Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4.

Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes. EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively. MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells. Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

Effects of Zuogui Pill () and Yougui Pill () on the expression of brain-derived neurotrophic factor and cyclic adenosine monophosphate/protein kinase A signaling transduction pathways of axonal regeneration in model rats with experimental autoimmune encephalomyelitis.

OBJECTIVE: To study the effects of Zuogui Pill (, ZGP) and Yougui Pill (, YGP) on the expressions of brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling of axonal regeneration in the Lewis rats with experimental autoimmune encephalomyelitis (EAE), in order to explore the possible mechanism of ZGP and YGP on promoting axonal regeneration. METHODS: The rats were randomly divided into normal control (NC), model (MO), prednisone acetate (PA), ZGP and YGP groups. The EAE model of rat was established by injecting antigen containing myelin basic protein (MBP)68-86. The brain and spinal cord were harvested on the 14th and 28th day post-immunization (PI), the protein and mRNA expression of BDNF and PKA in the brain and spinal cord of rats were detected by Western blot analysis and real-time quantitative polymerase chain reaction (PCR), and the cAMP levels were detected by using enzyme-immunoassay method. RESULTS: (1) On the 28th day PI, the mRNA expression of BDNF in brain white matter and spinal cord of rats in ZGP and YGP groups were up-regulated, especially in YGP group (P<0.05 or P<0.01). (2) On the 14th day PI, the cAMP levels in brain white matters significantly increased in PA and YGP groups compared with MO group (P<0.05 or P<0.01), and the cAMP level in YGP group was higher than that in ZGP group (P<0.05). The cAMP level in spinal cord also significantly increased in YGP group compared with MO, PA and ZGP groups, respectively (P<0.01). (3) On the 14th day PI, the PKA expression in spinal cord of rats in ZGP group was significantly decreased compared with MO and YGP groups, respectively (P<0.05). (4) On the 28th day PI, there was a positive correlation between cAMP and PKA expression in the brain white matter of YGP rats. CONCLUSIONS: The results suggest that ZGP and YGP may promote axonal regeneration by modulating cAMP/PKA signal transduction pathway, but the targets of molecular mechanism of ZGP may be different from those of YGP.

[Use of vitamins for correction of the functional state of cytochrome P450 systems in experimental allergic encephalomyelitis].

It is known that inflammatory cytokines, which level is significantly increased in the pathogenesis of multiple sclerosis (MS), as well as interferon-beta, which is used to treat autoimmune diseases, can inhibit cytochrome P450-dependent processes of detoxification and biotransformation. The uncontrolled decrease of the activity of these processes may have a negative affect on the state of patients, so it is urgent to study the functional state of the cytochrome P450 system and to develop effective means for its regulation in these conditions. The effect of vitamin D3 and efficiency of its composition with vitamins B1, B2, B6, PP, E, alpha-lipoic, alpha-linolenoic acid and mineral substances (Mg, Zn, Se) in prevention of a functional state changes of cytochrome P450- and b5-dependent systems of the rat brain and liver endoplasmic reticulum at EAE are investigated. It has been shown that the essential decrease of the level of these cytochromes is observed both in the brain and liver. In addition the level of activity of NADH- and NADPH-oxidoreductases, which are part of microsomal electron transport chain components and coupled with monooxigenases, was reduced. These changes confirm the disturbances of a redox state and functional activity of detoxication and biotransformation systems in the studied animal tissues. Supplement of vitamin D3 as well as the composition of biologically active substances, which we developed earlier, effectively eliminated the decrease of the level of cytochromes and activities of NADH-oxidoreductase in immunised rat tissues. Normalization of these disturbances can be explained by antioxidant and membrane-stabilizing properties of applied substances, and also by the ability to reduce the activity of inflammatory reactions by regulation of the level of inflammatory cytokines in rat organism at EAE. Thus the studied vitamin-mineral composition appeared to be more effective to normalize the found disturbances and it can be useful for prevention of exacerbations and for improvement of a status of patients with multiple sclerosis and other diseases, which are accompanied with hyperactivation of immune system.

In vitro screening of NADPH oxidase inhibitors and in vivo effects of L-leucinethiol on experimental autoimmune encephalomyelitis-induced mice.

