Extracellular vesicles as human therapeutics: A scoping review of the literature.

Extracellular vesicles (EVs) are released by all cells and contribute to cell-to-cell communication. The capacity of EVs to target specific cells and to efficiently deliver a composite profile of functional molecules have led researchers around the world to hypothesize their potential as therapeutics. While studies of EV treatment in animal models are numerous, their actual clinical benefit in humans has more slowly started to be tested. In this scoping review, we searched PubMed and other databases up to 31 December 2023 and, starting from 13,567 records, we selected 40 pertinent published studies testing EVs as therapeutics in humans. The analysis of those 40 studies shows that they are all small pilot trials with a large heterogeneity in terms of administration route and target disease. Moreover, the absence of a placebo control in most of the studies, the predominant local application of EV formulations and the inconsistent administration dose metric still impede comparison across studies and firm conclusions about EV safety and efficacy. On the other hand, the recording of some promising outcomes strongly calls out for well-designed larger studies to test EVs as an alternative approach to treat human diseases with no or few therapeutic options.

Caloric restriction for the immunometabolic control of human health.

Nutrition affects all physiological processes occurring in our body, including those related to the function of the immune system; indeed, metabolism has been closely associated with the differentiation and activity of both innate and adaptive immune cells. While excessive energy intake and adiposity have been demonstrated to cause systemic inflammation, several clinical and experimental evidence show that calorie restriction (CR), not leading to malnutrition, is able to delay aging and exert potent anti-inflammatory effects in different pathological conditions. This review provides an overview of the ability of different CR-related nutritional strategies to control autoimmune, cardiovascular, and infectious diseases, as tested by preclinical studies and human clinical trials, with a specific focus on the immunological aspects of these interventions. In particular, we recapitulate the state of the art on the cellular and molecular mechanisms pertaining to immune cell metabolic rewiring, regulatory T cell expansion, and gut microbiota composition, which possibly underline the beneficial effects of CR. Although studies are still needed to fully evaluate the feasibility and efficacy of the nutritional intervention in clinical practice, the experimental observations discussed here suggest a relevant role of CR in lowering the inflammatory state in a plethora of different pathologies, thus representing a promising therapeutic strategy for the control of human health.

Immunobiology of pregnancy: from basic science to translational medicine.

Embryo implantation failure and spontaneous abortions represent the main causes of infertility in developed countries. Unfortunately, incomplete knowledge of the multiple factors involved in implantation and fetal development keeps the success rate of medically assisted procreation techniques relatively low. According to recent literature, cellular and molecular mechanisms of 'immunogenic tolerance' towards the embryo are crucial to establish an 'anti-inflammatory' state permissive of a healthy pregnancy. In this review we dissect the role played by the immune system in the endometrial-embryo crosstalk, with a particular emphasis towards the fork-head-box-p3 (Foxp3+) CD4+CD25+ regulatory T (Treg) cells and discuss the most recent therapeutic advances in the context of early immune-mediated pregnancy loss.

Regulatory T cells as metabolic sensors.

Compelling experimental evidence links immunity and metabolism. In this perspective, we propose forkhead-box-P3 (FoxP3)+CD4+CD25+ regulatory T (Treg) cells as key metabolic sensors controlling the immunological state in response to their intrinsic capacity to perceive nutritional changes. Treg cell high anabolic state in vivo, residency in metabolically crucial districts, and recirculation between lymphoid and non-lymphoid sites enable them to recognize the metabolic cues and adapt their intracellular metabolism and anti-inflammatory function at the paracrine and systemic levels. As privileged regulators at the interface between neuroendocrine and immune systems, the role of Treg cells in maintaining metabolic homeostasis makes these cells promising targets of therapeutic strategies aimed at restoring organismal homeostasis not only in autoimmune but also metabolic disorders.

Repurposing SGLT-2 Inhibitors to Target Aging: Available Evidence and Molecular Mechanisms.

