Dysregulation of Systemic Immunity in Aging and Dementia

Neuroinflammation and the tissue-resident innate immune cells, the microglia, respond and contribute to neurodegenerative pathology. Although microglia have been the focus of work linking neuroinflammation and associated dementias like Alzheimer’s Disease, the inflammatory milieu of brain is a conglomerate of cross-talk amongst microglia, systemic immune cells and soluble mediators like cytokines. Age-related changes in the inflammatory profile at the levels of both the brain and periphery are largely orchestrated by immune system cells. Strong evidence indicates that both innate and adaptive immune cells, the latter including T cells and B cells, contribute to chronic neuroinflammation and thus dementia. Neurodegenerative hallmarks coupled with more traditional immune system stimuli like infection or injury likely combine to trigger and maintain persistent microglial and thus brain inflammation. This review summarizes age-related changes in immune cell function, with special emphasis on lymphocytes as a source of inflammation, and discusses how such changes may potentiate both systemic and central nervous system inflammation to culminate in dementia. We recap the understudied area of AD-associated changes in systemic lymphocytes in greater detail to provide a unifying perspective of inflammation-fueled dementia, with an eye toward evidence of two-way communication between the brain parenchyma and blood immune cells. We focused our review on human subjects studies, adding key data from animal models as relevant

Dysregulation of Systemic Immunity in Aging and Dementia

Neuroinflammation and the tissue-resident innate immune cells, the microglia, respond and contribute to neurodegenerative pathology. Although microglia have been the focus of work linking neuroinflammation and associated dementias like Alzheimer’s Disease, the inflammatory milieu of brain is a conglomerate of cross-talk amongst microglia, systemic immune cells and soluble mediators like cytokines. Age-related changes in the inflammatory profile at the levels of both the brain and periphery are largely orchestrated by immune system cells. Strong evidence indicates that both innate and adaptive immune cells, the latter including T cells and B cells, contribute to chronic neuroinflammation and thus dementia. Neurodegenerative hallmarks coupled with more traditional immune system stimuli like infection or injury likely combine to trigger and maintain persistent microglial and thus brain inflammation. This review summarizes age-related changes in immune cell function, with special emphasis on lymphocytes as a source of inflammation, and discusses how such changes may potentiate both systemic and central nervous system inflammation to culminate in dementia. We recap the understudied area of AD-associated changes in systemic lymphocytes in greater detail to provide a unifying perspective of inflammation-fueled dementia, with an eye toward evidence of two-way communication between the brain parenchyma and blood immune cells. We focused our review on human subjects studies, adding key data from animal models as relevant

Saturated Fatty Acid Activates T Cell Inflammation Through a Nicotinamide Nucleotide Transhydrogenase (NNT)-Dependent Mechanism

Circulating fatty acids (FAs) increase with obesity and can drive mitochondrial damage and inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that positively regulates nicotinamide adenine dinucleotide phosphate (NADPH), a key mediator of energy transduction and redox homeostasis. The role that NNT-regulated bioenergetics play in the inflammatory response of immune cells in obesity is untested. Our objective was to determine how free fatty acids (FFAs) regulate inflammation through impacts on mitochondria and redox homeostasis of peripheral blood mononuclear cells (PBMCs). PBMCs from lean subjects were activated with a T cell-specific stimulus in the presence or absence of generally pro-inflammatory palmitate and/or non-inflammatory oleate. Palmitate decreased immune cell expression of NNT, NADPH, and anti-oxidant glutathione, but increased reactive oxygen and proinflammatory Th17 cytokines. Oleate had no effect on these outcomes. Genetic inhibition of NNT recapitulated the effects of palmitate. PBMCs from obese (BMI \u3e 30) compared to lean subjects had lower NNT and glutathione expression, and higher Th17 cytokine expression, none of which were changed by exogenous palmitate. Our data identify NNT as a palmitate-regulated rheostat of redox balance that regulates immune cell function in obesity and suggest that dietary or therapeutic strategies aimed at increasing NNT expression may restore redox balance to ameliorate obesity-associated inflammation

Saturated Fatty Acid Activates T Cell Inflammation Through a Nicotinamide Nucleotide Transhydrogenase (NNT)-Dependent Mechanism

