Research advances in the immunomodulatory mechanism of galectin-9 and its role in liver diseases / 临床肝胆病杂志

Galectin-9 （Gal-9） is a member of the galectin family that can specifically recognize and bind to galactosides. Recent studies have shown that Gal-9 is highly expressed in the liver and can help to maintain intrahepatic immune homeostasis and perform biological functions in various liver diseases. This article reviews the immunomodulatory functions of Gal-9 and its role in different liver diseases. Studies have shown that Gal-9 has important biological functions in different liver diseases through multiple pathways. Research on the specific immunomodulatory mechanisms and functions of Gal-9 may help to discover the therapeutic role of Gal-9 in liver diseases.

Increased tumor-associated mast cells facilitate thyroid cancer progression by inhibiting CD8+ T cell function through galectin-9

As an important component of solid tumors, mast cells show specific phenotypes in various tumor microenvironments. However, the precise mechanism of mast cell accumulation and the phenotypic features of thyroid cancer (TC) remain largely unknown. Here, we found that mast cells were obviously recruited to tumor tissue by TC-derived stem cell factor (SCF). With tumor progression, mast cell levels increased gradually. In addition, intratumoral mast cells expressed higher levels of the immunosuppressive molecule galectin-9, which effectively suppresses CD8+ T-cell antitumor immunity in vitro. Blocking galectin-9 on tumor-infiltrating mast cells reversed the immunosuppression of CD8+ T cells. In conclusion, our data elucidated novel protumorigenic and immunosuppressive roles of mast cells in TC. In addition, our results indicated that blocking mast cells may impede tumor progression and ameliorate the prognosis of TC patients.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Expression and significance of serum soluble T cell immunoglobulin-domain and mucin-domain protein-3 and galectin-9 in patients with early acute pancreatitis / 中华消化杂志

Objective:To investigate the expression and significance of serum soluble T cell immunoglobulin-domain and mucin-domain protein-3 (sTIM-3) and galectin-9 (Gal-9) in patients with early acute pancreatitis (AP), so as to provide theoretical and clinical evidence for the early prediction and diagnosis of AP.Methods:From 15 September 2020 to 23 July 2021, a total of 94 AP patients with a time from onset to admission ≤48 h who were admitted to Changzhou No.2 People′s Hospital, Nanjing Medical University were selected, including 42 cases of mild acute pancreatitis (MAP), 35 cases of moderately severe acute pancreatitis (MSAP) and 17 cases of severe acute pancreatitis (SAP). The basic clinical features of AP patients were collected. Acute physiology and chronic health evaluation Ⅱ(APACHE Ⅱ), modified computed tomography severity index (MCTSI) and bedside index for severity in acute pancreatitis (BISAP) scores were evaluated in all AP patients. The levels of serum interleukin (IL)-6, IL-10, Gal-9 and sTIM-3 were detected with enzyme linked immunosorbent assay. Kruskal-Wallis test and Mann-Whitney U test were used for statistical analysis. Spearman rank correlation test and Pearson correlation analysis were used to analyze the correlation of sTIM-3, Gal-9 with inflammatory indicators and AP related scoring systems. Receiver operating characteristic curve (ROC) was performed for efficiency analysis of the combination of sTIM-3 and Gal-9 in predicting the severity of AP patients. Results:Serum sTIM-3, Gal-9 and IL-6 levels of SAP patients were higher than those of MAP patients (2 085.00 ng/L (1 628.00 ng/L, 2 673.00 ng/L) vs. 746.10 ng/L (514.50 ng/L, 1 303.00 ng/L); 466.60 ng/L (375.90 ng/L, 629.30 ng/L) vs. 108.10 ng/L (90.29 ng/L, 138.90 ng/L); (323.60±62.93) ng/L vs. (42.90±28.82) ng/L), while IL-10 level was lower than that of MAP patients ((760.30±200.40) ng/L vs. (1 206.00±566.30) ng/L), and the differences were statistically significant ( Z=45.00 and <0.01, t=23.62 and 3.15; all P<0.01). The APACHE Ⅱ and BISAP scores of SAP patients were higher than those of MAP and MSAP patients (12.00(6.00, 16.50) vs. 3.00(2.00, 5.00) and 6.00(3.00, 8.00); 3.00(3.00, 4.00) vs.1.00(1.00, 1.00) and 2.00(2.00, 3.00)), and the MCTSI score was higher than that of MAP patients (4.00(3.00, 6.00) vs. 2.00(0.00, 2.00)), and the differences were statistically significant ( Z=644.50, 704.00, 474.50, 492.50 and 664.00, all P<0.001). Serum sTIM-3 and Gal-9 were positively correlated with the pro-inflammatory factor IL-6 ( r=0.552 and 0.297, P<0.001 and =0.004). Serum sTIM-3 was negatively correlated with the anti-inflammatory factor IL-10 ( r=-0.397, P<0.001). There was no correlation between Gal-9 and the anti-inflammatory factor IL-10 ( P>0.05). Serum sTIM-3 and Gal-9 were positively correlated with APACHE Ⅱ, MCTSI and BISAP scores ( r=0.210, 0.271 and 0.363, P=0.042, =0.008 and <0.001; r=0.390, 0.448 and 0.440, all P<0.001). The areas under ROC curves (95% confidence interval) of serum sTIM-3 and Gal-9 detected alone and in combination was 0.805 (0.716 to 0.895), 0.814 (0.725 to 0.903) and 0.856 (0.773 to 0.939), respectively, and the sensitivity was 69.2%, 67.3%, 75.0%, respectively, and the specificity was 83.3%, 97.6%, 97.6%, respectively. Conclusions:The serum levels of sTIM-3 and Gal-9 increased in patients with early AP and are correlated with the severity of AP. The combined detection of sTIM-3 and Gal-9 has high sensitivity in predicting early AP, and the two indicators may be the reliable predictors of early AP.