Caloric restriction promotes longevity in multiple animal models. Compounds modulating nutrient-sensing pathways have been suggested to reproduce part of the beneficial effect of caloric restriction on aging. However, none of the commonly studied caloric restriction mimetics actually produce a decrease in calories. Sodium-glucose cotransporter 2 inhibitors (SGLT2-i) are a class of drugs which lower glucose by promoting its elimination through urine, thus inducing a net loss of calories. This effect promotes a metabolic shift at the systemic level, fostering ketones and fatty acids utilization as glucose-alternative substrates, and is accompanied by a modulation of major nutrient-sensing pathways held to drive aging, e.g., mTOR and the inflammasome, overall resembling major features of caloric restriction. In addition, preliminary experimental data suggest that SGLT-2i might also have intrinsic activities independent of their systemic effects, such as the inhibition of cellular senescence. Consistently, evidence from both preclinical and clinical studies have also suggested a marked ability of SGLT-2i to ameliorate low-grade inflammation in humans, a relevant driver of aging commonly referred to as inflammaging. Considering also the amount of data from clinical trials, observational studies, and meta-analyses suggesting a tangible effect on age-related outcomes, such as cardiovascular diseases, heart failure, kidney disease, and all-cause mortality also in patients without diabetes, here we propose a framework where at least part of the benefit provided by SGLT-2i is mediated by their ability to blunt the drivers of aging. To support this postulate, we synthesize available data relative to the effect of this class on: 1- animal models of healthspan and lifespan; 2- selected molecular pillars of aging in preclinical models; 3- biomarkers of aging and especially inflammaging in humans; and 4- COVID-19-related outcomes. The burden of evidence might prompt the design of studies testing the potential employment of this class as anti-aging drugs.

Epigenetics: An opportunity to shape innate and adaptive immune responses.

Epigenetics connects genetic and environmental factors: it includes DNA methylation, histone post-translational modifications and the regulation of chromatin accessibility by non-coding RNAs, all of which control constitutive or inducible gene transcription. This plays a key role in harnessing the transcriptional programs of both innate and adaptive immune cells due to its plasticity and environmental-driven nature, piloting myeloid and lymphoid cell fate decisions with no change in their genomic sequence. In particular, epigenetic marks at the site of lineage-specific transcription factors and maintenance of cell type-specific epigenetic modifications, referred to as 'epigenetic memory', dictate cell differentiation, cytokine production and functional capacity following repeated antigenic exposure in memory T cells. Moreover, metabolic and epigenetic reprogramming occurring during a primary innate immune response leads to enhanced responses to secondary challenges, a phenomenon known as 'trained immunity'. Here, we discuss how stable and dynamic epigenetic states control immune cell identity and plasticity in physiological and pathological conditions. Dissecting the regulatory circuits of cell fate determination and maintenance is of paramount importance for understanding the delicate balance between immune cell activation and tolerance, in healthy conditions and in autoimmune diseases.

A rapid and inexpensive genotyping method using dried blood spots for mutational analysis in a mutant mouse model: an update.

BACKGROUND: Dried blood spot (DBS) testing is a well-known method of bio-sampling by which blood samples are blotted and dried on filter paper. The dried samples can then be analyzed by several techniques such as DNA amplification and HPLC. We have developed a non-invasive sampling followed by an alternative protocol for genomic DNA extraction from a drop of blood adsorbed on paper support. This protocol consists of two separate steps: (1) organic DNA extraction from the DBS, followed by (2) DNA amplification by polymerase chain reaction (PCR). The PCR-restriction fragment length polymorphism (PCR-RFLP) is an advantageous and simple approach to detect single nucleotide polymorphisms (SNPs). RESULTS: We have evaluated the efficiency of our method for the extraction of genomic DNA from DBS by testing its performance in genotyping mouse models of obesity and herein discuss the specificity and feasibility of this novel procedure. CONCLUSIONS: Our protocol is easy to perform, fast and inexpensive and allows the isolation of pure DNA from a tiny amount of sample.

Anti-inflammatory effect of SGLT-2 inhibitors via uric acid and insulin.

