Identification of functional enhancer variants associated with type I diabetes in CD4+ T cells.

Type I diabetes is an autoimmune disease mediated by T-cell destruction of ß cells in pancreatic islets. Currently, there is no known cure, and treatment consists of daily insulin injections. Genome-wide association studies and twin studies have indicated a strong genetic heritability for type I diabetes and implicated several genes. As most strongly associated variants are noncoding, there is still a lack of identification of functional and, therefore, likely causal variants. Given that many of these genetic variants reside in enhancer elements, we have tested 121 CD4+ T-cell enhancer variants associated with T1D. We found four to be functional through massively parallel reporter assays. Three of the enhancer variants weaken activity, while the fourth strengthens activity. We link these to their cognate genes using 3D genome architecture or eQTL data and validate them using CRISPR editing. Validated target genes include CLEC16A and SOCS1. While these genes have been previously implicated in type 1 diabetes and other autoimmune diseases, we show that enhancers controlling their expression harbor functional variants. These variants, therefore, may act as causal type 1 diabetic variants.

Single-cell transcriptome reveals the heterogeneity of malignant ductal cells and the prognostic value of REG4 and SPINK1 in primary pancreatic ductal adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths, with very limited therapeutic options available. This study aims to comprehensively depict the heterogeneity and identify prognostic targets for PDAC with single-cell RNA sequencing (scRNA-seq) analysis. Methods: ScRNA-seq analysis was performed on 16 primary PDAC and three adjacent lesions. A series of analytical methods were applied for analysis in cell clustering, gene profiling, lineage trajectory analysis and cell-to-cell interactions. In vitro experiments including colony formation, wound healing and sphere formation assay were performed to assess the role of makers. Results: A total of 32,480 cells were clustered into six major populations, among which the ductal cell cluster expressing high copy number variants (CNVs) was defined as malignant cells. Malignant cells were further subtyped into five subgroups which exhibited specific features in immunologic and metabolic activities. Pseudotime trajectory analysis indicated that components of various oncogenic pathways were differentially expressed along tumor progression. Furthermore, intensive substantial crosstalk between ductal cells and stromal cells was identified. Finally, genes (REG4 and SPINK1) screened out of differentially expressed genes (DEGs) were upregulated in PDAC cell lines. Silencing either of them significantly impaired proliferation, invasion, migration and stemness of PDAC cells. Conclusions: Our findings offer a valuable resource for deciphering the heterogeneity of malignant ductal cells in PDAC. REG4 and SPINK1 are expected to be promising targets for PDAC therapy.

Removal of Double-Stranded RNA Contaminants During Template-Directed Synthesis of mRNA.

The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these contaminants are the result of the T7 RNA polymerase side activity. In this study, we used a modified method of mRNA purification based on the selective binding of dsRNA to cellulose in ethanol-containing buffer. It was shown both in vivo and in vitro that the cellulose-purified mRNA preparation leads neither to activation of the lymphocyte inflammatory marker CD69 nor to increased release of IFNα in mice, and does not contain impurities detectable by antibodies to dsRNA.

A prognostic model based on CLEC6A predicts clinical outcome of breast cancer patients.

CLEC6A, (C-type lectin domain family 6, member A), plays a prominent role in regulating innate immunity and adaptive immunity. CLEC6A has shown great potential as a target for cancer immunotherapy. This study aims to explore the prognostic value of CLEC6A, and analyze the relationship associated with the common hematological parameters in breast cancer patients. We performed a retrospective analysis on 183 breast cancer patients data in hospital information system from January 2013 to December 2015. The expression of CLEC6A was recorded via semiquantitative immunohistochemistry in breast cancer. The association between expression of CLEC6A and relative parameters were performed by Chi-square test and Fisher's exact test. Kaplan-Meier assay and Log-rank test were performed to evaluate the survival time. The Cox proportional hazards regression analysis was applied to identify prognostic factors. Nomograms were conducted to predict 1-, 3-, and 5-year disease free survival (DFS) and overall survival (OS) for breast cancer, which could be a good reference in clinical practice. The nomogram model was estimated by calibration curve analysis for its function of discrimination. The accuracy and benefit of the nomogram model were appraised by comparing it to only CLEC6A via decision curve analysis (DCA). The prediction accuracy of CLEC6A was also determined by time-dependent receiver operating characteristics (TDROC) curves, and the area under the curve (AUC) for different survival time. There were 94 cases in the CLEC6A low-expression group and 89 cases in CLEC6A high-expression group. Compared to CLEC6A low-expression group, the CLEC6A high-expression group had better survival (DFS: 56.95 vs. 70.81 months, P = 0.0078 and OS: 67.98 vs. 79.05 months, P = 0.0089). The CLEC6A was a potential prognostic factor in multivariate analysis (DFS: P = 0.023, hazard ratio (HR): 0.454, 95 % confidence interval (CI): 0.229-0.898; OS: P = 0.020, HR: 0.504, 95 %CI: 0.284-0.897). The nomogram in accordance with these potential prognostic factors was constructed to predict survival and the calibration curve analysis had indicated that the predicted line was well-matched with reference line in 1-, 3-, and 5-year DFS and OS category. The 1-, 3-, and 5-year DCA curves have revealed that nomogram model yielded larger net benefits than CLEC6A alone. Finally, the TDROC curve indicated that CLEC6A could better predict 1-year DFS and OS than others. Furthermore, we combined these potential independent prognostic factors to analyze the relationship among these hematologic index and oxidative stress indicators, and indicated that higher CLEC6A level, higher CO2 level or low CHOL level or high HDL-CHO level would have survived longer and better prognosis. In breast cancer, high expression of CLEC6A can independently predict better survival. Our nomogram consisted of CLEC6A and other indicators has good predictive performance and can facilitate clinical decision-making.

