Hypothyroidism-associated immunosuppression involves induction of galectin-1-producing regulatory T cells.

Hypothyroidism exerts deleterious effects on immunity, but the precise role of the hypothalamic-pituitary-thyroid (HPT) axis in immunoregulatory and tolerogenic programs is barely understood. Here, we investigated the mechanisms underlying hypothyroid-related immunosuppression by examining the regulatory role of components of the HPT axis. We first analyzed lymphocyte activity in mice overexpressing the TRH gene (Tg-Trh). T cells from Tg-Trh showed increased proliferation than wild-type (WT) euthyroid mice in response to polyclonal activation. The release of Th1 pro-inflammatory cytokines was also increased in Tg-Trh and TSH levels correlated with T-cell proliferation. To gain further mechanistic insights into hypothyroidism-related immunosuppression, we evaluated T-cell subpopulations in lymphoid tissues of hypothyroid and control mice. No differences were observed in CD3/CD19 or CD4/CD8 ratios between these strains. However, the frequency of regulatory T cells (Tregs) was significantly increased in hypothyroid mice, and not in Tg-Trh mice. Accordingly, in vitro Tregs differentiation was more pronounced in naïve T cells isolated from hypothyroid mice. Since Tregs overexpress galectin-1 (Gal-1) and mice lacking this lectin (Lgals1-/- ) show reduced Treg function, we investigated the involvement of this immunoregulatory lectin in the control of Tregs in settings of hypothyroidism. Increased T lymphocyte reactivity and reduced frequency of Tregs were found in hypothyroid Lgals1-/- mice when compared to hypothyroid WT animals. This effect was rescued by the addition of recombinant Gal-1. Finally, increased expression of Gal-1 was found in Tregs purified from hypothyroid WT mice compared with their euthyroid counterpart. Thus, a substantial increase in the frequency and activity of Gal-1-expressing Tregs underlies immunosuppression associated with hypothyroid conditions, with critical implications in immunopathology, metabolic disorders, and cancer.

Control of intestinal inflammation by glycosylation-dependent lectin-driven immunoregulatory circuits.

Diverse immunoregulatory circuits operate to preserve intestinal homeostasis and prevent inflammation. Galectin-1 (Gal1), a ß-galactoside-binding protein, promotes homeostasis by reprogramming innate and adaptive immunity. Here, we identify a glycosylation-dependent "on-off" circuit driven by Gal1 and its glycosylated ligands that controls intestinal immunopathology by targeting activated CD8+ T cells and shaping the cytokine profile. In patients with inflammatory bowel disease (IBD), augmented Gal1 was associated with dysregulated expression of core 2 ß6-N-acetylglucosaminyltransferase 1 (C2GNT1) and α(2,6)-sialyltransferase 1 (ST6GAL1), glycosyltransferases responsible for creating or masking Gal1 ligands. Mice lacking Gal1 exhibited exacerbated colitis and augmented mucosal CD8+ T cell activation in response to 2,4,6-trinitrobenzenesulfonic acid; this phenotype was partially ameliorated by treatment with recombinant Gal1. While C2gnt1-/- mice exhibited aggravated colitis, St6gal1-/- mice showed attenuated inflammation. These effects were associated with intrinsic T cell glycosylation. Thus, Gal1 and its glycosylated ligands act to preserve intestinal homeostasis by recalibrating T cell immunity.

Intracellular immune sensing promotes inflammation via gasdermin D-driven release of a lectin alarmin.

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

Characterization of a neutralizing anti-human galectin-1 monoclonal antibody with angioregulatory and immunomodulatory activities.

Galectins, a family of highly conserved ß-galactoside-binding proteins, control tumor progression by modulating different hallmarks of cancer. Galectin-1 (Gal-1), a proto-type member of this family, plays essential roles in tumor angiogenesis and immunosuppression by cross-linking glycosylated receptors on the surface of endothelial and immune cells. Targeted disruption of Gal-1 suppresses tumor growth by counteracting aberrant angiogenesis and reinforcing antitumor immunity in several experimental settings. Given the multiple therapeutic benefits associated with Gal-1 blockade, several Gal-1 inhibitors, including glycan-based competitors, antagonistic peptides, aptamers and neutralizing monoclonal antibodies, have been designed and evaluated in pre-clinical tumor models. Here we report the biochemical and functional characterization of a newly developed neutralizing anti-human Gal-1 monoclonal antibody (Gal-1-mAb3), which specifically recognizes a unique epitope in Gal-1 protein and exerts both angioregulatory and immunomodulatory activities. Blockade of Gal-1 function using Gal-1-mAb3, might be relevant not only in cancer but also in other pathologic conditions characterized by aberrant angiogenesis and uncontrolled immunosuppression.