Expressions of T-cell immunoglobulin mucin-3 and galectin-9 in acute lymphoblastic leukemia and their clinical significances / 白血病·淋巴瘤

Objective:To explore the expression levels, clinical significances and prognostic evaluation value of T-cell immunoglobulin mucin-3 (Tim-3) and galectin-9 (Gal-9) in bone marrow cells of patients with newly diagnosed acute lymphocytic leukemia (ALL).Methods:Bone marrow samples from 30 newly diagnosed ALL patients admitted to First Affiliated Hospital of Xinjiang Medical University from September 2016 to September 2018 were selected, and peripheral blood samples from 20 healthy volunteers during the same period in the First Affiliated Hospital of Xinjiang Medical University were treated as the controls. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect mRNA relative expression levels of Tim-3 and Gal-9. Differences in mRNA expression of Tim-3 and Gal-9 among ALL patients with varied clinicopathological characteristics were compared. Overall survival (OS) analysis was performed by using the Kaplan-Meier method, Cox proportional hazards model was used to make univariate and multivariate survival analysis.Results:mRNA relative expression levels of Tim-3 and Gal-9 in 30 newly diagnosed ALL patients were higher than those in the healthy control group (2.86±0.47 vs. 0.45±0.05, t = 21.65, P<0.05; 9.79±0.58 vs. 0.96±0.23, t = 63.24, P<0.05). mRNA relative expression level of Tim-3 had statistically significant differences in patients with different ages, France-America-Britain (FAB) Cooperative Group classification, hazard grades and central nervous system invasion (all P<0.01). There were statistically significant differences in mRNA relative expression level of Gal-9 for patients with different ages, FAB Cooperative Group classification, white blood cell count (WBC), central nervous system invasion and NOTCH1 mutation (all P<0.01). All patients were grouped by mRNA relative expression levels of Tim-3 and Gal-9, and patients in high Tim-3 expression group (≥2.86) had worse overall survival (OS) compared with that for patients in low Tim-3 expression group (<2.86) ( P = 0.048). Patients in high Gal-9 expression group (≥9.79) had worse OS compared with that for patients in low Gal-9 expression group (<9.79) ( P = 0.031). Moreover, the OS in Tim-3 and Gal-9 both high expression group was worse than that in Tim-3 and Gal-9 both low expression group and in the low expression group of either of them (all P<0.05). There was no statistically significant difference in OS between the high Tim-3 expression with low Gal-9 expression group and the high Gal-9 expression with low Tim-3 expression group ( P > 0.05). Multivariate Cox regression analysis revealed that peripheral blood WBC≥11.4×10 9/L, BCR-ABL gene mutation, central nervous system invasion, and high expression of Tim-3 and Gal-9 were independent risk prognostic factors of OS for newly diagnosed ALL patients (all P<0.05) . There was a positive correlation between the expression levels of Tim-3 and Gal-9 ( r = 0.788, P<0.01). Conclusions:The high expression of Tim-3 and its ligand Gal-9 are independent effecting factors of poor prognosis in newly diagnosed ALL patients. The expression levels of Tim-3 and Gal-9 can be served as a potential prognostic indicator for ALL patients.

