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.

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.

The role of KLRG1: a novel biomarker and new therapeutic target.

Killer cell lectin-like receptor G1 (KLRG1) is an immune checkpoint receptor expressed predominantly in NK and T-cell subsets that downregulates the activation and proliferation of immune cells and participates in cell-mediated immune responses. Accumulating evidence has demonstrated the importance of KLRG1 as a noteworthy disease marker and therapeutic target that can influence disease onset, progression, and prognosis. Blocking KLRG1 has been shown to effectively mitigate the effects of downregulation in various mouse tumor models, including solid tumors and hematologic malignancies. However, KLRG1 inhibitors have not yet been approved for human use, and the understanding of KLRG1 expression and its mechanism of action in various diseases remains incomplete. In this review, we explore alterations in the distribution, structure, and signaling pathways of KLRG1 in immune cells and summarize its expression patterns and roles in the development and progression of autoimmune diseases, infectious diseases, and cancers. Additionally, we discuss the potential applications of KLRG1 as a tool for tumor immunotherapy.

Effects of a ß-Glucan-Rich Blend of Medicinal Mushrooms and Botanicals on Innate Immune Cell Activation and Function Are Enhanced by a Very Low Dose of Bovine Colostrum Peptides.

Nutraceutical immune support offers potential for designing blends with complementary mechanisms of action for robust support of innate immune alertness. We documented enhanced immune activation when bovine colostrum peptides (BC-Pep) were added to an immune blend (IB) containing ß-glucans from yeast, shiitake, maitake, and botanical non-ß-glucan polysaccharides. Human peripheral blood mononuclear cells (PBMCs) were cultured with IB, BC-Pep, and IB + BC-Pep for 20 h, whereafter expression of the activation marker CD69 was evaluated on NK cells, NKT cells, and T cells. Cytokine levels were tested in culture supernatants. PBMCs were co-cultured with K562 target cells to evaluate T cell-mediated cytotoxicity. IB + BC-Pep triggered highly significant increases in IL-1ß, IL-6, and TNF-α, above that of cultures treated with matching doses of either IB or BC-Pep. NK cell and T cell activation was increased by IB + BC-Pep, reaching levels of CD69 expression several fold higher than either BC-Pep or IB alone. IB + BC-Pep significantly increased T cell-mediated cytotoxic killing of K562 target cells. This synergistic effect suggests unique amplification of signal transduction of NK cells and T cells due to modulation of IB-induced signaling pathways by BC-Pep and is of interest for further pre-clinical and clinical testing of immune defense activity against virally infected and transformed cells.

Circulating KLRG1+ long-lived effector memory T cells retain the flexibility to become tissue resident.

KLRG1+ CD8 T cells persist for months after clearance of acute infections and maintain high levels of effector molecules, contributing protective immunity against systemic pathogens. Upon secondary infection, these long-lived effector cells (LLECs) are incapable of forming other circulating KLRG1- memory subsets such as central and effector memory T cells. Thus, KLRG1+ memory T cells are frequently referred to as a terminally differentiated population that is relatively short lived. Here, we show that after viral infection of mice, effector cells derived from LLECs rapidly enter nonlymphoid tissues and reduce pathogen burden but are largely dependent on receiving antigen cues from vascular endothelial cells. Single-cell RNA sequencing reveals that secondary memory cells in nonlymphoid tissues arising from either KLRG1+ or KLRG1- memory precursors develop a similar resident memory transcriptional signature. Thus, although LLECs cannot differentiate into other circulating memory populations, they still retain the flexibility to enter tissues and establish residency.

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

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

Tissue-resident trained immunity in hepatocytes protects against septic liver injury in zebrafish.

Trained immunity is classically characterized by long-term functional reprogramming of innate immune cells to combat infectious diseases. Infection-induced organ injury is a common clinical severity phenotype of sepsis. However, whether the induction of trained immunity plays a role in protecting septic organ injury remains largely unknown. Here, through establishing an in vivo ß-glucan training and lipopolysaccharide (LPS) challenge model in zebrafish larvae, we observe that induction of trained immunity could inhibit pyroptosis of hepatocytes to alleviate septic liver injury, with an elevated trimethyl-histone H3 lysine 4 (H3K4me3) modification that targets mitophagy-related genes. Moreover, we identify a C-type lectin domain receptor in zebrafish, named DrDectin-1, which is revealed as the orchestrator in gating H3K4me3 rewiring-mediated mitophagy activation and alleviating pyroptosis-engaged septic liver injury in vivo. Taken together, our results uncover tissue-resident trained immunity in maintaining liver homeostasis at the whole-animal level and offer an in vivo model to efficiently integrate trained immunity for immunotherapies.