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.

Publisher Correction: Multiple concomitant mechanisms contribute to low platelet count in patients with immune thrombocytopenia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glioblastomas exploit truncated O-linked glycans for local and distant immune modulation via the macrophage galactose-type lectin.

Glioblastoma is the most aggressive brain malignancy, for which immunotherapy has failed to prolong survival. Glioblastoma-associated immune infiltrates are dominated by tumor-associated macrophages and microglia (TAMs), which are key mediators of immune suppression and resistance to immunotherapy. We and others demonstrated aberrant expression of glycans in different cancer types. These tumor-associated glycans trigger inhibitory signaling in TAMs through glycan-binding receptors. We investigated the glioblastoma glycocalyx as a tumor-intrinsic immune suppressor. We detected increased expression of both tumor-associated truncated O-linked glycans and their receptor, macrophage galactose-type lectin (MGL), on CD163+ TAMs in glioblastoma patient-derived tumor tissues. In an immunocompetent orthotopic glioma mouse model overexpressing truncated O-linked glycans (MGL ligands), high-dimensional mass cytometry revealed a wide heterogeneity of infiltrating myeloid cells with increased infiltration of PD-L1+ TAMs as well as distant alterations in the bone marrow (BM). Our results demonstrate that glioblastomas exploit cell surface O-linked glycans for local and distant immune modulation.

Multiple concomitant mechanisms contribute to low platelet count in patients with immune thrombocytopenia.

Mechanisms leading to low platelet count in immune thrombocytopenia (ITP) involves both decreased production and increased destruction of platelet. However, the contribution of these pathologic mechanisms to clinical outcome of individual patients is uncertain. Here we evaluated different pathogenic mechanisms including in vitro megakaryopoiesis, platelet/megakaryocyte (MK) desialylation and MK apoptosis, and compared these effects with thrombopoyesis and platelet apoptosis in the same cohort of ITP patients. Normal umbilical cord blood-CD34+ cells, mature MK derived cells or platelets were incubated with plasma from ITP patients. Despite inhibition of thrombopoiesis previously observed, megakaryopoiesis was normal or even increased. Plasma from ITP patients affected the sialylation pattern of control platelets and this effect occurred concomitantly with apoptosis in 35% ITP samples. However, none of these abnormalities were observed in control MKs incubated with ITP plasma. Addition of mononuclear cells as immune effectors did not lead to phosphatidylserine exposure in MK, ruling out an antibody-mediated cytotoxic effect. These results suggest that both desialylation and apoptosis may be relevant mechanisms leading to platelet destruction although, they do not interfere with MK function. Analysis of these thrombocytopenic factors in individual patients showed no specific distribution pattern. However, the presence of circulating antiplatelet autoantibodies was associated with higher incidence of abnormalities. In conclusion, the causes of thrombocytopenia are multifactorial and may occur together, providing a rational basis for the use of combination therapies targeting concomitant ITP mechanisms in patients with refractory disease.

Galectins: Multitask signaling molecules linking fibroblast, endothelial and immune cell programs in the tumor microenvironment.

Tumor cells corrupt surrounding normal cells instructing them to support proliferative, pro-angiogenic and immunosuppressive networks that favor tumorigenesis and metastasis. This dynamic cross-talk is sustained by a range of intracellular signals and extracellular mediators produced by both tumoral and non-tumoral cells. Galectins -whether secreted or intracellularly expressed- play central roles in the tumorigenic process by delivering regulatory signals that contribute to reprogram fibroblasts, endothelial and immune cell programs. Through glycosylation-dependent or independent mechanisms, these endogenous lectins control a variety of cellular events leading to tumor cell proliferation, survival, migration, inflammation, angiogenesis and immune escape. Here we discuss the role of galectin-driven pathways, particularly those activated in non-tumoral stromal cells, in modulating tumor progression.

