Examining the Impact of Galectin-9 on Latent HIV Transcription.

The ß-galactoside-binding protein Galectin-9 (Gal-9) functions as a double-edged sword during HIV infection. On the one hand, Gal-9 can reactivate HIV latently infected cells, the main barrier to achieving HIV eradication, making them visible to immune clearance. On the other hand, Gal-9 induces latent HIV transcription by activating T cell Receptor (TCR) signaling pathways. These signaling pathways induce undesirable pro-inflammatory responses. While these unwanted responses can be mitigated by rapamycin without impacting Gal-9-mediated latent HIV reactivation, this effect raises the concern that Gal-9 may play a role in the chronic immune activation/inflammation that persists in people living with HIV despite antiretroviral therapy. Together, these data highlight the need to understand the positive and negative impacts of galectin interactions on immunological functions during HIV infection. In this chapter, we describe methods that can be used to investigate the effects of galectins, in particular Gal-9, on latent HIV transcription in vitro and ex vivo.

Evaluation of the Role of Galectins in Parasite Immunity.

Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are ruminant specific galectins, first reported in sheep. Although their roles in parasite immunity are still being elucidated, it appears that they influence protection against parasites. In gastrointestinal infections with the nematode Haemonchus contortus, both galectin-11 and galectin-14 appear to be protective. However, in a chronic infection of liver fluke, Fasciola hepatica, these galectins may aid parasite survival. To unravel the structural, functional, and ligand profile of galectin-11 and galectin-14, recombinant production of these proteins is vital. Here we present the recombinant production of soluble galectin-11 and galectin-14 from domestic sheep for in vitro and structural biology studies. These methods include parasite cultivation and infection, galectin staining of host and parasite tissue, surface staining of parasites with recombinant galectins, pull-down assays to identify endogenous galectin binding proteins, and in vitro assays to monitor the effect of galectins on parasite development.

Methods for Assessing the Effects of Galectins on Leukocyte Trafficking and Clearance.

Numerous protocols exist for investigating leukocyte recruitment and clearance both in vitro and in vivo. Here we describe an in vitro flow chamber assay typically used for studying the mechanisms underpinning leukocyte movement through the endothelium and zymosan-induced peritonitis, an acute in vivo model of inflammation that enables both leukocyte trafficking and clearance to be monitored. Insight is given as to how these models can be used to study the actions of galectins on the inflammatory process.

Method to Study the Role of Galectins in Angiogenesis In Vivo Using the Chick Chorioallantoic Membrane Assay.

Angiogenesis is a complex multi-step process involving various activities of endothelial cells. These activities are influenced in vivo by environmental conditions like interactions with other cell types and the microenvironment. Galectins play a role in several of these interactions and are therefore required for proper execution of in vivo angiogenesis. This chapter describes a method to study galectins during physiologic and pathophysiologic angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay.

Examination of the Role of Galectins and Galectin Inhibitors in Endothelial Cell Biology.

The growth of new blood vessels is a key event in many (patho) physiological processes, including embryogenesis, wound healing, inflammatory diseases, and cancer. Neovascularization requires different, well-coordinated actions of endothelial cells, i.e., the cells lining the luminal side of all blood vessels. Galectins are involved in several of these activities. In this chapter, we describe methods to study galectins in three key functions of endothelial cells during angiogenesis, i.e., endothelial cell migration, endothelial cell sprouting, and endothelial cell network formation.

Impact of Galectin-Receptor Interactions on Liver Pathology During the Erythrocytic Stage of Plasmodium berghei Malaria.

