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Background/aim: Galectin-9 (Gal-9) induces tumor cell apoptosis in lymphoma and other malignant cell types. Duodenal adenocarcinoma is a rare malignancy, and there are insufficient data to determine a standard therapeutic approach. Here, we investigated the antitumor effect of Gal-9 in HuTu-80 duodenal adenocarcinoma cells., Materials and Methods: Cell proliferation was examined in HuTu-80 cells using a Cell Counting Kit-8 assay. Cell cycle analysis, apoptosis array, and microRNA expression analysis were performed to identify the effect of Gal-9 on HuTu-80 cells. The antitumor effect of Gal-9 was also examined using xenograft mouse models., Results: Gal-9 suppressed the proliferation of HuTu-80 via blockade of the G 0 to G 1 cell cycle transition. This blockade was accompanied by a strong decrease in cyclin D1 and phosphorylated Rb, suggesting a G 1 arrest. Additionally, Gal-9 induced apoptosis, and the expression of cleaved caspase-3 was increased in Gal-9-treated HuTu-80 cells according to the apoptosis array. MiRNA microarrays revealed that Gal-9 altered the expression of miRNAs in HuTu-80 cells., Conclusion: These data demonstrate the therapeutic potential of Gal-9 and provide molecular mechanistic insights into its antitumor effect in HuTu-80 cells., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9 knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of β2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9 significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Colon cancer is the second leading cause of cancer‑related mortality worldwide, and the prognosis of advanced colon cancer has remained poor in recent years. Galectin‑9 (Gal‑9) is a tandem‑repeat type galectin that has recently been shown to exert antiproliferative effects on various types of cancer cells. The present study aimed to assess the effects of Gal‑9 on human colon and colorectal cancer cells in vitro and in vivo , as well as to evaluate the microRNAs (miRNAs/miRs) associated with the antitumor effects of Gal‑9. We examined the ability of Gal‑9 to inhibit cell proliferation via apoptosis, and the effects of Gal‑9 on cell cycle‑related molecules in various human colon and colorectal cancer cell lines. In addition, Gal‑9‑mediated changes in activated tyrosine kinase receptors and angiogenic molecules were assessed using protein array chips in colon and colorectal cancer cells. Moreover, miRNA array analysis was performed to examine Gal‑9‑induced miRNA expression profiles. We also elucidated if Gal‑9 inhibited tumor growth in a murine in vivo model. We found that Gal‑9 suppressed the cell proliferation of colon cancer cell lines in vitro and in vivo . Our data further revealed that Gal‑9 increased caspase‑cleaved keratin 18 levels in Gal‑9‑treated colon cancer cells. In addition, Gal‑9 enhanced the phosphorylation of ALK, DDR1, and EphA10 proteins. Furthermore, the miRNA expression levels, such as miR‑1246, miR‑15b‑5p, and miR‑1237, were markedly altered by Gal‑9 treatment in vitro and in vivo . In conclusion, Gal‑9 suppresses the cell proliferation of human colon cancer by inducing apoptosis, and these findings suggest that Gal‑9 can be a potential therapeutic target in the treatment of colon cancer.

Galectin-9 (Gal-9) enhances tumor immunity mediated by T cells, macrophages, and dendritic cells. Its expression level in various cancers correlates with prognosis. Furthermore, Gal-9 directly induces apoptosis in various cancers; however, its mechanism of action and bioactivity has not been clarified. We evaluated Gal-9 antitumor effect against esophageal squamous cell carcinoma (ESCC) to analyze the dynamics of apoptosis-related molecules, elucidate its mechanism of action, and identify relevant changes in miRNA expressions. KYSE-150 and KYSE-180 cells were treated with Gal-9 and their proliferation was evaluated. Gal-9 inhibited cell proliferation in a concentration-dependent manner. The xenograft mouse model established with KYSE-150 cells was administered with Gal-9 and significant suppression in the tumor growth observed. Gal-9 treatment of KYSE-150 cells increased the number of Annexin V-positive cells, activation of caspase-3, and collapse of mitochondrial potential, indicating apoptosis induction. c-Jun NH 2 -terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) phosphorylation were activated and could be involved in apoptosis. Therefore, Gal-9 induces mitochondria-mediated apoptosis of ESCC and inhibits cell proliferation in vitro and in vivo with JNK and p38 activation.

Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immunosuppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9 -Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epitopes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models., Competing Interests: Several authors were employed by commercial companies: Cellvax (CL, CB, MW), GalPharma Co, Ltd (TN), H-Immune Therapeutics (LC). This does not alter our adherence to PLOS ONE policies on sharing data and materials. Our antibodies are the subject of the following patent entitled “Antibody which is directed against galectin-9 and is an inhibitor of the suppressor activity of regulatory T lymphocytes”, WO 2015/185875 A2. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: In systemic lupus erythematosus (SLE), an autoimmune disease associated with multiple organ involvement, the development of lupus nephritis determines prognosis, and arthritis impairs quality of life. Galectin 9 (Gal-9, Lgals9) is a β-galactoside-binding lectin that has been used for clinical application in autoimmune diseases, since recombinant Gal-9, as a ligand for T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), induces apoptosis of activated CD4+TIM-3+ Th1 cells. This study was undertaken to investigate whether deficiency of Lgals9 has beneficial or deleterious effects on lupus in a murine model., Methods: Gal-9 +/+ and Gal-9 -/- female BALB/c mice were injected with pristane, and the severity of arthritis, proteinuria, and levels of autoantibody production were assessed at several time points immediately following injection. At 7 months after pristane injection, renal pathologic features, the severity of joint inflammation, and formation of lipogranulomas were evaluated. Subsets of inflammatory cells in the spleen and peritoneal lavage were characterized, and expression levels of cytokines from peritoneal macrophages were analyzed., Results: Lgals9 deficiency protected against the development of immune complex glomerulonephritis, arthritis, and peritoneal lipogranuloma formation in BALB/c mice in this murine model of pristane-induced lupus. The populations of T cell subsets and B cells in the spleen and peritoneum were not altered by Lgals9 deficiency in pristane-injected BALB/c mice. Furthermore, Lgals9 deficiency protected against pristane-induced lupus without altering the Toll-like receptor 7-type I interferon pathway., Conclusion: Gal-9 is required for the induction and development of lupus nephritis and arthritis in this murine model of SLE. The results of the current investigation provide a potential new strategy in which antagonism of Gal-9 may be beneficial for the treatment of nephritis and arthritis in patients with SLE through targeting of activated macrophages., (© 2018, American College of Rheumatology.)

