Plasma extracellular superoxide dismutase concentration, allelic variations in the SOD3 gene and risk of myocardial infarction and all-cause mortality in people with type 1 and type 2 diabetes.

BACKGROUND: Oxidative stress is involved in development of diabetes complications. Extracellular superoxide dismutase (EC-SOD, SOD3) is a major extracellular antioxidant enzyme and is highly expressed in arterial walls. Advanced oxidation protein products (AOPP) and 8-iso-prostaglandin (isoprostane) are markers of oxidative stress. We investigated association of SOD3 gene variants, plasma concentrations of EC-SOD, AOPP and isoprostane with myocardial infarction and mortality in diabetic patients. METHODS: We studied three cohorts designed to evaluate the vascular complications of diabetes: the GENEDIAB study (469 participants with type 1 diabetes at baseline; follow-up data for 259 participants), the GENESIS study (603 participants with type 1 diabetes at baseline; follow-up data for 525 participants) and the DIABHYCAR study (3137 participants with type 2 diabetes at baseline and follow-up). Duration of follow-up was 9, 5, and 5 years, respectively. Main outcome measures were incidence of myocardial infarction, and cardiovascular and total mortality during follow-up. Six single nucleotide polymorphisms in the SOD3 locus were genotyped in the three cohorts. Plasma concentrations of EC-SOD, AOPP, and isoprostane were measured in baseline samples of GENEDIAB participants. RESULTS: In GENEDIAB/GENESIS pooled cohorts, the minor T-allele of rs2284659 variant was inversely associated with the prevalence at baseline (Odds Ratio 0.48, 95% CI 0.29-0.78, p = 0.004) and the incidence during follow-up of myocardial infarction (Hazard Ratio 0.58, 95% CI 0.40-0.83, p = 0.003) and with cardiovascular (HR 0.33, 95% CI 0.08-0.74, p = 0.004) and all-cause mortality (HR 0.44, 95% CI 0.21-0.73, p = 0.0006). The protective allele was associated with higher plasma EC-SOD and lower plasma AOPP concentrations in GENEDIAB. It was also inversely associated with incidence of myocardial infarction (HR 0.75, 95% CI 0.59-0.94, p = 0.01) and all-cause mortality (HR 0.87, 95% CI 0.79-0.97, p = 0.008) in DIABHYCAR. CONCLUSIONS: The T-allele of rs2284659 in the promoter of SOD3 was associated with a more favorable plasma redox status and with better cardiovascular outcomes in diabetic patients. Our results suggest that EC-SOD plays an important role in the mechanisms of vascular protection against diabetes-related oxidative stress.

Catalase activity, allelic variations in the catalase gene and risk of kidney complications in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Oxidative stress is involved in the pathogenesis of diabetic nephropathy. The antioxidant enzyme catalase plays a key role in redox regulation in the kidney. We investigated associations of catalase gene (CAT) polymorphisms and plasma catalase activity with diabetic nephropathy in type 1 diabetic patients. METHODS: We genotyped nine single nucleotide polymorphisms (SNPs) in the CAT region in participants from the Survival Genetic Nephropathy (SURGENE) (340 French participants, 10 year follow-up) and the Génétique de la Néphropathie Diabétique (GENEDIAB) (444 Belgian and French participants, 8 year follow-up) study cohorts. Replication was performed in a Brazilian cross-sectional cohort (n = 451). Baseline plasma catalase activity was measured in SURGENE (n = 120) and GENEDIAB (n = 391) participants. RESULTS: The A allele of rs7947841 was associated with the prevalence of incipient (OR 2.79, 95% CI 1.21, 6.24, p = 0.01) and established or advanced nephropathy (OR 5.72, 95% CI 1.62, 22.03, p = 0.007), and with the incidence of renal events, which were defined as new cases of microalbuminuria or progression to a more severe stage of nephropathy during follow-up (HR 1.82, 95% CI 1.13, 2.81, p = 0.01) in SURGENE participants. The same risk allele was associated with incipient nephropathy (OR 3.13, 95% CI 1.42, 7.24, p = 0.004) and with the incidence of end-stage renal disease (ESRD) (HR 2.11, 95% CI 1.23, 3.60, p = 0.008) in GENEDIAB participants. In both cohorts, the risk allele was associated with lower catalase activity. Associations with incipient and established or advanced nephropathy were confirmed in the replication cohort. CONCLUSIONS/INTERPRETATION: CAT variants were associated with the prevalence and incidence of diabetic nephropathy and ESRD in type 1 diabetic patients. Our results confirm the protective role of catalase against oxidative stress in the kidney.

