Subhajit Dasgupta

Human immunodeficiency virus type 1 (HIV-1) tat induces nitric-oxide synthase in human astroglia

Human immunodeficiency virus type 1 (HIV-1) infection is known to cause neuronal injury and dementia in a significant proportion of patients. However, the mechanism by which HIV-1 mediates its deleterious effects in the brain is poorly defined. The present study was undertaken to investigate the effect of the HIV-1tat gene on the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astroglia. Expression of the tat gene as RSV-tat but not that of the CAT gene as RSV-CAT in U373MG astroglial cells led to the induction of NO production and the expression of iNOS protein and mRNA. Induction of NO production by recombinant HIV-1 Tat protein and inhibition of RSV-tat-induced NO production by anti-Tat antibodies suggest that RSV-tat-induced production of NO is dependent on Tat and that Tat is secreted from RSV-tat-transfected astroglia. Similar to U373MG astroglial cells, RSV-tat also induced the production of NO in human primary astroglia. The induction of human iNOS promoter-derived luciferase activity by the expression of RSV-tat suggests that RSV-tat induces the transcription of iNOS. To understand the mechanism of induction of iNOS, we investigated the role of NF-κB and C/EBPβ, transcription factors responsible for the induction of iNOS. Activation of NF-κB as well as C/EBPβ by RSV-tat, stimulation of RSV-tat-induced production of NO by the wild type of p65 and C/EBPβ, and inhibition of RSV-tat-induced production of NO by Δp65, a dominant-negative mutant of p65, and ΔC/EBPβ, a dominant-negative mutant of C/EBPβ, suggest that RSV-tat induces iNOS through the activation of NF-κB and C/EBPβ. In addition, we show that extracellular signal-regulated kinase (ERK) but not that p38 mitogen-activated protein kinase (MAPK) is involved in RSV-tat induced production of NO. Interestingly, PD98059, an inhibitor of the ERK pathway, and ΔERK2, a dominant-negative mutant of ERK2, inhibited RSV-tat-induced production of NO through the inhibition of C/EBPβ but not that of NF-κB. This study illustrates a novel role for HIV-1 tat in inducing the expression of iNOS in human astrocytes that may participate in the pathogenesis of HIV-associated dementia.

Antineuroinflammatory Effect of NF-κB Essential Modifier-Binding Domain Peptides in the Adoptive Transfer Model of Experimental Allergic Encephalomyelitis

It has been shown that peptides corresponding to the NF-κB essential modifier-binding domain (NBD) of IκB kinase α or IκB kinase β specifically inhibit the induction of NF-κB activation without inhibiting the basal NF-κB activity. The present study demonstrates the effectiveness of NBD peptides in inhibiting the disease process in adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. Clinical symptoms of EAE were much lower in mice receiving wild-type (wt)NBD peptides compared with those receiving mutated (m)NBD peptides. Histological and immunocytochemical analysis showed that wtNBD peptides inhibited EAE-induced spinal cord mononuclear cell invasion and normalized p65 (the RelA subunit of NF-κB) expression within the spinal cord. Analysis of lymph node cells isolated from donor and recipient mice showed that wtNBD peptides but not mNBD peptides were able to shift the immune response from a Th1 to a Th2 profile. Consistently, wtNBD peptides but not mNBD peptides inhibited the encephalitogenicity of myelin basic protein-specific T cells. Furthermore, i.p. injection of wtNBD peptides but not mNBD peptides was also able to reduce LPS- and IFN-γ-induced expression of inducible NO synthase, IL-1β, and TNF-α in vivo in the cerebellum. Taken together, our results support the conclusion that NBD peptides are antineuroinflammatory, and that NBD peptides may have therapeutic effect in neuroinflammatory disorders such as multiple sclerosis.

Induction of tumor necrosis factor‐α (TNF‐α) by interleukin‐12 p40 monomer and homodimer in microglia and macrophages