Molecular mechanism of geniposide in regulating GLUT2 glycosylation in pancreatic β cells / 中国中药杂志

Type 2 diabetes mellitus( T2 DM) is a common chronic metabolic disease characterized by persistent hyperglycemia and insulin resistance. In pancreatic β-cells,glucose-stimulated insulin secretion( GSIS) plays a pivotal role in maintaining the balance of blood glucose level. Previous studies have shown that geniposide,one of the active components of Gardenia jasminoides,could quickly regulate the absorption and metabolism of glucose,and affect glucose-stimulated insulin secretion in pancreatic β cells,but the specific mechanism needs to be further explored. Emerging evidence indicated that glycosylation of glucose transporter( GLUT) has played a key role in sensing cell microenvironmental changes and regulating glucose homeostasis in eucaryotic cells. In this study,we studied the effects of geniposide on the key molecules of GLUT2 glycosylation in pancreatic β cells. The results showed that geniposide could significantly up-regulate the mRNA and protein levels of Glc NAc T-Ⅳa glycosyltransferase( Gn T-Ⅳa) and galectin-9 but had no signi-ficant effect on the expression of clathrin,and geniposide could distinctively regulate the protein level of Gn T-Ⅳa in a short time( 1 h) under the conditions of low and medium glucose concentrations,but had no significant effect on the protein level of galectin-9. In addition,geniposide could also remarkably affect the protein level of glycosylated GLUT2 in a short-time treatment. The above results suggested that geniposide could quickly regulate the protein level of Gn T-Ⅳa,a key molecule of protein glycosylation in INS-1 rat pancreatic βcells and affect the glycosylation of GLUT2. These findings suggested that the regulation of geniposide on glucose absorption,metabolism and glucose-stimulated insulin secretion might be associated with its efficacy in regulating GLUT2 glycosylation and affecting its distribution on the cell membrane and cytoplasm in pancreatic β cells.

Modulatory effect of Tim-3/Galectin-9 axis on T-cell-mediated immunity in pulmonary tuberculosis

Patients affected by pulmonary tuberculosis (PTB) manifest deficiencies in innate cellular immunity. The Tim3/Galectin-9 axis is an important regulator of Th1 cell immunity, leading to Th1 cell apoptosis. Herein, thisstudy aims to clarify the underlying roles of the Tim-3/Galectin-9 axis in T-cell immunity in PTB. Peripheralblood mononuclear cells (PBMCs) were extracted from subjects with and without PTB to examine theexpression of CD4, CD8, CD25, and Tim-3 under the stimulation of Mycobacterium tuberculosis (MTB) andpurified protein derivative (PPD). In addition, the expression of Tim-3 and Galectin-9 in PBMCs was determined. The Tim-3/Galectin-9 axis in the PBMCs was activated or blocked to detect the secreted levels of IFNc, TNF-a, IL-2, and IL-22. MTB stimulation increased the expression of CD4, CD8, CD25, Tim-3, andGalectin-9 in PBMCs. The blockade of Tim-3/Galectin-9 axis resulted in reduced secretion of IFN-c, TNF-a,IL-2, and IL-22 from T-cells. Moreover, Tim-3?CD4?T, Tim-3?CD8?, and Tim-3?CD25?T cells exhibited agreater ability to inhibit the replication of MTB in macrophages. Taken conjointly, the blockade of Tim-3/Galectin-9 axis inhibits the secretion of inflammatory cytokines in T-cells to regulate the T-cell immunity inPTB.

