Janet A. Chavis

Female sex steroids: effects upon microglial cell activation.

Multiple sclerosis occurs more commonly in females than males. However, the mechanisms resulting in gender differences in multiple sclerosis are unknown. Activated microglia are believed to contribute to multiple sclerosis pathology, perhaps in part due to production of nitric oxide (NO) and TNF-alpha, molecules which can be toxic to cells including oligodendrocytes. The current study demonstrates that the female sex steroids estriol, beta-estradiol and progesterone inhibit lipopolysaccharide (LPS) induction of nitric oxide (NO) production by primary rat microglia and by the mouse N9 microglial cell line. These hormones act by inhibiting the production of inducible nitric oxide synthase (iNOS) which catalyses the synthesis of NO. Estriol likely inhibits iNOS gene expression since the hormone blocks LPS induction of iNOS RNA levels. The pro-inflammatory cytokines IFN-gamma and TNF-alpha are believed to be important modulators of multiple sclerosis. Here, we demonstrate that estrogens and progesterone also inhibit NO production by microglial cells activated in response to these cytokines. Activated microglia elicit TNF-alpha in addition to NO and we further demonstrate that estrogens and progesterone repress TNF-alpha production by these cells. Finally, estriol and progesterone, at concentrations consistent with late pregnancy, inhibit NO and TNF-alpha production by activated microglia, suggesting that hormone inhibition of microglial cell activation may contribute to the decreased severity of multiple sclerosis symptoms commonly associated with pregnancy.

Agonists for the peroxisome proliferator-activated receptor-α and the retinoid X receptor inhibit inflammatory responses of microglia

The peroxisome proliferator‐activated receptor‐α (PPAR‐α) plays a key role in lipid metabolism and inflammation. Recently, we demonstrated that administration of the PPAR‐α agonists gemfibrozil and fenofibrate, inhibit the clinical signs of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). In the present study we investigated the effects of PPAR‐α agonists on primary mouse microglia, a cell type implicated in the pathology of MS and EAE. Our studies demonstrated that the PPAR‐α agonists ciprofibrate, fenofibrate, gemfibrozil, and WY 14,643 each inhibited NO production by cytokine‐stimulated microglia in a dose‐dependent manner. However, fenofibrate and WY 14,643 were more potent inhibitors than gemfibrozil and ciprofibrate. In LPS‐stimulated microglia, only fenofibrate and WY 14,643 significantly suppressed NO production. Additionally, PPAR‐α agonists inhibited the secretion of the proinflammatory cytokines IL‐1β, TNF‐α, IL‐6, and IL‐12 p40 and the chemokine MCP‐1 by LPS‐stimulated microglia. Retinoid X receptors (RXRs) physically interact with PPAR‐α receptors, and the resulting heterodimers regulate the expression of PPAR‐responsive genes. Interestingly, the RXR agonist 9‐cis retinoic acid (9‐cis RA) inhibited NO production by LPS‐stimulated microglia. Furthermore, a combination of 9‐cis RA and the PPAR‐α agonist fenofibrate cooperatively inhibited NO production by these cells. A combination of these agonists also selectively inhibited the expression of proinflammatory cytokines including IL‐1β, TNF‐α, and IL‐6 by LPS‐stimulated microglia. Collectively, these results raise the possibility that PPAR‐α and RXR agonists might have benefit as a therapy in MS, where activated microglia are believed to contribute to disease pathology.

Peroxisome proliferator-activated receptor agonist regulation of glial activation: Relevance to CNS inflammatory disorders

Peroxisome proliferator-activated receptors (PPARs) play key roles in lipid metabolism and inflammation. Recent studies indicated that PPARs are also capable of modulating immune responses. Microglia and astrocytes are cells resident to the central nervous system (CNS) that function to protect against environmental insults including pathogens. However, following CNS inflammation, reactive gliosis occurs which is characterized by astrocyte hypertrophy and increased glial proliferation. Under such conditions, glia can become chronically activated and may contribute to the neuropathology associated with a variety of neuroinflammatory disorders including multiple sclerosis (MS), Alzheimers disease (AD), Parkinsons disease (PD), amyotrophic lateral sclerosis (ALS), and stroke. A review of the role of PPAR agonists in modulating glial cell activation is presented. Included is a discussion of the molecular mechanisms of action of these PPAR agonists and the potential utility of these agents for the treatment of neuroinflammatory disorders.