The present study was undertaken to explore the role of interleukin‐12 (IL‐12) p40 in the expression of TNF‐α in microglia. Interestingly, we have found that IL‐12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose‐dependently induced the production of TNF‐α and the expression of TNF‐α mRNA in BV‐2 microglial cells. In addition to BV‐2 microglial cells, p70, p402 and p40 also induced the production of TNF‐α in mouse primary microglia and peritoneal macrophages. As the activation of both NF‐κB and CCAAT/enhancer binding protein β (C/EBPβ) is important for the expression of TNF‐α in microglial cells, we investigated the effect of p40 on the activation of NF‐κB as well as C/EBPβ. Activation of NF‐κB as well as C/EBPβ by p40 and inhibition of p40‐induced expression of TNF‐α by Δp65, a dominant‐negative mutant of p65, and ΔC/EBPβ, a dominant‐negative mutant of C/EBPβ, suggests that p40 induces the expression of TNF‐α through the activation of NF‐κB and C/EBPβ. In addition, we show that p40 induced the activation of both extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK). Interestingly, PD98059, an inhibitor of ERK, inhibited p40‐induced expression of TNF‐α through the inhibition of C/EBPβ, but not that of NF‐κB, whereas SB203580, an inhibitor of p38 MAPK, inhibited p40‐induced expression of TNF‐α through the inhibition of both NF‐κB and C/EBPβ. This study delineates a novel biological function of p40 in inducing TNF‐α in microglia and macrophages.

Gemfibrozil Ameliorates Relapsing-Remitting Experimental Autoimmune Encephalomyelitis Independent of Peroxisome Proliferator-Activated Receptor-α

The present study underlines the importance of gemfibrozil, a lipid-lowering drug and an activator of peroxisome proliferator-activated receptor-α (PPAR-α), in inhibiting the disease process of adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of relapsing-remitting multiple sclerosis. Clinical symptoms of EAE, infiltration of mononuclear cells, and demyelination were significantly lower in SJL/J female mice receiving gemfibrozil through food chow than those without gemfibrozil. It is noteworthy that the drug was equally effective in treating EAE in PPAR-α wild-type as well as knockout mice. Gemfibrozil also inhibited the encephalitogenicity of MBP-primed T cells and switched the immune response from a Th1 to a Th2 profile independent of PPAR-α. Gemfibrozil consistently inhibited the expression and DNA-binding activity of T-bet, a key regulator of interferon-γ (IFN-γ) expression and stimulated the expression and DNA-binding activity of GATA3, a key regulator of IL-4. Gemfibrozil treatment decreased the number of T-bet–positive T cells and increased the number of GATA3-positive T cells in spleen of donor mice. The histological and immunohistochemical analyses also demonstrate the inhibitory effect of gemfibrozil on the invasion of T-bet–positive T cells into the spinal cord of EAE mice. Furthermore, we demonstrate that the differential effect of gemfibrozil on the expression of T-bet and GATA3 was due to its inhibitory effect on NO production. Although excess NO favored the expression of T-bet, scavenging of NO stimulated the expression of GATA-3. Taken together, our results suggest gemfibrozil, an approved drug for hyperlipidemia in humans, may find further therapeutic use in multiple sclerosis.

Sodium Phenylacetate Inhibits Adoptive Transfer of Experimental Allergic Encephalomyelitis in SJL/J Mice at Multiple Steps

Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. The present study underlines the importance of sodium phenylacetate (NaPA), a drug approved for urea cycle disorders, in inhibiting the disease process of adoptively transferred EAE in female SJL/J mice at multiple steps. Myelin basic protein (MBP)-primed T cells alone induced the expression of NO synthase (iNOS) and the activation of NF-κB in mouse microglial cells through cell-cell contact. However, pretreatment of MBP-primed T cells with NaPA markedly inhibited its ability to induce microglial expression of iNOS and activation of NF-κB. Consistently, adoptive transfer of MBP-primed T cells, but not that of NaPA-pretreated MBP-primed T cells, induced the clinical symptoms of EAE in female SJL/J mice. Furthermore, MBP-primed T cells isolated from NaPA-treated donor mice were also less efficient than MBP-primed T cells isolated from normal donor mice in inducing iNOS in microglial cells and transferring EAE to recipient mice. Interestingly, clinical symptoms of EAE were much less in mice receiving NaPA through drinking water than those without NaPA. Similar to NaPA, sodium phenylbutyrate, a chemically synthesized precursor of NaPA, also inhibited the disease process of EAE. Histological and immunocytochemical analysis showed that NaPA inhibited EAE-induced spinal cord mononuclear cell invasion and normalized iNOS, nitrotyrosine, and p65 (the RelA subunit of NF-κB) expression within the spinal cord. Taken together, our results raise the possibility that NaPA or sodium phenylbutyrate taken through drinking water or milk may reduce the observed neuroinflammation and disease process in multiple sclerosis patients.