Prediction of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation by the level of galectin-9 in peripheral blood / 中华血液学杂志

Objective@#To evaluate possible effects of Gelctin-9 on acute graft versus host disease (aGVHD) development and clinical outcomes in patients before and afer allogeneic hematopoietic stem cell transplantation (allo-HSCT) .@*Methods@#Peripheral blood samples were obtained from 29 patients and 15 healthy volunteers with heparin anticoagulant tubes. Samples were analyzed using ELISA kits to measure the serum concentrations of Galectin-9.@*Results@#Patients developing aGVHD had significantly lower level of Galectin-9 [ (7.96±1.18) μg/L] before allo-HSCT compared with those not developing aGVHD [ (12.37±0.97) μg/L, P<0.001]. And after allo-HSCT, the consentration of Galectin-9 increased markedly in patients developing aGVHD [ (17.78±1.78) μg/L] compared with those not developing aGVHD [ (9.45±0.80) μg/L, P<0.001]. Patients developing 3-4 grade aGVHD had significantly higher level of Galectin-9 [ (23.25±2.59) μg/L] compared with those developing 1-2 grade aGVHD [ (14.37±1.45) μg/L, P=0.008] and those without aGVHD [ (9.45±0.80) μg/L, P<0.001]. The patients with lower level of Galectin-9 after allo-HSCT (<13.61 μg/L) showed more favorable clinical outcomes compared with those with higher level of Galectin-9 (≥13.61 μg/L) . The 3-year overall survival rates were (100.00±6.05) % and (69.23±12.80) %, respectively (P=0.009) . The cumulative incidence of non-relapse mortality was significantly higher in high Galectin-9 group [ (23.08±11.69) %] in comparison with low Gaelctin-9 group [ (0.00±7.39) %] (P=0.023) . There was no significant difference between the two groups in terms of the cumulative incidence of relapse. The cumulative incidence of relapse at 3 years were (8.33±7.98) % and (12.50±8.27) % in high and low Galectin-9 groups, respectively (P=0.708) .@*Conclusions@#The serum concentration of Galectin-9 at the time of engraftment after allo-HSCT may be used as a predictor for the development and severity of aGVHD. Galectin-9 might be considered as a potential new approach to regulate transplant rejection to achieve desirable survival.

Analysis of Tim-3/Gal-9 expression and T cell infiltration in adenocarcinoma of the esophagogastric junction / 上海交通大学学报（医学版）

Objective • To detect T cell immunoglobulin mucin 3 (Tim-3) and galectin 9 (Gal-9) expression as well as CD3+ T cells and CD8+ T cells infiltration in the tumor tissues of adenocarcinoma of the esophagogastric junction (AEG), and analyze their correlations with the patients' clinical characteristics and survival prognosis. Methods • A retrospective case study was used to collect clinical data and follow-up data of 116 AEG patients who were admitted to Renji Hospital, Shanghai Jiao Tong University School of Medicine from Dec. 2005 to Dec. 2013. Tim-3, Gal-9, CD3, and CD8 protein expressions were detected by immunohistochemistry in the tumor tissues, and the clinical characteristics and prognosis were compared among the patients with different levels of protein expression and T cells infiltration. Results • The results of immunohistochemistry showed that Tim-3 mainly expressed in the infiltrating immune cells, and Gal-9 mainly expressed in the tumor cells. The analysis on the clinical characteristics revealed that Tim-3 expression level was related to the Siewert classification (P=0.030) and CD8+ T cells infiltration level was related to the tumor TNM stage (P=0.042). The results of survival analysis showed that the patients with high level of CD8+ T cells infiltration had a better survival prognosis (P=0.047). However, there was no difference in the prognosis among the patients with different Tim-3 and/or Gal-9 expression levels or with different CD3+ T cell infiltration levels. Conclusion • AEG patients with high level of CD8+ T cells infiltration usually have earlier TNM stages and better prognosis. There is no significant difference in the prognosis of AEG patients with different Tim-3/Gal-9 expression levels.

Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Acute cellular rejection (ACR) remains a major concern after liver transplantation. Predicting and monitoring acute rejection by non-invasive methods are very important for guiding the use of immunosuppressive drugs. Many studies have shown that exosomes and their contents are potential biomarkers for various liver diseases. Here, we identify and validate the role of exosomes and galectin-9 in ACR after liver transplantation. Exosomes were isolated from three sets of paired patients, with and without ACR, and the proteins within the exosomes were isolated and identified. Candidate proteins were then validated using a tissue microarray containing resected liver samples from 73 ACR and 63 non-rejection patients. Finally, protein expression and clinical manifestations were included in Kaplan-Meier survival and Cox regression analyses. Circulating exosomes were isolated from ACR and non-rejection patients and characterized using transmission electron microscopy and western blotting for CD63/CD81. Western blotting experiments revealed higher levels of galectin-9 protein in circulating exosomes from ACR recipients. Immunohistochemical analysis of the tissue microarray showed that the expression of galectin-9 in resected liver was significantly higher in the ACR group than in the non-rejection group (P<0.05). Higher levels of galectin-9 expression in resected livers were associated with poorer prognosis (P<0.05). Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation.

Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Acute cellular rejection (ACR) remains a major concern after liver transplantation. Predicting and monitoring acute rejection by non-invasive methods are very important for guiding the use of immunosuppressive drugs. Many studies have shown that exosomes and their contents are potential biomarkers for various liver diseases. Here, we identify and validate the role of exosomes and galectin-9 in ACR after liver transplantation. Exosomes were isolated from three sets of paired patients, with and without ACR, and the proteins within the exosomes were isolated and identified. Candidate proteins were then validated using a tissue microarray containing resected liver samples from 73 ACR and 63 non-rejection patients. Finally, protein expression and clinical manifestations were included in Kaplan-Meier survival and Cox regression analyses. Circulating exosomes were isolated from ACR and non-rejection patients and characterized using transmission electron microscopy and western blotting for CD63/CD81. Western blotting experiments revealed higher levels of galectin-9 protein in circulating exosomes from ACR recipients. Immunohistochemical analysis of the tissue microarray showed that the expression of galectin-9 in resected liver was significantly higher in the ACR group than in the non-rejection group (P<0.05). Higher levels of galectin-9 expression in resected livers were associated with poorer prognosis (P<0.05). Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation.

Expression of PD-L1 and Gal-9 in human adipose-derived stem cells and its influencing factors / 中华整形外科杂志

Objective@#To explore whether human adipose-derived stem cells (ADSCs) express PD-L1 and Gal-9 and its potential influence factors.@*Methods@#ADSCs isolated from 28 healthy female donors who underwent liposuction of the abdomen or breast tissue were cultured and characterized. The expression of PD-L1 and Gal-9 were detected using flow cytometry. The impact of donor age, body mass index (BMI), donor site and interferon-γ (IFN-γ) on the expression of PD-L1 and Gal-9 was analyzed using multivariate linear regression analysis.@*Results@#The cultured cells fulfilled the criteria for defining MSCs according to the international standards and expressed PD-L1 and Gal-9. Breast-derived ADSCs had higher expression levels of PD-L1 (37.24%±8.20%) and Gal-9 ( 4.41%±2.65%) than those in abdomen-derived ADSCs (28.80%±8.59% and 2.51%±1.39%, respectively) (P=0.018, P=0.039). Multivariate linear regression analysis showed that donor age and site were independent risk factors affecting PD-L1 expression (P=0.009, P=0.006). The expression of PD-L1 decreased by 0.385% for every one year of age increase, and it was 8.58% higher in the breast-derived ADSCs than in the abdomen-derived ADSCs. Donor site was an independent risk factor for Gal-9 expression (P=0.041). Gal-9 positive expression rate in the breast-derived ADSCs was 1.898% higher than that in the abdomen-derived ADSCs. BMI was not a risk factor affecting PD-L1 or Gal-9 expression on ADSCs. The expression of PD-L1 and Gal-9 on ADSCs were significantly upregulated after 48 hours stimulation with 20 ng/ml IFN-γ in vitro.@*Conclusions@#Human ADSCs expressed PD-L1 and Gal-9. Donor age, site and IFN-γ treatment were independent risk factors affecting the expression of PD-L1 and Gal-9 on ADSCs.

