Targeting a novel apoptotic pathway in human disease.

Apoptotic pathways have always been regarded as a key-player in preserving tissue and organ homeostasis. Excessive activation or resistance to activation of cell death signaling may indeed be responsible for several mechanisms of disease, including malignancy and chronic degenerative diseases. Therefore, targeting apoptotic factors gained more and more attention in the scientific community and novel strategies emerged aimed at selectively blocking or stimulating cell death signaling. This is also the case for the TMEM219 death receptor, which is activated by a circulating ligand, the Insulin-like growth factor binding protein 3 (IGFBP3) and induces a caspase-8-dependent apoptosis of the target cells. Interestingly, stimulation of the IGFBP3/TMEM219 axis exerts an anti-proliferative effect, while blockade of the TMEM219 deleterious signal protects TMEM219-expressing cells of the endocrine pancreas, lung, and intestine from damage and death. Here, we summarize the most updated reports on the role of the IGFBP3/TMEM219 apoptotic axis in disease conditions, including intestinal disorders and diabetes, and we describe the advancements in designing and testing novel TMEM219-based targeting approaches in emerging potential clinical applications.

Broadening horizons in mechanisms, management, and treatment of diabetic kidney disease.

Diabetic kidney disease (DKD) is the first cause of end-stage kidney disease in patients with diabetes and its prevalence is increasing worldwide. It encompasses histological alterations that mainly affect the glomerular filtration unit, which include thickening of the basement membrane, mesangial cell proliferation, endothelial alteration, and podocyte injury. These morphological abnormalities further result in a persistent increase of urinary albumin-to-creatinine ratio and in a reduction of the estimated glomerular filtration rate. Several molecular and cellular mechanisms have been recognized, up to date, as major players in mediating such clinical and histological features and many more are being under investigation. This review summarizes the most recent advances in understanding cell death mechanisms, intracellular signaling pathways and molecular effectors that play a role in the onset and progression of diabetic kidney damage. Some of those molecular and cellular mechanisms have been already successfully targeted in preclinical models of DKD and, in some cases, strategies have been tested in clinical trials. Finally, this report sheds light on the relevance of novel pathways that may become therapeutic targets for future applications in DKD.

eATP and autoimmune diabetes.

PURPOSE OF REVIEW: The purine nucleotide adenosine triphosphate (ATP) is released into extracellular spaces as extracellular ATP (eATP) as a consequence of cell injury or death and activates the purinergic receptors. Once released, eATP may facilitate T-lymphocyte activation and differentiation. The purpose of this review is to elucidate the role of ATP-mediated signaling in the immunological events related to type 1 diabetes (T1D). RECENT FINDINGS: T lymphocytes mediate immune response during the onset of T1D and promote pancreatic islet or whole pancreas rejection in transplantation. Recent data suggest a potential role for eATP in early steps of T1D onset and of allograft rejection. In different preclinical experimental models and clinical trials, several drugs targeting purinergic signaling have been employed to abrogate lymphocyte activation and differentiation, thus representing an achievable treatment to prevent/revert T1D or to induce long-term islet allograft function. SUMMARY: In preclinical and clinical settings, eATP-signaling inhibition induces immune tolerance in autoimmune disease and in allotransplantation. In this view, the purinergic system may represent a novel therapeutic target for auto- and allo-immunity.

Autoantibodies against the glial glutamate transporter GLT1/EAAT2 in Type 1 diabetes mellitus-Clues to novel immunological and non-immunological therapies.

Islet cell surface autoantibodies were previously found in subjects with type 1 diabetes mellitus (T1DM), but their target antigens and pathogenic mechanisms remain elusive. The glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2) is expressed on the membrane of pancreatic ß-cells and physiologically controls extracellular glutamate concentrations thus preventing glutamate-induced ß-cell death. We hypothesized that GLT1 could be an immunological target in T1DM and that autoantibodies against GLT1 could be pathogenic. Immunoprecipitation and ELISA experiments showed that sera from T1DM subjects recognized GLT1 expressed in brain, pancreatic islets, and GLT1-transfected COS7-cell extracts. We validated these findings in two cohorts of T1DM patients by quantitative immunofluorescence assays. Analysis of the combined data sets indicated the presence of autoantibodies against GLT1 in 32 of the 87 (37%) T1DM subjects and in none of healthy controls (n = 64) (p < 0.0001). Exposure of pancreatic ßTC3 cells and human islets to purified IgGs from anti-GLT1 positive sera supplemented with complement resulted in plasma membrane ruffling, cell lysis and death. The cytotoxic effect was prevented when sera were depleted from IgGs. Furthermore, in the absence of complement, 6 out of 16 (37%) anti-GLT1 positive sera markedly reduced GLT1 transport activity in ßTC3 cells by inducing GLT1 internalization, also resulting in ß-cell death. In conclusion, we provide evidence that GLT1 is a novel T1DM autoantigen and that anti-GLT1 autoantibodies cause ß-cell death through complement-dependent and independent mechanisms. GLT1 seems an attractive novel therapeutic target for the prevention of ß-cell death in individuals with diabetes and prediabetes.

The anti-inflammatory and immunological properties of GLP-1 Receptor Agonists.

In the last few years, a great interest has emerged in investigating the pleiotropic effects of Glucagon Like Peptide-1 Receptor Agonists (GLP-1RAs). While GLP-1RAs ability to lower plasma glucose and to induce weight loss has allowed them to be approved for the treatment of diabetes and obesity, consistent evidences from in vitro studies and preclinical models suggested that GLP-1RAs have anti-inflammatory properties and that may modulate the immune-system. Notably, such anti-inflammatory effects target different pathways in different tissues, underling the broad spectrum of GLP-1RAs actions. This review examines some of the currently proposed molecular mechanisms of GLP-1RAs actions and explores their potential benefits in reducing inflammatory responses, which may well suggest a future therapeutic use of GLP-1RAs in new indications.

miR-21 antagonism reprograms macrophage metabolism and abrogates chronic allograft vasculopathy.

Despite much progress in improving graft outcome during cardiac transplantation, chronic allograft vasculopathy (CAV) remains an impediment to long-term graft survival. MicroRNAs (miRNAs) emerged as regulators of the immune response. Here, we aimed to examine the miRNA network involved in CAV. miRNA profiling of heart samples obtained from a murine model of CAV and from cardiac-transplanted patients with CAV demonstrated that miR-21 was most significantly expressed and was primarily localized to macrophages. Interestingly, macrophage depletion with clodronate did not significantly prolong allograft survival in mice, while conditional deletion of miR-21 in macrophages or the use of a specific miR-21 antagomir resulted in indefinite cardiac allograft survival and abrogated CAV. The immunophenotype, secretome, ability to phagocytose, migration, and antigen presentation of macrophages were unaffected by miR-21 targeting, while macrophage metabolism was reprogrammed, with a shift toward oxidative phosphorylation in naïve macrophages and with an inhibition of glycolysis in pro-inflammatory macrophages. The aforementioned effects resulted in an increase in M2-like macrophages, which could be reverted by the addition of L-arginine. RNA-seq analysis confirmed alterations in arginase-associated pathways associated with miR-21 antagonism. In conclusion, miR-21 is overexpressed in murine and human CAV, and its targeting delays CAV onset by reprogramming macrophages metabolism.

The Impact of Diabetes Mellitus on Cardiovascular Risk Onset in Children and Adolescents.

The prevalence of diabetes mellitus is rising among children and adolescents worldwide. Cardiovascular diseases are the main cause of morbidity and mortality in diabetic patients. We review the impact of diabetes on establishing, during childhood and adolescence, the premises for cardiovascular diseases later in life. Interestingly, it seems that hyperglycemia is not the only factor that establishes an increased cardiovascular risk in adolescence. Other factors have been recognized to play a role in triggering the onset of latent cardiovascular diseases in the pediatric population. Among these cardiovascular risk factors, some are modifiable: glucose variability, hypoglycemia, obesity, insulin resistance, waist circumference, hypertension, dyslipidemia, smoking alcohol, microalbuminuria and smoking. Others are unmodifiable, such as diabetes duration and family history. Among the etiological factors, subclinical endothelial dysfunction represents one of the earliest key players of atherosclerosis and it can be detected during early ages in patients with diabetes. A better assessment of cardiovascular risk in pediatric population still represents a challenge for clinicians, and thus further efforts are required to properly identify and treat pediatric patients who may suffer from cardiovascular disease later in early adulthood.