Hepatopathy is frequently observed in patients with severe malaria but its pathogenesis remains unclear. Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses, and exhibit pivotal roles during Plasmodium spp. infection. Here, we analyzed the impact of blockage of galectin-receptor interactions by treatment with alpha (α)-lactose on liver immunopathology during the erythrocytic stage of malaria in mice infected with Plasmodium berghei ANKA (PbANKA). Our results found that compared with PbANKA-infected mice (malarial mice), blockage of galectin-receptor interactions led to decreased host survival rate and increased peripheral blood parasitemia; exacerbated liver pathology, increased numbers of CD68+ macrophages and apoptotic cells, and increased parasite burden in the livers on days 5 and 7 post infection (p.i.) as well as increased mRNA expression levels of galectin-9 (Gal-9) and its receptor, the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), interferon (IFN)α, IFNγ, and the triggering receptor expressed on myeloid cells (TREM)-1 in the livers or spleens of PbANKA-infected mice on day 7 p.i. Observed by transmission electron microscopy, the peritoneal macrophages isolated from malarial mice with α-lactose treatment had more pseudopodia than those from malarial mice. Measured by using quantitative real-time reverse transcription-polymerase chain reaction assay, the mRNA expression levels of Gal-9, IFNα, IFNß, IFNγ, and TREM-1 were increased in the peritoneal macrophages isolated from malarial mice with α-lactose treatment in comparison of those from malarial mice. Furthermore, significant positive correlations existed between the mRNA levels of Gal-9 and Tim-3/IFNγ/TREM-1 in both the livers and the peritoneal macrophages, and between Gal-9 and Tim-3/TREM-1 in the spleens of malarial mice; significant positive correlations existed between the mRNA levels of Gal-9 and IFNγ in the livers and between Gal-9 and IFNα in the peritoneal macrophages from malarial mice treated with α-lactose. Our data suggest a potential role of galectin-receptor interactions in limiting liver inflammatory response and parasite proliferation by down-regulating the expressions of IFNα, IFNγ, and TREM-1 during PbANKA infection.

The role of Tim-3/Galectin-9 pathway in T-cell function and prognosis of patients with human papilloma virus-associated cervical carcinoma.

The interaction between Tim-3 on T cell and its ligand, Galectin-9, negatively regulates cellular immune responses. However, the role of Tim-3/Galectin-9 pathway in the immune evasion of cervical cancer remains unknown. This study is to investigate the expression, function, and regulation of Tim-3/Galectin-9 signaling pathway in human papilloma virus (HPV) positive cervical cancer. Flow cytometry showed that Tim-3 expression on T cell and Galectin-9 expression on monocytes in HPV positive cervical cancer patients were significantly higher compared to cervical intraepithelial neoplasia and benign uterine fibroids Tim-3 + CD4+ Th1 cells and Tim-3 + CD8+ T cells in HPV positive cervical cancer patients were significantly reduced after surgery. Serum TGF-ß and IL-10 levels were positively correlated with Tim-3 + Treg cells, while IFN-γ and IL-2 were negatively correlated with Tim-3 + Th1 cells. Additionally, Tim-3 + CD4+ T cells were positively correlated with Galectin-9 + monocytes. Survival curve analysis showed that Tim-3 + CD4+ T cells were negatively correlated with patient survival, and closely related to FIGO stage, degree of differentiation, and lymph node metastasis of HPV positive cervical cancer. In vitro experiments showed that by blocking the Tim-3/Galectin-9 pathway, the proliferation of T cells and their ability to express IFN-γ, IL-2, perforin, and granzyme B was significantly restored. In conclusion, high levels of Tim-3 and Galectin-9 in HPV positive cervical cancer patients play roles in the progression of disease by promoting Treg cells to inhibit the cytotoxic function of Th1 and CD8+ T cells. Tim-3/Galectin-9 may serve as a new immunotherapy target for patients with HPV positive cervical cancer.

Reprogramming the tumor metastasis cascade by targeting galectin-driven networks.

A sequence of interconnected events known as the metastatic cascade promotes tumor progression by regulating cellular and molecular interactions between tumor, stromal, endothelial, and immune cells both locally and systemically. Recently, a new concept has emerged to better describe this process by defining four attributes that metastatic cells should undergo. Every individual hallmark represents a unique trait of a metastatic cell that impacts directly in the outcome of the metastasis process. These critical features, known as the hallmarks of metastasis, include motility and invasion, modulation of the microenvironment, cell plasticity and colonization. They are hierarchically regulated at different levels by several factors, including galectins, a highly conserved family of ß-galactoside-binding proteins abundantly expressed in tumor microenvironments and sites of metastasis. In this review, we discuss the role of galectins in modulating each hallmark of metastasis, highlighting novel therapeutic opportunities for treating the metastatic disease.

