Increased frequencies of highly activated regulatory T cells skewed to a T helper 1-like phenotype with reduced suppressive capacity in dengue patients.

The pathogenesis of dengue involves a complex interplay between the viral factor and the host immune response. A mismatch between the infecting serotype and the adaptive memory response is hypothesized to lead to exacerbated immune responses resulting in severe dengue. Here, we aim to define in detail the phenotype and function of different regulatory T cell (Treg) subsets and their association with disease severity in a cohort of acute dengue virus (DENV)-infected Cambodian children. Treg frequencies and proliferation of Tregs are increased in dengue patients compared to age-matched controls. Tregs from dengue patients are skewed to a Th1-type Treg phenotype. Interestingly, Tregs from severe dengue patients produce more interleukin-10 after in vitro stimulation compared to Tregs from classical dengue fever patients. Functionally, Tregs from dengue patients have reduced suppressive capacity, irrespective of disease severity. Taken together, these data suggest that even though Treg frequencies are increased in the blood of acute DENV-infected patients, Tregs fail to resolve inflammation and thereby could contribute to the immunopathology of dengue. IMPORTANCE: According to the World Health Organization, dengue is the fastest-spreading, epidemic-prone infectious disease. The extent of dengue virus infections increased over the years, mainly driven by globalization-including travel and trade-and environmental changes. Dengue is an immunopathology caused by an imbalanced immune response to a secondary heterotypic infection. As regulatory T cells (Tregs) are essential in maintaining immune homeostasis and dampening excessive immune activation, this study addressed the role of Tregs in dengue immunopathology. We show that Tregs from dengue patients are highly activated, skewed to a Th1-like Treg phenotype and less suppressive compared to healthy donor Tregs. Our data suggest that Tregs fail to resolve ongoing inflammation during dengue infection and hence contribute to the immunopathology of severe dengue disease. These data clarify the role of Tregs in dengue immunopathogenesis, emphasizing the need to develop T cell-based vaccines for dengue.

Rapamycin rescues loss of function in blood-brain barrier-interacting Tregs.

In autoimmunity, FOXP3+ Tregs skew toward a proinflammatory, nonsuppressive phenotype and are, therefore, unable to control the exaggerated autoimmune response. This largely affects the success of autologous Treg therapy, which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the CNS during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss of function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential and a proinflammatory Th1/17 signature, and they upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less-suppressive CD49d+ Tregs in the cerebrospinal fluid of people with MS. In summary, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive proinflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.

Pyridoxamine Limits Cardiac Dysfunction in a Rat Model of Doxorubicin-Induced Cardiotoxicity.

The use of doxorubicin (DOX) chemotherapy is restricted due to dose-dependent cardiotoxicity. Pyridoxamine (PM) is a vitamin B6 derivative with favorable effects on diverse cardiovascular diseases, suggesting a cardioprotective effect on DOX-induced cardiotoxicity. The cardioprotective nature of PM was investigated in a rat model of DOX-induced cardiotoxicity. Six-week-old female Sprague Dawley rats were treated intravenously with 2 mg/kg DOX or saline (CTRL) weekly for eight weeks. Two other groups received PM via the drinking water next to DOX (DOX+PM) or saline (CTRL+PM). Echocardiography, strain analysis, and hemodynamic measurements were performed to evaluate cardiac function. Fibrotic remodeling, myocardial inflammation, oxidative stress, apoptosis, and ferroptosis were evaluated by various in vitro techniques. PM significantly attenuated DOX-induced left ventricular (LV) dilated cardiomyopathy and limited TGF-ß1-related LV fibrotic remodeling and macrophage-driven myocardial inflammation. PM protected against DOX-induced ferroptosis, as evidenced by restored DOX-induced disturbance of redox balance, improved cytosolic and mitochondrial iron regulation, and reduced mitochondrial damage at the gene level. In conclusion, PM attenuated the development of cardiac damage after DOX treatment by reducing myocardial fibrosis, inflammation, and mitochondrial damage and by restoring redox and iron regulation at the gene level, suggesting that PM may be a novel cardioprotective strategy for DOX-induced cardiomyopathy.

Balancing functions of regulatory T cells in mosquito-borne viral infections.