Background and Aim: Chronic liver diseases progress from chronic inflammation to fibrosis to tumorigenesis. Galectin-9, a β-galactoside-specific animal lectin, is indicated to contribute to all three steps of progression. The aim of this study was to determine which of the three steps was most dominant in elevating the serum galectin-9 concentration and to test the possibility of galectin-9 as a serum biomarker., Methods: Japanese patients with chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), non-alcoholic fatty liver disease, or alcoholic liver disease who provided informed consent were enrolled in this study. Serum galectin-9 levels were measured using a sandwich ELISA. Multiple regression analyses were performed using ezr to identify factors that determined serum galectin-9 concentration., Results: One hundred one patients with 50 of chronic hepatitis and 51 of liver cirrhosis were enrolled; the cohort included 45 cases of hepatitis C virus infection, 13 cases of hepatitis B virus infection, and 46 cases with HCC-related complications. The median serum galectin-9 concentration was 77.54 pg/mL (interquartile range: 18.89-241.9 pg/mL). Multiple linear regression analyses proved Fibrosis-4 index and aspartate aminotransferase to platelet ratio index, indexes of liver fibrosis, were able to predict the serum galectin-9 levels with statistical significance. A multiple logistic regression analysis determined 10 pg/mL increase in the serum galectin-9 concentration presented an odds ratio of 3.90 for liver fibrosis progression., Conclusions: The serum galectin-9 concentration represents a potential biomarker of liver fibrosis in patients with chronic liver diseases, regardless of chronic inflammation or the presence of HCC complications. Furthermore, higher serum galectin-9 levels are a predictor for liver fibrosis progression., (© 2017 The Authors Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background/aims: Galectin-9 is a soluble immune modulator with versatile functions, including a role as an immune checkpoint molecule. Therefore, the amount of galectin-9 in the blood may reflect an individual's immunological balance. Many studies have conducted galectin-9 measurements; however, the reported galectin-9 concentration in the blood varies greatly, even within healthy controls. This study investigates the variation between the reported and actual concentrations of galectin-9., Methods: A GalPharma ELISA and an R&D Systems ELISA kit were directly compared using the same set of plasma and a series of recombinant galectins, including degraded galectin-9. Furthermore, galectin-9 in plasma was concentrated using anti-galectin-9 antibody-conjugated beads, and subjected to western blotting to estimate the quantity and integrity of galectin-9 and assess the consistency of ELISA measurements., Results: The R&D Systems' ELISA indicated a 50-fold higher median concentration of plasma galectin-9 than that indicated by the GalPharma ELISA. This variation is due to aberrantly enhanced reactivity of the R&D Systems' ELISA to degraded galectin-9 present in small quantities in the plasma. The GalPharma ELISA could detect only intact galectin-9 and its results correlated well with the plasma galectin-9 level obtained by western blotting., Conclusion: ELISA kits from R&D Systems reacts aberrantly higher against degraded galectin-9 than the intact galectin-9. Therefore, the existence of a small amount of degraded galectin-9 in a test sample hinders the quantification. As galectin-9 is a fragile protein, this is a serious concern when using this kit. Based on quantifications from the GalPharma ELISA, the median (25th-75th percentiles) galectin-9 concentration in healthy subjects in the current study cohort was calculated as 110 pg/mL (67 -154 pg/mL)., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Stimulation of several TNF receptor family proteins has been shown to dampen inflammatory disease in murine models through augmenting the number and/or activity of regulatory T cells (Tregs). We recently found that one molecule, 4-1BB, used binding to Galectin-9 to exert its immunosuppressive effects and drive expansion of CD8 + Foxp3 - Tregs. We now show that ligation of another TNFR family molecule, DR3, which has previously been found to strongly expand CD4 + Foxp3 + Tregs and suppress inflammation, also requires Galectin-9. We found that the extracellular region of DR3 directly binds to Galectin-9, and that Galectin-9 associates with DR3 in Tregs. From studies in vitro with Galectin-9 -/- CD4 + T cells and Tregs, we found that stimulatory activity induced by ligating DR3 was in part dependent on Galectin-9. In vivo, in a model of experimental autoimmune encephalomyelitis, we show that an agonist of DR3 suppressed disease, correlating with expansion of CD4 + Foxp3 + Tregs, and this protective effect was lost in Galectin-9 -/- mice. Similar results were seen in an allergic lung inflammation model. Thus, we demonstrate a novel function of Galectin-9 in facilitating activity of DR3 related to Treg-mediated suppression., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Galectin-9 (G9) is a tandem-repeat type β-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Liver metastasis from gastrointestinal cancer defines a patient's prognosis. Despite medical developments, pancreatic cancer with liver metastasis confers a very poor prognosis. Galectin-9 (Gal‑9) is a tandem-repeat-type galectin that has recently been demonstrated to exert antitumor effects on various types of cancer cells by inducing apoptosis. However, the apoptotic pathway of Gal‑9 in solid tumors is unclear. The aim of the present study was to evaluate the effects of Gal‑9 on human liver metastasis from pancreatic cancer. Gal‑9 suppressed cell proliferation in metastatic liver cancer cell lines derived from pancreatic cancer (KMP2, KMP7, and KMP8) and increased the levels of caspase-cleaved keratin 18 and fluorescein isothiocyanate (FITC)-conjugated Annexin V. Furthermore, expression of apoptosis-related molecules such as caspase-7, cleaved caspase-3, cleaved PARP, cytochrome c, Smac/Diablo and HtrA2/Omi was enhanced. However, Gal‑9 did not affect expression of various cell cycle-related proteins. The microRNA (miRNA) expression profile was markedly altered by Gal‑9, and various miRNAs might contribute to tumor growth suppression. Our data reveal that Gal‑9 suppresses the growth of liver metastasis, possibly by inducing apoptosis through a mechanism involving mitochondria and changes in miRNA expression. Thus, Gal‑9 might serve as a therapeutic agent for the treatment of liver metastasis from pancreatic cancer.

Purpose: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow obstruction and pulmonary emphysema. Persistent inflammation and remodeling of the lungs and airways result in reduced lung function and a lower quality of life. Galectin (Gal)-9 plays a crucial role as an immune modulator in various diseases. However, its role in the pathogenesis of pulmonary emphysema is unknown. This study investigates whether Gal-9 is involved in pulmonary inflammation and changes in emphysema in a porcine pancreatic elastase (PPE)-induced emphysema model., Materials and Methods: Gal-9 was administered to mice subcutaneously once daily from 1 day before PPE instillation to day 5. During the development of emphysema, lung tissue and bronchoalveolar lavage fluid (BALF) were collected. Histological and cytological findings, concentrations of chemokines and matrix metalloproteinases (MMPs) in the BALF, and the influence of Gal-9 treatment on neutrophils were analyzed., Results: Gal-9 suppressed the pathological changes of PPE-induced emphysema. The mean linear intercept (Lm) of Gal-9-treated emphysema mice was significantly lower than that of PBS-treated emphysema mice (66.1 ± 3.3 μm vs. 118.8 ± 14.8 μm, respectively; p < 0.01). Gal-9 decreased the number of neutrophils and levels of MMP-9, MMP-2 and tissue inhibitor of metalloproteinases (TIMP)-1 in the BALF. The number of neutrophils in the BALF correlated significantly with MMPs levels. Interestingly, Gal-9 pretreatment in vitro inhibited the chemotactic activity of neutrophils and MMP-9 production from neutrophils. Furthermore, in Gal-9-deficient mice, PPE-induced emphysema progressed significantly compared with that in wild-type (WT) mice (108.7 ± 6.58 μm vs. 77.19 ± 6.97 μm, respectively; p < 0.01)., Conclusions: These results suggest that Gal-9 protects PPE-induced inflammation and emphysema by inhibiting the infiltration of neutrophils and decreasing MMPs levels. Exogenous Gal-9 could be a potential therapeutic agent for COPD.