Circulating fatty acids (FAs) increase with obesity and can drive mitochondrial damage and inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that positively regulates nicotinamide adenine dinucleotide phosphate (NADPH), a key mediator of energy transduction and redox homeostasis. The role that NNT-regulated bioenergetics play in the inflammatory response of immune cells in obesity is untested. Our objective was to determine how free fatty acids (FFAs) regulate inflammation through impacts on mitochondria and redox homeostasis of peripheral blood mononuclear cells (PBMCs). PBMCs from lean subjects were activated with a T cell-specific stimulus in the presence or absence of generally pro-inflammatory palmitate and/or non-inflammatory oleate. Palmitate decreased immune cell expression of NNT, NADPH, and anti-oxidant glutathione, but increased reactive oxygen and proinflammatory Th17 cytokines. Oleate had no effect on these outcomes. Genetic inhibition of NNT recapitulated the effects of palmitate. PBMCs from obese (BMI \u3e30) compared to lean subjects had lower NNT and glutathione expression, and higher Th17 cytokine expression, none of which were changed by exogenous palmitate. Our data identify NNT as a palmitate-regulated rheostat of redox balance that regulates immune cell function in obesity and suggest that dietary or therapeutic strategies aimed at increasing NNT expression may restore redox balance to ameliorate obesity-associated inflammation

Regulatory T Cells Control Effector T Cell Inflammation in Human Prediabetes

A disparate array of plasma/serum markers provides evidence for chronic inflammation in human prediabetes, a condition that is most closely replicated by standard mouse models of obesity and metaflammation. These remain largely nonactionable and contrast with our rich understanding of inflammation in human type 2 diabetes. New data show that inflammatory profiles produced by CD4+ T cells define human prediabetes as a unique inflammatory state. Regulatory T cells (Treg) control mitochondrial function and cytokine production by CD4+ effector T cells (Teff) in prediabetes and type 2 diabetes by supporting T helper (Th)17 or Th1 cytokine production, respectively. These data suggest that Treg control of Teff metabolism regulates inflammation differentially in prediabetes compared with type 2 diabetes. Queries of genes that impact mitochondrial function or pathways leading to transcription of lipid metabolism genes identified the fatty acid importer CD36 as highly expressed in Treg but not Teff from subjects with prediabetes. Pharmacological blockade of CD36 in Treg from subjects with prediabetes decreased Teff production of the Th17 cytokines that differentiate overall prediabetes inflammation. We conclude that Treg control CD4+ T cell cytokine profiles through mechanisms determined, at least in part, by host metabolic status. Furthermore, Treg CD36 uniquely promotes Th17 cytokine production by Teff in prediabetes

Type 1 Diabetes Alters Lipid Handling and Metabolism in Human Fibroblasts and Peripheral Blood Mononuclear Cells

Triggers of the autoimmune response that leads to type 1 diabetes (T1D) remain poorly understood. A possibility is that parallel changes in both T cells and target cells provoke autoimmune attack. We previously documented greater Ca2+ transients in fibroblasts from T1D subjects than non-T1D after exposure to fatty acids (FA) and tumor necrosis factor α (TNFα). These data indicate that metabolic and signal transduction defects present in T1D can be elicited ex vivo in isolated cells. Changes that precede T1D, including inflammation, may activate atypical responses in people that are genetically predisposed to T1D. To identify such cellular differences in T1D, we quantified a panel of metabolic responses in fibroblasts and peripheral blood cells (PBMCs) from age-matched T1D and non-T1D subjects, as models for non-immune and immune cells, respectively. Fibroblasts from T1D subjects accumulated more lipid, had higher LC-CoA levels and converted more FA to CO2, with less mitochondrial proton leak in response to oleate alone or with TNFα, using the latter as a model of inflammation. T1D-PBMCs contained and also accumulated more lipid following FA exposure. In addition, they formed more peroxidized lipid than controls following FA exposure. We conclude that both immune and non-immune cells in T1D subjects differ from controls in terms of responses to FA and TNFα. Our results suggest a differential sensitivity to inflammatory insults and FA that may precede and contribute to T1D by priming both immune cells and their targets for autoimmune reactions

14-color flow cytometry to determine the contribution of mitochondrial mass to differences in glycolytic capacity in human immune cell subsets

