Sanjay K. Garg

Extracellular redox modulation by regulatory T cells.

We demonstrate that the mechanism of redox remodeling during mouse T cell activation involves secretion of glutathione by dendritic cells and its subsequent cleavage to cysteine. Extracellular cysteine accumulation results in a lower redox potential, which is conducive to proliferation, and changes the net redox status of exofacial protein domains. Regulatory T cells inhibit this redox metabolite-signaling pathway, which represents a previously unrecognized mechanism for immunosuppression of effector T cells.

Nitrated alpha-synuclein and microglial neuroregulatory activities.

Microglial neuroinflammatory responses affect the onset and progression of Parkinson’s disease (PD). We posit that such neuroinflammatory responses are, in part, mediated by microglial interactions with nitrated and aggregated α-synuclein (α-syn) released from Lewy bodies as a consequence of dopaminergic neuronal degeneration. As disease progresses, secretions from α-syn-activated microglia can engage neighboring glial cells in a cycle of autocrine and paracrine amplification of neurotoxic immune products. Such pathogenic processes affect the balance between a microglial neurotrophic and neurotoxic signature. We now report that microglia secrete both neurotoxic and neuroprotective factors after exposure to nitrated α-syn (N-α-syn). Proteomic (surface enhanced laser desorption–time of flight, 1D sodium dodecyl sulfate electrophoresis, and liquid chromatography-tandem mass spectrometry) and limited metabolomic profiling demonstrated that N-α-syn-activated microglia secrete inflammatory, regulatory, redox-active, enzymatic, and cytoskeletal proteins. Increased extracellular glutamate and cysteine and diminished intracellular glutathione and secreted exosomal proteins were also demonstrated. Increased redox-active proteins suggest regulatory microglial responses to N-α-syn. These were linked to discontinuous cystatin expression, cathepsin activity, and nuclear factor-kappa B activation. Inhibition of cathepsin B attenuated, in part, N-α-syn microglial neurotoxicity. These data support multifaceted microglia functions in PD-associated neurodegeneration.

Aging Is Associated with an Increase in T Cells and Inflammatory Macrophages in Visceral Adipose Tissue

Age-related adiposity has been linked to chronic inflammatory diseases in late life. To date, the studies on adipose tissue leukocytes and aging have not taken into account the heterogeneity of adipose tissue macrophages (ATMs), nor have they examined how age impacts other leukocytes such as T cells in fat. Therefore, we have performed a detailed examination of ATM subtypes in young and old mice using state of the art techniques. Our results demonstrate qualitative changes in ATMs with aging that generate a decrease in resident type 2 (M2) ATMs. The profile of ATMs in old fat shifts toward a proinflammatory environment with increased numbers of CD206−CD11c− (double-negative) ATMs. The mechanism of this aging-induced shift in the phenotypic profile of ATMs was found to be related to a decrease in peroxisome proliferator-activated receptor-γ expression in ATMs and alterations in chemokine/chemokine receptor expression profiles. Furthermore, we have revealed a profound and unexpected expansion of adipose tissue T cells in visceral fat with aging that includes a significant induction of regulatory T cells in fat. Our findings demonstrate a unique inflammatory cell signature in the physiologic context of aging adipose tissue that differs from those induced in setting of diet-induced obesity.

Regulatory T cells interfere with glutathione metabolism in dendritic cells and T cells

Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (Tregs) suppress proliferation of CD4+CD25− effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation.

Na+ and K+ ion imbalances in Alzheimer's disease

Alzheimers disease (AD) is associated with impaired glutamate clearance and depressed Na(+)/K(+) ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na(+)] and [K(+)] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na(+)] in frontal (25%) and parietal cortex (20%) and in cerebellar [K(+)] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na(+)] and an 8-15% increase in intracellular [K(+)]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25-35 peptide increases intracellular levels of Na(+) (~2-3-fold) and K(+) (~1.5-fold), which were associated with reduced levels of Na(+)/K(+) ATPase and the Na(+)-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na(+) and K(+) levels were also caused by Aβ 1-40, but not by Aβ 1-42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.

Taurine Biosynthesis by Neurons and Astrocytes

The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [35S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [35S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.

The undertow of sulfur metabolism on glutamatergic neurotransmission

Metabolic interdependence between specialized cells in an organ represents a strategy for energy economy by requiring expression of only a subset of pathway genes in a given cell type. In brain, sulfur metabolism exemplifies this principle of metabolic cooperation between glial and neuronal cells and furnishes three key reagents: S-adenosylmethionine, glutathione and taurine. The pathways for glutathione and taurine syntheses depend on metabolic integration between astrocytes and neurons and intersect with the glutamine-glutamate cycle, which underlies glutamatergic synaptic transmission and requires cooperation between these cell types. We propose that underlying waves of glutamate clearance by astrocytes are activation of cystine import and taurine efflux that result, respectively, from a shared transporter and an increase in solute concentration that triggers osmoregulatory responses.

System xc- regulates microglia and macrophage glutamate excitotoxicity in vivo

It is widely believed that microglia and monocyte-derived macrophages (collectively referred to as central nervous system (CNS) macrophages) cause excitotoxicity in the diseased or injured CNS. This view has evolved mostly from in vitro studies showing that neurotoxic concentrations of glutamate are released from CNS macrophages stimulated with lipopolysaccharide (LPS), a potent inflammogen. We hypothesized that excitotoxic killing by CNS macrophages is more rigorously controlled in vivo, requiring both the activation of the glutamate/cystine antiporter (system x(c)(-)) and an increase in extracellular cystine, the substrate that drives glutamate release. Here, we show that non-traumatic microinjection of low-dose LPS into spinal cord gray matter activates CNS macrophages but without causing overt neuropathology. In contrast, neurotoxic inflammation occurs when LPS and cystine are co-injected. Simultaneous injection of NBQX, an antagonist of AMPA glutamate receptors, reduces the neurotoxic effects of LPS+cystine, implicating glutamate as a mediator of neuronal cell death in this model. Surprisingly, neither LPS nor LPS+cystine adversely affects survival of oligodendrocytes or oligodendrocyte progenitor cells. Ex vivo analyses show that redox balance in microglia and macrophages is controlled by induction of system x(c)(-) and that high GSH:GSSG ratios predict the neurotoxic potential of these cells. Together, these data indicate that modulation of redox balance in CNS macrophages, perhaps through regulating system x(c)(-), could be a novel approach for attenuating injurious neuroinflammatory cascades.

Aging is associated with increased regulatory T-cell function

Regulatory T‐cell (Treg, CD4+CD25+) dysfunction is suspected to play a key role in immune senescence and contributes to increased susceptibility to diseases with age by suppressing T‐cell responses. FoxP3 is a master regulator of Treg function, and its expression is under control of several epigenetically labile promoters and enhancers. Demethylation of CpG sites within these regions is associated with increased FoxP3 expression and development of a suppressive phenotype. We examined differences in FoxP3 expression between young (3–4 months) and aged (18–20 months) C57BL/6 mice. DNA from CD4+ T cells is hypomethylated in aged mice, which also exhibit increased Treg numbers and FoxP3 expression. Additionally, Treg from aged mice also have greater ability to suppress effector T‐cell (Teff) proliferation in vitro than Tregs from young mice. Tregs from aged mice exhibit greater redox remodeling–mediated suppression of Teff proliferation during coculture with DCs by decreasing extracellular cysteine availability to a greater extent than Tregs from young mice, creating an adverse environment for Teff proliferation. Tregs from aged mice produce higher IL‐10 levels and suppress CD86 expression on DCs more strongly than Tregs from young mice, suggesting decreased T‐cell activity. Taken together, these results reveal a potential mechanism of higher Treg‐mediated activity that may contribute to increased immune suppression with age.

IFN-γ and IL-4 differentially shape metabolic responses and neuroprotective phenotype of astrocytes

Astrocytes can either exacerbate or ameliorate secondary degeneration at sites of injury in the CNS but the contextual basis for eliciting these opposing phenotypes is poorly understood. In this study, we demonstrate that the two major cytokines produced by Th1 and Th2 cells, interferon‐γ (IFN‐γ), and interleukin‐4 (IL‐4), respectively, contribute differentially to shaping a neuroprotective response in astrocytes. While IFN‐γ protects the ability of oxidatively stressed murine astrocytes to clear extracellular glutamate in culture, IL‐4 has no effect at any concentration that was tested (10–100 ng/mL). The enhanced release of neuroprotective thiols and lactate by astrocytes in response to T cell stimulation is mimicked by both IL‐4 and IFN‐γ. When co‐administered, IL‐4 abrogated the protective effect of low IFN‐γ on the glutamate clearance function of oxidatively stressed astrocytes in a dose‐dependent manner. Astrocyte‐conditioned media obtained from cells cultured in the presence of IL‐4 (10 or 100 ng/mL) or IFN‐γ (10 ng/mL) decreased by ∼2‐fold, neuronal apoptosis induced by oxidative stress in vitro. However, unlike IL‐4, IFN‐γ at high concentrations (100 ng/mL) was not neuroprotective. Our studies with IFN‐γ and IL‐4 suggest that a balanced Th1 and Th2 cytokine response might be needed for protecting two key astrocytic functions, glutamate clearance and thiol secretion and might be pertinent to neuroprotective approaches that are aimed at inhibition of an initial pro‐inflammatory response to injury or its sustained boosting.