Fulminant hepatitis is a severe liver disease resulting in hepatocyte necrosis. Galectin‑9 (Gal‑9) is a tandem‑repeat‑type galectin that has been evaluated as a potential therapeutic agent for various diseases that regulate the host immune system. Concanavalin A (ConA) injection into mice results in serious, immune‑mediated liver injury similar to human viral, autoimmune and fulminant hepatitis. The present study investigated the effects of Gal‑9 treatment on fulminant hepatitis in vivo and the effect on the expression of microRNAs (miRNAs), in order to identify specific miRNAs associated with the immune effects of Gal‑9. A ConA‑induced mouse hepatitis model was used to investigate the effects of Gal‑9 treatment on overall survival rates, liver enzymes, histopathology and miRNA expression levels. Histological analyses, TUNEL assay, immunohistochemistry and miRNA expression characterization, were used to investigate the degree of necrosis, fibrosis, apoptosis and infiltration of neutrophils and macrophages. Overall survival rates following ConA administration were significantly higher in Gal‑9‑treated mice compared with control mice treated with ConA + PBS. Histological examination revealed that Gal‑9 attenuated hepatocellular damage, reduced local neutrophil infiltration and prevented the local accumulation of macrophages and liver cell apoptosis in ConA‑treated mice. In addition, various miRNAs induced by Gal‑9 may contribute to its anti‑apoptotic, anti‑inflammatory and pro‑proliferative effects on hepatocytes. The results of the present study demonstrate that Gal‑9 may be a candidate therapeutic target for the treatment of fulminant hepatitis.

The incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in western countries. The overall mortality of this disease remains high with a 5-year survival rate of less than 20%, despite remarkable advances in the care of patients with EAC. Galectin-9 (Gal-9) is a tandem-repeat type galectin that exerts anti-proliferative effects on various cancer cell types. The aim of the present study was to evaluate the effects of Gal-9 on human EAC cells and to assess the expression of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. Gal-9 suppressed the proliferation of the EAC cell lines OE19, OE33, SK-GT4, and OACM 5.1C. Additionally, Gal-9 treatment induced apoptosis and increased the expression levels of caspase-cleaved cytokeratin 18, activated caspase-3 and activated caspase-9. However, it did not promote cell cycle arrest by reducing cell cycle-related protein levels. Furthermore, Gal-9 increased the level of the angiogenesis-related protein interleukin-8 (IL-8) and markedly altered miRNA expression. Based on these findings, Gal-9 may be of clinical use for the treatment of EAC.

Dysregulation of apoptosis is a major hallmark in cancer biology that might equip tumors with a higher malignant potential and chemoresistance. The anti-cancer activities of lectin, defined as a carbohydrate-binding protein that is not an enzyme or antibody, have been investigated for over a century. Recently, galectin-9, which has two distinct carbohydrate recognition domains connected by a linker peptide, was noted to induce apoptosis in thymocytes and immune cells. The apoptosis of these cells contributes to the development and regulation of acquired immunity. Furthermore, human recombinant galectin-9, hG9NC (null), which lacks an entire region of the linker peptide, was designed to resist proteolysis. The hG9NC (null) has demonstrated anti-cancer activities, including inducing apoptosis in hematological, dermatological and gastrointestinal malignancies. In this review, the molecular characteristics, history and apoptosis-inducing potential of galectin-9 are described., Competing Interests: Mitsuomi Hirashima is one of the board members of GalPharma Co., Ltd. The author has the following patents related to material pertinent to this article: “Novel modified galectin 9 proteins and use thereof” which is applied by GalPharma and issued in Japan (4792390), the USA (8,268,324), EPC (1736541), Canada (2,561,696), India (239130), and Korea ((10-1222281) as of 2013.12.2). The author has the following products related to material pertinent to this article: stable-form Gal-9. This does not alter the two authors’ adherence to all the journal policies on sharing data and materials. The other authors declare no conflicts of interest.

Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART), and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9) potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002), more potently than vorinostat (p = 0.02). rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p<0.05). rGal-9 signals through N-linked oligosaccharides and O-linked hexasaccharides on the T cell surface, modulating the gene expression levels of key transcription initiation, promoter proximal-pausing, and chromatin remodeling factors that regulate HIV latency. Beyond latent viral reactivation, rGal-9 induces robust expression of the host antiviral deaminase APOBEC3G in vitro and ex vivo (FDR<0.006) and significantly reduces infectivity of progeny virus, decreasing the probability that the HIV reservoir will be replenished when latency is reversed therapeutically. Lastly, endogenous levels of soluble galectin-9 in the plasma of 72 HIV-infected ART-suppressed individuals were associated with levels of HIV RNA in CD4+ T cells (p<0.02) and with the quantity and binding avidity of circulating anti-HIV antibodies (p<0.009), suggesting a role of galectin-9 in regulating HIV transcription and viral production in vivo during therapy. Our data suggest that galectin-9 and the host glycosylation machinery should be explored as foundations for novel HIV cure strategies.