Mosquito-borne viral infections are on the rise worldwide and can lead to severe symptoms such as haemorrhage, encephalitis, arthritis or microcephaly. A protective immune response following mosquito-borne viral infections requires the generation of a controlled and balanced immune response leading to viral clearance without immunopathology. Here, regulatory T cells play a central role in restoring immune homeostasis. In current review, we aim to provide an overview and summary of the phenotypes of FOXP3+ Tregs in various mosquito-borne arboviral disease, their association with disease severity and their functional characteristics. Furthermore, we discuss the role of cytokines and Tregs in the immunopathogenesis of mosquito-borne infections. Lastly, we discuss possible novel lines of research which could provide additional insight into the role of Tregs in mosquito-borne viral infections in order to develop novel therapeutic approaches or vaccination strategies.

Sodium as an Important Regulator of Immunometabolism.

Salt sensitivity concerns blood pressure alterations after a change in salt intake (sodium chloride). The heart is a pump, and vessels are tubes; sodium can affect both. A high salt intake increases cardiac output, promotes vascular dysfunction and capillary rarefaction, and chronically leads to increased systemic vascular resistance. More recent findings suggest that sodium also acts as an important second messenger regulating energy metabolism and cellular functions. Besides endothelial cells and fibroblasts, sodium also affects innate and adaptive immunometabolism, immune cell function, and influences certain microbes and microbiota-derived metabolites. We propose the idea that the definition of salt sensitivity should be expanded beyond high blood pressure to cellular and molecular salt sensitivity.

Effects of Recombinant IL-13 Treatment on Gut Microbiota Composition and Functional Recovery after Hemisection Spinal Cord Injury in Mice.

In recent years, the gut-central nervous system axis has emerged as a key factor in the pathophysiology of spinal cord injury (SCI). Interleukin-13 (IL-13) has been shown to have anti-inflammatory and neuroprotective effects in SCI. The aim of this study was to investigate the changes in microbiota composition after hemisection injury and to determine whether systemic recombinant (r)IL-13 treatment could alter the gut microbiome, indirectly promoting functional recovery. The gut microbiota composition was determined by 16S rRNA gene sequencing, and correlations between gut microbiota alterations and functional recovery were assessed. Our results showed that there were no changes in alpha diversity between the groups before and after SCI, while PERMANOVA analysis for beta diversity showed significant differences in fecal microbial communities. Phylogenetic classification of bacterial families revealed a lower abundance of the Bacteroidales S24-7 group and a higher abundance of Lachnospiraceae and Lactobacillaceae in the post-SCI group. Systemic rIL-13 treatment improved functional recovery 28 days post-injury and microbiota analysis revealed increased relative abundance of Clostridiales vadin BB60 and Acetitomaculum and decreased Anaeroplasma, Ruminiclostridium_6, and Ruminococcus compared to controls. Functional assessment with PICRUSt showed that genes related to glyoxylate cycle and palmitoleate biosynthesis-I were the predominant signatures in the rIL-13-treated group, whereas sulfolactate degradation super pathway and formaldehyde assimilation-I were enriched in controls. In conclusion, our results indicate that rIL-13 treatment promotes changes in gut microbial communities and may thereby contribute indirectly to the improvement of functional recovery in mice, possibly having important implications for the development of novel treatment options for SCI.

Impact of High Salt-Intake on a Natural Gut Ecosystem in Wildling Mice.

The mammalian holobiont harbors a complex and interdependent mutualistic gut bacterial community. Shifts in the composition of this bacterial consortium are known to be a key element in host health, immunity and disease. Among many others, dietary habits are impactful drivers for a potential disruption of the bacteria-host mutualistic interaction. In this context, we previously demonstrated that a high-salt diet (HSD) leads to a dysbiotic condition of murine gut microbiota, characterized by a decrease or depletion of well-known health-promoting gut bacteria. However, due to a controlled and sanitized environment, conventional laboratory mice (CLM) possess a less diverse gut microbiota compared to wild mice, leading to poor translational outcome for gut microbiome studies, since a reduced gut microbiota diversity could fail to depict the complex interdependent networks of the microbiome. Here, we evaluated the HSD effect on gut microbiota in CLM in comparison to wildling mice, which harbor a natural gut ecosystem more closely mimicking the situation in humans. Mice were treated with either control food or HSD and gut microbiota were profiled using amplicon-based methods targeting the 16S ribosomal gene. In line with previous findings, our results revealed that HSD induced significant loss of alpha diversity and extensive modulation of gut microbiota composition in CLM, characterized by the decrease in potentially beneficial bacteria from Firmicutes phylum such as the genera Lactobacillus, Roseburia, Tuzzerella, Anaerovorax and increase in Akkermansia and Parasutterella. However, HSD-treated wildling mice did not show the same changes in terms of alpha diversity and loss of Firmicutes bacteria as CLM, and more generally, wildlings exhibited only minor shifts in the gut microbiota composition upon HSD. In line with this, 16S-based functional analysis suggested only major shifts of gut microbiota ecological functions in CLM compared to wildling mice upon HSD. Our findings indicate that richer and wild-derived gut microbiota is more resistant to dietary interventions such as HSD, compared to gut microbiota of CLM, which may have important implications for future translational microbiome research.