Pronociceptive Roles of Schwann Cell-Derived Galectin-3 in Taxane-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of taxanes such as paclitaxel and docetaxel. Despite the high medical needs, insufficient understanding of the complex mechanism underlying CIPN pathogenesis precludes any endorsed causal therapy to prevent or relieve CIPN. In this study, we report that elevation of plasma galectin-3 level is a pathologic change common to both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. Following multiple intraperitoneal injections of paclitaxel in mice, galectin-3 levels were elevated in Schwann cells within the sciatic nerve but not in other peripheral organs or cells expressing galectin-3. Consistent with this, paclitaxel treatment of primary cultures of rat Schwann cells induced upregulation and secretion of galectin-3. In vitro migration assays revealed that recombinant galectin-3 induced a chemotactic response of the murine macrophage cell line RAW 264.7. In addition, perineural administration of galectin-3 to the sciatic nerve of naive mice mimicked paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. By contrast, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity despite the higher level of plasma galectin-3. Deficiency (Galectin-3 -/- mice) or pharmacologic inhibition of galectin-3 inhibited paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. In conclusion, we propose that Schwann cell-derived galectin-3 plays a pronociceptive role via macrophage infiltration in the pathogenesis of taxane-induced peripheral neuropathy. Therapies targeting this phenomenon, which is common to patients with CIPN and mouse models, represent a novel approach to suppress taxane-related CIPN. SIGNIFICANCE: These findings demonstrate that the elevation of plasma galectin-3 is a CIPN-related pathologic change common to humans and mice, and that targeting galectin-3 is a therapeutic option to delay CIPN progression.

Medawar's PostEra: Galectins Emerged as Key Players During Fetal-Maternal Glycoimmune Adaptation.

Lectin-glycan interactions, in particular those mediated by the galectin family, regulate many processes required for a successful pregnancy. Over the past decades, increasing evidence gathered from in vitro and in vivo experiments indicate that members of the galectin family specifically bind to both intracellular and membrane bound carbohydrate ligands regulating angiogenesis, immune-cell adaptations required to tolerate the fetal semi-allograft and mammalian embryogenesis. Therefore, galectins play important roles in fetal development and placentation contributing to maternal and fetal health. This review discusses the expression and role of galectins during the course of pregnancy, with an emphasis on maternal immune adaptions and galectin-glycan interactions uncovered in the recent years. In addition, we summarize the galectin fingerprints associated with pathological gestation with particular focus on preeclampsia.

Neurobiochemical Cross-talk Between COVID-19 and Alzheimer's Disease.

COVID-19, the global threat to humanity, shares etiological cofactors with multiple diseases including Alzheimer's disease (AD). Understanding the common links between COVID-19 and AD would harness strategizing therapeutic approaches against both. Considering the urgency of formulating COVID-19 medication, its AD association and manifestations have been reviewed here, putting emphasis on memory and learning disruption. COVID-19 and AD share common links with respect to angiotensin-converting enzyme 2 (ACE2) receptors and pro-inflammatory markers such as interleukin-1 (IL-1), IL-6, cytoskeleton-associated protein 4 (CKAP4), galectin-9 (GAL-9 or Gal-9), and APOE4 allele. Common etiological factors and common manifestations described in this review would aid in developing therapeutic strategies for both COVID-19 and AD and thus impact on eradicating the ongoing global threat. Thus, people suffering from COVID-19 or who have come round of it as well as people at risk of developing AD or already suffering from AD, would be benefitted.

Galectin-3 Modulates Macrophage Activation and Contributes Smooth Muscle Cells Apoptosis in Abdominal Aortic Aneurysm Pathogenesis.

Galectin-3 (Gal-3) is a 26-kDa lectin that regulates many aspects of inflammatory cell behavior. We assessed the hypothesis that increased levels of Gal-3 contribute to abdominal aortic aneurysm (AAA) progression by enhancing monocyte chemoattraction through macrophage activation. We analyzed the plasma levels of Gal-3 in 76 patients with AAA (AAA group) and 97 controls (CTL group) as well as in angiotensin II (Ang-II)-infused ApoE knockout mice. Additionally, conditioned media (CM) were used to polarize THP-1 monocyte to M1 macrophages with or without Gal-3 inhibition through small interfering RNA targeted deletion to investigate whether Gal-3 inhibition could attenuate macrophage-induced inflammation and smooth muscle cell (SMC) apoptosis. Our results showed a markedly increased expression of Gal-3 in the plasma and aorta in the AAA patients and experimental mice compared with the CTL group. An in vitro study demonstrated that the M1 cells exhibited increased Gal-3 expression. Gal-3 inhibition markedly decreased the quantity of macrophage-induced inflammatory regulators, including IL-8, TNF-α, and IL-1ß, as well as messenger RNA expression and MMP-9 activity. Moreover, Gal-3-deficient CM weakened SMC apoptosis through Fas activation. These findings prove that Gal-3 may contribute to AAA progression by the activation of inflammatory macrophages, thereby promoting SMC apoptosis.

Oral Administration of Fucoidan Can Exert Anti-Allergic Activity after Allergen Sensitization by Enhancement of Galectin-9 Secretion in Blood.