CLEC7A regulates M2 macrophages to suppress the immune microenvironment and implies poorer prognosis of glioma.

Background: Gliomas constitute a category of malignant tumors originating from brain tissue, representing the majority of intracranial malignancies. Previous research has demonstrated the pivotal role of CLEC7A in the progression of various cancers, yet its specific implications within gliomas remain elusive. The primary objective of this study was to investigate the prognostic significance and immune therapeutic potential of CLEC7A in gliomas through the integration of bioinformatics and clinical pathological analyses. Methods: This investigation involved examining and validating the relationship between CLEC7A and glioma using samples from Hospital, along with data from TCGA, GEO, GTEx, and CGGA datasets. Subsequently, we explored its prognostic value, biological functions, expression location, and impact on immune cells within gliomas. Finally, we investigated its potential impact on the chemotaxis and polarization of macrophages. Results: The expression of CLEC7A is upregulated in gliomas, and its levels escalate with the malignancy of tumors, establishing it as an independent prognostic factor. Functional enrichment analysis revealed a significant correlation between CLEC7A and immune function. Subsequent examination of immune cell differential expression demonstrated a robust association between CLEC7A and M2 macrophages. This conclusion was further substantiated through single-cell analysis, immunofluorescence, and correlation studies. Finally, the knockout of CLEC7A in M2 macrophages resulted in a noteworthy reduction in macrophage chemotaxis and polarization factors. Conclusion: CLEC7A expression is intricately linked to the pathology and molecular characteristics of gliomas, establishing its role as an independent prognostic factor for gliomas and influencing macrophage function. It could be a promising target for immunotherapy in gliomas.

Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.

Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

Immune function of a C-type lectin with long tandem repeats and abundant threonine in the ridgetail white prawn Exopalaemon carinicauda.

C-type lectins (CTLs) are an important class of pattern recognition receptors (PRRs) that exhibit structural and functional diversity in invertebrates. Repetitive DNA sequences are ubiquitous in eukaryotic genomes, representing distinct modes of genome evolution and promoting new gene generation. Our study revealed a new CTL that is composed of two long tandem repeats, abundant threonine, and one carbohydrate recognition domain (CRD) in Exopalaemon carinicauda and has been designated EcTR-CTL. The full-length cDNA of EcTR-CTL was 1242 bp long and had an open reading frame (ORF) of 999 bp that encoded a protein of 332 amino acids. The genome structure of EcTR-CTL contains 4 exons and 3 introns. The length of each repeat unit in EcTR-CTL was 198 bp, which is different from the short tandem repeats reported previously in prawns and crayfish. EcTR-CTL was abundantly expressed in the intestine and hemocytes. After Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge, the expression level of EcTR-CTL in the intestine was upregulated. Knockdown of EcTR-CTL downregulated the expression of anti-lipopolysaccharide factor, crustin, and lysozyme during Vibrio infection. The recombinant CRD of EcTR-CTL (rCRD) could bind to bacteria, lipopolysaccharides, and peptidoglycans. Additionally, rCRD can directly bind to WSSV. These findings indicate that 1) CTLs with tandem repeats may be ubiquitous in crustaceans, 2) EcTR-CTL may act as a PRR to participate in the innate immune defense against bacteria via nonself-recognition and antimicrobial peptide regulation, and 3) EcTR-CTL may play a positive or negative role in the process of WSSV infection by capturing virions.