Fatty acid desaturation by stearoyl-CoA desaturase-1 controls regulatory T cell differentiation and autoimmunity.

The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis (MS). Emerging evidence indicates that endogenous and dietary-induced changes in fatty acid metabolism have a major impact on both T cell fate and autoimmunity. To date, however, the molecular mechanisms that underlie the impact of fatty acid metabolism on T cell physiology and autoimmunity remain poorly understood. Here, we report that stearoyl-CoA desaturase-1 (SCD1), an enzyme essential for the desaturation of fatty acids and highly regulated by dietary factors, acts as an endogenous brake on regulatory T-cell (Treg) differentiation and augments autoimmunity in an animal model of MS in a T cell-dependent manner. Guided by RNA sequencing and lipidomics analysis, we found that the absence of Scd1 in T cells promotes the hydrolysis of triglycerides and phosphatidylcholine through adipose triglyceride lipase (ATGL). ATGL-dependent release of docosahexaenoic acid enhanced Treg differentiation by activating the nuclear receptor peroxisome proliferator-activated receptor gamma. Our findings identify fatty acid desaturation by SCD1 as an essential determinant of Treg differentiation and autoimmunity, with potentially broad implications for the development of novel therapeutic strategies and dietary interventions for autoimmune disorders such as MS.

Sodium perturbs mitochondrial respiration and induces dysfunctional Tregs.

FOXP3+ regulatory T cells (Tregs) are central for peripheral tolerance, and their deregulation is associated with autoimmunity. Dysfunctional autoimmune Tregs display pro-inflammatory features and altered mitochondrial metabolism, but contributing factors remain elusive. High salt (HS) has been identified to alter immune function and to promote autoimmunity. By investigating longitudinal transcriptional changes of human Tregs, we identified that HS induces metabolic reprogramming, recapitulating features of autoimmune Tregs. Mechanistically, extracellular HS raises intracellular Na+, perturbing mitochondrial respiration by interfering with the electron transport chain (ETC). Metabolic disturbance by a temporary HS encounter or complex III blockade rapidly induces a pro-inflammatory signature and FOXP3 downregulation, leading to long-term dysfunction in vitro and in vivo. The HS-induced effect could be reversed by inhibition of mitochondrial Na+/Ca2+ exchanger (NCLX). Our results indicate that salt could contribute to metabolic reprogramming and that short-term HS encounter perturb metabolic fitness and long-term function of human Tregs with important implications for autoimmunity.

Dissecting the role of CSF2RB expression in human regulatory T cells.

Colony stimulating factor 2 receptor subunit beta (CSF2RB; CD131) is the common subunit of the type I cytokine receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5. Interestingly, FOXP3+ regulatory T cells (Tregs), which play a pivotal role in prevention of autoimmunity have been demonstrated to highly overexpress CSF2RB and genome-wide association studies (GWAS) identified CSF2RB as being linked to autoimmune diseases like multiple sclerosis (MS). However, the exact biological role of CD131 in human Tregs has not been defined yet. Here we investigated CD131 importance on Treg phenotype and function in a broad range of in vitro studies. Although we could not recognize a specific function of CSF2RB; CD131 in human Tregs, our data show that CD131 expression is vastly restricted to Tregs even under stimulatory conditions, indicating that CD131 could aid as a potential marker to identify Treg subpopulations from pools of activated CD4+ T cells. Importantly, our analysis further demonstrate the overexpression of CSF2RB in Tregs of patients with autoimmune diseases like MS and systemic lupus erythematosus (SLE) in comparison to healthy controls, thereby indicating that CSF2RB expression in Tregs could serve as a potential novel biomarker for disease.

Phloretin enhances remyelination by stimulating oligodendrocyte precursor cell differentiation.

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Why endogenous repair mechanisms frequently fail in these disorders is poorly understood. However, there is now evidence indicating that this is related to an overly inflammatory microenvironment combined with the intrinsic inability of oligodendrocyte precursor cells (OPCs) to differentiate into mature myelinating cells. Previously, we found that phloretin, a flavonoid abundantly present in apples and strawberries, reduces neuroinflammation by driving macrophages toward an antiinflammatory phenotype. Here, we show that phloretin also markedly stimulates remyelination in ex vivo and in vivo animal models. Improved remyelination was attributed to a direct impact of phloretin on OPC maturation and occurred independently from alterations in microglia function and inflammation. We found, mechanistically, that phloretin acts as a direct ligand for the fatty acid sensing nuclear receptor peroxisome proliferator-activated receptor gamma, thereby promoting the maturation of OPCs. Together, our findings indicate that phloretin has proregenerative properties in central nervous system disorders, with potentially broad implications for the development of therapeutic strategies and dietary interventions aimed at promoting remyelination.

Glutathione-dependent redox balance characterizes the distinct metabolic properties of follicular and marginal zone B cells.

The metabolic principles underlying the differences between follicular and marginal zone B cells (FoB and MZB, respectively) are not well understood. Here we show, by studying mice with B cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that glutathione synthesis affects homeostasis and differentiation of MZB to a larger extent than FoB, while glutathione-dependent redox control contributes to the metabolic dependencies of FoB. Specifically, Gclc ablation in FoB induces metabolic features of wild-type MZB such as increased ATP levels, glucose metabolism, mTOR activation, and protein synthesis. Furthermore, Gclc-deficient FoB have a block in the mitochondrial electron transport chain (ETC) due to diminished complex I and II activity and thereby accumulate the tricarboxylic acid cycle metabolite succinate. Finally, Gclc deficiency hampers FoB activation and antibody responses in vitro and in vivo, and induces susceptibility to viral infections. Our results thus suggest that Gclc is required to ensure the development of MZB, the mitochondrial ETC integrity in FoB, and the efficacy of antiviral humoral immunity.

High-Salt Diet Induces Depletion of Lactic Acid-Producing Bacteria in Murine Gut.

Dietary habits are amongst the main factors that influence the gut microbiome. Accumulating evidence points to the impact of a high-salt diet (HSD) on the composition and function of the intestinal microbiota, immune system and disease. In the present study, we thus investigated the effects of different NaCl content in the food (0.03%/sodium deficient, 0.5%/control, 4% and 10% NaCl) on the gut microbiome composition in mice. The bacterial composition was profiled using the 16S ribosomal RNA (rRNA) gene amplicon sequencing. Our results revealed that HSD led to distinct gut microbiome compositions compared to sodium-deficient or control diets. We also observed significant reduction in relative abundances of bacteria associated with immuno-competent short-chain fatty acid (SCFA) production (Bifidobacterium, Faecalibaculum, Blautia and Lactobacillus) in HSD-fed mice along with significant enrichment of Clostridia, Alistipes and Akkermansia depending on the sodium content in food. Furthermore, the predictive functional profiling of microbial communities indicated that the gut microbiota found in each category presents differences in metabolic pathways related to carbohydrate, lipid and amino acid metabolism. The presented data show that HSD cause disturbances in the ecological balance of the gastrointestinal microflora primarily through depletion of lactic acid-producing bacteria in a dose-dependent manner. These findings may have important implications for salt-sensitive inflammatory diseases.

Skin Sodium Accumulates in Psoriasis and Reflects Disease Severity.

Sodium can accumulate in the skin at concentrations exceeding serum levels. A high sodium environment can lead to pathogenic T helper 17 cell expansion. Psoriasis is a chronic inflammatory skin disease in which IL-17‒producing T helper 17 cells play a crucial role. In an observational study, we measured skin sodium content in patients with psoriasis and in age-matched healthy controls by Sodium-23 magnetic resonance imaging. Patients with PASI > 5 showed significantly higher sodium and water content in the skin but not in other tissues than those with lower PASI or healthy controls. Skin sodium concentrations measured by Sodium-23 spectroscopy or by atomic absorption spectrometry in ashed-skin biopsies verified the findings with Sodium-23 magnetic resonance imaging. In vitro T helper 17 cell differentiation of naive CD4+ cells from patients with psoriasis markedly induced IL-17A expression under increased sodium chloride concentrations. The imiquimod-induced psoriasis mouse model replicated the human findings. Extracellular tracer Chromium-51-EDTA measurements in imiquimod- and sham-treated skin showed similar extracellular volumes, rendering excessive water of intracellular origin. Chronic genetic IL-17A‒driven psoriasis mouse models underlined the role of IL-17A in dermal sodium accumulation and inflammation. Our data describe skin sodium as a pathophysiological feature of psoriasis, which could open new avenues for its treatment.