A previous study revealed that fucoidan inhibited mast cell degranulation through the upregulation of galectin-9 in blood. The purpose of this study is to elucidate its mechanism using ovalbumin (OVA) induced anaphylaxis model mice (BALB/c, Female, 5-week-old) and mast cell line (RBL-2H3 cells). Oral administration of fucoidan after sensitization with OVA/Al(OH)3 inhibited reduction of rectal temperature induced by activation of mast cells. Fucoidan increased galectin-9 mRNA expression only in colonic epithelial cells. These results suggested that fucoidan could suppress the allergic symptoms in sensitized mice by inducing galectin-9 production from colonic epithelial cells. In addition, to check the influence of galectin 9 on the degranulation of mast cells, RBL-2H3 cell lines were treated directly with recombinant galectin-9. As expected, galectin-9 inhibited degranulation of RBL-2H3 cells pre-bound with IgE. Moreover, the residual amounts of IgE on RBL-2H3 cells were decreased by an addition of galectin-9. It was demonstrated that galectin-9 could remove IgE even if IgE was already bound to mast cells and suppress the mast cells degranulation induced by antigen. This study shows that fucoidan might become an effective therapeutic agent for patients already developed type I allergic diseases.

Structural, molecular, functional and immunological characterization of Wuchereria bancrofti-galectin.

Galectins regulate growth and differentiation of immune cells and inflammation through their carbohydrate-binding function in humans, while also play a role in parasite survival. This study focused on the galectin of lymphatic filarial parasite Wuchereria bancrofti (Wb-Gal). The multiple sequence alignment with other galectins showed that the Wb-Gal belonged to galactoside binding lectin family, particularly tandem repeat type galectin-9. A homology model of Wb-Gal was developed in the I-TASser server using high similarity 3D structures with a quality score of 89.5. Molecular docking and dynamics studies revealed that the CCRD and NCRD of Wb-Gal bind with galactose and lactose. Further, Wb-Gal was cloned into the pET28 vector, expressed in E. coli Rosetta strain and purified by affinity chromatography. In the hemagglutination assays, the rWb-Gal bound to lactose, galactose, and glucose. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) using different clinical filarial sera showed that the IgG and IgM response was against Wb-Gal x very high in all filarial clinical groups, whereas the IgA and IgG2 response was minimum to negligible. There was an enhanced response of IgG1 and IgG4 antibodies in Microfilaremics (MF) cases compared to Chronic Pathology (CP) and Endemic Normal (EN) individuals. Interestingly, the IgE response was comparatively higher in EN than MF and CP. These studies show that Wb-Gal is a member of the lectin family of proteins binding to different carbohydrates and may have an important role in the pathophysiology of filarial infection which needs to be investigated in greater detail.

Galectin-1-driven T cell exclusion in the tumor endothelium promotes immunotherapy resistance.

Immune checkpoint inhibitors (ICIs), although promising, have variable benefit in head and neck cancer (HNC). We noted that tumor galectin-1 (Gal1) levels were inversely correlated with treatment response and survival in patients with HNC who were treated with ICIs. Using multiple HNC mouse models, we show that tumor-secreted Gal1 mediates immune evasion by preventing T cell migration into the tumor. Mechanistically, Gal1 reprograms the tumor endothelium to upregulate cell-surface programmed death ligand 1 (PD-L1) and galectin-9. Using genetic and pharmacological approaches, we show that Gal1 blockade increases intratumoral T cell infiltration, leading to a better response to anti-PD1 therapy with or without radiotherapy. Our study reveals the function of Gal1 in transforming the tumor endothelium into an immune-suppressive barrier and that its inhibition synergizes with ICIs.

Pregnancy Galectinology: Insights Into a Complex Network of Glycan Binding Proteins.

Galectins are a phylogenetically conserved family of soluble ß-galactoside binding proteins, consisting of 15 different types, each with a specific function. Galectins contribute to placentation by regulating trophoblast development, migration, and invasion during early pregnancy. In addition, galectins are critical players regulating maternal immune tolerance to the embedded embryo. Recently, the role of galectins in angiogenesis during decidualization and in placenta formation has gained attention. Altered expression of galectins is associated with abnormal pregnancies and infertility. This review focuses on the role of galectins in pregnancy-associated processes and discusses the relevance of galectin-glycan interactions as potential therapeutic targets in pregnancy disorders.

Galectins in prostate and bladder cancer: tumorigenic roles and clinical opportunities.

