases. Kue-Tama is a recently established hybrid grouper derived from the longtooth grouper Epinephelus bruneus (♀) × giant grouper E. lanceolatus (♂). In our previous study, this hybrid showed significantly higher resistance against the skin fluke Benedenia epinepheli, a problematic parasite in grouper farming, than the longtooth grouper. In the present study, we explored lectins in the skin mucus of hybrids and their parent species. While C-type lectins of approximately 15 kDa were obtained from longtooth groupers, additional C-type lectins with molecular masses of approximately 20 and 30 kDa, as well as 45-kDa F-type lectin, were also detected in Kue-Tama and giant groupers. Semi-quantitative reverse transcript-polymerase chain reaction (RT-PCR) demonstrated that the gene expression levels of both C-type and F-type lectins were significantly higher in the skin of the hybrid and giant groupers than that of the longtooth grouper. In addition, some skin mucus lectins of the hybrid and giant groupers were bound to the fluke, suggesting that these lectins conferred resistance to parasitic infections.

Genome-wide identification and expression analysis of C-type lectins in discus fish (Symphysodon aequifasciatus) during parental care.

Discus fish (Symphysodon aequifasciatus) exhibit a unique parental care behavior: adult discus produces secretion through their skin, on which the larvae live after birth. The immune components in the skin mucus of parental discus would change during different parental care. C-type lectins (CTLs) could identify and eliminate pathogenic microorganisms and play important roles in innate immunity. Studies on CTLs of discus fish especially during parental care, however, are scarce. Here, we identified 186 CTL genes that distributed in 27 linkage groups based on discus genome. Phylogenetic analysis showed that S. aequifasciatus CTL (SaCTL) members were grouped into 14 subfamilies. A total of 80 gene replication events occurred, of which 15 pairs were subjected to segmental duplication and 65 pairs underwent tandem duplication. Ka/Ks ranged from 0.11 (SaCTL25/SaCTL158) to 0.68 (SaCTL36/SaCTL69), all undergoing purifying selection. RNA-seq analysis revealed that SaCTL members, including duplicated genes, in the skin of parental discus show distinct expression patterns in different care stages and between male and female parents. The SaCTL11 was differentially expressed in most care stages and reached the maximum after eggs spawned, but the expression of its paired SaCTL14 was low in each stage. The SaCTL39 increased first and then decreased, reaching a peak in eggs spawned, while paired SaCTL48 first decreased and then increased, reaching a peak in hatched eggs. The SaCTL50 was differentially expressed only in female fish during care, but not in male fish. These results provide new insights into the evolution and potential functional differentiation of CTLs in discus fish during parental care.

DECTIN-1: A modifier protein in CTLA-4 haploinsufficiency.

Autosomal dominant loss-of-function (LoF) variants in cytotoxic T-lymphocyte associated protein 4 (CTLA4) cause immune dysregulation with autoimmunity, immunodeficiency and lymphoproliferation (IDAIL). Incomplete penetrance and variable expressivity are characteristic of IDAIL caused by CTLA-4 haploinsufficiency (CTLA-4h), pointing to a role for genetic modifiers. Here, we describe an IDAIL proband carrying a maternally inherited pathogenic CTLA4 variant and a paternally inherited rare LoF missense variant in CLEC7A, which encodes for the ß-glucan pattern recognition receptor DECTIN-1. The CLEC7A variant led to a loss of DECTIN-1 dimerization and surface expression. Notably, DECTIN-1 stimulation promoted human and mouse regulatory T cell (Treg) differentiation from naïve αß and γδ T cells, even in the absence of transforming growth factor-ß. Consistent with DECTIN-1's Treg-boosting ability, partial DECTIN-1 deficiency exacerbated the Treg defect conferred by CTL4-4h. DECTIN-1/CLEC7A emerges as a modifier gene in CTLA-4h, increasing expressivity of CTLA4 variants and acting in functional epistasis with CTLA-4 to maintain immune homeostasis and tolerance.