Peroxisome proliferator-activated receptor-α and retinoid X receptor agonists inhibit inflammatory responses of astrocytes

The peroxisome proliferator-activated receptor-alpha (PPAR-alpha) plays a key role in lipid metabolism and inflammation. Recently, we demonstrated that administration of the PPAR-alpha agonists gemfibrozil and fenofibrate, inhibit the clinical signs of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In the present study, we investigated the effects of PPAR-alpha agonists on primary mouse astrocytes, a cell type implicated in the pathology of MS and EAE. Our studies demonstrated that the PPAR-alpha agonists fenofibrate, and WY 14643 inhibited NO production by LPS-stimulated astrocytes in a dose-dependent manner. Additionally, PPAR-alpha agonists inhibited the secretion of the pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6 by LPS-stimulated astrocytes. Fenofibrate inhibited NF-kappaB DNA binding activity, suggesting a mechanism by which PPAR-alpha agonists may regulate the expression of genes encoding these pro-inflammatory molecules. Retinoid X receptors (RXRs) physically interact with PPAR-alpha receptors, and the resulting heterodimers regulate the expression of PPAR-responsive genes. Interestingly, a combination of 9-cis RA and the PPAR-alpha agonists fenofibrate or gemfibrozil cooperatively inhibited NO, TNF-alpha, IL-1beta, IL-6, and MCP-1 production by these cells. Collectively, these results raise the possibility that PPAR-alpha and RXR agonists might be effective in the treatment of MS, where activated astrocytes are believed to contribute to disease pathology.

The cyclopentone prostaglandin 15-deoxy-Δ12,14 prostaglandin J2 represses nitric oxide, TNF-α, and IL-12 production by microglial cells

Abstract Prostaglandins are generally considered pro-inflammatory molecules that contribute to the pathology associated with a variety of immune-mediated diseases including multiple sclerosis. However, recently it has been demonstrated that specific cyclopentone prostaglandin metabolites including 15-deoxy-Δ 12,14 prostaglandin J2 (15d-PGJ2) are capable of repressing the production of pro-inflammatory molecules by cells of the monocyte/macrophage lineage. Activated microglia produce nitric oxide (NO) and TNF-α, molecules which can be toxic to cells including oligodendrocytes, thus potentially contributing to the pathology associated with multiple sclerosis. The current study demonstrates that 15d-PGJ2 inhibits lipopolysachharide (LPS) induction of NO and TNF-α production by rat primary microglia and mouse N9 microglial cells. 15d-PGJ2 also inhibits NO production by microglial cells activated in response to IFN-γ and TNF-α, cytokines believed to be important modulators of multiple sclerosis. IL-12 plays a critical role in stimulating the production of Th1 cells, which are believed to contribute to the pathology associated with multiple sclerosis. The current studies demonstrate that 15d-PGJ2 represses the production of IL-12 by microglial cells. Collectively, these studies demonstrate that the prostaglandin metabolite 15d-PGJ2 represses microglial production of potentially cytotoxic molecules, as well as molecules capable of altering T-cell phenotype. These in vitro studies suggest the possibility that the prostaglandin 15d-PGJ2 may modulate inflammatory diseases including multiple sclerosis.

Inhibition of microglial cell activation by cortisol

Cortisol is a steroid hormone produced in response to stress. This glucocorticoid can be toxic to neurons, and thus may be important in neurodegenerative diseases including Alzheimers disease. Activated microglia produce molecules including nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) which can also be toxic to neurons. The current study was designed to determine the effect of cortisol upon the activation of primary cultured microglia and transformed N9 microglial cells. The studies indicate that cortisol represses lipopolysaccharide (LPS) induction of nitric oxide production in these microglial cells. The hormone acts by inhibiting the production of inducible nitric oxide synthase (iNOS) which catalyses the synthesis of NO. Cortisol likely acts by blocking transcription of iNOS gene expression since the hormone represses LPS induction of iNOS RNA levels in these cells. Activated microglia produce increased TNF-alpha, in addition to increased NO. The current studies demonstrate that cortisol inhibits release of TNF-alpha from LPS-treated microglial cells. Collectively, these data suggest that although cortisol may be directly toxic to neurons, the hormone may indirectly protect neurons by blocking the production of cytotoxic molecules by microglia.

