Macrophages are activated during microbial infection to coordinate inflammatory responses and host defense. Here we found that in macrophages activated by bacterial lipopolysaccharide (LPS), mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) regulated glucose oxidation to drive inflammatory responses. GPD2, a component of the glycerol phosphate shuttle, boosted glucose oxidation to fuel the production of acetyl-coA, acetylation of histones and induction of genes encoding inflammatory mediators. While acute exposure to LPS drove macrophage activation, prolonged exposure to LPS triggered tolerance to LPS, in which macrophages induce immunosuppression to limit the detrimental effects of sustained inflammation. The shift in the inflammatory response was modulated by GPD2, which coordinated a shutdown of oxidative metabolism; this limited the availability of acetyl-coA for histone acetylation at genes encoding inflammatory mediators and thus contributed to the suppression of inflammatory responses. Therefore, GPD2 and the glycerol-phosphate shuttle integrate the extent of microbial stimulation with glucose oxidation to balance the beneficial and detrimental effects of the inflammatory response.

Macrophages are found in most tissues of the body, where they have tissue- and context-dependent roles in maintaining homeostasis as well as coordinating adaptive responses to various stresses. Their capacity for specialized functions is controlled by polarizing signals, which activate macrophages by upregulating transcriptional programs that encode distinct effector functions. An important conceptual advance in the field of macrophage biology, emerging from recent studies, is that macrophage activation is critically supported by metabolic shifts. Metabolic shifts fuel multiple aspects of macrophage activation, and preventing these shifts impairs appropriate activation. These findings raise the exciting possibility that macrophage functions in various contexts could be regulated by manipulating their metabolism. Here, we review the rapidly evolving field of macrophage metabolism, discussing how polarizing signals trigger metabolic shifts and how these shifts enable appropriate activation and sustain effector activities. We also discuss recent studies indicating that the mitochondria are central hubs in inflammatory macrophage activation.

Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Dietary interventions based on protein restriction (PR) reduce circulating triglycerides (TGs), but underlying mechanisms and clinical relevance remain unclear. Here, we show that 1 week of a protein-free diet without enforced calorie restriction significantly lowered circulating TGs in both lean and diet-induced obese mice. Mechanistically, the TG-lowering effect of PR was due, in part, to changes in very low-density lipoprotein (VLDL) metabolism both in liver and peripheral tissues. In the periphery, PR stimulated VLDL-TG consumption by increasing VLDL-bound APOA5 expression and promoting VLDL-TG hydrolysis and clearance from circulation. The PR-mediated increase in Apoa5 expression was controlled by the transcription factor CREBH, which coordinately regulated hepatic expression of fatty acid oxidation-related genes, including Fgf21 and Ppara. The CREBH-APOA5 axis activation upon PR was intact in mice lacking the GCN2-dependent amino acid-sensing arm of the integrated stress response. However, constitutive hepatic activation of the amino acid-responsive kinase mTORC1 compromised CREBH activation, leading to blunted APOA5 expression and PR-recalcitrant hypertriglyceridemia. PR also contributed to hypotriglyceridemia by reducing the rate of VLDL-TG secretion, independently of activation of the CREBH-APOA5 axis. Finally, a randomized controlled clinical trial revealed that 4-6 weeks of reduced protein intake (7%-9% of calories) decreased VLDL particle number, increased VLDL-bound APOA5 expression, and lowered plasma TGs, consistent with mechanistic conservation of PR-mediated hypotriglyceridemia in humans with translational potential as a nutraceutical intervention for dyslipidemia.

Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8+ T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8+ T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8+ T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8+ T cells. Finally, CSFT levels on effector CD8+ T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8+ T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.

Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22-and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4 ؉ T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.A ntibodies are a critical component of the immune system's defense to infectious microorganisms. In order to initiate an antibody response to a pathogen, naïve B cells must first be activated through recognition of antigen by the B cell receptor (BCR). Following antigen stimulation, activated B cells enlist cognate CD4 ϩ T cell help to stimulate clonal expansion (1). Upon activation and proliferation, B cells embark on two distinct differentiation pathways (2). First, the initial production of antibody to a pathogen is accomplished through the differentiation of activated B cells into extrafollicular plasmablasts (3). These short-lived cells are essential in generating low-affinity antibodies early during the infection. However, to generate long-lived humoral immunity, activated B cells must migrate to the germinal center, undergo affinity maturation by somatic hypermutation, and undergo isotype switching to produce memory B cells or antibody-secreting plasma cells (ASC) (3). Memory B cells are long-lived and rapidly respond to pathogen re-encounter by proliferating and differentiating into ASC (4). High-affinity, long-lived ASC migrate to the bone marrow, where they continuously secrete antibody and persist for a year or more in mice (5) and decades in humans (6). Therefore, determining the factors that modulate these pathways is critical not only for understanding the generation and maintenance of serological memory but also for optimizing vaccines and therapeutics for autoimmune disorders.Following antigen receptor ligation, reactive oxygen intermediates (ROI) are generated and required for B cell function (7-9). Previous work has demonstrated that antioxidant treatment decreased lipo...

Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of EpsteinBarr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice, and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8؉ T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4؉ T cells were increased in latently infected mice compared to those in mice infected solely with LCMV. When the memory phase was reached, latently infected mice had an LCMVspecific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8؉ T cells had decreased CD27 and increased killer cell lectin-like receptor G1 (KLRG1) expression. Upon secondary challenge, LCMV-specific secondary effector CD8 ؉ T cells expanded and cleared the infection. However, the LCMV-specific secondary memory CD8 ؉ T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together, these results demonstrate that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8 ؉ T cells during acute infection. IMPORTANCE CD8 ؉ T cells are critical for the clearance of intracellular pathogens, including viruses, certain bacteria, and tumors. However, current models for memory CD8؉ T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella-zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8 ؉ T cells during acute infection. These results suggest that unlike pathogen-free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.

Although T cells play a critical role in protection from viruses, bacteria and tumors, they also cause autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). Unwanted T cell responses during organ transplant, graft versus host disease (GVHD), and allergies are also major clinical problems. While drugs are available to suppress unwanted immune responses they have limited efficacy with serious side effects. Thus new therapeutics limiting T cell activation, proliferation and function can make an immediate clinical impact. To identify new suppressors of lymphocyte activation, proliferation and function, we examined the immunosuppressive activity of gold(I) analogues of platinum-acridine antitumor agents. We found that the gold complex, Au-ACRAMTU-PEt3 is a potent suppressor of murine and human T cell activation. Preincubation with Au-ACRAMTU-PEt3 suppresses the proliferation of CD4+ and CD8+ T cells at a similar concentration as pharmaceutical grade cyclosporine A. Au-ACRAMTU-PEt3 pretreatment decreases the production of IFNγ, TNFα, IL-2, and IL-17 by human and murine CD4+ and CD8+ T cells. When mice were treated with Au-ACRAMTU-PEt3 during viral infection the expansion of virus-specific CD8+ T cells was decreased 10-fold and viral load was elevated. Taken together these results demonstrate that Au-ACRAMTU-PEt3 has potent immunosuppressive activity that could be used to suppress immune responses during transplantation and autoimmunity.

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