Immune-Mediated and Hypoxia-Regulated Programs: Accomplices in Resistance to Anti-angiogenic Therapies.

In contrast to mechanisms taking place during resistance to chemotherapies or other targeted therapies, compensatory adaptation to angiogenesis blockade does not imply a mutational alteration of genes encoding drug targets or multidrug resistance mechanisms but instead involves intrinsic or acquired activation of compensatory angiogenic pathways. In this article we highlight hypoxia-regulated and immune-mediated mechanisms that converge in endothelial cell programs and preserve angiogenesis in settings of vascular endothelial growth factor (VEGF) blockade. These mechanisms involve mobilization of myeloid cell populations and activation of cytokine- and chemokine-driven circuits operating during intrinsic and acquired resistance to anti-angiogenic therapies. Particularly, we focus on findings underscoring a role for galectins and glycosylated ligands in promoting resistance to anti-VEGF therapies and discuss possible strategies to overcome or attenuate this compensatory pathway. Finally, we highlight emerging evidence demonstrating the interplay between immunosuppressive and pro-angiogenic programs in the tumor microenvironment (TME) and discuss emerging combinatorial anticancer strategies aimed at simultaneously potentiating antitumor immune responses and counteracting aberrant angiogenesis.

Clinical Relevance of Galectin-1 and Galectin-3 in Rheumatoid Arthritis Patients: Differential Regulation and Correlation With Disease Activity.

Galectins, a family of animal lectins, play central roles in immune system regulation, shaping both innate and adaptive responses in physiological and pathological processes. These include rheumatoid arthritis (RA), a chronic multifactorial autoimmune disease characterized by inflammatory responses that affects both articular and extra-articular tissues. Galectins have been reported to play central roles in RA and its experimental animal models. In this perspective article we present new data highlighting the regulated expression of galectin-1 (Gal-1) and galectin-3 (Gal-3) in sera from RA patients under disease-modifying anti-rheumatic drugs (DMARDs) and/or corticoid treatment in the context of a more comprehensive discussion that summarizes the roles of galectins in joint inflammation. We found that Gal-1 levels markedly increase in sera from RA patients and positively correlate with erythrocyte sedimentation rate (ERS) and disease activity score 28 (DAS-28) parameters. On the other hand, Gal-3 is downregulated in RA patients, but positively correlates with health assessment questionnaire parameter (HAQ). Finally, by generating receiver-operator characteristic (ROC) curves, we found that Gal-1 and Gal-3 serum levels constitute good parameters to discriminate patients with RA from healthy individuals. Our findings uncover a differential regulation of Gal-1 and Gal-3 which might contribute to the anti-inflammatory effects elicited by DMARDs and corticoid treatment in RA patients.

Galectin-1-Driven Tolerogenic Programs Aggravate Yersinia enterocolitica Infection by Repressing Antibacterial Immunity.

Yersinia enterocolitica is an enteropathogenic bacterium that causes gastrointestinal disorders, as well as extraintestinal manifestations. To subvert the host's immune response, Y. enterocolitica uses a type III secretion system consisting of an injectisome and effector proteins, called Yersinia outer proteins (Yops), that modulate activation, signaling, and survival of immune cells. In this article, we show that galectin-1 (Gal-1), an immunoregulatory lectin widely expressed in mucosal tissues, contributes to Y. enterocolitica pathogenicity by undermining protective antibacterial responses. We found higher expression of Gal-1 in the spleen and Peyer's patches of mice infected orogastrically with Y. enterocolitica serotype O:8 compared with noninfected hosts. This effect was prevented when mice were infected with Y. enterocolitica lacking YopP or YopH, two critical effectors involved in bacterial immune evasion. Consistent with a regulatory role for this lectin during Y. enterocolitica pathogenesis, mice lacking Gal-1 showed increased weight and survival, lower bacterial load, and attenuated intestinal pathology compared with wild-type mice. These protective effects involved modulation of NF-κB activation, TNF production, and NO synthesis in mucosal tissue and macrophages, as well as systemic dysregulation of IL-17 and IFN-γ responses. In vivo neutralization of these proinflammatory cytokines impaired bacterial clearance and eliminated host protection conferred by Gal-1 deficiency. Finally, supplementation of recombinant Gal-1 in mice lacking Gal-1 or treatment of wild-type mice with a neutralizing anti-Gal-1 mAb confirmed the immune inhibitory role of this endogenous lectin during Y. enterocolitica infection. Thus, targeting Gal-1-glycan interactions may contribute to reinforce antibacterial responses by reprogramming innate and adaptive immune mechanisms.