Resveratrol effects on astrocyte function: Relevance to neurodegenerative diseases

Inflammatory molecules have been implicated in the pathogenesis of neurodegenerative diseases such as Parkinsons disease, Alzheimers disease, and multiple sclerosis. Resveratrol is an anti-fungal compound found in the skins of red grapes and other fruits and nuts. We examined the ability of resveratrol to inhibit lipopolysaccharide (LPS)-induced production of inflammatory molecules from primary mouse astrocytes. Resveratrol inhibited LPS-induced production of nitric oxide (NO); the cytokines tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), and IL-6; and the chemokine monocyte chemotactic protein-1 (MCP-1), which play critical roles in innate immunity, by astrocytes. Resveratrol also suppressed astrocyte production of IL-12p40 and IL-23, which are known to alter the phenotype of T cells involved in adaptive immunity. Finally resveratrol inhibited astrocyte production of C-reactive protein (CRP), which plays a role in a variety of chronic inflammatory disorders. Collectively, these studies suggest that resveratrol may be an effective therapeutic agent in neurodegenerative diseases initiated or maintained by inflammatory processes.

Cyclopentenone Prostaglandins PGA2 and 15-Deoxy-Δ12,14 PGJ2 Suppress Activation of Murine Microglia and Astrocytes: Implications for Multiple Sclerosis

The cyclopentenone prostaglandin (cPG) 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2) has been identified as a potent antiinflammatory agent that is able to inhibit the activation of macrophages and microglia. Additionally, 15d‐PGJ2 is able to ameliorate the clinical manifestations of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Many biological effects of 15d‐PGJ2 have been attributed to the peroxisome proliferator activated receptor‐gamma (PPAR‐γ). PGA2, like 15d‐PGJ2, is a cPG. The aim of this study is to compare the relative effectiveness of these two cPGs in inhibiting the inflammatory response of mouse microglia and astrocytes, two cell types that upon activation may contribute to the pathology of EAE and MS. Purified primary mouse microglia and astrocytes were treated with either 15d‐PGJ2 or PGA2 and then stimulated with either lipopolysaccharide (LPS) or a combination of interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α. The results show that 15d‐PGJ2 and PGA2 both potently inhibited the production of nitrite, as well as proinflammatory cytokines and chemokines, from microglia and astrocytes. Generally, regulation of NO production was more sensitive to 15d‐PGJ2, however, cytokine and chemokine production was more sensitive to PGA2 treatment. These results demonstrate for the first time that PGA2 is a potent antiinflammatory mediator.

Dehydroepiandrosterone inhibits microglial nitric oxide production in a stimulus-specific manner

Dehydroepiandrosterone (DHEA) is a steroid that circulates in abundance in the form of a sulfated reserve (DHEA‐S). The levels of DHEA decline with age and further in age‐related neuropathologies, including Alzheimer disease. Because of their reported anti‐inflammatory effects, we tested the actions of these compounds on microglia. At concentrations of 3−9 to 1−6 M, DHEA and DHEA‐S inhibited the production of nitrite and morphological changes stimulated by lipopolysaccharide. DHEA and DHEA‐S also inhibited LPS induction of iNOS protein, but neither inhibited LPS‐induced iNOS mRNA or the activation of NF‐κB. These data suggest that the hormone regulates nitrite production through a post‐transcriptional mechanism. Interestingly, microglial nitrite production in response to a secreted form of the β‐amyloid precursor protein (sAPP) was unaffected by DHEA. Another Alzheimer‐related factor, amyloid β‐peptide, also stimulated microglial nitrite production but in a manner dependent on the co‐stimulus interferon‐γ. DHEA was found to inhibit only the interferon‐γ component of the microglial response. These data add to a growing body of evidence for differences in the profiles of mononuclear phagocytes activated by distinct stimuli. J. Neurosci. Res. 62:503–509, 2000.

Sex Steroid Regulation of Microglial Cell Activation

Abstract: Multiple sclerosis (MS) occurs more commonly in females than males. However, the mechanisms resulting in gender differences in MS are unknown. Several studies have suggested that sex steroids influence the development and severity of MS. For example, pregnancy influences MS symptoms, with remission in the third trimester of gestation, followed by exacerbation in the postpartum period. In addition, oral contraceptives containing female sex steroids have been associated with a lower risk of developing MS and decreased disability. Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disorder initiated by T cells reactive against central nervous system (CNS) antigens. EAE is characterized by inflammation and demyelination of the CNS, and by remittent paralysis—features consistent with MS. Recent studies have suggested that female sex steroids may modulate EAE, at least in part, through effects on T cells. For example, sex steroids shift T cells toward a Th2 phenotype in vitro, and cytokines produced by Th2 cells generally suppress EAE. Activated microglia also are believed to contribute to MS pathology; perhaps due in part to production of nitric oxide (NO) and TNF‐α, molecules which can be toxic to CNS cells, including oligodendrocytes. We are currently investigating the role of sex steroids in modulating microglial cell function in relation to MS. It is hoped that elucidation of the mechanisms by which sex steroids modulate CNS inflammation will lead to future therapies in the treatment of MS.