Sodium-glucose cotransporter 2 (SGLT-2) inhibitors (i) reduce cardiovascular and renal events in patients with and without type 2 diabetes (T2D). However, the underlying mechanisms are debated. Low-grade inflammation (LGI) is a key driver of vascular complications, suggested to be attenuated by SGLT-2i in animal models. Based on a specific working hypothesis, here we investigated the net effect of SGLT-2i on LGI in patients with T2D and the possible underlying mechanism. We enrolled patients with T2D treated either with a stable therapy with SGLT-2i or with other glucose-lowering drugs (GLD) (n = 43 per group after matching for a range of pro-inflammatory variables), and tested hs-CRP and interleukin (IL)-6 as primary variables of interest. Patients treated with SGLT-2i had lower circulating levels of IL-6, a prototypical marker of LGI, but also of uric acid and fasting insulin, compared with patients treated with other GLD. Then, to explore whether uric acid and insulin might mediate the effect of SGLT-2i on IL-6, we tested physiologically pertinent doses of these two molecules (i.e. 0.5 mM uric acid and 1 nM insulin) in two in vitro models of LGI, i.e. monocytes (THP-1) treated with LPS and endothelial cells (HUVEC) exposed to hyperglycaemia. Results from in vitro models supported a pro-inflammatory role for uric acid and its combination with insulin in monocytes and for uric acid alone in hyperglycaemia-stimulated endothelial cells. On the contrary, we observed no drug-intrinsic, anti-inflammatory effect for dapagliflozin, empagliflozin, and canagliflozin in the same models. Overall, these results suggest that SGLT-2i possess a tangible activity against LGI, an effect possibly mediated by their ability to lower uric acid and insulin concentrations and that juxtaposes other proposed mechanisms in explaining the observed benefit of this class on cardiovascular and renal endpoints.

High levels of blood circulating immune checkpoint molecules in children with new-onset type 1 diabetes are associated with the risk of developing an additional autoimmune disease.

AIMS/HYPOTHESIS: We assessed the levels of blood circulating immune checkpoint molecules (ICMs) at diagnosis of type 1 diabetes, and determined their association with the risk of developing an additional autoimmune disorder over time. METHODS: Children with new-onset type 1 diabetes (n = 143), without biological and/or clinical signs of additional autoimmune disorders, and healthy children (n = 75) were enrolled, and blood circulating levels of 14 ICMs were measured. The children with type 1 diabetes were divided into two groups on the basis of the development of an additional autoimmune disease in the 5 years after diabetes onset. Differences in soluble ICM levels between the groups were assessed, and a Cox regression analysis was used to evaluate their association with the risk of development of an additional autoimmune disease over time. To validate the data, circulating ICMs were measured in an independent cohort of 60 children with new-onset type 1 diabetes stratified into two groups. RESULTS: We found that the levels of circulating ICMs were significantly higher in children with new-onset diabetes compared with healthy children. Further, we observed that children with type 1 diabetes who developed a second autoimmune disease over time (T1D-AAD+ children) had higher levels of soluble ICMs than children with type 1 diabetes who did not (T1D-AAD- children). Cox regression models revealed that high circulating levels of CD137/4-1BB and PD-1 molecules at diabetes diagnosis were associated with the risk of developing an additional autoimmune disease in both type 1 diabetes cohorts. CONCLUSIONS/INTERPRETATION: Our findings suggest that soluble CD137/4-1BB and PD-1 molecules may be used as prognostic biomarkers in children with type 1 diabetes, and may pave the way for novel immunological screening at diabetes onset, allowing early identification of children at higher risk of developing other autoimmune conditions over time.

The folate way to T cell fate.

The role of folate-dependent one carbon (1C) metabolism in CD4+ T cell polarization is incompletely understood. In this issue of Immunity, Sugiura et al. (2021) provide evidence that blocking the 1C metabolic enzyme MTHFD2 may curb pro-inflammatory CD4+ T cells, while redirecting them toward a regulatory T cell phenotype.

Type 1 Diabetes and Associated Cardiovascular Damage: Contribution of Extracellular Vesicles in Tissue Crosstalk.

Significance: Type 1 diabetes (T1D) is characterized by the autoimmune destruction of the insulin secreting ß-cells, with consequent aberrant blood glucose levels. Hyperglycemia is the common denominator for most of the chronic diabetic vascular complications, which represent the main cause of life reduction in T1D patients. For this disease, three interlaced medical needs remain: understanding the underlying mechanisms involved in pancreatic ß-cell loss; identifying biomarkers able to predict T1D progression and its related complications; recognizing novel therapeutic targets. Recent Advances: Extracellular vesicles (EVs), released by most cell types, were discovered to contain a plethora of different molecules (including microRNAs) with regulatory properties, which are emerging as mediators of cell-to-cell communication at the paracrine and endocrine level. Recent knowledge suggests that EVs may act as pathogenic factors, and be developed into disease biomarkers and therapeutic targets in the context of several human diseases. Critical Issues: EVs have been recently shown to sustain a dysregulated cellular crosstalk able to exacerbate the autoimmune response in the pancreatic islets of T1D; moreover, EVs were shown to be able to monitor and/or predict the progression of T1D and the insurgence of vasculopathies. Future Directions: More mechanistic studies are needed to investigate whether the dysregulation of EVs in T1D patients is solely reflecting the progression of diabetes and related complications, or EVs also directly participate in the disease process, thus pointing to a potential use of EVs as therapeutic targets/tools in T1D. Antioxid. Redox Signal. 36, 631-651.

