Summary data-based Mendelian randomization and single-cell RNA sequencing analyses identify immune associations with low-level LGALS9 in sepsis.

Sepsis is one of the major challenges in intensive care units, characterized by the complexity of the host immune status. To gain a deeper understanding of the pathogenesis of sepsis, it is crucial to study the phenotypic changes in immune cells and their underlying molecular mechanisms. We conducted Summary data-based Mendelian randomization analysis by integrating genome-wide association studies data for sepsis with expression quantitative trait locus data, revealing a significant decrease in the expression levels of 17 biomarkers in sepsis patients. Furthermore, based on single-cell RNA sequencing data, we elucidated potential molecular mechanisms at single-cell resolution and identified that LGALS9 inhibition in sepsis patients leads to the activation and differentiation of monocyte and T-cell subtypes. These findings are expected to assist researchers in gaining a more in-depth understanding of the immune dysregulation in sepsis.

The Variations' in Genes Encoding TIM-3 and Its Ligand, Galectin-9, Influence on ccRCC Risk and Prognosis.

Renal cell cancer is the most common type of kidney cancer in adults, and clear cell renal cell carcinoma (ccRCC) is the most diagnosed type. T cell immunoglobulin and mucin-domain-containing-3 (TIM-3) belongs to immunological checkpoints that are key regulators of the immune response. One of the known TIM-3 ligands is galectin-9 (LGALS9). A limited number of studies have shown an association between TIM-3 polymorphisms and cancer risk in the Asian population; however, there is no study on the role of LGALS9 polymorphisms in cancer. The present study aimed to analyze the influence of TIM-3 and LGALS9 polymorphisms on susceptibility to ccRCC and patient overall survival (OS), with over ten years of observations. Using TaqMan probes, ARMS-PCR, and RFPL-PCR, we genotyped two TIM-3 single-nucleotide polymorphisms (SNPs): rs1036199 and rs10057302, and four LGALS9 SNPs: rs361497, rs3751093, rs4239242, and rs4794976. We found that the presence of the rs10057302 A allele (AC + AA genotypes) as well as the rs4794976 T allele (GT + TT genotypes) decreased susceptibility to ccRCC by two-fold compared to corresponding homozygotes. A subgroup analysis showed the association of some SNPs with clinical features. Moreover, TIM-3 rs1036199 significantly influenced OS. Our results indicate that variations within TIM-3 and LGALS9 genes are associated with ccRCC risk and OS.

Gene polymorphisms of LGALS2, LGALS3 and LGALS9 in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complicated rheumatic autoimmune disease. Lectin, galactoside-binding soluble, 2 (LGALS2), LGALS3 and LGALS9, three members of the galectin family, play potential roles in autoimmune diseases, including RA. However, association of genetic polymorphisms of LGALS2, LGALS3 and LGALS9 with RA risk in a Southern Chinese Han population has not been elucidated. A case-control study was conducted herein, including 500 RA patients and 650 healthy individuals of Southern Chinese Han origin. Twelve single nucleotide polymorphisms (SNPs), including rs7291467 for the LGALS2 gene, rs4644, rs4652, rs1009977, rs2274273 and rs17128183 for the LGALS3 gene, and rs4795835, rs3763959, rs4239242, rs3751093, rs732222 and rs4794976 for the LGALS9 gene, were genotyped. Polymorphisms were genotyped using the KASP method. Frequencies of rs1009977 genotype TG and rs3751093 genotype GA of LGALS3 gene were significantly different between RA patients and healthy controls (P = 0.049, P = 0.033). Allele T and genotypes TT and TT + TG of rs4794976 for LGALS9 gene were significantly correlated with RA risk (P = 0.017, P = 0.012, P = 0.041). Subgroup analysis revealed that rs1009977, rs2274273 and rs17128183 polymorphisms of LGALS3 gene and rs4795835 polymorphism of LGALS9 gene were correlated with several RA clinical manifestations (all P < 0.05). In addition, haplotype GCGTT showed an increased risk for RA (OR = 1.216, 95% CI: 1.028-1.438, P = 0.023), whereas haplotype GCGTG showed a reduced risk for RA susceptibility (OR = 0.779, 95% CI: 0.625-0.971, P = 0.026). In conclusion, LGALS3 and LGALS9 gene polymorphisms may associate with RA predisposition in a Southern Chinese Han population.

Secretion of Galectin-9 as a DAMP during Dengue Virus Infection in THP-1 Cells.

Damage-associated molecular patterns (DAMPs) are endogenous cellular molecules released to the extracellular environment in response to stress conditions such as virus infection. Galectins are ß-galactoside-binding proteins that are widely expressed in cells and tissues of the immune system, are localized in the cell cytoplasm, and have roles in inflammatory responses and immune responses against infection. Elevated levels of galectin-9 (Gal-9) in natural human infections have been documented in numerous reports. To investigate the effect of dengue virus (DENV) infection on expression of endogenous Gal-9, monocytic THP-1 cells were infected with varying doses of DENV-3 (multiplicity of infection (MOI) 0.01, 0.03 and 0.1) and incubated at varying time points (Day 1, Day 2, Day 3). Results showed augmentation of Gal-9 levels in the supernatant, reduction of Gal-9 levels in the cells and decreased expression of LGALS9 mRNA, while DENV-3 mRNA copies for all three doses remained stable through time. Dengue virus induced the secretion of Gal-9 as a danger response; in turn, Gal-9 and other inflammatory factors, and stimulated effector responses may have limited further viral replication. The results in this pilot experiment add to the evidence of Gal-9 as a potential DAMP.

Integrative analysis of single-cell and bulk transcriptome data reveal the significant role of macrophages in lupus nephritis.

OBJECTIVE: We attempted to identify abnormal immune cell components and signaling pathways in lupus nephritis (LN) and to identify potential therapeutic targets. METHODS: Differentially expressed genes (DEGs) between LN and normal kidney tissues were identified from bulk transcriptome data, and functional annotation was performed. The phenotypic changes in macrophages and aberrant intercellular signaling communications within immune cells were imputed from LN scRNA-seq data using trajectory analysis and verified using immunofluorescence staining. Finally, lentivirus-mediated overexpression of LGALS9, the gene encoding Galectin 9, in THP-1 cells was used to study the functional effect of this gene on monocytic cells. RESULTS: From bulk transcriptome data, a significant activation of interferon (IFN) signaling was observed, and its intensity showed a significantly positive correlation with the abundance of infiltrating macrophages in LN. Analysis of scRNA-seq data revealed 17 immune cell clusters, with macrophages showing the highest enrichment of intercellular signal communication in LN. Trajectory analysis revealed macrophages in LN undergo a phenotypic change from inflammatory patrolling macrophages to phagocytic and then to antigen-presenting macrophages, and secrete various pro-inflammatory factors and complement components. LGALS9 was found significantly upregulated in macrophages in LN, which was confirmed by the immunofluorescence assay. Gene functional study showed that LGALS9 overexpression in THP-1 cells significantly elicited pro-inflammatory activation, releasing multiple immune cell chemoattractants. CONCLUSION: Our results present an important pathophysiological role for macrophages in LN, and our preliminary results demonstrate significant pro-inflammatory effects of LGALS9 gene in LN macrophages.

Potential Plasma Proteins (LGALS9, LAMP3, PRSS8 and AGRN) as Predictors of Hospitalisation Risk in COVID-19 Patients.