Characterization of the genetic variation and evolutionary divergence of the CLEC18 family.

BACKGROUND: The C-type lectin family 18 (CLEC18) with lipid and glycan binding capabilities is important to metabolic regulation and innate immune responses against viral infection. However, human CLEC18 comprises three paralogous genes with highly similar sequences, making it challenging to distinguish genetic variations, expression patterns, and biological functions of individual CLEC18 paralogs. Additionally, the evolutionary relationship between human CLEC18 and its counterparts in other species remains unclear. METHODS: To identify the sequence variation and evolutionary divergence of human CLEC18 paralogs, we conducted a comprehensive analysis using various resources, including human and non-human primate reference genome assemblies, human pangenome assemblies, and long-read-based whole-genome and -transcriptome sequencing datasets. RESULTS: We uncovered paralogous sequence variants (PSVs) and polymorphic variants (PVs) of human CLEC18 proteins, and identified distinct signatures specific to each CLEC18 paralog. Furthermore, we unveiled a novel segmental duplication for human CLEC18A gene. By comparing CLEC18 across human and non-human primates, our research showed that the CLEC18 paralogy probably occurred in the common ancestor of human and closely related non-human primates, and the lipid-binding CAP/SCP/TAPS domain of CLEC18 is more diverse than its glycan-binding CTLD. Moreover, we found that certain amino acids alterations at variant positions are exclusive to human CLEC18 paralogs. CONCLUSIONS: Our findings offer a comprehensive profiling of the intricate variations and evolutionary characteristics of human CLEC18.

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

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

KLRG1-expressing CD8+ T cells are exhausted and polyfunctional in patients with chronic hepatitis B.

Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1- CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1-E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.

Relationship between the Number of Repeats in the Neck Regions of L-SIGN and Augmented Virus Replication and Immune Responses in Dengue Hemorrhagic Fever.

C-type lectins play a crucial role as pathogen-recognition receptors for the dengue virus, which is responsible for causing both dengue fever (DF) and dengue hemorrhagic fever (DHF). DHF is a serious illness caused by the dengue virus, which exists in four different serotypes: DEN-1, DEN-2, DEN-3, and DEN-4. We conducted a genetic association study, during a significant DEN-2 outbreak in southern Taiwan, to explore how variations in the neck-region length of L-SIGN (also known as CD209L, CD299, or CLEC4M) impact the severity of dengue infection. PCR genotyping was utilized to identify polymorphisms in variable-number tandem repeats. We constructed L-SIGN variants containing either 7- or 9-tandem repeats and transfected these constructs into K562 and U937 cells, and cytokine and chemokine levels were evaluated using enzyme-linked immunosorbent assays (ELISAs) following DEN-2 virus infection. The L-SIGN allele 9 was observed to correlate with a heightened risk of developing DHF. Subsequent results revealed that the 9-tandem repeat was linked to elevated viral load alongside predominant T-helper 2 (Th2) cell responses (IL-4 and IL-10) in K562 and U937 cells. Transfecting K562 cells in vitro with L-SIGN variants containing 7- and 9-tandem repeats confirmed that the 9-tandem repeat transfectants facilitated a higher dengue viral load accompanied by increased cytokine production (MCP-1, IL-6, and IL-8). Considering the higher prevalence of DHF and an increased frequency of the L-SIGN neck's 9-tandem repeat in the Taiwanese population, individuals with the 9-tandem repeat may necessitate more stringent protection against mosquito bites during dengue outbreaks in Taiwan.

Reduced Expression of CLEC4G in Neurons Is Associated with Alzheimer's Disease.

CLEC4G, a glycan-binding receptor, has previously been demonstrated to inhibit Aß generation, yet its brain localization and functions in Alzheimer's disease (AD) are not clear. We explored the localization, function, and regulatory network of CLEC4G via experiments and analysis of RNA-seq databases. CLEC4G transcripts and proteins were identified in brain tissues, with the highest expression observed in neurons. Notably, AD was associated with reduced levels of CLEC4G transcripts. Bioinformatic analyses revealed interactions between CLEC4G and relevant genes such as BACE1, NPC1, PILRA, TYROBP, MGAT1, and MGAT3, all displaying a negative correlation trend. We further identified the upstream transcriptional regulators NR2F6 and XRCC4 for CLEC4G and confirmed a decrease in CLEC4G expression in APP/PS1 transgenic mice. This study highlights the role of CLEC4G in protecting against AD progression and the significance of CLEC4G for AD research and management.