Elevated HbA1c levels in pre-Covid-19 infection increases the risk of mortality: A sistematic review and meta-analysis.

AIMS: Diabetes is emerging as a risk factor for coronavirus disease (COVID)-19 prognosis. However, contradictory findings have been reported regarding the impact of glycaemic control on COVID-19 outcome. The aim of this meta-analysis was to explore the impact of hospital pre-admission or at-admission values of HbA1c on COVID-19 mortality or worsening in patients with diabetes. MATERIALS AND METHODS: We searched PubMed, Embase and Scopus up to 30th December 2020. Eligibility criteria for study selection were the following: (1)enrolling patients with any form of diabetes mellitus and hospitalized for COVID-19 and (2) reporting data regarding HbA1c values before infection or at hospital admission in relation to COVID-19 mortality or worsening. Descriptive statistics, HbA1c values, odds ratios (ORs) and hazard ratios were extracted from seven observational studies and generic inverse variance (random effects) of OR was used to estimate the effect of HbA1c on COVID-19 outcome. RESULTS: HbA1c was linearly associated with an increased COVID-19 mortality or worsening when considered as a continuous variable (OR 1.01 [1.01, 1.01]; p < 0.00001). Similarly, when analysing studies providing the number of events according to the degree of glycaemic control among various strata, a significantly increased risk was observed with poor glycaemic control (OR 1.15 [1.11, 1.19]; p < 0.00001), a result corroborated by sensitivity analysis. CONCLUSIONS: Notwithstanding the large heterogeneity in study design and patients' characteristics in the few available studies, data suggest that patients with diabetes and poor glycaemic control before infection might have an increased risk of COVID-19 related mortality.

CD4+ T-Cell Activation Prompts Suppressive Function by Extracellular Vesicle-Associated MicroRNAs.

MicroRNAs (miRNAs), small non-coding molecules targeting messenger RNAs and inhibiting protein translation, modulate key biological processes, including cell growth and development, energy utilization, and homeostasis. In particular, miRNAs control the differentiation, survival, and activation of CD4 + T conventional (Tconv) cells, key players of the adaptive immunity, and regulate the physiological response to infections and the pathological loss of immune homeostasis in autoimmunity. Upon T-cell receptor (TCR) stimulation, the described global miRNA quantitative decrease occurring in T cells is believed to promote the acquisition of effector functions by relaxing the post-transcriptional repression of genes associated with proliferation and cell activity. MiRNAs were initially thought to get downregulated uniquely by intracellular degradation; on the other hand, miRNA secretion via extracellular vesicles (EVs) represents an additional mechanism of rapid downregulation. By focusing on molecular interactions by means of graph theory, we have found that miRNAs released by TCR-stimulated Tconv cells are significantly enriched for targeting transcripts upregulated upon stimulation, including those encoding for crucial proteins associated with Tconv cell activation and function. Based on this computational approach, we present our perspective based on the following hypothesis: a stimulated Tconv cell will release miRNAs targeting genes associated with the effector function in the extracellular space in association with EVs, which will thus possess a suppressive potential toward other Tconv cells in the paracrine environment. We also propose possible future directions of investigation aimed at taking advantage of these phenomena to control Tconv cell effector function in health and autoimmunity.

Effect of time and titer in convalescent plasma therapy for COVID-19.