Background: The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has posed unprecedented challenges to healthcare systems worldwide. Here, we have identified proteomic and genetic signatures for improved prognosis which is vital for COVID-19 research. Methods: We investigated the proteomic and genomic profile of COVID-19-positive patients (n = 400 for proteomics, n = 483 for genomics), focusing on differential regulation between hospitalised and non-hospitalised COVID-19 patients. Signatures had their predictive capabilities tested using independent machine learning models such as Support Vector Machine (SVM), Random Forest (RF) and Logistic Regression (LR). Results: This study has identified 224 differentially expressed proteins involved in various inflammatory and immunological pathways in hospitalised COVID-19 patients compared to non-hospitalised COVID-19 patients. LGALS9 (p-value < 0.001), LAMP3 (p-value < 0.001), PRSS8 (p-value < 0.001) and AGRN (p-value < 0.001) were identified as the most statistically significant proteins. Several hundred rsIDs were queried across the top 10 significant signatures, identifying three significant SNPs on the FSTL3 gene showing a correlation with hospitalisation status. Conclusions: Our study has not only identified key signatures of COVID-19 patients with worsened health but has also demonstrated their predictive capabilities as potential biomarkers, which suggests a staple role in the worsened health effects caused by COVID-19.

The mechano-chemical circuit in fibroblasts and dendritic cells drives basal cell proliferation in psoriasis.

Psoriasis is an intractable immune-mediated disorder that disrupts the skin barrier. While studies have dissected the mechanism by which immune cells directly regulate epidermal cell proliferation, the involvement of dermal fibroblasts in the progression of psoriasis remains unclear. Here, we identified that signals from dendritic cells (DCs) that migrate to the dermal-epidermal junction region enhance dermal stiffness by increasing extracellular matrix (ECM) expression, which further promotes basal epidermal cell hyperproliferation. We analyzed cell-cell interactions and observed stronger interactions between DCs and fibroblasts than between DCs and epidermal cells. Using single-cell RNA (scRNA) sequencing, spatial transcriptomics, immunostaining, and stiffness measurement, we found that DC-secreted LGALS9 can be received by CD44+ dermal fibroblasts, leading to increased ECM expression that creates a stiffer dermal environment. By employing mouse psoriasis and skin organoid models, we discovered a mechano-chemical signaling pathway that originates from DCs, extends to dermal fibroblasts, and ultimately enhances basal cell proliferation in psoriatic skin.

Study on pro-inflammatory effect and mechanism of galectin-9 (LGALS9) in osteoarthritis: Exacerbating inflammatory response by activating JNK and ERK1/2 pathways.

Galectin-9 (LGALS9) plays an important role in the occurrence and development of many diseases, including immunity, infection, cancer, etc. Studies have found that LGALS9 can phosphorylate ERK1/2 in the MAPK pathway. However, there is currently no clear conclusion on the role of LGALS9 in OA, and it is worth further exploring the regulatory role and mechanism of LGALS9 in OA in this study. In the initial stage, we collected 6 cases of hip joint soft tissue from normal individuals and 6 cases from OA patients clinically to analyze the differential expression of LGALS9 between normal individuals and OA patients; Subsequently, RNAi technology was used to preliminarily clarify the regulatory role of LGALS9 in an in vitro OA model; Then, lentivirus was used to knock down and overexpress LGALS9, and in vivo and in vitro OA models were constructed. QRT-PCR, western blot, safranin fast green staining (SO), immunofluorescence and other experimental methods were used to quantitatively analyze inflammatory and signaling pathway indicators, further improving the regulatory effect of LGALS9 on inflammation and the pathogenesis of OA.

Intercellular Molecular Crosstalk Networks within Invasive and Immunosuppressive Tumor Microenvironment Subtypes Associated with Clinical Outcomes in Four Cancer Types.