Galectins: emerging regulatory checkpoints linking tumor immunity and angiogenesis.

Immune checkpoints, a plethora of inhibitory pathways aimed at maintaining immune cell homeostasis, may be co-opted by cancer cells to evade immune destruction. Therapies targeting immune checkpoints have reached a momentum yielding significant clinical benefits in patients with various malignancies by unleashing anti-tumor immunity. Galectins, a family of glycan-binding proteins, have emerged as novel regulatory checkpoints that promote immune evasive programs by inducing T-cell exhaustion, limiting T-cell survival, favoring expansion of regulatory T cells, de-activating natural killer cells and polarizing myeloid cells toward an immunosuppressive phenotype. Concomitantly, galectins can trigger vascular signaling programs, serving as bifunctional messengers that couple tumor immunity and angiogenesis. Thus, targeting galectin-glycan interactions may halt tumor progression by simultaneously augmenting antitumor immunity and suppressing aberrant angiogenesis.

Melatonin Contributes to the Seasonality of Multiple Sclerosis Relapses.

Seasonal changes in disease activity have been observed in multiple sclerosis, an autoimmune disorder that affects the CNS. These epidemiological observations suggest that environmental factors influence the disease course. Here, we report that melatonin levels, whose production is modulated by seasonal variations in night length, negatively correlate with multiple sclerosis activity in humans. Treatment with melatonin ameliorates disease in an experimental model of multiple sclerosis and directly interferes with the differentiation of human and mouse T cells. Melatonin induces the expression of the repressor transcription factor Nfil3, blocking the differentiation of pathogenic Th17 cells and boosts the generation of protective Tr1 cells via Erk1/2 and the transactivation of the IL-10 promoter by ROR-α. These results suggest that melatonin is another example of how environmental-driven cues can impact T cell differentiation and have implications for autoimmune disorders such as multiple sclerosis.

Re-wiring regulatory cell networks in immunity by galectin-glycan interactions.

Programs that control immune cell homeostasis are orchestrated through the coordinated action of a number of regulatory cell populations, including regulatory T cells, regulatory B cells, myeloid-derived suppressor cells, alternatively-activated macrophages and tolerogenic dendritic cells. These regulatory cell populations can prevent harmful inflammation following completion of protective responses and thwart the development of autoimmune pathology. However, they also have a detrimental role in cancer by favoring escape from immune surveillance. One of the hallmarks of regulatory cells is their remarkable plasticity as they can be positively or negatively modulated by a plethora of cytokines, growth factors and co-stimulatory signals that tailor their differentiation, stability and survival. Here we focus on the emerging roles of galectins, a family of highly conserved glycan-binding proteins in regulating the fate and function of regulatory immune cell populations, both of lymphoid and myeloid origins. Given the broad distribution of circulating and tissue-specific galectins, understanding the relevance of lectin-glycan interactions in shaping regulatory cell compartments will contribute to the design of novel therapeutic strategies aimed at modulating their function in a broad range of immunological disorders.

Study of galectins in tumor immunity: strategies and methods.

During the past decade, a better understanding of the cellular and molecular mechanisms underlying tumor immunity has provided the appropriate framework for the development of therapeutic strategies for cancer immunotherapy. Under this complex scenario, galectins have emerged as promising molecular targets for cancer therapy responsible of creating immunosuppressive microenvironments at sites of tumor growth and metastasis. Galectins, expressed in tumor, stromal, and endothelial cells, contribute to thwart the development of immune responses by favoring the expansion of T regulatory cells and contributing to their immunosuppressive activity, driving the differentiation of tolerogenic dendritic cells, limiting T cell viability, and maintaining T cell anergy. The emerging data promise a future scenario in which the selective blockade of individual members of the galectin family, either alone or in combination with other therapeutic regimens, will contribute to halt tumor progression by counteracting tumor-immune escape. Here we describe a selection of methods used to investigate the role of galectin-1 in tumor-immune escape.

