Role of inflammasome activation in tumor immunity triggered by immune checkpoint blockers.

Immune checkpoint blockers improve the overall survival of a limited number of patients among different cancers. Identifying pathways that influence the immunological and clinical response to treatment is critical to improve the therapeutic efficacy and predict clinical responses. Recently, a key role has been assigned to innate immune mechanisms in checkpoint blockade-driven anti-tumor responses. However, inflammatory pathways can both improve and impair anti-tumor immunity. In this review, we discuss how different inflammatory pathways, particularly inflammasome activation, can influence the clinical outcome of immune checkpoint blockers. Inflammasome activation may reinforce anti-tumor immunity by boosting CD8+ T cell priming as well as by enhancing T helper type 17 (Th17) responses. In particular, we focus on the modulation of the cation channel transmembrane protein 176B (TMEM176B) and the ectonucleotidase CD39 as potential targets to unleash inflammasome activation leading to reinforced anti-tumor immunity and improved efficacy of immune checkpoint blockers. Future studies should be aimed at investigating the mechanisms and cell subsets involved in inflammasome-driven anti-tumor responses.

The Role of Galectin-3: From Oligodendroglial Differentiation and Myelination to Demyelination and Remyelination Processes in a Cuprizone-Induced Demyelination Model.

The aim of this work was to combine our previously published results with our new data to show how galectin-3 (Gal-3) controls myelin integrity and function, promotes oligodendroglial cell differentiation, and regulates microglial responses to limit cuprizone- (CPZ)-induced demyelination and foster remyelination. In this study, 8-week-old Gal-3-deficient (Lgals3 -/-) and wild type (WT) mice were fed a diet containing 0.2 % CPZ w/w for 6 weeks, after which CPZ was withdrawn in order to allow remyelination. Our results show that remyelination was less efficient in Lgals3 -/- than in WT mice. Electron microscopic images from remyelinated sections in Lgals3 -/- mice revealed collapsed axons with a defective myelin wrap, while remyelinated WT mice had normal axons without relevant myelin wrap disruption. MMP-3 expression increased during remyelination in WT but not in Lgals3 -/- mice. The number of CD45+, TNFα+ and TREM-2b+ cells decreased only in WT mice only, with no alterations in Lgals3 -/- mice during demyelination and remyelination. Therefore, Gal-3 influences remyelination by mechanisms involving the tuning of microglial cells, modulation of MMP activity, and changes in myelin architecture.

The thyroid hormone triiodothyronine reinvigorates dendritic cells and potentiates anti-tumor immunity.

Dendritic cell (DC) cancer vaccines have shown limited clinical benefit. Thus, the identification of signals and molecular pathways that potentiate the immunogenicity of DCs has become a major challenge in cancer research. Our studies demonstrate that triiodothyronine endows DCs with enhanced ability to stimulate cytotoxic T-cell responses with implications in DC-based immunotherapy.

The thyroid status reprograms T cell lymphoma growth and modulates immune cell frequencies.

UNLABELLED: In spite of considerable evidence on the regulation of immunity by thyroid hormones, the impact of the thyroid status in tumor immunity is poorly understood. Here, we evaluated the antitumor immune responses evoked in mice with different thyroid status (euthyroid, hyperthyroid, and hypothyroid) that developed solid tumors or metastases after inoculation of syngeneic T lymphoma cells. Hyperthyroid mice showed increased tumor growth along with increased expression of cell cycle regulators compared to hypothyroid and control tumor-bearing mice. However, hypothyroid mice showed a higher frequency of metastases than the other groups. Hyperthyroid mice bearing tumors displayed a lower number of tumor-infiltrating T lymphocytes, lower percentage of functional IFN-γ-producing CD8(+) T cells, and higher percentage of CD19(+) B cells than euthyroid tumor-bearing mice. However, no differences were found in the distribution of lymphocyte subpopulations in tumor-draining lymph nodes (TDLNs) or spleens among different experimental groups. Interestingly, hypothyroid TDLN showed an increased percentage of regulatory T (Treg) cells, while hyperthyroid mice displayed increased number and activity of splenic NK cells, which frequency declined in spleens from hypothyroid mice. Moreover, a decreased number of splenic myeloid-derived suppressor cells (MDSCs) were found in tumor-bearing hyperthyroid mice as compared to hypothyroid or euthyroid mice. Additionally, hyperthyroid mice showed increased cytotoxic activity, which declined in hypothyroid mice. Thus, low levels of intratumoral cytotoxic activity would favor tumor local growth in hyperthyroid mice, while regional and systemic antitumor response may contribute to tumor dissemination in hypothyroid animals. Our results highlight the importance of monitoring the thyroid status in patients with T cell lymphomas. KEY MESSAGES: T cell lymphoma phenotype is paradoxically influenced by thyroid status. Hyperthyroidism favors tumor growth and hypothyroidism rises tumor dissemination. Thyroid status affects the distribution of immune cell types in the tumor milieu. Thyroid status also modifies the nature of local and systemic immune responses.

