Chandramohan Natarajan

Curcumin Inhibits Experimental Allergic Encephalomyelitis by Blocking IL-12 Signaling Through Janus Kinase-STAT Pathway in T Lymphocytes

Experimental allergic encephalomyelitis (EAE) is a CD4+ Th1 cell-mediated inflammatory demyelinating autoimmune disease of the CNS that serves as an animal model for multiple sclerosis (MS). IL-12 is a proinflammatory cytokine that plays a crucial role in the induction of neural Ag-specific Th1 differentiation and pathogenesis of CNS demyelination in EAE and MS. Curcumin (1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a naturally occurring polyphenolic phytochemical isolated from the rhizome of the medicinal plant Curcuma longa. It has profound anti-inflammatory activity and been traditionally used to treat inflammatory disorders. In this study we have examined the effect and mechanism of action of curcumin on the pathogenesis of CNS demyelination in EAE. In vivo treatment of SJL/J mice with curcumin significantly reduced the duration and clinical severity of active immunization and adoptive transfer EAE. Curcumin inhibited EAE in association with a decrease in IL-12 production from macrophage/microglial cells and differentiation of neural Ag-specific Th1 cells. In vitro treatment of activated T cells with curcumin inhibited IL-12-induced tyrosine phosphorylation of Janus kinase 2, tyrosine kinase 2, and STAT3 and STAT4 transcription factors. The inhibition of Janus kinase-STAT pathway by curcumin resulted in a decrease in IL-12-induced T cell proliferation and Th1 differentiation. These findings highlight the fact that curcumin inhibits EAE by blocking IL-12 signaling in T cells and suggest its use in the treatment of MS and other Th1 cell-mediated inflammatory diseases.

Peroxisome proliferator-activated receptor-gamma agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling and Th1 differentiation.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear receptor transcription factor that regulates adipocyte differentiation and glucose homeostasis. PPARγ agonists are potent therapeutic agents for the treatment of type 2 diabetes and obesity. PPARγ agonists also prevent inflammation in animal models, suggesting their use for the treatment of human inflammatory diseases. Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated inflammatory demyelinating disease model of multiple sclerosis (MS) and IL-12 plays a crucial role in the pathogenesis of EAE and MS. In this study we have examined the effect of PPARγ agonists on the pathogenesis of EAE. In vivo treatment of SJL/J mice with PPARγ agonists, 15-deoxyΔ12,14 prostaglandin J2 or Ciglitazone, decreased the duration and clinical severity of active immunization and adoptive transfer models of EAE. PPARγ agonists inhibited EAE in association with a decrease in IL-12 production and differentiation of neural antigen-specific Th1 cells. In vitro treatment of activated T cells with PPARγ agonists inhibited IL-12-induced activation of JAK-STAT signaling pathway and Th1 differentiation. These findings highlight the fact that PPARγ agonists regulate central nervous system inflammation and demyelination by inhibiting IL-12 production, IL-12 signaling and Th1 differentiation in EAE.

Peroxisome Proliferator-Activated Receptor-γ-Deficient Heterozygous Mice Develop an Exacerbated Neural Antigen-Induced Th1 Response and Experimental Allergic Encephalomyelitis

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor transcription factor that regulates cell growth, differentiation, and homeostasis. PPARγ agonists are potent therapeutic agents for type 2 diabetes, obesity, and inflammation. Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated inflammatory demyelinating autoimmune disease model of multiple sclerosis. We have shown recently that PPARγ agonists inhibit EAE by blocking IL-12 production, IL-12 signaling, and neural Ag-induced Th1 differentiation. In this study, we show that the PPARγ-deficient heterozygous mice develop an exacerbated EAE with prolonged clinical symptoms than the wild-type littermates, following immunization with myelin oligodendrocyte glycoprotein (MOG) p35–55 peptide. The exacerbation of EAE in PPARγ+/− mice associates with an increased expansion of CD4+ and CD8+ T cells and expression of CD40 and MHC class II molecules in response to MOGp35–55 Ag. The PPARγ+/− mice also showed an increase in T cell proliferation and Th1 response to MOGp35–55 Ag than the wild-type littermates. These findings suggest that PPARγ be a critical physiological regulator of CNS inflammation and demyelination in EAE and perhaps multiple sclerosis and other Th1 cell-mediated autoimmune diseases.