Sodium glucose cotransporters inhibitors in type 1 diabetes.

Sodium glucose cotransporter inhibitors (SGLTi) are oral hypoglycemic drugs that reduce renal glucose re-uptake and induce glycosuria. SGLTi have been successfully tested in large randomized clinical trials for type 2 diabetes, and several molecules have been approved in this setting by the international pharmaceutical agencies. Additionally, recent evidence has shown that SGLTi may be useful also in type 1 diabetes (T1D). Indeed, these drugs can be used as an ancillary to insulin to improve glycemic control and reduce insulin dosage, and such regimens have been associated with a lower rate of hypoglycemic episodes. The pharmacological effects of SGLTi therapy are described herein, and we also discuss the future use of SGLTi in T1D.

The emerging role of acid sphingomyelinase in autophagy.

Autophagy, the main intracellular process of cytoplasmic material degradation, is involved in cell survival and death. Autophagy is regulated at various levels and novel modulators of its function are being continuously identified. An intriguing recent observation is that among these modulators is the sphingolipid metabolising enzyme, Acid Sphingomyelinase (A-SMase), already known to play a fundamental role in apoptotic cell death participating in several pathophysiological conditions. In this review we analyse and discuss the relationship between autophagy and A-SMase describing how A-SMase may regulate it and defining, for the first time, the existence of an A-SMase-autophagy axis. The imbalance of this axis plays a role in cancer, nervous system, cardiovascular, and hepatic disorders.

The thyroid hormone triiodothyronine controls macrophage maturation and functions: protective role during inflammation.

The endocrine system participates in regulating macrophage maturation, although little is known about the modulating role of the thyroid hormones. In vitro results demonstrate a negative role of one such hormone, triiodothyronine (T3), in triggering the differentiation of bone marrow-derived monocytes into unpolarized macrophages. T3-induced macrophages displayed a classically activated (M1) signature. A T3-induced M1-priming effect was also observed on polarized macrophages because T3 reverses alternatively activated (M2) activation, whereas it enhances that of M1 cells. In vivo, circulating T3 increased the content of the resident macrophages in the peritoneal cavity, whereas it reduced the content of the recruited monocyte-derived cells. Of interest, T3 significantly protected mice against endotoxemia induced by lipopolysaccharide i.p. injection; in these damaged animals, decreased T3 levels increased the recruited (potentially damaging) cells, whereas restoring T3 levels decreased recruited and increased resident (potentially beneficial) cells. These data suggest that the anti-inflammatory effect of T3 is coupled to the modulation of peritoneal macrophage content, in a context not fully explained by the M1/M2 framework. Thyroid hormone receptor expression analysis and the use of different thyroid hormone receptor antagonists suggest thyroid hormone receptor ß1 as the major player mediating T3 effects on macrophages. The novel homeostatic link between thyroid hormones and the pathophysiological role of macrophages opens new perspectives on the interactions between the endocrine and immune systems.

Nitric oxide drives embryonic myogenesis in chicken through the upregulation of myogenic differentiation factors.

The muscle-specific variant of neuronal nitric oxide (NO) synthase (NOS-I), is developmentally regulated in mouse suggesting a role of NO during myogenesis. In chick embryo, a good model of development, we found that the expression of NOS-I is up-regulated, but only in the early phase of development. Through a pharmacological intervention in ovo we found that NO signalling plays a relevant role during embryonic development. The inhibition of NOS-I decreased the growth of embryo, in particular of muscle tissue, while the restoring of physiological NO levels, via administration of a NO donor, reversed this effect. We found a selective action of NO, produced by NOS-I, on regulatory factors involved in myogenic differentiation in the early phase of chick embryo development: inhibition of NO generation leads to a decreased expression of the Myocyte enhancer factor 2a (Mef2a), Mef2c, Myogenin and Myosin, which was reversed by the administration of a NO donor. NO had no effects on Myf5 and MyoD, the myogenic regulatory factors necessary for myogenic determination. The action of NO on the myogenic regulatory factors was mediated via generation of cyclic GMP (cGMP) and activation of the cGMP-dependent protein kinase G (PKG). Finally we found in myoblasts in vitro that the activation of Mef2c was the key event mediating the NO-induced modulation of myogenesis. Our results identify NO produced by NOS-I as a key messenger in the early phase of embryonic development of chicken, acting as a critical determinant of myogenesis through its physiological cGMP/PKG pathway.

Modulation of Acid Sphingomyelinase in Melanoma Reprogrammes the Tumour Immune Microenvironment.

The inflammatory microenvironment induces tumours to acquire an aggressive and immunosuppressive behaviour. Since acid sphingomyelinase (A-SMase) downregulation in melanoma was shown to determine a malignant phenotype, we aimed here to elucidate the role of A-SMase in the regulation of tumour immunogenic microenvironment using in vivo melanoma models in which A-SMase was either downregulated or maintained at constitutively high levels. We found high levels of inflammatory factors in low A-SMase expressing tumours, which also displayed an immunosuppressive/protumoural microenvironment: high levels of myeloid-derived suppressor cells (MDSCs) and regulatory T lymphocytes (Tregs), as well as low levels of dendritic cells (DCs). In contrast, the restoration of A-SMase in melanoma cells not only reduced tumour growth and immunosuppression, but also induced a high recruitment at tumour site of effector immune cells with an antitumoural function. Indeed, we observed a poor homing of MDSCs and Tregs and the increased recruitment of CD8(+) and CD4(+) T lymphocytes as well as the infiltration of DCs and CD8(+)/CD44(high) T lymphocytes. This study demonstrates that change of A-SMase expression in cancer cells is sufficient per se to tune in vivo melanoma growth and that A-SMase levels modulate immune cells at tumour site. This may be taken into consideration in the setting of therapeutic strategies.

TMEM219 regulates the transcription factor expression and proliferation of beta cells.

Pancreatic beta cells replenishment is considered the next therapeutic option for type 1 diabetes; while stimulating endogenous beta cells proliferation is the "holy grail" for those patients with exhausted beta cell mass. Here we are demonstrating that the pro-apoptotic receptor TMEM219 is expressed in fetal pancreas, in beta cell precursors and in in vitro embryonic-derived endocrine progenitors. TMEM219 signaling negatively regulates beta cells at early stages and induces Caspase 8-mediated cell death. Pharmacological blockade of TMEM219 further rescued beta cell precursor and proliferation markers, and decreased cell death, both in islets and in in vitro-derived endocrine progenitors, allowing for beta cell preservation. While addressing the upstream controlling TMEM219 expression, we determined the TMEM219 miRNet; indeed, one of those miRNAs, miR-129-2, is highly expressed in human islets, particularly in patients at risk or with established type 1 diabetes. miR-129-2 mimic downregulated TMEM219 expression in islets, in in vitro embryonic-derived endocrine progenitors and in highly proliferating insulinoma-derived cells. Moreover, miR-129-2 inhibitor induced a TMEM219 overexpression in insulinoma-derived cells, which restored cell proliferation and functional markers, thus acting as endogenous regulator of TMEM219 expression. The TMEM219 upstream regulator miR129-2 controls the fate of beta cell precursors and may unleash their regenerative potentials to replenish beta cells in type 1 diabetes.

Glucagon-like peptide 1 receptor is a T cell-negative costimulatory molecule.

Glucagon-like peptide-1 receptor (GLP-1R) is a key regulator of glucose metabolism known to be expressed by pancreatic ß cells. We herein investigated the role of GLP-1R on T lymphocytes during immune response. Our data showed that a subset of T lymphocytes expresses GLP-1R, which is upregulated during alloimmune response, similarly to PD-1. When mice received islet or cardiac allotransplantation, an expansion of GLP-1Rpos T cells occurred in the spleen and was found to infiltrate the graft. Additional single-cell RNA sequencing (scRNA-seq) analysis conducted on GLP-1Rpos and GLP-1Rneg CD3+ T cells unveiled the existence of molecular and functional dissimilarities between both subpopulations, as the GLP-1Rpos are mainly composed of exhausted CD8 T cells. GLP-1R acts as a T cell-negative costimulatory molecule, and GLP-1R signaling prolongs allograft survival, mitigates alloimmune response, and reduces T lymphocyte graft infiltration. Notably, GLP-1R antagonism triggered anti-tumor immunity when tested in a preclinical mouse model of colorectal cancer.

Sphingolipids and brain resident macrophages in neuroinflammation: an emerging aspect of nervous system pathology.

Sphingolipid metabolism is deeply regulated along the differentiation and development of the central nervous system (CNS), and the expression of a peculiar spatially and temporarily regulated sphingolipid pattern is essential for the maintenance of the functional integrity of the nervous system. Microglia are resident macrophages of the CNS involved in general maintenance of neural environment. Modulations in microglia phenotypes may contribute to pathogenic forms of inflammation. Since defects in macrophage/microglia activity contribute to neurodegenerative diseases, it will be essential to systematically identify the components of the microglial cell response that contribute to disease progression. In such complex processes, the sphingolipid systems have recently emerged to play important roles, thus appearing as a key new player in CNS disorders. This review provides a rationale for harnessing the sphingolipid metabolic pathway as a potential target against neuroinflammation.

Possible pathogenetic relationship between Fabry disease and renal cell carcinoma.

The occurrence of renal cell carcinoma (RCC) in Fabry disease (FD) is a rare event. We report a deep ultrastructural study of RCC in a patient with a previous histological diagnosis of FD. In order to highlight analogies and differences between the two histological samples, we used the nephrectomy specimen as a 'repeat biopsy', making a dynamic analysis of the evolution of the disease-related kidney damage. Secondly, a comparative ultrastructural analysis between non-neoplastic tissue and cancer demonstrated for the first time the presence of zebra bodies in the tumor cells. Finally, a hypothetical speculation about the relationship between the lysosomal accumulation, the oxidative damage and the genesis of the tumor was performed. The link connected the accumulation of glycosphingolipid globotriaosylceramide, characteristic of FD, with the expression of CD74 and macrophage migration inhibitory factor that may play an important role in tumorigenesis regulated by the Von Hippel-Lindau/hypoxia-inducible factor 1α pathway.

Abnormalities of the oculomotor function in type 1 diabetes and diabetic neuropathy.

AIMS: Abnormalities in the oculomotor system may represent an early sign of diabetic neuropathy and are currently poorly studied. We designed an eye-tracking-based test to evaluate oculomotor function in patients with type 1 diabetes. METHODS: We used the SRLab-Tobii TX300 Eye tracker®, an eye-tracking device, coupled with software that we developed to test abnormalities in the oculomotor system. The software consists of a series of eye-tracking tasks divided into 4 classes of parameters (Resistance, Wideness, Pursuit and Velocity) to evaluate both smooth and saccadic movement in different directions. We analyzed the oculomotor system in 34 healthy volunteers and in 34 patients with long-standing type 1 diabetes. RESULTS: Among the 474 parameters analyzed with the eye-tracking-based system, 11% were significantly altered in patients with type 1 diabetes (p < 0.05), with a higher proportion of abnormalities observed in the Wideness (24%) and Resistance (10%) parameters. Patients with type 1 diabetes without diabetic neuropathy showed more frequently anomalous measurements in the Resistance class (p = 0.02). The classes of Velocity and Pursuit were less frequently altered in patients with type 1 diabetes as compared to healthy subjects, with anomalous measurements mainly observed in patients with diabetic neuropathy. CONCLUSIONS: Abnormalities in oculomotor system function can be detected in patients with type 1 diabetes using a novel eye-tracking-based test. A larger cohort study may further determine thresholds of normality and validate whether eye-tracking can be used to non-invasively characterize early signs of diabetic neuropathy. TRIAL: NCT04608890.

Indirect and Direct Effects of SARS-CoV-2 on Human Pancreatic Islets.

Recent studies have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may induce metabolic distress, leading to hyperglycemia in patients affected by coronavirus disease 19 (COVID-19). We investigated the potential indirect and direct effects of SARS-CoV-2 on human pancreatic islets in 10 patients who became hyperglycemic after COVID-19. Although there was no evidence of peripheral anti-islet autoimmunity, the serum of these patients displayed toxicity on human pancreatic islets, which could be abrogated by the use of anti-interleukin-1ß (IL-1ß), anti-IL-6, and anti-tumor necrosis factor α, cytokines known to be highly upregulated during COVID-19. Interestingly, the receptors of those aforementioned cytokines were highly expressed on human pancreatic islets. An increase in peripheral unmethylated INS DNA, a marker of cell death, was evident in several patients with COVID-19. Pathology of the pancreas from deceased hyperglycemic patients who had COVID-19 revealed mild lymphocytic infiltration of pancreatic islets and pancreatic lymph nodes. Moreover, SARS-CoV-2-specific viral RNA, along with the presence of several immature insulin granules or proinsulin, was detected in postmortem pancreatic tissues, suggestive of ß-cell-altered proinsulin processing, as well as ß-cell degeneration and hyperstimulation. These data demonstrate that SARS-CoV-2 may negatively affect human pancreatic islet function and survival by creating inflammatory conditions, possibly with a direct tropism, which may in turn lead to metabolic abnormalities observed in patients with COVID-19.

Immunogenicity and Safety of SARS-CoV-2 mRNA Vaccines in a Cohort of Patients With Type 1 Diabetes.

Patients with type 1 diabetes (T1D) may develop severe outcomes during coronavirus disease 2019 (COVID-19), but their ability to generate an immune response against the SARS-CoV-2 mRNA vaccines remains to be established. We evaluated the safety, immunogenicity, and glycometabolic effects of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in patients with T1D. A total of 375 patients (326 with T1D and 49 subjects without diabetes) who received two doses of the SARS-CoV-2 mRNA vaccines (mRNA-1273, BNT162b2) between March and April 2021 at ASST Fatebenefratelli Sacco were included in this monocentric observational study. Local and systemic adverse events were reported in both groups after SARS-CoV-2 mRNA vaccination, without statistical differences between them. While both patients with T1D and subjects without diabetes exhibited a parallel increase in anti-SARS-CoV-2 spike titers after vaccination, the majority of patients with T1D (70% and 78%, respectively) did not show any increase in the SARS-CoV-2-specific cytotoxic response compared with the robust increase observed in all subjects without diabetes. A reduced secretion of the T-cell-related cytokines interleukin-2 and tumor necrosis factor-α in vaccinated patients with T1D was also observed. No glycometabolic alterations were evident in patients with T1D using continuous glucose monitoring during follow-up. Administration of the SARS-CoV-2 mRNA vaccine is associated with an impaired cellular SARS-CoV-2-specific cytotoxic immune response in patients with T1D.

The IGFBP3/TMEM219 pathway regulates beta cell homeostasis.

Loss of pancreatic beta cells is a central feature of type 1 (T1D) and type 2 (T2D) diabetes, but a therapeutic strategy to preserve beta cell mass remains to be established. Here we show that the death receptor TMEM219 is expressed on pancreatic beta cells and that signaling through its ligand insulin-like growth factor binding protein 3 (IGFBP3) leads to beta cell loss and dysfunction. Increased peripheral IGFBP3 was observed in established and at-risk T1D/T2D patients and was confirmed in T1D/T2D preclinical models, suggesting that dysfunctional IGFBP3/TMEM219 signaling is associated with abnormalities in beta cells homeostasis. In vitro and in vivo short-term IGFBP3/TMEM219 inhibition and TMEM219 genetic ablation preserved beta cells and prevented/delayed diabetes onset, while long-term IGFBP3/TMEM219 blockade allowed for beta cell expansion. Interestingly, in several patients' cohorts restoration of appropriate IGFBP3 levels was associated with improved beta cell function. The IGFBP3/TMEM219 pathway is thus shown to be a physiological regulator of beta cell homeostasis and is also demonstrated to be disrupted in T1D/T2D. IGFBP3/TMEM219 targeting may therefore serve as a therapeutic option in diabetes.