Effect of Galectin-9/ Tim-3 pathway on the polarization of M1/M2 subtype in murine macrophages induced by lipopolysaccharide / 中华危重病急救医学

Objective To investigate the meaning and molecular mechanisms of Galectin-9/ T-cell immunoglobulin mucin-3 (Tim-3) pathway on lipopolysaccharide (LPS) induced murine macrophage M1/M2 subtype polarization.Methods The murine peritoneal macrophages RAW264.7 were cultured in vitro until the cells had matured with 80％-90％ fusion rate. ① The cells were cultured in serum-free medium and treated with 0 (blank control), 0.01, 0.1, 1, 10 and 100 mg/L LPS for 24 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) or Western Blot was used to determine the expressions of M1 macrophage markers such as interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and M2 macrophage markers such as arginase-1 (Arg-1), leukocyte differentiation antigen 206 (CD206), as well as Tim-3 and Galectin-9 in the cells. ② The other mice peritoneal macrophages were divided into blank control group (cultured in serum-free DMEM medium for 24 hours), LPS treatment group (cultured in serum-free DMEM medium containing 0.1 mg/L LPS for 24 hours) and α-lactose pretreatment group (pretreated with serum-free DMEM containing 40μmol/L Galectin-9 signal antagonist 1 hour before LPS stimulation). Over closed Galectin-9 signal was used to verify the role of Galectin-9 in macrophage M1/M2 subtype polarization.Results ① After stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L) for 24 hours, the expression of M1 markers was only slightly increased such as iNOS mRNA or not significantly changed such as IL-6 mRNA in macrophages, while the expressions of M2 markers such as Arg-1 mRNA and CD206 mRNA were significantly increased and peaked at LPS concentrations of 0.1 mg/L and 0.01 mg/L [compared with blank control group:Arg-1 mRNA (2-ΔΔCt) was 1.85±0.07 vs. 1.00±0.02, CD206 mRNA (2-ΔΔCt) was 2.03±0.11 vs.1.00±0.05, both P < 0.01]. With the increase of LPS concentration, the expressions of IL-6 mRNA and iNOS mRNA continued to increase, while the expressions of Arg-1 mRNA and CD206 mRNA were gradually decreased, and the macrophage M1/M2 subtype polarization status changed. At the same time, the level of Tim-3 protein in macrophages was significantly up-regulated after stimulation with 0.01 mg/L LPS as compared with that of blank control group (Tim-3/GAPDH:0.84±0.04 vs. 0.69±0.02,P < 0.01), peaked at LPS concentrations of 0.1 mg/L, and then decreased with increasing LPS concentration. The intracellular Galectin-9 and supernatant secreted Galectin-9 (s-Galectin-9) protein levels showed no significant change after stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L), while then gradually decreased with the increase of LPS concentration. ② Compared with blank control group, the mRNA expressions of M1 marker iNOS and M2 markers Arg-1 and CD206 were significantly increased in LPS treatment group, but IL-6 mRNA level was not changed significantly. The mRNA levels of IL-6 and iNOS were further elevated after pretreatment with α-lactose as compared with that of the LPS treatment group [IL-6 mRNA (2-ΔΔCt): 1.44±0.02 vs. 1.14±0.11, iNOS mRNA (2-ΔΔCt):2.45±0.04 vs. 2.01±0.08, bothP < 0.01], while the mRNA levels of Arg-1 and CD206 were significantly decreased [Arg-1 mRNA (2-ΔΔCt): 0.75±0.01 vs. 1.85±0.02, CD206 mRNA (2-ΔΔCt): 0.58±0.02 vs. 2.03±0.14, bothP < 0.01]. Meanwhile, the blocking of Galectin-9 signaling could also reduce the extracellular s-Galectin-9 (compared with LPS treatment group: s-Galectin-9/GAPDH was 0.10±0.01 vs. 0.23±0.02,P < 0.01), down-regulated the expressions of Tim-3 and Galectin-9 (Tim-3/GAPDH: 0.28±0.01 vs. 0.43±0.01, Galectin-9/GAPDH: 0.21±0.01 vs. 0.43±0.01, bothP < 0.01).Conclusions LPS regulates macrophage M1/M2 subtype polarization via Galectin-9/ Tim-3 signaling pathway. Low-doses of LPS can limit the development of inflammation by accommodating the expression and secretion of Galectin-9 to polarize macrophages to M2. High-doses of LPS promotes the development of inflammation by down-regulating the expression and secretion of Galectin-9 to polarize macrophages to M1.