Immunological characteristics of CD103+CD161+ T lymphocytes on chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNPs) is a heterogeneous disease characterized by local inflammation of the upper airway and sinus mucosa. T cell-mediated immune responses play irreplaceable roles in the pathogenesis of nasal polyps. CD161+ T cells have been implicated in the pathology of several diseases through cytokine production and cytotoxic activity. However, the immunological characteristics of CD161+ T cells in nasal mucosa are still not well understood, particularly in CRSwNPs. Our research revealed a notable enrichment of CD161+ T cells in nasal tissues compared to peripheral blood, with a significantly more infiltration of CD161+ T cells in CRSwNPs compared to control nasal samples. Phenotypical analysis found that CD161+ T cells predominantly co-expressed tissue-resident memory surface markers CD103, CD69, and CD45RO. CD161+CD103+ T cells demonstrated complicated effector functions, marked by elevated levels of PD-1, CTLA-4, IL-17, and IFN-γ and diminished expression of FoxP3 and CD25. Interestingly, despite CD161+ T cells was more abundant in polyp tissues compared to normal control tissues, and then further categorizing polyp samples into distinct groups based on clinical characteristics, only the recurrent CRSwNP group showed a significant reduction in CD161+CD8+ T cells compared to the primary CRSwNP group. This finding suggested the necessity for further research to comprehensively understand the underlying mechanisms and the broader significance of CD161+ T cells in the advancement and relapse of CRSwNPs.

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.

Yeast ß-glucan promotes antiviral type I interferon response via dectin-1.

Pseudorabies virus (PRV), an alphaherpesvirus, is a neglected zoonotic pathogen. Dectin-1 sensing of ß-glucan (BG) induces trained immunity, which can possibly form a new strategy for the prevention of viral infection. However, alphaherpesvirus including PRV have received little to no investigation in the context of trained immunity. Here, we found that BG pretreatment improved the survival rate, weight loss outcomes, alleviated histological injury and decreased PRV copy number of tissues in PRV-infected mice. Type I interferons (IFNs) including IFN-α/ß levels in serum were significantly increased by BG. However, these effects were abrogated in the presence of Dectin-1 antagonist. Dectin-1-mediated effect of BG was also confirmed in porcine and murine macrophages. These results suggested that BG have effects on type I IFNs with antiviral property involved in Dectin-1. In piglets, oral or injected immunization with BG and PRV vaccine could significantly elevated the level of PRV-specific IgG and type I IFNs. And it also increased the antibody levels of porcine reproductive and respiratory syndrome virus vaccine and classical swine fever vaccine that were later immunized, indicating a broad-spectrum effect on improving vaccine immunity. On the premise that the cost was greatly reducing, the immunological effect of oral was better than injection administration. Our findings highlighted that BG induced type I IFNs related antiviral effect against PRV involved in Dectin-1 and potential application value as a feed additive to help control the spread of PRV and future emerging viruses.

IL-17A and TNF-α-induced Dectin-1 expression may promote keratinocyte proliferation in psoriatic lesions.

Psoriasis is a common skin disease with a high recurrence rate. Aberrant keratinocyte proliferation is a significant pathogenic characteristic of psoriatic lesions, and studies have revealed that the development of psoriasis is significantly influenced by pro-inflammatory cytokines, such as IL-17A and TNF-α. Biologics targeting these cytokines have been widely used in psoriasis treatment and achieve remarkable effects, however, the underlying mechanism of how IL-17A and TNF-α specifically regulate keratinocyte proliferation has not been fully elucidated. Dectin-1 is an essential membrane protein that is directly related to the immune microenvironment and the proliferation of multiple cell types. To elucidate how IL-17A and TNF-α may promote keratinocyte proliferation in psoriatic lesions and whether Dectin-1 is involved. The expression of Dectin-1 in keratinocytes from psoriatic lesions was detected by real-time PCR, western blot and immunofluorescence. Correlation analysis and cytological experiments were then performed to determine the relationship between Dectin-1 and IL-17A/TNF-α in psoriatic lesions. Finally, we investigated the signalling pathway through which Dectin-1 may promote keratinocyte proliferation. Dectin-1 was significantly increased in keratinocytes from psoriatic lesions. Moreover, IL-17A and TNF-α effectively induced the expression of Dectin-1 in HaCaT cells, which was shown to activate the Syk/NF-κB signalling pathway and promote the proliferation of keratinocytes. IL-17A and TNF-α may promote the proliferation of keratinocytes in psoriatic lesions through induction of Dectin-1, indicating that Dectin-1 could be a potential therapeutic target for the treatment of psoriasis.

