Peroxisome Proliferator-Activated Receptor-δ Acts within Peripheral Myeloid Cells to Limit Th Cell Priming during Experimental Autoimmune Encephalomyelitis.

Peroxisome proliferator-activated receptor (PPAR)-δ is a fatty acid-activated transcription factor that regulates metabolic homeostasis, cell growth, and differentiation. Previously, we reported that mice with a global deficiency of PPAR-δ develop an exacerbated course of experimental autoimmune encephalomyelitis (EAE), highlighting a role for this nuclear receptor in limiting the development of CNS inflammation. However, the cell-specific contribution of PPAR-δ to the more severe CNS inflammatory response remained unclear. In this study, we studied the specific involvement of PPAR-δ in myeloid cells during EAE using mice that had Cre-mediated excision of floxed Ppard driven by the lysozyme M (LysM) promoter (LysM Cre :Ppard fl/fl). We observed that LysM Cre :Ppard fl/fl mice were more susceptible to EAE and developed a more severe course of this disease compared with Ppard fl/fl controls. The more severe EAE in LysM Cre :Ppard fl/fl mice was associated with an increased accumulation of pathogenic CD4+ T cells in the CNS and enhanced myelin-specific Th1 and Th17 responses in the periphery. Adoptive transfer EAE studies linked this EAE phenotype in LysM Cre :Ppard fl/fl mice to heightened Th responses. Furthermore, studies using an in vitro CD11b+ cell:Th cell coculture system revealed that CD11b+CD11c+ dendritic cells (DC) from LysM Cre :Ppard fl/fl mice had a heightened capacity to prime myelin oligodendrocyte glycoprotein (MOG)-specific Th cells compared with Ppard fl/fl counterparts; the effects of DC on Th1 cytokine production were mediated through production of the IL-12p40 homodimer. These studies revealed a role for PPAR-δ in DC in limiting Th cell priming during EAE.

Peroxisome Proliferator-Activated Receptor-δ Supports the Metabolic Requirements of Cell Growth in TCRß-Selected Thymocytes and Peripheral CD4+ T Cells.

During T cell development, progenitor thymocytes undergo a large proliferative burst immediately following successful TCRß rearrangement, and defects in genes that regulate this proliferation have a profound effect on thymus cellularity and output. Although the signaling pathways that initiate cell cycling and nutrient uptake after TCRß selection are understood, less is known about the transcriptional programs that regulate the metabolic machinery to promote biomass accumulation during this process. In this article, we report that mice with whole body deficiency in the nuclear receptor peroxisome proliferator-activated receptor-δ (PPARδmut) exhibit a reduction in spleen and thymus cellularity, with a decrease in thymocyte cell number starting at the double-negative 4 stage of thymocyte development. Although in vivo DNA synthesis was normal in PPARδmut thymocytes, studies in the OP9-delta-like 4 in vitro system of differentiation revealed that PPARδmut double-negative 3 cells underwent fewer cell divisions. Naive CD4+ T cells from PPARδmut mice also exhibited reduced proliferation upon TCR and CD28 stimulation in vitro. Growth defects in PPAR-δ-deficient thymocytes and peripheral CD4+ T cells correlated with decreases in extracellular acidification rate, mitochondrial reserve, and expression of a host of genes involved in glycolysis, oxidative phosphorylation, and lipogenesis. By contrast, mice with T cell-restricted deficiency of Ppard starting at the double-positive stage of thymocyte development, although exhibiting defective CD4+ T cell growth, possessed a normal T cell compartment, pointing to developmental defects as a cause of peripheral T cell lymphopenia in PPARδmut mice. These findings implicate PPAR-δ as a regulator of the metabolic program during thymocyte and T cell growth.

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-γ by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-γ production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-γ production have an impact on IFN-γ responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-γ production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-γ responses to L. monocytogenes and survival of mice from this infection.

Antagonizing Peroxisome Proliferator-Activated Receptor α Activity Selectively Enhances Th1 Immunity in Male Mice.

Females exhibit more robust Th1 responses than males. Our previous work suggested that this sex disparity is a consequence of higher activity of the androgen-induced gene peroxisome proliferator-activated receptor α (PPARα) in male CD4(+) T cells. The objective of this study was to elucidate the cellular and molecular mechanism of how PPARα inhibits Th1 responses in male mice. In this study, we found that PPARα functions within CD4(+) and CD8(+) T lymphocytes and NKT cells to negatively regulate IFN-γ responses in male mice and identified Ifng as the gene target of PPARα repression. Treatment of male CD4(+) T cells with the PPARα agonist fenofibrate induced the recruitment of PPARα and the nuclear receptor-interacting protein, nuclear receptor corepressor 1, to specific cis-regulatory elements in the Ifng locus. This recruitment associated with reduced histone acetylation at these sites. Knockdown of nuclear receptor corepressor 1 in primary male T cells abolished the effect of fenofibrate in reducing IFN-γ production. In contrast, treatment of male T cells with IS001, a novel antagonist of PPARα, increased Ifng gene expression and histone acetylation across the Ifng locus. Finally, we investigated the effects of IS001 on IFN-γ responses in mice during infection with the Th1-associated pathogen Listeria monocytogenes and observed that IS001 enhanced IFN-γ production by NKT, CD4(+), and CD8(+) T cells and improved the survival of male, but not female, mice. Our findings provide a novel mechanism of why IFN-γ responses are more robust in females and introduce a small-molecule IS001 that can be used to enhance Th1 immunity in males.

Puberty in females enhances the risk of an outcome of multiple sclerosis in children and the development of central nervous system autoimmunity in mice.

BACKGROUND: For reasons that remain unclear, three times more women develop multiple sclerosis (MS) than men. This preponderance among women is evident only after 12 years of age, implicating pubertal factors in the risk of MS. OBJECTIVE: To investigate the influence of female puberty on central nervous system (CNS) autoimmunity. METHODS: We examined the relationship between age of menarche on MS outcomes in 116 female children (< 16 years old) whom presented with incident 'acquired demyelinating syndromes' (ADS) and were followed prospectively in the national Canadian Pediatric Demyelinating Disease Study, from 2004-2013. Furthermore, we directly investigated the effects of puberty on susceptibility to experimental autoimmune encephalomyelitis (EAE) in two groups of female mice that differed only in their pubertal status. RESULTS: In the ADS children, a later age of menarche was associated with a decreased risk of subsequent MS diagnosis. This relationship persisted, after accounting for patient age at ADS presentation and the presence of ≥1 T2 lesions on brain magnetic resonance imaging (MRI), with a hazard ratio (HR) of 0.64; and additional factors that associate with MS outcomes in ADS children, including low vitamin D levels. Furthermore, we found female mice that had transitioned through puberty were more susceptible to EAE than age-matched, pre-pubertal mice. CONCLUSION: Puberty in females enhances CNS autoimmune mechanisms that lead to MS in humans and EAE in mice.