Galectins: major signaling modulators inside and outside the cell.

Galectins control cell behavior by acting on different signaling pathways. Most of the biological activities ascribed to these molecules rely upon recognition of extracellular glycoconjugates and establishment of multivalente interactions, which trigger adaptive biological responses. However, galectins are also detected within the cell in different compartments, where their regulatory functions still remain poorly understood. A deeper understanding of the entire galectin signalosome and its impact in cell behavior is therefore essential in order to delineate new strategies to specifically manipulate both galectin expression and function. This review summarizes our current knowledge of the signaling pathways activated by galectins, their glycan dependence and the cellular compartment where they become activated and are biologically relevant.

Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury.

Following spinal cord injury (SCI), semaphorin 3A (Sema3A) prevents axonal regeneration through binding to the neuropilin-1 (NRP-1)/PlexinA4 receptor complex. Here, we show that galectin-1 (Gal-1), an endogenous glycan-binding protein, selectively bound to the NRP-1/PlexinA4 receptor complex in injured neurons through a glycan-dependent mechanism, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. Although both Gal-1 and its monomeric variant contribute to de-activation of microglia, only high concentrations of wild-type Gal-1 (which co-exists in a monomer-dimer equilibrium) bind to the NRP-1/PlexinA4 receptor complex and promote axonal regeneration. Our results show that Gal-1, mainly in its dimeric form, promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A binding to NRP-1/PlexinA4 complex, supporting the use of this lectin for the treatment of SCI patients.

Galectin-3 controls the response of microglial cells to limit cuprizone-induced demyelination.

Galectin-3 (Gal-3) is a ß-galactoside-binding lectin that plays an important role in inflammatory and neurodegenerative diseases. Cuprizone (CPZ)-induced demyelination is characterized by the loss of mature oligodendrocytes (OLG) by apoptosis, myelin sheath degeneration and recruitment of microglia and astrocytes to the lesioned area. We compared CPZ-induced demyelination of 8-week-old Lgals3(-/-) vs WT mice. Lgals3(-/-) mice displayed a similar susceptibility to CPZ-induced demyelination up to the fifth week, as evaluated by MBP immunostaining and electronic microscopy. However, OLG progenitors (OPC) generated in CPZ-treated Lgals3(-/-) mice showed diminished arborization, suggesting decreased ability of these cells to differentiate. Surprisingly, while WT mice experienced spontaneous remyelination in the fifth week of CPZ treatment-even though the CPZ diet was maintained up to sixth week-Lgals3(-/-) mice lacked this capacity and suffered continuous demyelination up to the sixth week, accompanied by pronounced astroglial activation. Moreover, after 2weeks of CPZ treatment, WT and Lgals3(-/-) mice showed lower innate anxiety as compared with respective naive mice, but only CPZ-treated Lgals3(-/-) mice showed decreased locomotor activity and exhibited spatial working memory impairment. Expression of Gal-3 increased during CPZ-induced demyelination in microglia but not in astrocytes. While CPZ-treated WT mice displayed heightened microglial activation associated with ED1 expression and pronounced upregulation of the phagocytic receptor TREM-2b, this effect was not observed in CPZ-treated Lgals3(-/-) mice which, in spite of showing an increased number of microglia, these cells evidenced caspase-3 activation. Our results indicate that Gal-3 is expressed in microglial cells to modulate their phenotype, facilitating the onset of remyelination and OLG differentiation.