Galanin Coordinates Macrophage-Associated Fibro-Inflammatory Response and Mitochondrial Integrity in Myocardial Infarction Reperfusion Injury.

Myocardial infarction activates an intense fibro-inflammatory reaction that is essential for cardiac remodeling and heart failure (HF). Bioactive peptide galanin plays a critical role in regulating cardiovascular homeostasis; however, its specific functional relevance in post-infarction fibro-inflammatory reprogramming remains obscure. Here, we show that galanin coordinates the fibro-inflammatory trajectory and mitochondrial integrity in post-infarction reperfusion injury. Aberrant deposition of collagen was associated with a marked increase in CD68-positive macrophage infiltration in cardiac tissue in mice subjected to myocardial ischemia/reperfusion (I/R) for 14 days compared to sham controls. Furthermore, we found that the myocardial expression level of a specific marker of M2 macrophages, CD206, was significantly down-regulated in I/R-challenged mice. In contrast, galanin treatment started during the reperfusion phase blunted the fibro-inflammatory responses and promoted the expression of CD206 in I/R-remodeled hearts. In addition, we found that the anti-apoptotic and anti-hypertrophic effects of galanin were associated with the preservation of mitochondrial integrity and promotion of mitochondrial biogenesis. These findings depict galanin as a key arbitrator of fibro-inflammatory responses to cardiac I/R injury and offer a promising therapeutic trajectory for the treatment of post-infarct cardiovascular complications.

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.

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.

Systematic analysis of mucosal-associated invariant T cells in haematological malignancies.

Mucosal-associated invariant T-cells (MAIT) are unconventional T-cells with cytotoxic and pro-inflammatory properties. Previous research has reported contradictory findings on their role in cancerogenesis with data being even scarcer in haematological malignancies. Here, we report the results of a systematic analysis of MAIT cells in treatment-naïve patients with a broad range of haematological malignancies. We analysed peripheral blood of 204 patients and 50 healthy subjects. The pool of haematological patients had a statistically significant lower both the absolute value (median values, 0.01 × 109/L vs. 0.05 × 109/L) of MAIT cells and their percentage (median values 0.94% vs. 2.56%) among T-cells compared to the control group. Separate analysis showed that the decrease in the absolute number of MAIT cells is significant in patients with acute myeloid leukaemia, myeloproliferative neoplasms, plasma cell myeloma, B-cell non-Hodgkin lymphomas, otherwise not specified, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma compared to the control population. Furthermore, in haematological malignancies, MAIT cells overexpress PD-1 (average values, 51.7% vs. 6.7%), HLA-DR (average values, 40.2% vs. 7%), CD38 (average values, 25.9% vs. 4.9%) and CD69 (average values, 40.2% vs. 9.2%). Similar results were obtained when comparing patients with individual malignancies to the control population. Our data show that the depletion of circulating MAIT cells is a common observation in a broad spectrum of haematological malignancies. In addition to their reduced numbers, MAIT cells acquire an activated/exhausted phenotype.

Immunogenicity and efficacy of CNA25 as a potential whole-cell vaccine against systemic candidiasis.

Disseminated fungal infections account for ~1.5 million deaths per year worldwide, and mortality may increase further due to a rise in the number of immunocompromised individuals and drug-resistance fungal species. Since an approved antifungal vaccine is yet to be available, this study explored the immunogenicity and vaccine efficacy of a DNA polymerase mutant strain of Candida albicans. CNA25 is a pol32ΔΔ strain that exhibits growth defects and does not cause systemic candidiasis in mice. Immunized mice with live CNA25 were fully protected against C. albicans and C. parapsilosis but partially against C. tropicalis and C. glabrata infections. CNA25 induced steady expression of TLR2 and Dectin-1 receptors leading to a faster recognition and clearance by the immune system associated with the activation of protective immune responses mostly mediated by neutrophils, macrophages, NK cells, B cells, and CD4+ and CD8+ T cells. Molecular blockade of Dectin-1, IL-17, IFNγ, and TNFα abolished resistance to reinfection. Altogether, this study suggested that CNA25 collectively activates innate, adaptive, and trained immunity to be a promising live whole-cell vaccine against systemic candidiasis.

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.

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.

Hydatid fluid from Echinococcus granulosus induces autophagy in dendritic cells and promotes polyfunctional T-cell responses.

Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. Echinococcus granulosus is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of beclin-1, atg16l1 and atg12, indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of il-6, il-10, il-12, tnf-α, ifn-γ and tgf-ß genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.