Myelin Basic Protein-primed T Cells Induce Nitric Oxide Synthase in Microglial Cells IMPLICATIONS FOR MULTIPLE SCLEROSIS

The presence of autoreactive T cells recognizing self myelin antigens is necessary for the development of central nervous system autoimmune diseases such as multiple sclerosis (MS). The present study was undertaken to investigate the role of myelin basic protein (MBP)-primed T cells in the expression of inducible nitric oxide synthase (iNOS) in microglial cells. MBP-primed T cells alone markedly induced the production of NO and the expression of iNOS protein and mRNA in mouse BV-2 microglial cells. Similarly, MBP-primed T cells also induced the production of NO in mouse primary microglia. This induction of NO production was primarily dependent on the contact between MBP-primed T cells and microglia. The expression of very late antigen-4 (VLA-4) on the surface of MBP-primed T cells and inhibition of MBP-primed T cell-induced microglial NO production by functional blocking of antibodies to the α4 chain of VLA-4 (CD49d) suggest that VLA-4 integrin on MBP-primed T cells plays an important role in contact-mediated induction of iNOS. Since IFN-β has been used to treat MS patients, we examined the effect of IFN-β on MBP-primed T cell-induced the production of NO. Surprisingly, IFN-β alone induced the production of NO in microglial cells. However, the pretreatment of MBP-primed T cells with IFN-β inhibited the expression of VLA-4 integrin on the surface of MBP-primed T cells and thereby inhibited the ability of those T cells to induce the production of NO in microglial cells. This study illustrates a novel role of neuroantigen-primed T cells in inducing contact-mediated expression of iNOS in microglial cells that may participate in the pathogenesis of MS.

Regulation of tumor necrosis factor-α expression by CD40 ligation in BV-2 microglial cells

Ligation of CD40 has been shown to induce/stimulate the expression of tumor necrosis factor‐α (TNF‐α) in microglial cells. This study delineates the mechanism by which CD40 ligation regulates the expression of TNF‐α in BV‐2 microglial cells. There was very little induction of TNF‐α by ligation of CD40 alone by either cross‐linking antibodies against CD40 or␣recombinant CD40 ligand (CD154). The absence of any increase in TNF‐α production by CD40 ligation alone even in CD40‐overexpressed BV‐2 microglial cells suggest that signal transduced by the ligation of CD40 alone is not sufficient for strong induction of TNF‐α. However, CD40 ligation markedly induced the production of TNF‐α as well as the expression of TNF‐α mRNA in interferon‐γ (IFN‐γ)‐stimulated BV‐2 glial cells. Ligation of CD40 in CD40‐overexpressed cells markedly enhanced the expression of TNF‐α in the presence of IFN‐γ. To understand the mechanism of CD40 ligation‐mediated induction/stimulation of TNF‐α, we investigated the role of nuclear factor‐κB (NF‐κB) and C/EBPβ. IFN‐γ alone was able to induce the activation of NF‐κB as well as C/EBPβ. However, CD40 ligation alone in the presence or absence of CD40 overexpression induced the activation of only NF‐κB and not that of C/EBPβ, suggesting that the activation of NF‐κB alone by CD40 ligation is not sufficient to induce the expression of TNF‐α and that the activation of C/EBPβ is also necessary for strong induction of TNF‐α. Consistently, a dominant‐negative mutant of p65 (Δp65) and that of C/EBPβ (ΔC/EBPβ) inhibited the expression of TNF‐α in BV‐2 microglial cells stimulated with the combination of IFN‐γ and CD40 ligand. Taken together, these studies suggest that activation of both NF‐κB and C/EBPβ is important for strong induction of TNF‐α and that CD40 ligation regulates the expression of TNF‐α by modulating the activation of only NF‐κB but not that of C/EBPβ.

Myelin Basic Protein-primed T Cells of Female but Not Male Mice Induce Nitric-oxide Synthase and Proinflammatory Cytokines in Microglia IMPLICATIONS FOR GENDER BIAS IN MULTIPLE SCLEROSIS