Background & Aims: Fewer than 50% of patients with acute liver failure (ALF) recover spontaneously, and ALF has high mortality without liver transplantation. Kupffer cells have been reported to mediate liver inflammation during drug-induced injury. Galectin-9 is produced by Kupffer cells and has diverse roles in regulating immunity. We investigated whether plasma levels of galectin-9 are associated with outcomes of patients with ALF., Methods: We analyzed plasma samples (collected at time of hospital admission) and clinical data from 149 patients included in the Acute Liver Failure Study Group from July 2006 through November 2010 (110 had acetaminophen-induced hepatotoxicity and 39 had nonacetaminophen drug-induced liver injury). We compared data with those from all patients enrolled in the study (from July 1, 2006 through October 30, 2013), and from healthy individuals of similar ages with no evidence of liver disease (control subjects). Plasma levels of galectin-9 were measured using a polyclonal antibody and colorimetric assay., Results: Patients with ALF had statistically higher plasma levels of galectin-9 than control subjects, but levels did not differ significantly between patients with acetaminophen-induced liver injury and drug-induced liver injury. A level of galectin-9 above 690 pg/mL was associated with a statistically significant increase in risk for mortality or liver transplantation caused by ALF. Competing risk analyses associated level of galectin-9 with transplant-free survival, independently of Model For End-Stage Liver Disease score or systemic inflammatory response syndrome., Conclusions: A one-time measurement of plasma galectin-9 level can be used to assign patients with ALF to high-, intermediate-, and low-risk groups. The combination of galectin-9 level and Model For End-Stage Liver Disease score was more closely associated with patient outcome than either value alone. These data might be used to determine patient prognoses and prioritize patients for liver transplantation. ClinicalTrials.gov ID NCT00518440., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Gallbladder cancer (GBC) is the most common and aggressive type of biliary tract cancer. There are various histological types of GBC, and the vast majority of GBC cases are adenocarcinomas. Squamous and adenosquamous carcinomas are rare GBC subtypes that are traditionally considered to be more aggressive and to be associated with a poorer prognosis than adenocarcinoma. Galectin-9 (Gal-9), a tandem-repeat-type galectin, has been reported to induce apoptosis-mediated elimination of various cancers, including hepatocellular carcinoma, cholangiocarcinoma, and hematologic malignancies. Therefore, we investigated the antitumor effects of Gal-9 on GBC in vitro and in vivo. In our in vitro experiments, Gal-9 suppressed cell proliferation in various GBC cell lines but not in the OCUG-1 cell line, which represents a poorly differentiated type of adenosquamous carcinoma. Gal-9 induced the apoptosis of Gal-9-sensitive GBC cells by increasing the levels of caspase-cleaved keratin 18 and phosphorylated p53. However, Gal-9 did not affect the expression of various cell cycle-related proteins. In addition, Gal-9 suppressed tumor growth by implanted human GBC cells in a xenograft model. Furthermore, Gal-9 induced the phosphorylation of the Ephrin type-B receptor, and the microRNA (miRNA) expression profile was markedly altered by Gal-9. Based on these results, various miRNAs might contribute to the suppression of tumor growth. Our data reveal that Gal-9 suppresses the growth of GBC, possibly by inducing apoptosis and altering miRNA expression. Thus, Gal-9 might serve as a therapeutic agent for the treatment of GBC.

Gastric cancer is the second-leading cause of cancer-related mortality worldwide, and the prognosis of advanced gastric cancer remains poor. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been demonstrated to exert anti-proliferative effects on various types of cancer cells. The aim of our present study was to evaluate the effects of Gal-9 on human gastric cancer cells and the expression levels of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. In our initial experiments, Gal-9 suppressed the proliferation of gastric cancer cell lines in vitro. Our data further revealed that Gal-9 increased caspase-cleaved keratin 18 (CCK18) levels in gastric cancer cells. Additionally, Gal-9 reduced the phosphorylation of vascular endothelial growth factor receptor-3 (VEGFR-3) and insulin-like growth factor-1 receptor (IGF-1R). Furthermore, miRNA expression levels were markedly altered with Gal-9 treatment in vitro. In conclusion, Gal-9 suppressed the proliferation of human gastric cancer cells by inducing apoptosis. These findings suggest that Gal-9 could be a potential therapeutic target in the treatment of gastric cancer.

Cholangiocarcinoma is the most common biliary malignancy and the second most common hepatic malignancy after hepatocellular carcinoma (HCC). Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been shown to exert antiproliferative effects on cancer cells. Therefore, the present study evaluated the effects of Gal-9 on the proliferation of human cholangiocarcinoma cells in vitro as well as the microRNAs (miRNAs) associated with the antitumor effects of Gal-9. Gal-9 suppressed the proliferation of cholangiocarcinoma cell lines in vitro and the growth of human cholangiocarcinoma cell xenografts in nude mice. Our data further revealed that Gal-9 increased caspase‑cleaved keratin 18 (CCK18) levels, and the expression of cytochrome c increased in Gal-9-treated cholangiocarcinoma cell lines. These data suggested that Gal-9 induced cholangiocarcinoma cell apoptosis via the intrinsic apoptosis pathway mediated by caspase-dependent or -independent pathways. In addition, Gal-9 reduced the phosphorylation of the epidermal growth factor receptor (EGFR), insulin-like growth factor and insulin-like growth factor-1 receptor (IGF-1R), hepatocyte growth factor receptor and fibroblast growth factor receptor 3 (FGFR3). These findings suggest that Gal-9 can be a candidate of therapeutic target in the treatment of cholangiocarcinoma.

Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca(2+)) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca(2+) mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca(2+) mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4(+) T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca(2+) mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4(+) T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRAS(mut)) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRAS(mut) CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAF(mut)). In KRAS(mut) CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRAS(mut) cancer cells. Thus, rLGALS9 has potent antitumor activity toward refractory KRAS(mut) CRC cells that may be exploitable for therapeutic use.

Galectin-9, a soluble β-galactoside-binding animal lectin, evokes apoptosis in various human cancer cell lines. The galectin-9 antitumor effect against hepatocellular carcinoma (HCC) is, however, unknown. We investigated whether galectin-9 suppresses HCC growth in vitro and in vivo. We assessed the antitumor effect of galectin-9 on HCC cells by conducting WST-8 assay in vitro and xenograft model analysis in vivo. Galectin-9-induced apoptosis was evaluated by FACS and ELISA in vitro and by TUNEL stain in vivo. Cell cycle alteration was profiled by FACS. Caspases were profiled by colorimetry. MicroRNAs related to the galectin-9 antitumor effects were determined using microarrays, and their antitumor effect was confirmed in a transfection study in vitro. The expression levels of the target proteins of the miRNAs extracted above were analyzed by western blot analysis. To summarize the results, galectin-9 inhibited the growth of the HCC cell lines HLE and Li-7 in vitro and Li-7 in vivo inducing apoptosis. Cell cycle turnover was not arrested in HLE and Li-7 cells in vitro. miR-1246 was similarly extracted both in vitro and in vivo, which sensitized Li-7 cells to apoptosis when transfected into the cells. DYRK1A, a target protein of miR-1246 was downregulated in Li-7 cells. Caspase-9 was upregulated in Li-7 cells in vitro and in vivo. In conclusion, galectin-9 inhibited the growth of HCC cells by apoptosis, but not cell cycle arrest, in vitro and in vivo. miR-1246 mediated signals of galectin-9, possibly through miR-1246-DYRK1A-caspase-9 axis. Galectin-9 might be a candidate agent for HCC chemotherapy.