Improving the Efficacy of Regulatory T Cell Therapy.

Autoimmunity is caused by an unbalanced immune system, giving rise to a variety of organ-specific to system disorders. Patients with autoimmune diseases are commonly treated with broad-acting immunomodulatory drugs, with the risk of severe side effects. Regulatory T cells (Tregs) have the inherent capacity to induce peripheral tolerance as well as tissue regeneration and are therefore a prime candidate to use as cell therapy in patients with autoimmune disorders. (Pre)clinical studies using Treg therapy have already established safety and feasibility, and some show clinical benefits. However, Tregs are known to be functionally impaired in autoimmune diseases. Therefore, ex vivo manipulation to boost and stably maintain their suppressive function is necessary when considering autologous transplantation. Similar to autoimmunity, severe coronavirus disease 2019 (COVID-19) is characterized by an exaggerated immune reaction and altered Treg responses. In light of this, Treg-based therapies are currently under investigation to treat severe COVID-19. This review provides a detailed overview of the current progress and clinical challenges of Treg therapy for autoimmune and hyperinflammatory diseases, with a focus on recent successes of ex vivo Treg manipulation.

Fast and Efficient Genome Editing of Human FOXP3+ Regulatory T Cells.

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.

Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes.

BACKGROUND: Dietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional, and functional adaption of human and murine mononuclear phagocytes. METHODS: Using Seahorse technology, pulsed stable isotope-resolved metabolomics, and enzyme activity assays, we characterize the central carbon metabolism and mitochondrial function of human and murine mononuclear phagocytes under HS in vitro. HS as well as pharmacological uncoupling of the electron transport chain under normal salt is used to analyze mitochondrial function on immune cell activation and function (as determined by Escherichiacoli killing and CD4+ T cell migration capacity). In 2 independent clinical studies, we analyze the effect of a HS diet during 2 weeks (URL: http://www.clinicaltrials.gov. Unique identifier: NCT02509962) and short-term salt challenge by a single meal (URL: http://www.clinicaltrials.gov. Unique identifier: NCT04175249) on mitochondrial function of human monocytes in vivo. RESULTS: Extracellular sodium was taken up into the intracellular compartment, followed by the inhibition of mitochondrial respiration in murine and human macrophages. Mechanistically, HS reduces mitochondrial membrane potential, electron transport chain complex II activity, oxygen consumption, and ATP production independently of the polarization status of macrophages. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like macrophages and diminished CD4+ T cell migration in HS-treated M2-like macrophages. Pharmacological uncoupling of the electron transport chain under normal salt phenocopies HS-induced transcriptional changes and bactericidal function of human and murine mononuclear phagocytes. Clinically, also in vivo, rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of [Formula: see text] and [Formula: see text] respectively, that correlated with decreased monocytic mitochondrial oxygen consumption. CONCLUSIONS: Our data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. Although these functional changes might help to resolve bacterial infections, a shift toward proinflammation could accelerate inflammatory cardiovascular disease.

Fasting alters the gut microbiome reducing blood pressure and body weight in metabolic syndrome patients.

Periods of fasting and refeeding may reduce cardiometabolic risk elevated by Western diet. Here we show in the substudy of NCT02099968, investigating the clinical parameters, the immunome and gut microbiome exploratory endpoints, that in hypertensive metabolic syndrome patients, a 5-day fast followed by a modified Dietary Approach to Stop Hypertension diet reduces systolic blood pressure, need for antihypertensive medications, body-mass index at three months post intervention compared to a modified Dietary Approach to Stop Hypertension diet alone. Fasting alters the gut microbiome, impacting bacterial taxa and gene modules associated with short-chain fatty acid production. Cross-system analyses reveal a positive correlation of circulating mucosa-associated invariant T cells, non-classical monocytes and CD4+ effector T cells with systolic blood pressure. Furthermore, regulatory T cells positively correlate with body-mass index and weight. Machine learning analysis of baseline immunome or microbiome data predicts sustained systolic blood pressure response within the fasting group, identifying CD8+ effector T cells, Th17 cells and regulatory T cells or Desulfovibrionaceae, Hydrogenoanaerobacterium, Akkermansia, and Ruminococcaceae as important contributors to the model. Here we report that the high-resolution multi-omics data highlight fasting as a promising non-pharmacological intervention for the treatment of high blood pressure in metabolic syndrome patients.