Highly conserved and extremely variable: The paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae).

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

A unique Ca2+-inhibited C-type lectin in shrimp Fenneropenaeuschinensis.

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight ß-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates.

Innate phase production of IFN-γ by memory and effector T cells expressing early activation marker CD69 during infection with Cryptococcus deneoformans in the lungs.

Cryptococcus deneoformans is a yeast-type fungus that causes fatal meningoencephalitis in immunocompromised patients and evades phagocytic cell elimination through an escape mechanism. Memory T (Tm) cells play a central role in preventing the reactivation of this fungal pathogen. Among these cells, tissue-resident memory T (TRM) cells quickly respond to locally invaded pathogens. This study analyzes the kinetics of effector T (Teff) cells and Tm cells in the lungs after cryptococcal infection. Emphasis is placed on the kinetics and cytokine expression of TRM cells in the early phase of infection. CD4+ Tm cells exhibited a rapid increase by day 3, peaked at day 7, and then either maintained their levels or exhibited a slight decrease until day 56. In contrast, CD8+ Tm cells reached their peak on day 3 and thereafter decreased up to day 56 post-infection. These Tm cells were predominantly composed of CD69+ TRM cells and CD69+ CD103+ TRM cells. Disruption of the CARD9 gene resulted in reduced accumulation of these TRM cells and diminished interferon (IFN) -γ expression in TRM cells. TRM cells were derived from T cells with T cell receptors non-specific to ovalbumin in OT-II mice during cryptococcal infection. In addition, TRM cells exhibited varied behavior in different tissues. These results underscore the importance of T cells, which produce IFN-γ in the lungs during the early stage of infection, in providing early protection against cryptococcal infection through CARD9 signaling.

DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway.

C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

The macrophage polarization in allergic responses induced by tropomyosin of Macrobrachium nipponense in cell and murine models.

Macrophages are one of the important immune cells, which play important roles in innate and adaptive immune. However, the roles of macrophages in food allergy are not thoroughly understood. To investigate the roles of macrophages during food allergy, we focused on the relationship between macrophage polarization and allergic responses induced by tropomyosin (TM) in the present study. Arg 1 and CD206 expressions in the TM group were significantly higher than those of the PBS group, while iNOS and TNF-α expressions were no obvious difference, moreover, the morphology of macrophages stimulated by TM was similar to that of M2 macrophages. These results indicated macrophages were mainly polarized toward M2 phenotypes in vitro. The antibodies, mMCP-1, histamine and cytokines, revealed that macrophages could participate in food allergy, and macrophage polarization was associated with changes in allergic-related factors. The cytokine levels of M2 phenotypes were significantly higher than those of M1 phenotypes in peripheral blood. The mRNA expressions and protein levels of Arg1 and iNOS in the jejunum and peritoneal cells indicated that M2 phenotypes were the major macrophage in these tissues compared with M1 phenotypes. Hence, macrophage polarization plays an important role in food allergy.

C-type lectin 12B/4E of black rockfish (Sebastes schlegelii) macrophages as pattern recognition receptors in the antibacterial mechanism of exploration.

As lower vertebrates, fish have both innate and adaptive immune systems, but the role of the adaptive immune system is limited, and the innate immune system plays an important role in the resistance to pathogen infection. C-type lectins (CLRs) are one of the major pattern recognition receptors (PRRs) of the innate immune system. CLRs can combine with pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to trigger NF-κB signaling pathway and exert immune efficacy. In this study, Ssclec12b and Ssclec4e of the C-type lectins, were found to be significantly up-regulated in the transcripts of Sebastes schlegelii macrophages stimulated by bacteria. The identification, expression and function of these lectins were studied. In addition, the recombinant proteins of the above two CLRs were obtained by prokaryotic expression. We found that rSsCLEC12B and rSsCLEC4E could bind to a variety of bacteria in a Ca2+-dependent manner, and promoted the agglutination of bacteria and blood cells. rSsCLEC12B and rSsCLEC4E assisted macrophages to recognize PAMPs and activate the NF-κB signaling pathway, thereby promoting the expression of inflammatory factors (TNF-α, IL-1ß, IL-6, IL-8) and regulating the early immune inflammation of macrophages. These results suggested that SsCLEC12B and SsCLEC4E could serve as PRRs in S. schlegelii macrophages to recognize pathogens and participate in the host antimicrobial immune process, and provided a valuable reference for the study of CLRs involved in fish innate immunity.