Glycobiology of cell death: when glycans and lectins govern cell fate.

Although one typically thinks of carbohydrates as associated with cell growth and viability, glycosylation also has an integral role in many processes leading to cell death. Glycans, either alone or complexed with glycan-binding proteins, can deliver intracellular signals or control extracellular processes that promote initiation, execution and resolution of cell death programs. Herein, we review the role of glycans and glycan-binding proteins as essential components of the cell death machinery during physiologic and pathologic settings.

Mast cells rescue implantation defects caused by c-kit deficiency.

Various physiologically relevant processes are regulated by the interaction of the receptor tyrosine kinase (c-Kit) and its ligand stem cell factor (SCF), with SCF known to be the most important growth factor for mast cells (MCs). In spite of their traditional role in allergic disorders and innate immunity, MCs have lately emerged as versatile modulators of a variety of physiologic and pathologic processes. Here we show that MCs are critical for pregnancy success. Uterine MCs presented a unique phenotype, accumulated during receptivity and expanded upon pregnancy establishment. Kit(W-sh/W-sh) mice, whose MC deficiency is based on restricted c-Kit gene expression, exhibited severely impaired implantation, which could be completely rescued by systemic or local transfer of wild-type bone marrow-derived MCs. Transferred wild-type MCs favored normal implantation, induced optimal spiral artery remodeling and promoted the expression of MC proteases, transforming growth factor-ß and connective tissue growth factor. MCs contributed to trophoblast survival, placentation and fetal growth through secretion of the glycan-binding protein galectin-1. Our data unveil unrecognized roles for MCs at the fetomaternal interface with critical implications in reproductive medicine.

Endogenous galectin-3 controls experimental malaria in a species-specific manner.

Galectins are evolutionarily conserved glycan-binding proteins with pleiotropic roles in innate and adaptive immune responses. Galectin-3 has been implicated in several immunological processes as well as in pathogen recognition through specific binding to glycosylated receptors on the surface of host cells or microorganisms. In spite of considerable evidence supporting a role for galectin-3 in host-pathogen interactions, the relevance of this lectin in the regulation of the host defence mechanisms in vivo is poorly understood. In this study, we analysed the impact of galectin-3 deficiency during infection with three distinct species of rodent malaria parasites, Plasmodium yoelii 17XNL, Plasmodium berghei ANKA and Plasmodium chabaudi AS. We found that galectin-3 deficiency showed a marginal effect on the course of parasitaemia during P. chabaudi infection, but did not alter the course of parasitaemia during P. berghei infection. However, lack of galectin-3 significantly reduced P. yoelii parasitaemia. This reduced parasitaemia in Lgals3(-/-) mice was consistent with higher titres of anti-P. yoelii MSP1(19) IgG2b isotype antibodies when compared with their wild-type counterparts. Our results reflect the complexity and singularity of host-pathogen interactions, indicating a species-specific role of endogenous galectin-3 in the control of parasite infections and the modulation of antibody responses.

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine.

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1(-/-)) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1(-/-) mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.

Galectin-3 drives oligodendrocyte differentiation to control myelin integrity and function.