Experimental autoimmune encephalomyelitis (EAE), a Th1 polarized demyelinating disease of the central nervous system, shares many pathological and clinical similarities with multiple sclerosis (MS). The objectives of this study were i) to evaluate the suppressive effects of L-leucinethiol (LeuSH), a metalloprotease inhibitor on EAE-induced mice and ii) to study the effects of LeuSH on matrix metalloproteinase-9 (MMP-9), NADPH oxidase and cytokines (IFN-γ, IL-5 and IL-10) in tissues and plasma of EAE mice as a measure of potential markers associated with EAE disease. C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) peptide in complete Freund's adjuvant to induce EAE. A significant difference was observed in body weights and clinical signs of LeuSH (8 mg/kg) administered EAE-induced mice compared to control mice. The findings of this study include alterations in the enzymatic expression of MMP-9, NADPH oxidase and cytokine levels in the brain, spinal cord, spleen, thymus and plasma of inhibitor-treated EAE mice as well as EAE-induced mice. The enzyme activities of NADPH oxidase were inhibited by LeuSH. From these results, it can be considered that LeuSH acts as one of the antigen candidates in ameliorating the clinical symptoms of EAE disease in mice.

Amelioration of experimental autoimmune encephalomyelitis by plumbagin through down-regulation of JAK-STAT and NF-κB signaling pathways.

Plumbagin (PL), a herbal compound derived from roots of the medicinal plant Plumbago zeylanica, has been shown to have immunosuppressive properties. Present report describes that PL is a potent novel agent in control of encephalitogenic T cell responses and amelioration of mouse experimental autoimmune encephalomyelitis (EAE), through down-regulation of JAK-STAT pathway. PL was found to selectively inhibit IFN-γ and IL-17 production by CD4(+) T cells, which was mediated through abrogated phosphorylation of JAK1 and JAK2. Consistent with IFN-γ and IL-17 reduction was suppressed STAT1/STAT4/T-bet pathway which is critical for Th1 differentiation, as well as STAT3/ROR pathway which is essential for Th17 differentiation. In addition, PL suppressed pro-inflammatory molecules such as iNOS, IFN-γ and IL-6, accompanied by inhibition of IκB degradation as well as NF-κB phosphorylation. These data give new insight into the novel immune regulatory mechanism of PL and highlight the great value of this kind of herb compounds in probing the complex cytokine signaling network and novel therapeutic targets for autoimmune diseases.

Cyclooxygenase expression and prostaglandin levels in central nervous system tissues during the course of chronic relapsing experimental autoimmune encephalomyelitis (EAE).

OBJECTIVE: Multiple sclerosis (MS) and its animal counterpart experimental autoimmune encephalomyelitis (EAE) have a major inflammatory component that drives and orchestrates both diseases. One particular group of mediators are the prostaglandins (PGs), which we have previously shown, through quantitation and pharmacological intervention, to be closely involved in the pathology of MS and EAE. The aim of the current study was to determine the expression of the PG-generating cyclooxygenase (COX) enzymes and the profile of PGE(2) and PGD(2), in selected central nervous system (CNS) tissues, with the development of the chronic relapsing (CR) form of EAE. In particular, the work investigates the possible relationship between the expression of COX isoenzymes and PG levels during the neurological phases of CR EAE. METHODS: CR EAE was induced in Biozzi mice with inoculum containing lyophilised, syngeneic spinal cord emulsified in complete Freund's adjuvant. The cerebral cortex, cerebellum and spinal cord were dissected from mice during the acute, remission and relapse stages of disease with a minimum of five animals per treatment. The expression of COX-1, COX-1b variant and COX-2, in pooled samples, was determined by Western blotting. PGE(2) and PGD(2) levels in extracted samples were measured using commercial enzyme immunoassay kits. RESULTS: COX-2 expression in spinal cords during acute disease remained unaltered and was in contrast to an enhancement of the enzyme, together with COX-1 and COX-1b, in all other sampled areas. PGE(2) and PGD(2) levels remained unchanged during the acute phase and the subsequent remission of symptoms. COX-1 and COX-1b expression was elevated in tissues during the relapse stage of CR EAE and concentrations of the prostanoids were markedly increased. CONCLUSIONS: The study examines the implications of COX isoenzyme expression over the course of CR EAE and discusses the reported relationship between PGE(2) and PGD(2) in the instigation and resolution of CNS inflammation. Consideration is also given to the treatment of CR EAE and suggests that drugs designed to limit the inflammatory effects of the PGs should be administered prior to or during the relapse phase of the disease.

Time-course changes in ectonucleotidase activities during experimental autoimmune encephalomyelitis.

The aim of the present study was to analyze the activities of extracellular purine metabolizing enzymes, CD39 (apyrase, EC 3.6.1.5) and CD73 (ecto-5' nucleotidase, EC 3.1.3.5) in experimental autoimmune encephalomyelitis (EAE). The levels of ATP, ADP and AMP hydrolysis were analyzed in the blood serum and in the rat spinal cord plasma membrane preparation 8, 15 and 25 days after induction of EAE. The animals were divided in three groups: control (saline), CFA (adjuvant-only) and EAE (CFA and homogenate of spinal cords). Eight days after immunization, ATP, ADP and AMP hydrolysis in the blood serum and spinal cord membrane preparations were unaffected in EAE compared to both, control and CFA group. In the peak of disease, ATP, ADP and AMP hydrolysis in EAE group showed significant decrease in the blood serum and prominent increase in the spinal cord membrane preparation compared to CFA and control group. At the end of illness, as judged by disappearance of clinical manifestation of EAE, ATP, ADP and AMP hydrolysis, although closer to CFA levels, were still significantly different in respect to the CFA group. Modulation of ATP, ADP and AMP hydrolysis suggests that they operate during EAE and might represent the basis of novel therapeutic strategies in immune-mediated diseases, such as MS.

Sodium salicylate-induced amelioration of experimental autoimmune encephalomyelitis in Lewis rats is associated with the suppression of inducible nitric oxide synthase and cyclooxygenases.

We studied the effects of oral administration of sodium salicylate on the expression of the pro-inflammatory mediators, nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1 and 2, in rats with experimental autoimmune encephalomyelitis (EAE). Sodium salicylate (200 mg/kg) was administered orally for 13 days after the induction of EAE by immunization with guinea pig myelin basic protein and complete Freund's adjuvant. The onset (P<0.0001) and severity (P<0.05) of EAE paralysis in salicylate-treated animals were delayed and suppressed significantly compared with vehicle-treated controls. Western blot analysis showed that expression of COX-2 and iNOS, but not COX-1, decreased significantly in the spinal cords of salicylate-treated rats compared with vehicle-treated controls (P<0.05) and this finding was paralleled by immunohistochemical observations. These results suggest that the amelioration by salicylate of paralysis in rats with EAE is mediated in part by the suppression of COX and iNOS.

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.

Targeting dipeptidyl peptidase IV (CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo.

CD26 or dipeptidyl peptidase IV (DP IV) is expressed on various cell types, including T cells. Although T cells can receive activating signals via CD26, the physiological role of CD26/DP IV is largely unknown. We used the reversible DP IV inhibitor Lys[Z(NO(2))]-pyrrolidide (I40) to dissect the role of DP IV in experimental autoimmune encephalomyelitis (EAE) and to explore the therapeutic potential of DP IV inhibition for autoimmunity. I40 administration in vivo decreased and delayed clinical and neuropathological signs of adoptive transfer EAE. I40 blocked DP IV activity in vivo and increased the secretion of the immunosuppressive cytokine TGF-beta1 in spinal cord tissue and plasma during acute EAE. In vitro, while suppressing autoreactive T cell proliferation and TNF-alpha production, I40 consistently up-regulated TGF-beta1 secretion. A neutralizing anti-TGF-beta1 Ab blocked the inhibitory effect of I40 on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not generally immunosuppressive, neither eliminating encephalitogenic T cells nor inhibiting T cell priming. These data suggest that DP IV inhibition represents a novel and specific therapeutic approach protecting from autoimmune disease by a mechanism that includes an active TGF-beta1-mediated antiinflammatory effect at the site of pathology.

Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis.

Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression.

Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis.

In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.

Nitric oxide and the immunomodulation of experimental allergic encephalomyelitis.