The β-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression within iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop, which further promoted Foxp3 expression. Galectin-9 increased iTreg cell stability and function by directly binding to its receptor CD44, which formed a complex with transforming growth factor-β (TGF-β) receptor I (TGF-βRI), and activated Smad3. Galectin-9 signaling was further found to regulate iTreg cell induction by dominantly acting through the CNS1 region of the Foxp3 locus. Our data suggest that exogenous galectin-9, in addition to being an effector molecule for Treg cells, acts synergistically with TGF-β to enforce iTreg cell differentiation and maintenance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Galectin-9 (Gal-9) is a β-galactosidase-binding lectin that promotes apoptosis, tissue inflammation, and T cell immune exhaustion, and alters HIV infection in part through engagement with the T cell immunoglobulin mucin domain-3 (Tim-3) receptor and protein disulfide isomerases (PDI). Gal-9 was initially thought to be an eosinophil attractant, but is now known to mediate multiple complex signaling events that affect T cells in both an immunosuppressive and inflammatory manner. To understand the kinetics of circulating Gal-9 levels during HIV infection we measured Gal-9 in plasma during HIV acquisition, in subjects with chronic HIV infection with differing virus control, and in uninfected individuals. During acute HIV infection, circulating Gal-9 was detected as early as 5 days after quantifiable HIV RNA and tracked plasma levels of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and IL-1β. In chronic HIV infection, Gal-9 levels positively correlated with plasma HIV RNA levels (r=0.29; p=0.023), and remained significantly elevated during suppressive antiretroviral therapy (median: 225.3 pg/ml) and in elite controllers (263.3 pg/ml) compared to age-matched HIV-uninfected controls (54 pg/ml). Our findings identify Gal-9 as a novel component of the first wave of the cytokine storm in acute HIV infection that is sustained at elevated levels in virally suppressed subjects and suggest that Gal-9:Tim-3 crosstalk remains active in elite controllers and antiretroviral (ARV)-suppressed subjects, potentially contributing to ongoing inflammation and persistent T cell dysfunction.

Biologics to TNF family receptors are prime candidates for therapy of immune disease. Whereas recent studies have highlighted a requirement for Fcγ receptors in enabling the activity of CD40, TRAILR, and GITR when engaged by antibodies, other TNFR molecules may be controlled by additional mechanisms. Antibodies to 4-1BB (CD137) are currently in clinical trials and can both augment immunity in cancer and promote regulatory T cells that inhibit autoimmune disease. We found that the action of agonist anti-4-1BB in suppressing autoimmune and allergic inflammation was completely dependent on Galectin-9 (Gal-9). Gal-9 directly bound to 4-1BB, in a site distinct from the binding site of antibodies and the natural ligand of 4-1BB, and Gal-9 facilitated 4-1BB aggregation, signaling, and functional activity in T cells, dendritic cells, and natural killer cells. Conservation of the Gal-9 interaction in humans has important implications for effective clinical targeting of 4-1BB and possibly other TNFR superfamily molecules., (© 2014 Madireddi et al.)

Antiglomerular basement membrane glomerulonephritis (anti-GBM GN) is a Th1- and Th17-predominant autoimmune disease. Galectin-9 (Gal-9), identified as the ligand of Tim-3, functions in diverse biological processes and leads to the apoptosis of CD4(+)Tim-3(+) T cells. It is still unclear how Gal-9 regulates the functions of Th1 and Th17 cells and prevents renal injury in anti-GBM GN. In this study, Gal-9 was administered to anti-GBM GN mice for 7 days. We found that Gal-9 retarded the increase of Scr, ameliorated renal tubular injury, and reduced the formation of crescents. The infiltration of Th1 and Th17 cells into the spleen and kidneys significantly decreased in Gal-9-treated nephritic mice. The reduced infiltration of Th1 and Th17 cells might be associated with the downregulation of CCL-20, CXCL-9, and CXCL-10 mRNAs in the kidney. In parallel, the blood levels of IFN-γ and IL-17A declined in Gal-9-treated nephritic mice at days 21 and 28. In addition, an enhanced Th2 cell-mediated immune response was observed in the kidneys of nephritic mice after a 7-day injection of Gal-9. In conclusion, the protective role of Gal-9 in anti-GBM GN is associated with the inhibition of Th1 and Th17 cell-mediated immune responses and enhanced Th2 immunity in the kidney.

Galectin-9 (Gal-9) is a β-galactoside-binding protein involved in various biologic processes, including cell aggregation, adhesion, chemoattraction, and apoptosis. Little is known, however, about the regulation mechanisms of Gal-9 production. Recent studies reported high plasma Gal-9 levels in humans infected with human immunodeficiency virus-1 and dengue virus. Viral respiratory infections such as influenza are common human illnesses. A synthetic double-stranded RNA, polyinosinic-polycytidylic acid (PolyIC), mimics the effects of viruses in various cell types and induces the expression of Gal-9 in endothelial cells. To examine the potential link between viral infection and Gal-9 expression, we measured plasma Gal-9 concentrations in patients with influenza. Subjects were 43 patients with influenza virus infection, 20 with pneumococcal pneumonia, and 20 healthy adults. Gal-9 concentrations in the plasma and in culture supernatants of human airway epithelial cells were measured using an enzyme-linked immunosorbent assay. Plasma Gal-9 concentrations were higher in patients with influenza infection than in patients with pneumococcal pneumonia and healthy subjects (p < 0.05). Patients with influenza were effectively differentiated from those with pneumococcal pneumonia or healthy subjects, based on the plasma levels of Gal-9 (p < 0.0001). Furthermore, using a human airway epithelial cell line, we showed that the presence of PolyIC but not lipopolysaccharides increased the Gal-9 concentration in the culture medium (p < 0.05), suggesting that PolyIC enhanced Gal-9 production. These findings support our proposal that Gal-9 production is induced by influenza virus infection in humans. In conclusion, plasma Gal-9 could be a new biomarker for patients with influenza infection.

The T cell Ig and mucin domain-containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment.

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

Introduction: Galectin-9 ameliorates various inflammatory conditions including autoimmune diseases by regulating T cell and macrophage/dendritic cell (DC) functions. However, the effect of galectin-9 on polymicrobial sepsis has not been assessed., Methods: We induced polymicrobial sepsis by cecal ligation and puncture (CLP) in mice. The survival rate was compared between galectin-9- and PBS-treated CLP mice. An ELISA was used to compare the levels of various cytokines in the plasma and culture supernatants. Fluorescence-activated cell sorting analysis was further performed to compare the frequencies of subpopulations of spleen cells., Results: Galectin-9 exhibited a protective effect in polymicrobial sepsis as demonstrated in galetin-9 transgenic mice and therapeutic galectin-9 administration. In contrast, such effect was not observed in nude mice, indicating the involvement of T cells in galectin-9-mediated survival prolongation. Galectin-9 decreased TNFα, IL-6, IL-10 and, high mobility group box 1 (HMGB1) and increased IL-15 and IL-17 plasma and spleen levels. Galectin-9 increased the frequencies of natural killer T (NKT) cells and PDCA-1+ CD11c+ macrophages (pDC-like macrophages) but did not change the frequency of CD4 or CD8 T cells, γδT cells or conventional DC. As expected, galectin-9 decreased the frequency of Tim-3+ CD4 T cells, most likely Th1 and Th17 cells. Intriguingly, many spleen NK1.1+ NKT cells and pDC-like macrophages expressed Tim-3. Galectin-9 increased the frequency of Tim-3-expressing NK1.1+ NKT cells and pDC-like macrophages. Galectin-9 further increased IL-17+ NK1.1+ NKT cells., Conclusion: These data suggest that galectin-9 exerts therapeutic effects on polymicrobial sepsis, possibly by expanding NKT cells and pDC-like macrophages and by modulating the production of early and late proinflammatory cytokines.