Signaling through JAK2-STAT5 pathway is essential for IL-3-induced activation of microglia

Microglia, the resident macrophage of the brain, mediates immune and inflammatory responses in the central nervous system (CNS). Activation of microglia and secretion of inflammatory cytokines associate with the pathogenesis of CNS diseases, including multiple sclerosis (MS), Alzheimers disease (AD), Parkinsons disease, prion disease, and AIDS dementia. Microbial pathogens, cytokines, chemokines, and costimulatory molecules are potent inducers of microglial activation in the CNS. Signaling through its receptor, IL‐3 induces the activation of JAK‐STAT and MAP kinase pathways in microglial cells. In this study, we found that in vitro treatment of EOC‐20 microglial cells with tyrphostin AG490 blocked IL‐3‐induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B signaling proteins. Stable transfection of EOC‐20 cells with a dominant negative JAK2 mutant also blocked IL‐3‐induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B in microglia. The blockade of JAK2‐STAT5 pathway resulted in a decrease in IL‐3‐induced proliferation and expression of CD40 and major histocompatibility complex class II molecules in microglia. These findings highlight the fact that JAK2‐STAT5 signaling pathway plays a critical role in mediating IL‐3‐induced activation of microglia.

nNOS mediated mitochondrial injury in LPS stimulated oligodendrocytes

Products of inflammation and the activation of nitric oxide synthase have been proposed as a mechanism of oligodendrocyte injury in CNS inflammation. There are currently three well described and known isoforms of NOS. Of these, neuronal NOS (nNOS) was initially discovered in neurons, endothelial NOS (eNOS) in vascular endothelium, while the inducible form of NOS (iNOS) is known to be activated in oligodendrocytes, astrocytes and microglia. We examined the activation of nNOS and the down stream effects of NO production in oligodendrocyte precursor cells (OPC) and MO3.13 cell line following culture with LPS. Our studies show that both MO3.13 cells and OPC are susceptible to the cellular injury resulting from LPS mediated activation and NO production. Activation of the TLR4 receptor with LPS led to decrease in cell viability that was associated with loss of mitochondrial membrane potential and impaired enzymatic activity of complex I and complex IV protein of the respiratory chain. 7-NI, a known inhibitor of nNOS was able to rescue of cells from LPS mediated mitochondrial damage. Loss of mitochondrial function was associated with translocation of cytochrome C and apoptosis inducing factor to the cytosol, setting the stage for apoptosis. Phosphorylation of PI3K and Akt was required for optimal activation of NOS. These studies provide a biochemical basis for nNOS mediated oligodendrocyte injury and suggest similar mechanisms may play a role in diseases characterized by oligodendrocyte loss and demyelination.

Prostaglandin I2 signaling drives Th17 differentiation and exacerbates experimental autoimmune encephalomyelitis.

Background Prostaglandin I2 (PGI2), a lipid mediator currently used in treatment of human disease, is a critical regulator of adaptive immune responses. Although PGI2 signaling suppressed Th1 and Th2 immune responses, the role of PGI2 in Th17 differentiation is not known. Methodology/Principal Findings In mouse CD4+CD62L+ naïve T cell culture, the PGI2 analogs iloprost and cicaprost increased IL-17A and IL-22 protein production and Th17 differentiation in vitro. This effect was augmented by IL-23 and was dependent on PGI2 receptor IP signaling. In mouse bone marrow-derived CD11c+ dendritic cells (BMDCs), PGI2 analogs increased the ratio of IL-23/IL-12, which is correlated with increased ability of BMDCs to stimulate naïve T cells for IL-17A production. Moreover, IP knockout mice had delayed onset of a Th17-associated neurological disease, experimental autoimmune encephalomyelitis (EAE), and reduced infiltration of IL-17A-expressing mononuclear cells in the spinal cords compared to wild type mice. These results suggest that PGI2 promotes in vivo Th17 responses. Conclusion The preferential stimulation of Th17 differentiation by IP signaling may have important clinical implications as PGI2 and its analogs are commonly used to treat human pulmonary hypertension.