Characterization of Dendritic Cell Metabolism by Flow Cytometry.

In response to different stimuli, dendritic cells (DCs) undergo metabolic reprogramming to support their function. Here we describe how fluorescent dyes and antibody-based approaches can be used to assess various metabolic parameters of DCs including glycolysis, lipid metabolism, mitochondrial activity, and the activity of important sensors and regulators of cellular metabolism, mTOR and AMPK. These assays can be performed using standard flow cytometry and will allow for the determination of metabolic properties of DC populations at single-cell level and to characterize metabolic heterogeneity within them.

Metabolic heterogeneity of tissue-resident macrophages in homeostasis and during helminth infection.

Tissue-resident macrophage populations constitute a mosaic of phenotypes, yet how their metabolic states link to the range of phenotypes and functions in vivo is still poorly defined. Here, using high-dimensional spectral flow cytometry, we observe distinct metabolic profiles between different organs and functionally link acetyl CoA carboxylase activity to efferocytotic capacity. Additionally, differences in metabolism are evident within populations from a specific site, corresponding to relative stages of macrophage maturity. Immune perturbation with intestinal helminth infection increases alternative activation and metabolic rewiring of monocyte-derived macrophage populations, while resident TIM4+ intestinal macrophages remain immunologically and metabolically hyporesponsive. Similar metabolic signatures in alternatively-activated macrophages are seen from different tissues using additional helminth models, but to different magnitudes, indicating further tissue-specific contributions to metabolic states. Thus, our high-dimensional, flow-based metabolic analyses indicates complex metabolic heterogeneity and dynamics of tissue-resident macrophage populations at homeostasis and during helminth infection.

Dendritic cell-intrinsic LKB1-AMPK/SIK signaling controls metabolic homeostasis by limiting the hepatic Th17 response during obesity.

Obesity-associated metabolic inflammation drives the development of insulin resistance and type 2 diabetes, notably through modulating innate and adaptive immune cells in metabolic organs. The nutrient sensor liver kinase B1 (LKB1) has recently been shown to control cellular metabolism and T cell priming functions of DCs. Here, we report that hepatic DCs from high-fat diet-fed (HFD-fed) obese mice display increased LKB1 phosphorylation and that LKB1 deficiency in DCs (CD11cΔLKB1) worsened HFD-driven hepatic steatosis and impaired glucose homeostasis. Loss of LKB1 in DCs was associated with increased expression of Th17-polarizing cytokines and accumulation of hepatic IL-17A+ Th cells in HFD-fed mice. Importantly, IL-17A neutralization rescued metabolic perturbations in HFD-fed CD11cΔLKB1 mice. Mechanistically, deficiency of the canonical LKB1 target AMPK in HFD-fed CD11cΔAMPKα1 mice recapitulated neither the hepatic Th17 phenotype nor the disrupted metabolic homeostasis, suggesting the involvement of other and/or additional LKB1 downstream effectors. We indeed provide evidence that the control of Th17 responses by DCs via LKB1 is actually dependent on both AMPKα1 salt-inducible kinase signaling. Altogether, our data reveal a key role for LKB1 signaling in DCs in protection against obesity-induced metabolic dysfunctions by limiting hepatic Th17 responses.

mTORC1 signaling in antigen-presenting cells of the skin restrains CD8+ T cell priming.

How mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of cellular metabolism, affects dendritic cell (DC) metabolism and T cell-priming capacity has primarily been investigated in vitro, but how mTORC1 regulates this in vivo remains poorly defined. Here, using mice deficient for mTORC1 component raptor in DCs, we find that loss of mTORC1 negatively affects glycolytic and fatty acid metabolism and maturation of conventional DCs, particularly cDC1s. Nonetheless, antigen-specific CD8+ T cell responses to infection are not compromised and are even enhanced following skin immunization. This is associated with increased activation of Langerhans cells and a subpopulation of EpCAM-expressing cDC1s, of which the latter show an increased physical interaction with CD8+ T cells in situ. Together, this work reveals that mTORC1 limits CD8+ T cell priming in vivo by differentially orchestrating the metabolism and immunogenicity of distinct antigen-presenting cell subsets, which may have implications for clinical use of mTOR inhibitors.