Research progress and mechanism of Galectin-9 and various malignant tumors / 实用肿瘤学杂志

Galectin-9 is a member of galectin,with chemotaxis eosinophils,regulating cell aggregation and adhesion,im-mune regulation and other functions.In recent years,many studies have shown that Galectin-9 plays an important role in a variety of malignant tumors,affecting tumor cell proliferation,apoptosis and other activities.This article focuses on the relationship between Ga-lectin-9 and various malignant tumors,and explores its possible mechanism of action.

Express of TIM-3 and Galectin-9 Genes in Peripheral Blood Mononuclear Cells from Patients with Asthma / 现代检验医学杂志

Objective To investigate the levels of the mRNA expression of TIM-3 and Galectin-9 in peripheral blood mono-cytes (PBMCs)of acute exacerbation asthma patients and their clinical significances.Methods 60 patients with acute exac-erbation asthma (eliminating 15 cases of non-conform to the regulations)and 30 cases of healthy subjects were collected from January to October of 2014.Used fluorescence quantitative real-time reverse transcription-polymerase chain reaction to measure the mRNA expression of TIM-3 and Galectin-9 in PBMCs of patients with asthma and healthy controls.Results The expression of TIM-3,Galectin-9 and IFN-γmRNA in the PBMCs from acute exacerbation asthma patients were all ab-normally higher than healthy controls (U =458.5,P =0.019;U =437.5,P =0.010;U =260,P <0.001).There were statis-tically significant differences between them.Conclusion TIM-3/Galectin-9 pathway may participate in the occurrence,devel-opment of asthma.TIM-3 or (and)Galectin-9 may prove to be an important target for treatments to asthma.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the beta-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

Increased expression of galectin-9 in experimental autoimmune encephalomyelitis / 대한수의학회지

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), reflects pathophysiologic steps in MS such as the influence of T cells and antibodies reactive to the myelin sheath, and the cytotoxic effect of cytokines. Galectin-9 (Gal-9) is a member of animal lectins that plays an essential role in various biological functions. The expression of Gal-9 is significantly enhanced in MS lesions; however, its role in autoimmune disease has not been fully elucidated. To identify the role of Gal-9 in EAE, we measured changes in mRNA and protein expression of Gal-9 as EAE progressed. Expression increased with disease progression, with a sharp rise occurring at its peak. Gal-9 immunoreactivity was mainly expressed in astrocytes and microglia of the central nervous system (CNS) and macrophages of spleen. Flow cytometric analysis revealed that Gal-9+CD11b+ cells were dramatically increased in the spleen at the peak of disease. Increased expression of tumor necrosis factor (TNF)-R1 and p-Jun N-terminal kinase (JNK) was observed in the CNS of EAE mice, suggesting that TNF-R1 and p-JNK might be key regulators contributing to the expression of Gal-9 during EAE. These results suggest that identification of the relationship between Gal-9 and EAE progression is critical for better understanding Gal-9 biology in autoimmune disease.