TLR3 Agonist Poly-IC Induces IL-33 and Promotes Myelin Repair

Background Impaired remyelination of demyelinated axons is a major cause of neurological disability. In inflammatory demyelinating disease of the central nervous system (CNS), although remyelination does happen, it is often incomplete, resulting in poor clinical recovery. Poly-IC a known TLR3 agonist and IL-33, a cytokine which is induced by poly-IC are known to influence recovery and promote repair in experimental models of CNS demyelination. Methodology and Principal Findings We examined the effect of addition of poly-IC and IL-33 on the differentiation and maturation of oligodendrocyte precursor cells (OPC) cultured in vitro. Both Poly-IC and IL-33 induced transcription of myelin genes and the differentiation of OPC to mature myelin forming cells. Poly-IC induced IL-33 in OPC and addition of IL-33 to in vitro cultures, amplified further, IL-33 expression suggesting an autocrine regulation of IL-33. Poly-IC and IL-33 also induced phosphorylation of p38MAPK, a signaling molecule involved in myelination. Following the induction of gliotoxic injury with lysolecithin to the corpus callosum (CC), treatment of animals with poly-IC resulted in greater recruitment of OPC and increased staining for myelin in areas of demyelination. Also, poly-IC treated animals showed greater expression of IL-33 and higher expression of M2 phenotype macrophages in the CC. Conclusion/Significance Our studies suggest that poly-IC and IL-33 play a role in myelin repair by enhancing expression of myelin genes and are therefore attractive therapeutic agents for use as remyelinating agents in human demyelinating disease.

Activation of NOD2/RIPK2 pathway induces mitochondrial injury to oligodendrocyte precursor cells in vitro and CNS demyelination in vivo

We examined the activation of innate immune pathway mediated by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in oligodendrocyte precursor cells (OPCs). We show that activation of NOD2 by ligand peptidoglycan (PGN) leads to the recruitment and phosphorylation of receptor-interacting serine/threonine kinase 2 (RIPK2). Phosphorylation of RIPK2 is followed by phosphorylation of neuronal nitric oxide synthase (nNOS), increase in NOS activity and subsequent accumulation of nitric oxide (NO) mediated N-tyrosinylated compounds in OPCs. The reversal of NOS activity by the nNOS inhibitor 7-nitroindazole (7-NI), but not by the iNOS inhibitor L-canavanine, supported the conclusion that the increased NOS activity was due to the selective activation of nNOS in OPCs. In addition, NO mediated injury to OPC was reflected in reduction in activity of respiratory enzymes such as complex I and IV, decrease in mitochondrial membrane potential and release of cytochrome-C from mitochondria. Furthermore, intracerebral injection of PGN into corpus callosum (CC) of rats led to the development of demyelination, which appeared as early as by day 3 post-injection, and involved the trunk of the CC by day 14. Accumulation of N-tyrosinylated proteins was seen in oligodendrocytes in regions of the CC which were in close proximity to the injection site. Taken together, these results suggest that PGN induced formation of NO, mitochondrial dysfunction and accumulation of N-tyrosinylated proteins in oligodendrocytes are likely mediators of central nervous system demyelination.

Extracellular loop II modulates GTP sensitivity of the prostaglandin EP3 receptor.

Unlike the majority of G protein–coupled receptors, the prostaglandin E2 (PGE2) E-prostanoid 3 (EP3) receptor binds agonist with high affinity that is insensitive to the presence of guanosine 5[prime]-O-(3-thio)triphosphate (GTPγS). We report the identification of mutations that confer GTPγS sensitivity to agonist binding. Seven point mutations were introduced into the conserved motif in the second extracellular loop (ECII) of EP3, resulting in acquisition of GTP-sensitive agonist binding. One receptor mutation W203A was studied in detail. Loss of agonist binding was observed on intact human embryonic kidney 293 cells expressing the W203A receptor, conditions where high GTP levels are present; however, high affinity binding [3H]PGE2 was observed in broken cell preparations washed free of GTP. The [3H]PGE2 binding of W203A in broken cell membrane fractions was inhibited by addition of GTPγS (IC50 21 ± 1.8 nM). Taken together, these results suggest that the wild-type EP3 receptor displays unusual characteristics of the complex coupled equilibria between agonist-receptor and receptor–G protein interaction. Moreover, mutation of ECII can alter this coupled equilibrium from GTP-insensitive agonist binding to more conventional GTP-sensitive binding. This suggests that for the mutant receptors, ECII plays a critical role in linking the agonist bound receptor conformation to the G protein nucleotide bound state.