Increased stromal PFKFB3-mediated glycolysis in inflammatory bowel disease contributes to intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the intestinal tract with currently not well-understood pathogenesis. In addition to the involvement of immune cells, increasing studies show an important role for fibroblasts in the pathogenesis of IBD. Previous work showed that glycolysis is the preferred energy source for fibroblasts in fibrotic diseases. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is a key kinase supporting glycolysis. Increased expression of PFKFB3 in several cancers and inflammatory diseases has been previously reported, but the metabolic status of fibroblasts and the role of PFKFB3 in patients with IBD are currently unknown. Therefore, in this study, we evaluated the role of glycolysis and PFKFB3 expression in IBD. Single-sample gene set enrichment analysis (ssGSEA) revealed that glycolysis was significantly higher in IBD intestinal samples, compared to healthy controls, which was confirmed in the validation cohorts of IBD patients. Single-cell sequencing data indicated that PFKFB3 expression was higher in IBD-derived stromal cells. In vitro, PFKFB3 expression in IBD-derived fibroblasts was increased after the stimulation with pro-inflammatory cytokines. Using seahorse real-time cell metabolic analysis, inflamed fibroblasts were shown to have a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reversed by inhibition of JAK/STAT pathway. Furthermore, increased expression of pro-inflammatory cytokines and chemokines in fibroblasts could be reverted by PFK15, a specific inhibitor of PFKFB3. In vivo experiments showed that PFK15 reduced the severity of dextran sulfate sodium (DSS)- and Tcell transfer induced colitis, which was accompanied by a reduction in immune cell infiltration in the intestines. These findings suggest that increased stromal PFKFB3 expression contributes to inflammation and the pathological function of fibroblasts in IBD. Inhibition of PFKFB3 suppressed their inflammatory characteristics.

Fasciola hepatica Fatty Acid Binding Protein 1 Modulates T cell Polarization by Promoting Dendritic Cell Thrombospondin-1 Secretion Without Affecting Metabolic Homeostasis in Obese Mice.

Background: The parasitic trematode Fasciola hepatica evades host immune defenses through secretion of various immunomodulatory molecules. Fatty Acid Binding Proteins (fhFABPs) are among the main excreted/secreted proteins and have been shown to display anti-inflammatory properties. However, little is currently known regarding their impact on dendritic cells (DCs) and their subsequent capacity to prime specific CD4+ T cell subsets. Methodology/Principal Findings: The immunomodulatory effects of both native F. hepatica extracts and recombinant fhFABPs were assessed on monocyte-derived human DCs (moDCs) and the underlying mechanism was next investigated using various approaches, including DC-allogenic T cell co-culture and DC phenotyping through transcriptomic, proteomic and FACS analyses. We mainly showed that fhFABP1 induced a tolerogenic-like phenotype in LPS-stimulated moDCs characterized by a dose-dependent increase in the cell-surface tolerogenic marker CD103 and IL-10 secretion, while DC co-stimulatory markers were not affected. A significant decrease in secretion of the pro-inflammatory cytokines IL-12p70 and IL-6 was also observed. In addition, these effects were associated with an increase in both Th2-on-Th1 ratio and IL-10 secretion by CD4+ T cells following DC-T cell co-culture. RNA sequencing and targeted proteomic analyses identified thrombospondin-1 (TSP-1) as a non-canonical factor highly expressed and secreted by fhFABP1-primed moDCs. The effect of fhFABP1 on T cell skewing was abolished when using a TSP-1 blocking antibody during DC-T cell co-culture. Immunomodulation by helminth molecules has been linked to improved metabolic homeostasis during obesity. Although fhFABP1 injection in high-fat diet-fed obese mice induced a potent Th2 immune response in adipose tissue, it did not improved insulin sensitivity or glucose homeostasis. Conclusions/Significance: We show that fhFABP1 modulates T cell polarization, notably by promoting DC TSP-1 secretion in vitro, without affecting metabolic homeostasis in a mouse model of type 2 diabetes.

Dendritic cells are what they eat: how their metabolism shapes T helper cell polarization.