Background: Dengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties., Objective: To investigate plasma Gal-9 levels as a biomarker for DENV infection., Study Design: We enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness., Results: During the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P<0.01)., Conclusion: Gal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Viral respiratory infection is the most common cause of acute asthma exacerbation in patients with stable asthma. The replication of most respiratory viruses requires the generation of double-stranded RNA (dsRNA), resulting in the activation of host immune responses. Synthetic dsRNA, polyinosinic-polycytidylic acid (PolyIC), mimics the effects of viruses in various cell types. To evaluate new therapies for mite antigen-induced chronic asthma, we developed an acute exacerbation model of mouse chronic asthma using mite antigen and PolyIC. We also examined the preventive effects of recombinant galectin-9 (Gal-9) on acute asthma exacerbation in this model., Methods: Airway hyperresponsiveness (AHR) was examined to evaluate the exacerbation of chronic asthma. To analyze airway inflammation, the numbers of inflammatory cells and concentrations of cytokines in the bronchoalveolar lavage fluid (BALF) were estimated by flow cytometry and enzyme-linked immunosorbent assay, respectively., Results: AHR was accelerated by intranasal administration of PolyIC in addition to mite antigen. Levels of cytokines that contribute to AHR, including interferon-γ, tumor necrosis factor-α, and RANTES (CCR5), and of Gal-9 in the BALF were elevated in this acute asthma exacerbation mouse model. Intranasal administration of recombinant Gal-9 reduced the PolyIC-induced AHR and levels of these cytokines in the BALF. Further, Gal-9 suppressed the production of cytokines induced by PolyIC in the alveolar macrophages. CONCLUSIONS. Our findings demonstrated that exogenous Gal-9 suppressed dsRNA-induced AHR in an acute exacerbation model of chronic asthma in mice, and suggest that recombinant Gal-9 could be therapeutically effective for preventing acute asthma exacerbation.

Background: Galectin-9 serves opposing roles in the innate and adaptive immune systems. Galectin-9 triggers T-cell immunoglobulin mucin-3 (Tim-3) on T helper type 1 (Th1) cells, thereby terminating Th1 immunity and protecting allografts from host immune attacks. Meanwhile, galectin-9 promotes the maturation of dendritic cells (DCs) that deliver proinflammatory signals. We previously showed that galectin-9 significantly prolongs cardiac allograft survival in mice but failed to induce tolerance. This study aimed at improving the administration protocol to induce allograft tolerance. We examined whether rapamycin can reverse the proinflammatory effects of galectin-9 on DCs and whether rapamycin synergizes with galectin-9 to induce cardiac allograft tolerance., Methods: Monocytes/DCs from cardiac allografts were assessed for Tim-3 expression by flow cytometry. Costimulatory molecules CD80/CD86 were measured on galectin-9/rapamycin-treated bone marrow-derived DCs by flow cytometry. We performed heterotopic cervical cardiac transplantation using BALB/c donors and C57BL/6 recipients and assessed graft survival time. T cells of long-term surviving recipients were immunoassayed for interferon-γ and interleukin-4 secretion., Results: Allograft-infiltrating monocytes/DCs expressed high Tim-3 levels (47.3%±5.6%). Expression of CD80/CD86 was up-regulated on galectin-9-treated bone marrow-derived DCs, which was reversed by rapamycin. Combined treatment with galectin-9 and rapamycin promoted the permanent acceptance of fully mismatched grafts (survival time >180 days; n=6). However, treatment with galectin-9 or rapamycin alone was not sufficient to induce tolerance. Galectin-9/rapamycin-induced tolerance was associated with low donor-specific interferon-γ and interleukin-4 secretion., Conclusions: Rapamycin inhibits proinflammatory effects of galectin-9 on DCs. Combined treatment of galectin-9 and rapamycin promotes allografts tolerance, which is associated with reduced Th1 and Th2 responses.

Human monocytes/macrophages (M/M(Ф)) of the innate immunity sense and respond to microbial products via specific receptor coupling with stimulatory (such as TLR) and inhibitory (such as Tim-3) receptors. Current models imply that Tim-3 expression on M/M(Ø) can deliver negative signaling to TLR-mediated IL-12 expression through trans association with its ligand Galectin-9 (Gal-9) presented by other cells. However, Gal-9 is also expressed within M/M(Ø), and the effect of intracellular Gal-9 on Tim-3 activities and inflammatory responses in the same M/M(Ø) remains unknown. In this study, our data suggest that Tim-3 and IL-12/IL-23 gene transcriptions are regulated by enhanced or silenced Gal-9 expression within monocytes through synergizing with TLR signaling. Additionally, TLR activation facilitates Gal-9/Tim-3 cis association within the same M/M(Ø) to differentially regulate IL-12/IL-23 expressions through STAT-3 phosphorylation. These results reveal a ligand (Gal-9) compartment-dependent regulatory effect on receptor (Tim-3) activities and inflammatory responses via TLR pathways--a novel mechanism underlying cellular responses to external or internal cues.

Galectin-9 (Gal-9) is known for induction of apoptosis in IFN-γ and IL-17 producing T-cells and amelioration of autoimmunity in murine models. On the other hand, Gal-9 induced IFN-γ positive T-cells in a sarcoma mouse model and in food allergy, suggesting that Gal-9 can have diametric effects on T-cell immunity. Here, we aimed to delineate the immunomodulatory effect of Gal-9 on human resting and ex vivo activated peripheral blood lymphocytes. Treatment of resting lymphocytes with low concentrations of Gal-9 (5-30 nM) induced apoptosis in ∼60% of T-cells after 1 day, but activated the surviving T-cells. These viable T-cells started to expand after 4 days with up to 6 cell divisions by day 7 and an associated shift from naïve towards central memory and IFN-γ producing phenotype. In the presence of T-cell activation signals (anti-CD3/IL-2) Gal-9 did not induce T-cell expansion, but shifted the CD4/CD8 balance towards a CD4-dominated T-cell response. Thus, Gal-9 activates resting T-cells in the absence of typical T-cell activating signals and promotes their transition to a TH1/C1 phenotype. In the presence of T-cell activating signals T-cell immunity is directed towards a CD4-driven response by Gal-9. Thus, Gal-9 may specifically enhance reactive immunological memory.

Galectin-9 is a lectin, which has various biological functions such as T-cell differentiation and apoptosis. Multivalency of carbohydrate binding is required for galectin-9 to function. Although galectin-1 (a proto-type galectin) forms an oligomer to obtain its multivalency, galectin-9 (a tandem-repeat-type one) has two carbohydrate recognition domains (CRD) in one polypeptide. However, a single CRD of galectin-9, especially the C-terminal one, exhibited pro-apoptotic activity suggesting oligomer formation capability. In this study, we monitored the nuclear magnetic resonance (NMR) signals of the backbone atoms of the galectin-9 C-terminal CRD (G9CCRD). Protein concentration dependence of the signals suggested that a region (F1-F4 strands) opposite to the ligand-binding site was involved in the self-association of G9CCRD. Site-directed mutagenesis in this region (Leu210, Trp277 and Leu279 to Thr; G9CCRD-3T) inhibited the self-association of G9CCRD, and improved the solubility, whereas it reduced its pro-apoptotic activity towards T cells. The high pro-apoptotic activity of G9CCRD seems to be due to the ability to form an oligomer. In addition, the same substitution in two-CRD-containing galectin-9 (G9Null-3T) also diminished the self-association and improved its solubility, although it hardly reduced the anti-proliferative and pro-apoptotic activities. G9CCRD contributes the self-association of full-length galectin-9 at high protein concentrations.

Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-γ)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-γ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-γ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

Galectin-9 is a tandem-repeat type galectin with two carbohydrate-recognition domains, and it was first identified as an eosinophil chemoattractant and activation factor. Subsequent studies revealed that galectin-9, similar to other galectins, modulates a variety of biological functions including cell aggregation and adhesion, as well as apoptosis of tumor cells. Galectin-9 has recently been shown to have an anti-proliferative effect on cancer cells. Recent studies have uncovered additional mechanisms by which T cell immunoglobulin mucin-3 (Tim-3), a receptor for galectin-9, negatively regulates T cell responses by promoting CD8+ T cell exhaustion and inducing expansion of myeloid-derived suppressor cells. These mechanisms are involved in tumor growth and escape from immunity. In many solid cancers, the loss of galectin-9 expression is closely associated with metastatic progression, and treatment with recombinant galectin-9 prevents metastatic spread in various preclinical cancer models. Here, we review the biology and physiological role of galectin-9, and discuss the therapeutic potential of galectin-9 in cancer as well as relevant patents.

Galectin-9 ameliorates various murine autoimmune disease models by regulating T cells and macrophages, although it is not known what role it may have in B cells. The present experiment shows that galectin-9 ameliorates a variety of clinical symptoms, such as proteinuria, arthritis, and hematocrit in MRL/lpr lupus-prone mice. As previously reported, galectin-9 reduces the frequency of Th1, Th17, and activated CD8(+) T cells. Although anti-dsDNA antibody was increased in MRL/lpr lupus-prone mice, galectin-9 suppressed anti-dsDNA antibody production, at least partly, by decreasing the number of plasma cells. Galectin-9 seemed to decrease the number of plasma cells by inducing plasma cell apoptosis, and not by suppressing BAFF production. Although about 20% of CD19(-/low) CD138(+) plasma cells expressed Tim-3 in MRL/lpr lupus-prone mice, Tim-3 may not be directly involved in the galectin-9-induced apoptosis, because anti-Tim-3 blocking antibody did not block galectin-9-induced apoptosis. This is the first report of plasma cell apoptosis being induced by galectin-9. Collectively, it is likely that galectin-9 attenuates the clinical severity of MRL lupus-prone mice by regulating T cell function and inducing plasma cell apoptosis.

Background: Galectin-9 (Gal-9) is a β-galactoside-binding protein that induces biological reactions, such as cell activation, chemoattraction, and apoptosis. We evaluated the role of Gal-9 in the pathogenesis of interstitial pneumonia (IP)., Methods: Gal-9 levels in the bronchoalveolar lavage fluid (BALF) of patients with IP associated with collagen vascular disease (CVD-IP) and with idiopathic interstitial pneumonias (IIPs), including idiopathic pulmonary fibrosis (IPF) and idiopathic nonspecific interstitial pneumonia (idiopathic NSIP), were estimated by enzyme-linked immunosorbent assay. Gal-9 expression in the lungs of these patients was evaluated using immunohistochemistry. The effect of Gal-9 on the growth and apoptosis of human lung fibroblast cells was assessed in vitro., Results: Gal-9 levels in the BALF were significantly higher in patients with CVD-IP than in patients with IIPs. Gal-9 levels significantly correlated with both the total cell count and the absolute number of lymphocytes in the BALF of patients with IIPs and CVD-IP. Strong reactivity with anti-Gal-9 antibody was observed in the cytoplasm of alveolar macrophages, lymphocytes, and type II pneumocytes in the lungs of patients with IP. Gal-9 expression in those cells was more remarkable in patients with CVD-IP than in patients with IPF and idiopathic NSIP. Gal-9 suppressed the growth of human lung fibroblast cells in a dose- dependent manner. Gal-9 induced apoptosis of human lung fibroblast cells in a dose-dependent manner., Conclusions: Our findings suggest that the expression level of Gal-9 in the lung is increased in patients with CVD-IP and that Gal-9 may have a protective role against pulmonary fibrosis of these patients.

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-β, and upregulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured human CD4(+) T cells, driving conventional CD4(+) T cells into CD25(+) Foxp3(+) Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4(+) T cells into Foxp3(+) Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3(+) Treg-cell development and function during HCV infection., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acute HIV-1 infection is often diagnosed as infectious mononucleosis and the symptoms resolve spontaneously after varying periods of time. After the infection of HIV-1 through the mucosa, the characteristic clinical symptoms and laboratory markers of acute HIV-1 infection appear in each patient through a complicated virus-host interaction. To understand the host responses, we measured two unique proinflammatory cytokines, galectin-9 (Gal-9) and osteopontin (OPN). A β-galactoside-binding mammalian lectin, Gal-9, reduces pro-inflammatory type-1 helper T (Th1) cells and Th17 cells and increases anti-inflammatory regulatory T cells. The plasma level of Gal-9 is known to be associated with HIV-1 viral load in chronic HIV-1 infection. On the contrary, osteopontin induces Th1/Th17 cells and promotes tissue inflammation. OPN is synthesized by variety of cells in the body, and dendritic cells are known to synthesize OPN in HIV-1 infected individuals. It was hypothesized that Gal-9 and/or OPN could be not only immune-modulators but also novel biomarkers of acute HIV-1 infection. We experienced 3 patients with acute HIV-1 and measured the levels of Gal-9 and OPN periodically before and after antiretroviral treatment. The results showed that the plasma levels of Gal-9 were extremely elevated [more than 2,300 pg/ml (normal range < 46 pg/ml)] in all three acute HIV-1 infected individuals and decreased rapidly after treatment. The changes in the OPN levels were less marked. In conclusion, the plasma levels of Gal-9 may be predictive of a severe inflammation status during the acute phase of HIV-1 infection and could be a potential biomarker during acute infection.