Females are more susceptible than males to multiple sclerosis (MS). However, the underlying mechanism behind this gender difference is poorly understood. Because the presence of neuroantigen-primed T cells within the CNS is necessary for the development of MS, the present study was undertaken to investigate the activation of microglia by myelin basic protein (MBP)-primed T cells of male, female, and castrated male mice. Interestingly, MBP-primed T cells isolated from female and castrated male but not from male mice induced the expression of inducible nitric-oxide synthase (iNOS) and proinflammatory cytokines (interleukin-1β (IL-1β), IL-1α, IL-6, and tumor necrosis factor-α) in microglia by cell-cell contact. Again there was no apparent defect in male microglia, because MBP-primed T cells isolated from female and castrated male but not male mice were capable of inducing the production of NO in male primary microglia. Inhibition of female T cell contact-mediated microglial expression of proinflammatory molecules by dominant-negative mutants of p65 and C/EBPβ suggest that female MBP-primed T cells induce microglial expression of proinflammatory molecules through the activation of NF-κB and C/EBPβ. Interestingly, MBP-primed T cells of male, female, and castrated male mice were able to induce microglial activation of NF-κB. However, MBP-primed T cells of female and castrated male but not male mice induced microglial activation of C/EBPβ. These studies suggest that microglial activation of C/EBPβ but not NF-κB by T cell:microglial contact is a gender-specific event and that male MBP-primed T cells are not capable of inducing microglial expression of proinflammatory molecules due to their inability to induce the activation of C/EBPβ in microglia. This novel gender-sensitive activation of microglia by neuroantigen-primed T cell contact could be one of the mechanisms behind the female-loving nature of MS.

Generation of Functional Blocking Monoclonal Antibodies Against Mouse Interleukin-12 p40 Homodimer and Monomer

The role of interleukin (IL)-12 (p40:p35) and IL-23 (p40:p19) is becoming clear in immune response and inflammation. However, biological functions of IL-12 p40 homodimer (p40(2)) and monomer (p40) remain poorly understood due to the lack of specific monoclonal antibodies (MAb). Earlier we have demonstrated that both p40(2) and p40 activate microglia and macrophages to induce the expression of iNOS and TNF-alpha. To facilitate the studies on p40(2) and p40 further, we here describe the production of neutralizing MAb against mouse p40(2) and p40 for the first time after immunization of Armenian hamsters with recombinant p40(2). Antibodies produced from clones a3-1d and d7-12c specifically recognized p40(2) but not p40, IL-12, and IL-23. These MAbs also inhibited p40(2)- but not p40-, IL-12-, and IL-23-induced production of inflammatory molecules and activation of NF-kappaB. On the other hand, antibodies produced from clones a3-3a and a3-7g specifically recognized p40 and inhibited p40- but not p40(2)-, IL-12-, and IL-23-induced production of inflammatory molecules and activation of NF-kappaB. While MAbs a3-1d and d7-12c were used to establish p40(2)-specific ELISA, we utilized MAbs a3-3a and a3-7g to develop p40-specific ELISA. Interestingly, the production of p40(2) and p40 but not IL-12 in mouse peritoneal macrophages and primary microglia was an immediate early response to bacterial lipopolysaccharides. Furthermore, double-stranded RNA, the active component of a viral infection, induced the production of p40(2) and p40 but not IL-12 in macrophages and microglia. These results indicate the presence of different regulatory mechanisms for the production of IL-12p40(2)/p40 and IL-12p70.

Therapeutic Impact of Sphingosine 1-phosphate Receptor Signaling in Multiple Sclerosis

Multiple sclerosis (MS) is a female predominant autoimmune demyelinating disease of central nervous system. The proper etiology is not clear. The existing therapies with interferon beta (Betaseron, Rebif), glatiramer acetate (copolymer 1, copaxone) are found to be promising for MS patients. The alpha-4 integrin antagonist monoclonal antibody Natalizumab has been found to decrease brain inflammation in relapsing-remitting MS via inhibition of alpha-4 beta- 1 integrinmediated mode of action of antigen -primed T cells to enter into central nervous system through blood brain barrier. The advancement of drug development introduced prospects of CD52 monoclonal antibody Alemtuzumab and CD20 monoclonal antibody Rituximab in MS therapy. The benefit versus risk ratios of these therapeutic monoclonal antibodies are currently under clinical trial. The ongoing researches demonstrated the importance of HMG-CoA reductase inhibitor statins, NF-κBp65 inhibitor NBD peptide, and antagonist of poly-ADP-ribose polymerase (PARP) in experimental autoimmune encephalomyelitis (EAE), animal model for MS. Recently, the clinical trials indicated the therapeutic prospect of G-protein coupled sphingosine 1-phosphate receptor (S1PR) in MS patients. Recent studies showed remyelination through selective activation of oligodendrocyte progenitor cells. In the context, role of S1PR-mediated signals following interaction with natural ligand S1P and agonist Fingolimod (FTY720) gain profound therapeutic importance in prevention of demyelination in MS brain. The S1PR agonist Fingolimod (FTY 720) has recently been approved by Food and Drug Administration for MS therapy. In the review, we provided an insight on S1PR mode of action in the aspect of treatment of autoimmune disorder, re-myelination and regeneration of axons in damaged central nervous system in multiple sclerosis.