Galectins control critical pathophysiological processes, including the progression and resolution of central nervous system (CNS) inflammation. In spite of considerable progress in dissecting their role within lymphoid organs, their functions within the inflamed CNS remain elusive. Here, we investigated the role of galectin-glycan interactions in the control of oligodendrocyte (OLG) differentiation, myelin integrity and function. Both galectin-1 and -3 were abundant in astrocytes and microglia. Although galectin-1 was abundant in immature but not in differentiated OLGs, galectin-3 was upregulated during OLG differentiation. Biochemical analysis revealed increased activity of metalloproteinases responsible for cleaving galectin-3 during OLG differentiation and modulating its biological activity. Exposure to galectin-3 promoted OLG differentiation in a dose- and carbohydrate-dependent fashion consistent with the 'glycosylation signature' of immature versus differentiated OLG. Accordingly, conditioned media from galectin-3-expressing, but not galectin-3-deficient (Lgals3(-/-)) microglia, successfully promoted OLG differentiation. Supporting these findings, morphometric analysis showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio in the corpus callosum and striatum of Lgals3(-/-) compared with wild-type (WT) mice. Moreover, the myelin structure was loosely wrapped around the axons and less smooth in Lgals3(-/-) mice versus WT mice. Behavior analysis revealed decreased anxiety in Lgals3(-/-) mice similar to that observed during early demyelination induced by cuprizone intoxication. Finally, commitment toward the oligodendroglial fate was favored in neurospheres isolated from WT but not Lgals3(-/-) mice. Hence, glial-derived galectin-3, but not galectin-1, promotes OLG differentiation, thus contributing to myelin integrity and function with critical implications in the recovery of inflammatory demyelinating disorders.

Meat-specific IgG and IgA antibodies coexist with IgE antibodies in sera from allergic patients: clinical association and modulation by exclusion diet.

IgE-mediated responses play a pivotal role in allergic patients with food intolerance. However, the association of food-specific IgG and IgA antibodies with the clinical outcome of allergic patients is still a matter of controversy. In this study we investigate whether beef-specific IgG and IgA antibodies may coexist with beef-specific IgE antibodies in food-allergic patients and examined their clinical relevance in different allergic settings. Beef-specific IgE, IgG and IgA antibodies were determined by solid-phase enzymoimmunoassay (ELISA) in a population of allergic patients (N=125) classified into patients with asthma, skin disease or gastrointestinal disorders, as well as in control subjects (N=80). IgE antibodies specific for citric fruits, tomato, cows milk, chickens egg and wheat were also determined. Beef was the predominant allergenic food in the whole population, not only for IgE (57.6 percent; P less than 0.001), but also for IgG and IgA isotypes (53.6 percent and 34.0 percent, respectively, P less than 0.001). Beef-specific IgE, IgG and IgA antibodies increased significantly in sera from patients with asthma, gastrointestinal disorders and skin allergy compared to sera from control subjects (P less than 0.001). Remarkably, IgG and IgA isotypes were significantly detected, even in the absence of IgE, in the three allergic conditions. All allergic patients, including those showing only IgG and IgA antibodies, significantly ameliorated their symptoms, and their levels of beef-specific antibodies were considerably reduced in response to a cow meat exclusion diet. While patients with gastrointestinal or skin allergic diseases were capable of tolerating beef following an established period of diet exclusion, asthmatic patients experienced a relapse of symptoms and showed a considerable increase in IgE, IgG and IgA-specific antibodies when re-challenged with a beef-enriched diet. Thus, beef-specific IgG and IgA antibodies coexist with IgE antibodies in sera from allergic patients and are significantly associated with the clinical course of allergic disorders, particularly asthma.

Duodenal intraepithelial lymphocytes of children with cow milk allergy preferentially bind the glycan-binding protein galectin-3.

A breakdown in intestinal homeostasis results in inflammatory bowel diseases including coeliac disease and allergy. Galectins, evolutionarily conserved beta-galactoside-binding proteins, can modulate immune-epithelial cell interactions by influencing immune cell fate and cytokine secretion. In this study we investigated the glycosylation signature, as well as the regulated expression of galectin-1 and -3 in human duodenal samples of allergic and non-allergic children. Whereas galectin-1 was predominantly localized in the epithelial compartment (epithelial cells and intraepithelial lymphocytes) and the underlying lamina propria (T cells, macrophages and plasma cells), galectin-3 was mainly expressed by crypt epithelial cells and macrophages in the lamina propria. Remarkably, expression of these galectins was not significantly altered in allergic versus non-allergic patients. Investigation of the glycophenotype of the duodenal inflammatory microenvironment revealed substantial alpha2-6-linked sialic acid bound to galactose in lamina propria plasma cells, macrophages and intraepithelial lymphocytes and significant levels of asialo core 1 O-glycans in CD68+ macrophages and enterocytes. Galectin-1 preferentially bound to neutrophils, plasma cells and enterocytes, while galectin-3 binding sites were mainly distributed on macrophages and intraepithelial lymphocytes. Notably, galectin-3, but not galectin-1 binding, was substantially increased in intraepithelial gut lymphocytes of allergic patients compared to non-allergic subjects, suggesting a potential role of galectin-3-glycan interactions in shaping epithelial-immune cell connections during allergic inflammatory processes.