Previous studies examining the effect of nitric oxide synthase (NOS) inhibition on the course of experimental allergic encephalomyelitis (EAE) have yielded conflicting results. This may relate to the use of nonspecific inhibitors and to differences between active and adoptive EAE. We examined the effect of treatment with L-N-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of the cytokine-inducible isoform of NOS, on the clinical course of active and adoptive EAE in Lewis rats. We find that while L-NIL treatment of recipients is protective in adoptive EAE, treatment of active EAE with L-NIL leads to a marked accentuation of disease expression. In L-NIL-treated animals treated with myelin basic protein/complete Freund's adjuvant (MBP/CFA), disease onset is accelerated and clinical symptoms are more severe. Accentuation of integrated disease scores is seen even if L-NIL treatment is started 5 days following immunization. The histological findings in involved spinal cords from L-NIL-treated animals with active EAE are similar to those from untreated animals with similar clinical scores. L-NIL treatment of MBP/CFA-immunized animals does not prevent recovery from clinical symptoms, nor does it allow for reinduction of disease in animals previously immunized with MBP/CFA. Treatment of F344 rats, a strain which is relatively nonsusceptible for EAE, with L-NIL results in consistent evidence of EAE following immunization with MBP/CFA. These findings, together with our previous work on interstitial nephritis, support a role for endogenously generated NO in immunoregulation of T cell responses following immunization with antigen in CFA, and suggest that inducibility of NOS expression may be an important susceptibility factor for autoimmunity.

Inhibition of brain macrophage/microglial respiratory chain enzyme activity in experimental autoimmune encephalomyelitis of the Lewis rat.

We measured the activities of five respiratory chain enzymes in brain macrophages/microglial cells from Lewis rats with experimental autoimmune encephalomyelitis (EAE) and found a significant reduction of the activity of nicotinamide adenine dinucleotide-dehydrogenase (NADH-DH), succinate cytochrome c reductase (SCCR) and succinate dehydrogenase (SDH) when compared with age-matched healthy control animals. The inhibition of NADH-DH (complex I) was specific for EAE, while we also found a reduction of SCCR and SDH activities (complex II) in newborn rats and adjuvant-immunised rats. Activities of NADH cytochrome c reductase (NCCR) and cytochrome c oxidase (COX) were not significantly changed. These observations demonstrate an impairment of brain macrophage/microglial respiratory chain function in central nervous system inflammation.

Anti-oxidant capacity of spinal cord and erythrocytes of guinea pigs in case of experimental allergic encephalomyelitis and after disease suppression.

In the lumbar spinal cord of EAE guinea pigs a significant increase in SOD activity, lipid hydroperoxides content (more than 60%) and Fe2(+)-ascorbate-induced lipid peroxidation was observed. Multiple injections of cytochrome C-vitamin B2-vitamin PP (CV-combination) during the disease latent period resulted in suppression of EAE development. Supplementation with vitamin C, vitamin B12 or ATP eliminated this suppressive effect. Upon treatment with CV-combination beginning on the day of the first EAE clinical signs a half of the sick animals recovered. In their erythrocytes the ratio between SOD and catalase activities was normalized, though on a higher level. In the lumbar spinal cord the concentration of lipid hydroperoxides was decreased to the control one. Oxidative damage of the central nervous system is one of the mechanisms underlying the pathogenesis of lethal EAE.

Selenium and experimental allergic encephalomyelitis. The effects of different levels of dietary selenium on clinico-pathological findings.

There are reports in which multiple sclerosis (MS) seems to be associated with abnormalities in selenium (Se) metabolism and erythrocyte glutathione-peroxidase (GSH-px) activity. Ordinary experimental allergic encephalomyelitis (EAE), which reflects some features of human MS, was induced in guinea pigs maintained with high, low and normal levels of Se in the diet. Evidence was obtained to indicate the following results: (i) a direct correlation between dietary Se levels and whole blood Se levels. (ii) Erythrocyte GSH-px activity was not found to be correlated with the blood Se content. (iii) The animals fed with low or normal levels of Se showed the same survival rates and developed EAE in a similar way and percentage. (iv) The animals fed with high non-toxic levels of Se showed a high incidence of death and some developed EAE with a subacute course, when compared with the other experimental groups. The results are discussed on the basis of findings in the literature.