Heterogeneity is a critical basis for understanding how the tumor microenvironment (TME) contributes to tumor progression. However, an understanding of the specific characteristics and functions of TME subtypes (subTMEs) in the progression of cancer is required for further investigations into single-cell resolutions. Here, we analyzed single-cell RNA sequencing data of 250 clinical samples with more than 200,000 cells analyzed in each cancer datum. Based on the construction of an intercellular infiltration model and unsupervised clustering analysis, four, three, three, and four subTMEs were revealed in breast, colorectal, esophageal, and pancreatic cancer, respectively. Among the subTMEs, the immune-suppressive subTME (subTME-IS) and matrix remodeling with malignant cells subTME (subTME-MRM) were highly enriched in tumors, whereas the immune cell infiltration subTME (subTME-ICI) and precancerous state of epithelial cells subTME (subTME-PSE) were less in tumors, compared with paracancerous tissues. We detected and compared genes encoding cytokines, chemokines, cytotoxic mediators, PD1, and PD-L1. The results showed that these genes were specifically overexpressed in different cell types, and, compared with normal tissues, they were upregulated in tumor-derived cells. In addition, compared with other subTMEs, the expression levels of PDCD1 and TGFB1 were higher in subTME-IS. The Cox proportional risk regression model was further constructed to identify possible prognostic markers in each subTME across four cancer types. Cell-cell interaction analysis revealed the distinguishing features in molecular pairs among different subTMEs. Notably, ligand-receptor gene pairs, including COL1A1-SDC1, COL6A2-SDC1, COL6A3-SDC1, and COL4A1-ITGA2 between stromal and tumor cells, associated with tumor invasion phenotypes, poor patient prognoses, and tumor advanced progression, were revealed in subTME-MRM. C5AR1-RPS19, LGALS9-HAVCR2, and SPP1-PTGER4 between macrophages and CD8+ T cells, associated with CD8+ T-cell dysfunction, immunosuppressive status, and tumor advanced progression, were revealed in subTME-IS. The spatial co-location information of cellular and molecular interactions was further verified by spatial transcriptome data from colorectal cancer clinical samples. Overall, our study revealed the heterogeneity within the TME, highlighting the potential pro-invasion and pro-immunosuppressive functions and cellular infiltration characteristics of specific subTMEs, and also identified the key cellular and molecular interactions that might be associated with the survival, invasion, immune escape, and classification of cancer patients across four cancer types.

MicroRNA Bta-miR-24-3p Suppressed Galectin-9 Expression through TLR4/NF-ĸB Signaling Pathway in LPS-Stimulated Bovine Endometrial Epithelial Cells.

Endometritis is a major infectious disease affecting dairy development. MicroRNAs are recognized as critical regulators of the innate immune response. However, the role and mechanism of Bta-miR-24-3p in the development of endometritis are still unclear. This study aimed to investigate the effect of Bta-miR-24-3p on the inflammatory response triggered by lipopolysaccharide (LPS) and to clarify the possible mechanism. LPS-treated bovine endometrial epithelial cells (BEECs) were cultured to investigate the role of Bta-miR-24-3p. The expression levels of Bta-miR-24-3p were downregulated, and galectin-9 (LGALS9) were measured by quantitative real-time polymerase chain reaction. The LPS-induced inflammatory response was assessed by the elevated secretion of inflammatory cytokines measured by using enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Activation of nuclear factor-κB (NF-κB) and TLR4 pathway was assessed by Western blot. The interaction between Bta-miR-24-3p and LGALS9 was validated by bioinformatics analysis and a luciferase reporter assay. LPS-induction in BEECs with Bta-miR-24-3p was overexpressed leads inhibition of pro-inflammatory cytokines, LGALS9 expression, and TLR4/NF-ĸB pathway deactivation. Knockdown of LGALS9 inhibited the LPS-induced inflammatory response in BEECs. LGALS9 was validated as a target of Bta-miR-24-3p. Cloned overexpression of LGALS9 failed to alter the effect of Bta-miR-24-3p on the inflammatory response in BEECs. Overall, Bta-miR-24-3p attenuated the LPS-induced inflammatory response via targeting LGALS9. The immunotherapeutic stabilisation of Bta-miR-24-3p could give a therapeutic option for endometritis and other disorders commonly associated with endometritis, suggesting a novel avenue for endometritis treatment.

Histone H3K9 and H3K14 acetylation at the promoter of the LGALS9 gene is associated with mRNA levels in cervical cancer cells.