An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer.

Inflammation is a critical process for eliminating pathogens, but can lead to serious deleterious effects if left unchecked. Identifying the endogenous factors that control immune tolerance and inflammation is a key goal in the field of immunology. Galectins, a family of endogenous lectins with affinity for beta-galactoside-containing oligosaccharides, are expressed by several cells of the immune system and tissue-resident stromal cells. According to their architecture, this family of glycan-binding proteins is classified in those containing one-carbohydrate-recognition domain (CRD) (proto-type), those containing two-CRD joined by a linker non-lectin domain (tandem-repeat) and those that have one-CRD attached to an N-terminal peptide (chimera-type). Accumulating evidence indicates that galectins play critical regulatory roles in immune cell response and homeostasis. In this review, we summarize recent developments in our understanding of the galectins' roles within different immune cell compartments, and in the broader context of the inflammatory microenvironments. In particular we illustrate the immunoregulatory role of three representative members of each galectin subfamily: galectin-1, -3 and -9. This body of knowledge, documenting the coming of age of galectins as potential immunosuppressive agents or targets for anti-inflammatory drugs, represents a sound basis to further explore their potential as novel therapies for autoimmune diseases, chronic inflammation and cancer.

Galectins: matricellular glycan-binding proteins linking cell adhesion, migration, and survival.

Galectins are a taxonomically widespread family of glycan-binding proteins, defined by at least one conserved carbohydrate-recognition domain with a canonical amino acid sequence and affinity for beta-galactosides. Because of their anti-adhesive as well as pro-adhesive extracellular functions, galectins appear to be a novel class of adhesion-modulating proteins collectively known as matricellular proteins (which include thrombospondin, SPARC, tenascin, hevin, and disintegrins). Accordingly, galectins can display de-adhesive effects when presented as soluble proteins to cells in a strong adhesive state. In this context, the de-adhesive properties of galectins should be considered as physiologically relevant as the proadhesive effects of these glycan-binding proteins. This article focuses on the roles of mammalian galectins in cell adhesion, spreading, and migration, and the crossregulation of these functions. Although careful attention should be paid when examining individual galectin functions due to overlapping distributions, these intriguing glycan-binding proteins offer promising possibilities for the treatment and intervention of a wide variety of pathological processes, including cancer, inflammation, and autoimmunity.

The coming of age of galectins as immunomodulatory agents: impact of these carbohydrate binding proteins in T cell physiology and chronic inflammatory disorders.

Immune cell homoeostasis is attributed to multiple distinct safety valves that are interconnected and intervene at defined checkpoints of the life cycle of immunocytes to guarantee clonal expansion and functional inactivation of self-reactive potentially autoaggressive lymphocytes. Galectins, animal lectins defined by shared consensus amino acid sequence and affinity for beta-galactose containing oligosaccharides, are found on various cells of the immune system, and their expression is associated with the differentiation and activation status of these cells. Over the past few years, galectins have been implicated in the regulation of many aspects of T cell physiology such as cell activation, differentiation, and apoptosis. In addition, a growing body of experimental evidence indicates that galectins may play critical roles in the modulation of chronic inflammatory disorders, autoimmunity, and cancer. Given the increased interest of immunologists in this field, the growing body of information raised during the past few years and the potential use of galectins as novel anti-inflammatory agents or targets for immunosuppressive drugs, we will summarise recent advances on the role of galectins in different aspects of T cell physiology and their impact in the development and/or resolution of chronic inflammatory disorders, autoimmunity, and cancer.