Dendritic cells (DCs) are professional antigen-presenting cells that play a crucial role in the priming and differentiation of CD4+ T cells into several distinct subsets including effector T helper (Th) 1, Th17 and Th2 cells, as well as regulatory T cells (Tregs). It is becoming increasingly clear that cellular metabolism shapes the functional properties of DCs. Specifically, the ability of DCs to drive polarization of different Th cell subsets may be orchestrated by the engagement of distinct metabolic pathways. In this review, we will discuss the recent advances in the DC metabolism field, by focusing on how cellular metabolism of DCs shapes their priming and polarization of distinct Th cell responses.

LKB1 expressed in dendritic cells governs the development and expansion of thymus-derived regulatory T cells.

Liver Kinase B1 (LKB1) plays a key role in cellular metabolism by controlling AMPK activation. However, its function in dendritic cell (DC) biology has not been addressed. Here, we find that LKB1 functions as a critical brake on DC immunogenicity, and when lost, leads to reduced mitochondrial fitness and increased maturation, migration, and T cell priming of peripheral DCs. Concurrently, loss of LKB1 in DCs enhances their capacity to promote output of regulatory T cells (Tregs) from the thymus, which dominates the outcome of peripheral immune responses, as suggested by increased resistance to asthma and higher susceptibility to cancer in CD11cΔLKB1 mice. Mechanistically, we find that loss of LKB1 specifically primes thymic CD11b+ DCs to facilitate thymic Treg development and expansion, which is independent from AMPK signalling, but dependent on mTOR and enhanced phospholipase C ß1-driven CD86 expression. Together, our results identify LKB1 as a critical regulator of DC-driven effector T cell and Treg responses both in the periphery and the thymus.

Human Dendritic Cells: Their Heterogeneity and Clinical Application Potential in Cancer Immunotherapy.

Dendritic cells (DC) are professional antigen presenting cells, uniquely able to induce naïve T cell activation and effector differentiation. They are, likewise, involved in the induction and maintenance of immune tolerance in homeostatic conditions. Their phenotypic and functional heterogeneity points to their great plasticity and ability to modulate, according to their microenvironment, the acquired immune response and, at the same time, makes their precise classification complex and frequently subject to reviews and improvement. This review will present general aspects of the DC physiology and classification and will address their potential and actual uses in the management of human disease, more specifically cancer, as therapeutic and monitoring tools. New combination treatments with the participation of DC will be also discussed.

Glutathione peroxidase-1 gene (GPX1) variants, oxidative stress and risk of kidney complications in people with type 1 diabetes.

BACKGROUND AND AIM: Glutathione peroxidase (GPX) is a class of antioxidant enzymes that catalyze the reduction of hydrogen peroxide to water. GPX1 is the most abundant isoform and is expressed in all kidney cells. Isoprostane and advanced oxidation protein products (AOPP) were identified as markers of oxidative stress in patients with kidney disease. We investigated associations of GPX1 genotypes with kidney complications, and with plasma concentrations of isoprostane and AOPP in type 1 diabetic patients. METHODS: Four SNPs in the GPX1 gene region were genotyped in SURGENE (n=340; 10-year follow-up); GENEDIAB (n=461) and GENESIS (n=584) cohorts of type 1 diabetic patients. Subsets of GENEDIAB (n=237) and GENESIS (n=466) participants were followed up for 9 and 5years, respectively. Plasma concentrations of isoprostane and AOPP were measured at baseline in GENEDIAB. Hazard ratios (HR) were estimated for incidence of kidney complications. RESULTS: In SURGENE, 98 renal events (new cases of microalbuminuria or progression to more severe stage of diabetic nephropathy) occurred during follow-up. The minor T-allele of rs3448 was associated with the incidence of renal events (HR 1.81, 95% CI 1.16-2.84, p=0.008). In GENESIS/GENEDIAB pooled study, end stage renal disease (ESRD) occurred during follow-up in 52 individuals. The same variant was associated with the incidence of ESRD (HR 3.34, 95% CI, 1.69-6.98, p=0.0004). The variant was also associated with higher plasma isoprostane concentration in GENEDIAB cohort: 2.02±0.12 (TT+CT) vs 1.75±0.13 (CC) ng/mL (p=0.009), and with higher plasma AOPP in the subset of participants with the baseline history of ESRD (TT+CT 67±6 vs CC 48±6µmol/L, p=0.006). CONCLUSIONS: The minor T-allele of rs3448 was associated with kidney complications (incidences of microalbuminuria, renal events and ESRD) in patients with type 1 diabetes. The risk allele was associated with higher plasma concentrations of isoprostane and AOPP. Our results are consistent with the implication of GPX1 in the mechanism of renal protection against oxidative stress in type 1 diabetic patients.