The clinical benefit of convalescent plasma (CP) for patients with coronavirus disease (COVID)-19 is still debated. In this systematic review and meta-analysis, we selected 10 randomized clinical trials (RCTs) and 15 non-randomized studies (total number of patients = 22,591) of CP treatment and evaluated two different scenarios: (1) disease stage of plasma recipients and (2) donated plasma antibody titer, considering all-cause mortality at the latest follow-up. Our results show that, when provided at early stages of the disease, CP significantly reduced mortality: risk ratio (RR) 0.72 (0.68, 0.77), p < 0.00001, while provided in severe or critical conditions, it did not (RR: 0.94 [0.86, 1.04], p = 0.22). On the other hand, the benefit on mortality was not increased by using plasma with a high-antibody titer compared with unselected plasma. This meta-analysis might promote CP usage in patients with early-stage COVID-19 in further RCTs to maximize its benefit in decreasing mortality, especially in less affluent countries.

Signals of pseudo-starvation unveil the amino acid transporter SLC7A11 as key determinant in the control of Treg cell proliferative potential.

Human CD4+CD25hiFOXP3+ regulatory T (Treg) cells are key players in the control of immunological self-tolerance and homeostasis. Here, we report that signals of pseudo-starvation reversed human Treg cell in vitro anergy through an integrated transcriptional response, pertaining to proliferation, metabolism, and transmembrane solute carrier transport. At the molecular level, the Treg cell proliferative response was dependent on the induction of the cystine/glutamate antiporter solute carrier (SLC)7A11, whose expression was controlled by the nuclear factor erythroid 2-related factor 2 (NRF2). SLC7A11 induction in Treg cells was impaired in subjects with relapsing-remitting multiple sclerosis (RRMS), an autoimmune disorder associated with reduced Treg cell proliferative capacity. Treatment of RRMS subjects with dimethyl fumarate (DMF) rescued SLC7A11 induction and fully recovered Treg cell expansion. These results suggest a previously unrecognized mechanism that may account for the progressive loss of Treg cells in autoimmunity and unveil SLC7A11 as major target for the rescue of Treg cell proliferation.

Diabetes and kidney disease: emphasis on treatment with SGLT-2 inhibitors and GLP-1 receptor agonists.

Kidney disease is a frequent microvascular complication of both type 1 and type 2 diabetes. Historic trials have demonstrated that a tight glycaemic control is the most powerful approach to decrease the chances of developing diabetic nephropathy. However, having an HbA1c < 7% does not completely suppress the risk of kidney disease. The observed residual risk is likely ascribable to two phenomena: 1- the presence of risk factors and alterations additive to and independent of glycaemia, and 2- the activation of long-lasting imbalances by periods of exposure to uncontrolled glycemia, a phenomenon referred to as metabolic memory or legacy effect. Long-lasting oxidative stress, epigenetic alterations, cellular senescence, and the resulting chronic low-grade inflammation are all candidate mechanisms explaining the development of nephropathy despite proper control of risk factors. Recently, two classes of drugs, i.e. glucagon-like peptide (GLP) 1 receptor agonists (RA) and sodium-glucose transporter 2 inhibitors (SGLT-i) have changed this scenario. Indeed, cardiovascular outcome and other trials have clearly shown a renoprotective effect for these drugs, well-beyond their glucose-lowering properties. In this review, we summarize: 1- selected key trials and mechanisms underlying the development of diabetic kidney disease and 2- the results relative to renal endpoints in clinical trials of GLP-1 RA and SGLT-2i. Then, we briefly discuss some of the hypotheses posited to explain the marked renoprotective properties of these two classes, evidencing the still existing gaps in knowledge and proposing future directions to further implement the use of these powerful, disease-modifying drugs.

The pleiotropic roles of leptin in metabolism, immunity, and cancer.

The discovery of the archetypal adipocytokine leptin and how it regulates energy homeostasis have represented breakthroughs in our understanding of the endocrine function of the adipose tissue and the biological determinants of human obesity. Investigations on leptin have also been instrumental in identifying physio-pathological connections between metabolic regulation and multiple immunological functions. For example, the description of the promoting activities of leptin on inflammation and cell proliferation have recognized the detrimental effects of leptin in connecting dysmetabolic conditions with cancer and with onset and/or progression of autoimmune disease. Here we review the multiple biological functions and complex framework of operations of leptin, discussing why and how the pleiotropic activities of this adipocytokine still pose major hurdles in the development of effective leptin-based therapeutic opportunities for different clinical conditions.

Metabolomics, Lipidomics, and Immunometabolism.

Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.