The mechanism of Galectin-9 in the immune regulation of the active CD4+T cells / 中华微生物学和免疫学杂志

Objective To study the immune regulation of Galectin-9 on the active CD4+T cells and demonstrate the mechanisms.Methods Lymphocytes were harvested from wild-type C57BL/6 mouse,from which na(i)ve CD4+T cells were separated via MACS and then stimulated with anti-CD3 antibody(Ab) (2.5 μg/ml),anti-CD28 Ab(5 μg/ml) and IL-2(100 ng/ml) for 3 days.The active CD4+T cells were divided into 3 groups:Control group,Galectin-9 group and Galectin-9+α-lactose group.We detected the cell proliferation level by CFSE fluorescence intensity and then dynamically observed the cell morphological changes.The proportion of CD4+CD69+T cell,Th1,Th2 and Th17 cell was valued; Meanwhile,ELISA was used to detect the cytokine levels of IFN-γ,IL-4,IL-10,IL-12,IL-17A and TGF-β1 secreted by lymphocytes.Also Western blot was used to observe the changes of T cell differentiation regulatory protein such as T-bet,GATA-3 and ROR-γt.Results Compared with control group and Galectin-9+α-lactose group,in Galectin-9 group,the cell morphology began to change at 2 h.Moreover the proportions of CD4+CD69+ T cell,Th1 and Th17 cells decreased (P＜0.05),but no significant differences in Th2 cells.The level of IFN-γ,IL-12,IL-17A and TGF-β1 from the supernatant decreased (P＜0.05),while Th2-type cytokines IL-4 and IL-1O did not change.In addition,the expressions of T-bet and ROR-γt were significantly down-regulated (P＜0.05).Conclusion Galectin-9 inhibited Th1 and Th17-type immune response,while had no effect on Th2-type immune response.The mechanism of the immune regulation may be related to affect the expression of Th1 and Th17 specific transcription factors at transcription level.

Galectin-9 and inflammation / 国际儿科学杂志

Galectin-9 is a novel eosinophil chemoattractant,which belong to Galectin family.Galectin-9exhibits a variety of biological functions,such as cell differentiation,maturation,adhesion and aggregation,cell chemotaxis,activity,and cell death.Galectin-9 may play important roles in the development of acute inflammation as well as chronic inflammation associated with allergies,autoimmune diseases,infectious processes.This review summarizes the advances in Galectin-9 study.

Influence of adenovirus carrying Galectin-9 gene on differentiation and proliferation of CD4+CD25+ regulatory T cells in mice with heart transplantation / 第二军医大学学报

Objective: To explore the influence of adenovirus carrying Galectin-9 gene on the survival of cardiac transplant and the differentiation and proliferation of CD4+ CD25+ Treg cells in mice receiving heterotopic heart transplantation. Methods: Neck heterotopic heart transplantation models were established with male BALB/c→C57BL/6 mice by Cuff method, and they were randomly divided into 3 groups (n=10): control group (received 0.9% sodium chloride solution after operation), adenovirus group, and pAdeasy™ Galectin-9 treatment group. The survival time of the graft hearts was recorded. The spleens of mice were collected when the graft hearts stopped beating or 14 days after operation) the mononuclear cells were isolated and the proportion of CD4+ CD25+ Treg cells was determined by flow cytometry. Treg cell related cytokines (IL-10, TGF-β1) in the peripheral blood were assayed by ELISA. Results: The cardiac allografts in pAdeasy™ Galectin-9 group survived significantly longer than those in the other two groups (P<0.01), and the proportion of CD4+ CD25+ Treg cells in CD4+ T cells was significantly higher (P<0.01). The levels of IL-10 and TGF-β1 were increased in pAdeasy™ Galectin-9 group compared with control group and adenovirus group (P < 0.01). Conclusion: pAdeasy™ Galectin-9 can promote the proliferation and differentiation of CD4+ CD25+ Treg cells in vivo, and can greatly improve the survival time of cardiac allograft after heart transplantation in mice.