Allelic variations in the CYBA gene of NADPH oxidase and risk of kidney complications in patients with type 1 diabetes.

Oxidative stress plays a pivotal role in the pathophysiology of diabetic nephropathy, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system is an important source of reactive oxygen species in hyperglycemic conditions in the kidney. Plasma concentration of advanced oxidation protein products (AOPP), a marker of oxidative stress, is increased in patients with diabetic nephropathy. We investigated associations of variants in the CYBA gene, encoding the regulatory subunit p22(phox) of NADPH oxidase, with diabetic nephropathy and plasma AOPP and myeloperoxidase (MPO) concentrations in type 1 diabetic patients. Seven SNPs in the CYBA region were analyzed in 1357 Caucasian subjects with type 1 diabetes from the SURGENE (n=340), GENEDIAB (n=444), and GENESIS (n=573) cohorts. Duration of follow-up was 10, 9, and 6 years, respectively. Cox proportional hazards and logistic regression analyses were used to estimate hazard ratios (HR) or odds ratios (OR) for incidence and prevalence of diabetic nephropathy. The major G-allele of rs9932581 was associated with the incidence of renal events defined as new cases of microalbuminuria or the progression to a more severe stage of nephropathy during follow-up (HR 1.59, 95% CI 1.17-2.18, P=0.003) in SURGENE. The same allele was associated with established/advanced nephropathy (OR 1.52, 95% CI 1.22-1.92, P=0.0001) and with the incidence of end-stage renal disease (ESRD) (HR 2.01, 95% CI 1.30-3.24, P=0.001) in GENEDIAB/GENESIS pooled studies. The risk allele was also associated with higher plasma AOPP concentration in subsets of SURGENE and GENEDIAB, with higher plasma MPO concentration in a subset of GENEDIAB, and with lower estimated glomerular filtration rate (eGFR) in the three cohorts. In conclusion, a functional variant in the promoter of the CYBA gene was associated with lower eGFR and with prevalence and incidence of diabetic nephropathy and ESRD in type 1 diabetic patients. These results are consistent with a role for NADPH oxidase in the pathophysiology of kidney complications of diabetes.

Linkage disequilibrium with HLA-DRB1-DQB1 haplotypes explains the association of TNF-308G>A variant with type 1 diabetes in a Brazilian cohort.

BACKGROUND: A functional variant in the promoter region of the gene encoding tumor necrosis factor (TNF; rs1800629, -308G>A) showed to confer susceptibility to T1D. However, TNF rs1800629 was found, in several populations, to be in linkage disequilibrium with HLA susceptibility haplotypes to T1D. We evaluated the association of TNF rs1800629 with T1D in a cohort of Brazilian subjects, and assessed the impact of HLA susceptibility haplotypes in this association. METHODS: 659 subjects with T1D and 539 control subjects were genotyped for TNF-308G>A variant. HLA-DRB1 and HLA-DQB1 genes were genotyped in a subset of 313 subjects with T1D and 139 control subjects. RESULTS: Associations with T1D were observed for the A-allele of rs1800629 (OR 1.69, 95% CI 1.33-2.15, p<0.0001, in a codominant model) and for 3 HLA haplotypes: DRB1*03:01-DQB1*02:01 (OR 5.37, 95% CI 3.23-8.59, p<0.0001), DRB1*04:01-DQB1*03:02 (OR 2.95, 95% CI 1.21-7.21, p=0.01) and DRB1*04:02-DQB1*03:02 (OR 2.14, 95% CI 1.02-4.50, p=0.04). Linkage disequilibrium was observed between TNF rs1800629 and HLA-DRB1 and HLA-DQB1 alleles. In a stepwise regression analysis HLA haplotypes, but not TNF rs1800629, remained independently associated with T1D. CONCLUSION: Our results do not support an independent effect of allelic variations of TNF in the genetic susceptibility to T1D.