Galectin-9 levels have been reported to be altered in several cancer types, but the mechanism that regulates the expression of Galectin-9 has not been clarified. Galectin-9 is encoded by the LGALS9 gene, which gives rise to eight mRNA variants. The aims of this study were: (a) to identify the mRNA variants of LGALS9, (b) to characterize CpG methylation and H3K9 and H3K14 histone acetylation at the promoter of the LGALS9 gene, and (c) to characterize the relationship between these modifications and LGALS9 expression level in cervical cancer cells. All mRNA variants were detected in HaCaT (nontumoural keratinocytes) and SiHa cells, and seven were observed in HeLa cells. The promoter region of LGALS9 contains eight CpG dinucleotides. No hypermethylation pattern related to low LGALS9 expression was identified in tumour cells. Chromatin immunoprecipitation analysis demonstrated higher acetylation of H3K9ac and H3K14ac in HaCaT cells, which was related to higher mRNA levels. The presence of the mRNA variants suggests that alternative splicing may regulate the expression of galectin-9 isoforms. The results of this study suggest that histone acetylation, but not promoter CpG methylation, may be involved in the transcriptional regulation of the LGALS9 gene.

Profiling of inhibitory immune checkpoints in glioblastoma: Potential pathogenetic players.

Glioblastoma (GBM) represents the most frequent glial tumor, with almost 3 new cases per 100,000 people per year. Despite treatment, the prognosis for GBM patients remains extremely poor, with a median survival of 14.6 months, and a 5-year survival less than 5%. It is generally believed that GBM creates a highly immunosuppressive microenvironment, sustained by the expression of immune-regulatory factors, including inhibitory immune checkpoints, on both infiltrating cells and tumor cells. However, the trials assessing the efficacy of current immune checkpoint inhibitors in GBM are still disappointing. In the present study, the expression levels of several inhibitory immune checkpoints in GBM (CD276, VTCN1, CD47, PVR, TNFRSF14, CD200, LGALS9, NECTIN2 and CD48) were characterized in order to evaluate their potential as prognostic and eventually, therapeutic targets. Among the investigated immune checkpoints, TNFRSF14 and NECTIN2 were identified as the most promising targets in GBM. In particular, a higher TNFRSF14 expression was associated with worse overall survival and disease-free survival, and with a lower Th1 response.

Molecular and immune correlates of TIM-3 (HAVCR2) and galectin 9 (LGALS9) mRNA expression and DNA methylation in melanoma.

BACKGROUND: The T cell immunoglobulin and mucin-domain containing-3 receptor TIM-3 (also known as hepatitis A virus cellular receptor 2, encoded by HAVCR2) and its ligand galectin 9 (LGALS9) are promising targets for immune checkpoint inhibition immunotherapies. However, little is known about epigenetic regulation of the encoding genes. This study aimed to investigate the association of TIM-3 and LGALS9 DNA methylation with gene expression, patients' survival, as well as molecular and immune correlates in malignant melanoma. RESULTS: Methylation of all six TIM-3 CpGs correlated significantly with TIM-3 mRNA levels (P ≤ 0.05). A strong inverse correlation (Spearman's ρ = - 0.49) was found in promoter regions, while a strong positive correlation (ρ = 0.63) was present in the gene body of TIM-3. High TIM-3 mRNA expression (hazard ratio (HR) = 0.88, 95% confidence interval (CI) [0.81-0.97], P = 0.007) was significantly associated with better overall survival. Seven of the eight LGALS9 CpG sites correlated significantly with LGALS9 mRNA levels (P ≤ 0.003). Methylation at five CpG sites showed a strong inverse correlation (Spearman's ρ = - 0.67) and at two sites a weak positive correlation (Spearman's ρ = 0.15). High LGALS9 mRNA expression was significantly associated with increased overall survival (HR = 0.83, 95%CI [0.75-0.93], P = 0.001). In addition, we found significant correlations between TIM-3 and LGALS9 methylation and mRNA expression with immune cell infiltrates and significant differences among distinct immune cell subsets. CONCLUSIONS: Our study points toward an epigenetic regulation of TIM-3 and LGALS9 via DNA methylation and might provide an avenue for the development of a predictive biomarker for response to immune checkpoint blockade.