Regulation of galectins by hypoxia and their relevance in angiogenesis: strategies and methods.

Formation of an aberrant and heterogeneous vascular network is a key pathological event in the multistep process of tumor growth and metastasis. Pro-angiogenic factors are synthesized and released from tumor, stromal, endothelial, and myeloid cells in response to hypoxic and immunosuppressive microenvironments which are commonly found during cancer progression. Emerging data indicate key roles for galectins, particularly galectin-1, -3, -8, and -9 in the regulation of angiogenesis in different pathophysiologic settings. Each galectin interacts with a preferred set of glycosylated receptors, triggers different signaling pathway, and promotes sprouting angiogenesis through different mechanisms. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current clinical approaches. Here we describe selected strategies and methods used to study the galectin-1 regulation by hypoxia and its role in blood vessel formation.

Integration of lectin-glycan recognition systems and immune cell networks in CNS inflammation.

Multiple sclerosis (MS) is a progressive degenerative disorder of the central nervous system (CNS), characterized by inflammation, demyelination and axonal loss. While the majority of MS patients experience relapsing-remitting symptoms followed by a secondary progressive phase, about 10-15% patients exhibit a primary progressive disease involving continuous progression from its onset. Here we review the role of lectin-glycan recognition systems, including those concerning siglecs, C-type lectins and galectins in the pathogenesis of MS and experimental autoimmune encephalomyelitis. Particularly, we will focus on the role of galectins in the fate of T cells, dendritic cells and CNS cell populations. Understanding the regulatory circuits governed by lectin-glycan interactions and their association with disease-associated cytokine networks will contribute to develop novel therapeutic strategies in MS.

Glycosylation-dependent lectin-receptor interactions preserve angiogenesis in anti-VEGF refractory tumors.

The clinical benefit conferred by vascular endothelial growth factors (VEGF)-targeted therapies is variable, and tumors from treated patients eventually reinitiate growth. Here, we identify a glycosylation-dependent pathway that compensates for the absence of cognate ligand and preserves angiogenesis in response to VEGF blockade. Remodeling of the endothelial cell (EC) surface glycome selectively regulated binding of galectin-1 (Gal1), which upon recognition of complex N-glycans on VEGFR2, activated VEGF-like signaling. Vessels within anti-VEGF-sensitive tumors exhibited high levels of α2-6-linked sialic acid, which prevented Gal1 binding. In contrast, anti-VEGF refractory tumors secreted increased Gal1 and their associated vasculature displayed glycosylation patterns that facilitated Gal1-EC interactions. Interruption of ß1-6GlcNAc branching in ECs or silencing of tumor-derived Gal1 converted refractory into anti-VEGF-sensitive tumors, whereas elimination of α2-6-linked sialic acid conferred resistance to anti-VEGF. Disruption of the Gal1-N-glycan axis promoted vascular remodeling, immune cell influx and tumor growth inhibition. Thus, targeting glycosylation-dependent lectin-receptor interactions may increase the efficacy of anti-VEGF treatment.

Structural basis of redox-dependent modulation of galectin-1 dynamics and function.

Galectin-1 (Gal-1), a member of a family of multifunctional lectins, plays key roles in diverse biological processes including cell signaling, immunomodulation, neuroprotection and angiogenesis. The presence of an unusual number of six cysteine residues within Gal-1 sequence prompted a detailed analysis of the impact of the redox environment on the functional activity of this lectin. We examined the role of each cysteine residue in the structure and function of Gal-1 using both experimental and computational approaches. Our results show that: (i) only three cysteine residues present in each carbohydrate recognition domain (CRD) (Cys2, Cys16 and Cys88) were important in protein oxidation, (ii) oxidation promoted the formation of the Cys16-Cys88 disulfide bond, as well as multimers through Cys2, (iii) the oxidized protein did not bind to lactose, probably due to poor interactions with Arg48 and Glu71, (iv) in vitro oxidation by air was completely reversible and (v) oxidation by hydrogen peroxide was relatively slow (1.7 ± 0.2 M(-1) s(-1) at pH 7.4 and 25°C). Finally, an analysis of key cysteines in other human galectins is also provided in order to predict their behaviour in response to redox variations. Collectively, our data provide new insights into the structural basis of Gal-1 redox regulation with critical implications in physiology and pathology.