Ocular infection with herpes simplex virus 1 (HSV-1) results in a chronic immunoinflamammtory reaction in the cornea, which is primarily orchestrated by CD4(+) T cells. Hence, targeting proinflammatory CD4(+) T cells or increasing the representation of cells that regulate their function is a relevant therapeutic strategy. In this report, we demonstrate that effective therapeutic control can be achieved using a combination of approaches under circumstances where monotherapy is ineffective. We use a convenient and highly effective monoclonal antibody (MAb) approach with MAbT25 to expand cells that express the tumor necrosis factor receptor superfamily member 25 (TNFRSF25). In naïve animals, these are predominantly cells that are Foxp3-positive regulatory T cells. MAbT25 treatment before or at the time of initial HSV infection was an effective means of reducing the severity of subsequent stromal keratitis lesions. However, MAbT25 treatment was not effective if given 6 days after infection since it expanded proinflammatory effector T cells, which also express TNFRSF25. Therefore, the MAbT25 procedure was combined with galectin-9 (Gal-9), an approach that compromises the activity of T cells involved in tissue damage. The combination therapy provided highly effective lesion control over that achieved by treatment with one of them. The beneficial outcome of the combination therapy was attributed to the expansion of the regulatory T cell population that additionally expressed activation markers such as CD103 needed to access inflammatory sites. Additionally, there was a marked reduction of CD4(+) gamma interferon-producing effector T cells responsible for orchestrating the tissue damage. The approach that we describe has potential application to control a wide range of inflammatory diseases, in addition to stromal keratitis, an important cause of human blindness.

T-cell immunoglobulin mucin-3 (Tim-3) is expressed on pathogenic T cells, and its ligand galectin-9 (gal-9) is up-regulated in inflamed tissues. When Tim-3(+) T cells encounter high gal-9 levels, they are deleted. Tim-3 is up-regulated on activated T cells during GVHD. Inhibition of Tim-3/gal-9 binding by infusion of a Tim-3-Ig fusion protein or Tim-3(-/-) donor T cells increased T-cell proliferation and GVHD lethality. When the Tim-3/gal-9 pathway engagement was augmented using gal-9 transgenic recipients, GVHD lethality was slowed. Together, these data indicate a potential for modulating this pathway to reduce disease by increasing Tim-3 or gal-9 engagement. Paradoxically, when Tim-3/gal-9 was inhibited in the absence of donor T-regulatory cells (Tregs), GVHD was inhibited. GVHD reduction was associated with decreased colonic inflammatory cytokines as well as epithelial barrier destruction. CD25-depleted Tim-3(-/-) donor T cells underwent increased activation-induced cell death because of increased IFN-γ production. To our knowledge, these studies are the first to show that although the absence of Tim-3/gal-9 pathway interactions augments systemic GVHD, concurrent donor Treg depletion paradoxically and surprisingly inhibits GVHD. Thus, although donor Tregs typically inhibit GVHD, under some conditions, such Tregs actually may contribute to GVHD by reducing activation-induced T-cell death.

Galectin-9 (Gal-9) has been identified as a Tim-3 ligand (L). The Tim-3-Tim-3L interaction serves as a specific down-regulator of the Th1 immune response. It has been reported that Tim-3 expression is higher in patients with inflammatory disorders such as rheumatoid arthritis compared to controls. In a herpes simplex virus-induced Behcet's disease (BD) mouse model, Tim-3 was expressed in a similarly high level. The expression of Gal-9 in macrophages from BD-like mice was lower than in asymptomatic BD normal mice; therefore, we injected 100 μg of Gal-9 into BD-like mice five times at 3 day intervals and subsequently observed changes in symptoms over 15 days. Gal-9 improved the symptoms of inflammation, decreased the severity score, and increased regulatory T cell expression in treated mice. Moreover, pro-inflammatory cytokine levels were lower in the Gal-9-treated group compared to the control group. Therefore, in the present study, Tim-3-Tim-3L interaction was found to influence inflammatory symptoms in BD-like mice., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Galectin-9 (Gal-9) is a tandem repeat-type member of the galectin family and is a ligand for T-cell immunoglobulin mucin domain 3 (Tim-3), a type-I glycoprotein that is persistently expressed on dysfunctional T cells during chronic infection. Studies in autoimmune diseases and chronic viral infections show that Tim-3 is a regulatory molecule that inhibits Th1 type immune responses. Here we show that soluble Gal-9 interacts with Tim-3 expressed on the surface of activated CD4(+) T cells and renders them less susceptible to HIV-1 infection and replication. The Gal-9/Tim-3 interaction on activated CD4(+) T cells, leads to down-regulation of HIV-1 coreceptors and up-regulation of the cyclin-dependent kinase inhibitor p21 (also known as cip-1 and waf-1). We suggest that higher expression of Tim-3 during chronic infection has evolved to limit persistent immune activation and associated tissue damage. These data demonstrate a novel mechanism for Gal-9/Tim-3 interactions to induce resistance of activated CD4(+) T cells to HIV-1 infection and suggest that Gal-9 may play a role in HIV-1 pathogenesis and could be used as a novel microbicide to prevent HIV-1 infection.

Galectin-9 (Gal-9) ameliorates autoimmune reactions by suppressing Th17 cells while augmenting Foxp3(+) regulatory T cells (Tregs). However, the exact mechanism of Gal-9-mediated immune modulation has been elusive. In a MOG-induced experimental allergic encephalomyelitis model using Gal-9(-/-) mice, we observed exacerbated inflammation and an increase in IL-17-producing Th17 cells balanced by a decrease in Foxp3+ Tregs. During in vitro Th17 skewing using TGF-β1 and IL-6, exogenous Gal-9 suppressed Th17 cell development and expanded Foxp3(+) Tregs from naïve CD4 T cells in an IL-2-dependent manner. Although Gal-9 induced cell death in Tim3-expressing differentiated Th17 cells, Gal-9 suppressed Th17 development in a Tim-3-independent. Benzyl-α-GalNAc (an O-glycan biosynthesis inhibitor), but not swainsonine (a complex-type N-glycan biosynthesis inhibitor) abrogated Gal-9-mediated inhibition of Th17 development indicating that there is a linkage between Gal-9 and an unidentified glycoprotein(s) with O-linked β-galactosides that suppress Th17 development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.