Advanced prostate and bladder cancer are two outstanding unmet medical needs for urological oncologists. The high prevalence of these tumours, lack of effective biomarkers and limited effective treatment options highlight the importance of basic research in these diseases. Galectins are a family of ß-galactoside-binding proteins that are frequently altered (upregulated or downregulated) in a wide range of tumours and have roles in different stages of tumour development and progression, including immune evasion. In particular, altered expression levels of different members of the galectin family have been reported in prostate and bladder cancers, which, together with the aberrant glycosylation patterns found in tumour cells and the constituent cell types of the tumour microenvironment, can result in malignant transformation and tumour progression. Understanding the roles of galectin family proteins in the development and progression of prostate and bladder cancer could yield key insights to inform the clinical management of these diseases.

Targeting galectin-1 inhibits pancreatic cancer progression by modulating tumor-stroma crosstalk.

Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal tumor types, with extremely low survival rates due to late diagnosis and resistance to standard therapies. A more comprehensive understanding of the complexity of PDA pathobiology, and especially of the role of the tumor microenvironment in disease progression, should pave the way for therapies to improve patient response rates. In this study, we identify galectin-1 (Gal1), a glycan-binding protein that is highly overexpressed in PDA stroma, as a major driver of pancreatic cancer progression. Genetic deletion of Gal1 in a Kras-driven mouse model of PDA (Ela-KrasG12Vp53-/- ) results in a significant increase in survival through mechanisms involving decreased stroma activation, attenuated vascularization, and enhanced T cell infiltration leading to diminished metastasis rates. In a human setting, human pancreatic stellate cells (HPSCs) promote cancer proliferation, migration, and invasion via Gal1-driven pathways. Moreover, in vivo orthotopic coinjection of pancreatic tumor cells with Gal1-depleted HPSCs leads to impaired tumor formation and metastasis in mice. Gene-expression analyses of pancreatic tumor cells exposed to Gal1 reveal modulation of multiple regulatory pathways involved in tumor progression. Thus, Gal1 hierarchically regulates different events implicated in PDA biology including tumor cell proliferation, invasion, angiogenesis, inflammation, and metastasis, highlighting the broad therapeutic potential of Gal1-specific inhibitors, either alone or in combination with other therapeutic modalities.

The immunological effect of Galectin-9/TIM-3 pathway after low dose Mifepristone treatment in mice at 14.5 day of pregnancy.

The abortifacient Mifepristone (RU486) has proven to be a safe, effective, acceptable option for millions of women seeking abortion during the first and second trimester of pregnancy although its precise mechanism of action is not well understood. The main objective of this study was to investigate the impact of low dose Mifepristone administration on placental Galectin-9 (Gal-9) expression, as well as its effect on the cell surface expression of Gal-9, TIM-3 and CD107a molecules by different T and NK cell subsets. A model of Mifepristone-induced immunological changes was established in syngeneic pregnant BALB/c mice. RU486-induced alteration in placental Gal-9 expression was determined by immunohistochemistry. For immunophenotypic analysis, mid-pregnancy decidual lymphocytes and peripheral mononuclear cells were obtained from Mifepristone treated and control mice at the 14.5 day of gestation. TIM-3 and Gal-9 expression by peripheral and decidual immune cells were examined by flow cytometry. Our results revealed a dramatically decreased intracellular Gal-9 expression in the spongiotrophoblast layer of the haemochorial placenta in Mifepristone treated pregnant mice. Although low dose RU486 treatment did not cause considerable change in the phenotypic distribution of decidual and peripheral immune cells, it altered the Gal-9 and TIM-3 expression by different NK and T cell subsets. In addition, the treatment significantly decreased the CD107a expression by decidual TIM-3+ NK cells, but increased its expression by decidual NKT cell compared to the peripheral counterparts. These findings suggest that low dose Mifepristone administration might induce immune alterations in both progesterone dependent and independent way.

Interleukin-10 Directly Inhibits CD8+ T Cell Function by Enhancing N-Glycan Branching to Decrease Antigen Sensitivity.

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity of CD8+ T cells was decreased during chronic infection and that this was directly mediated by IL-10. Mechanistically, we showed that IL-10 induced the expression of Mgat5, a glycosyltransferase that enhances N-glycan branching on surface glycoproteins. Increased N-glycan branching on CD8+ T cells promoted the formation of a galectin 3-mediated membrane lattice, which restricted the interaction of key glycoproteins, ultimately increasing the antigenic threshold required for T cell activation. Our study identified a regulatory loop in which IL-10 directly restricts CD8+ T cell activation and function through modification of cell surface glycosylation allowing the establishment of chronic infection.