Mitochondrial metabolism controls immune cell function, but comprehensive tools to assess human primary immune cell metabolic capacity remain rudimentary. We previously demonstrated that CD19+ B cells rely more heavily on anaerobic glycolysis (i.e. are more glycolytic) than CD4+ T cells. Furthermore, both PBMCs and CD4+ T cells from subjects with type 2 diabetes (T2D) are more glycolytic than their counterparts from BMI-matched non-T2D controls. The contribution of mitochondrial mass, an indicator of non-glycolytic metabolism, to the various metabolic phenotypes is untested. To assess the contribution of immune cell subset identity and mitochondrial mass to the enhanced glycolytic capacity of resting B cells and PBMCs from T2D subjects, we designed a 13-color panel based on standard immune cell subset markers and chemokine receptors, and included MitoTracker Green FM (MTG), which quantitatively indicates mitochondrial mass. We used this novel panel to phenotype 63 total samples from BMI-matched subjects in three groups: non-T2D, pre-T2D, and fulminant T2D, as defined by American Diabetes Association guidelines. The panel was built in several iterations to accommodate spillover of MTG fluorescence into neighboring channels and includes, besides MTG and live-dead discriminator, the following surface markers: CD4, CD8, CD19, CD45RA, CD25, CD127, CD14, CCR4, CCR5, CCR6, CXCR3, and CD161. The PBMC samples were run on a 4-laser BD FACSARIA II SORP with pre-established panel-specific PMT voltages tracked using 6-peak Ultrarainbow beads. To normalize MTG fluorescence intensity and thus minimize batch effects, each of 5 total batches included a reference donor PBMC sample that was frozen in multiple aliquots from one blood draw. Using this approach, we quantified the percentages of immune cell populations (CD19+ B cells, CD8+ naïve and memory/effector T cells, and CD4+ cells including Tregs and populations enriched in Th1, Th2 and Th17) along with the relative mitochondrial mass in each subset. We found that CD19+ B cells in PBMCs from both ND and T2D subjects had significantly less mitochondrial mass than CD4+ cells, supporting the demonstration that B cells are more glycolytic than CD4+ T cells. Of all the CD4+ T cell subsets, Th17 cells consistently had the lowest mitochondrial mass, consistent with the interpretation that Th17s are more dependent on glycolysis than previously appreciated. Our results validate the utility of our 13-color panel to simultaneously quantify relative mitochondrial mass in numerous immune cell subsets and thereby provide a new tool to explore metabolism in human primary cells

Obesity and Fatty Acids Promote Mitochondrial Translocation of STAT3 Through ROS-Dependent Mechanisms

Obesity promotes the onset and progression of metabolic and inflammatory diseases such as type 2 diabetes. The chronic low-grade inflammation that occurs during obesity triggers multiple signaling mechanisms that negatively affect organismal health. One such mechanism is the persistent activation and mitochondrial translocation of STAT3, which is implicated in inflammatory pathologies and many types of cancers. STAT3 in the mitochondria (mitoSTAT3) alters electron transport chain activity, thereby influencing nutrient metabolism and immune response. PBMCs and CD4+ T cells from obese but normal glucose-tolerant (NGT) middle-aged subjects had higher phosphorylation of STAT3 on residue serine 727 and more mitochondrial accumulation of STAT3 than cells from lean subjects. To evaluate if circulating lipid overabundance in obesity is responsible for age- and sex-matched mitoSTAT3, cells from lean subjects were challenged with physiologically relevant doses of the saturated and monounsaturated fatty acids, palmitate and oleate, respectively. Fatty acid treatment caused robust accumulation of mitoSTAT3 in all cell types, which was independent of palmitate-induced impairments in autophagy. Co-treatment of cells with fatty acid and trehalose prevented STAT3 phosphorylation and mitochondrial accumulation in an autophagy-independent but cellular peroxide–dependent mechanism. Pharmacological blockade of mitoSTAT3 either by a mitochondria-targeted STAT3 inhibitor or ROS scavenging prevented obesity and fatty acid–induced production of proinflammatory cytokines IL-17A and IL-6, thus establishing a mechanistic link between mitoSTAT3 and inflammatory cytokine production

Fatty Acid Metabolites Combine with Reduced β Oxidation to Activate Th17 Inflammation in Human Type 2 Diabetes

Mechanisms that regulate metabolites and downstream energy generation are key determinants of T cell cytokine production, but the processes underlying the Th17 profile that predicts the metabolic status of people with obesity are untested. Th17 function requires fatty acid uptake, and our new data show that blockade of CPT1A inhibits Th17-associated cytokine production by cells from people with type 2 diabetes (T2D). A low CACT:CPT1A ratio in immune cells from T2D subjects indicates altered mitochondrial function and coincides with the preference of these cells to generate ATP through glycolysis rather than fatty acid oxidation. However, glycolysis was not critical for Th17 cytokines. Instead, β oxidation blockade or CACT knockdown in T cells from lean subjects to mimic characteristics of T2D causes cells to utilize 16C-fatty acylcarnitine to support Th17 cytokines. These data show long-chain acylcarnitine combines with compromised β oxidation to promote disease-predictive inflammation in human T2D. Although glycolysis generally fuels inflammation, Nicholas, Proctor, and Agrawal et al. report that PBMCs from subjects with type 2 diabetes use a different mechanism to support chronic inflammation largely independent of fuel utilization. Loss- and gain-of-function experiments in cells from healthy subjects show mitochondrial alterations combine with increases in fatty acid metabolites to drive chronic T2D-like inflammation

Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation

Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes