Berberine attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice

Background

Berberine, an isoquinoline derivative alkaloid, has a wide range of pharmacological properties and is considered to have anti-inflammatory and neuroprotective effects. However, there are no reports about the effects and mechanisms of berberine in experimental autoimmune encephalomyelitis (EAE), an established model of multiple sclerosis (MS).

Methodology/Principal Findings

Female C57 BL/6 mice immunized with myelin oligodendrocyte glycoprotein 35–55 amino acid peptide were treated with berberine at the day of disease onset and medication was administered daily until mice were sacrificed. Blood–brain barrier (BBB) permeability and the alteration of matrix metalloproteinase-2 (MMP-2, 72 kDa) and matrix metalloproteinase-9 (MMP-9, 92 kDa) in the brain and cerebrospinal fluid (CSF) of EAE mice were detected by quantitative measurement for Evan''s blue (EB) content, Western blot and gelatin zymography respectively. The results showed that berberine attenuated clinical and pathological parameters of EAE, reduced the permeability of BBB, inhibited the activity and expression of MMP-9 but not MMP-2 in the CSF and brain of EAE mice.

Conclusions/Significance

These findings suggest that berberine is effective to attenuate the clinical severity of EAE in C57 BL/6 mice by reducing the permeability of BBB, decreasing the expression and activity of MMP-9, and decreasing the inflammatory infiltration. We think that berberine might be a potential therapeutic agent for MS.     

Naringenin attenuates experimental autoimmune encephalomyelitis by protecting the intact of blood-brain barrier and controlling inflammatory cell migration

Targeting pathogenic immune cell trafficking poses an attractive opportunity to attenuate autoimmune disorders such as multiple sclerosis (MS). MS and its animal model, experimental autoimmune encephalomyelitis (EAE), are characterized by the immune cells-mediated demyelination and neurodegeneration of the central nervous system (CNS). Our previous study has proven that dietary naringenin ameliorates EAE clinical symptoms via reducing the CNS cell infiltration. The present study examined the beneficial effects of naringenin on maintaining the blood-brain barrier in EAE mice via dietary naringenin intervention. The results showed that naringenin-treated EAE mice had an intact blood-CNS barrier by increasing tight junction-associated factors and decreasing Evans Blue dye in the CNS. Naringenin decreased the accumulation and maturation of conventional dendritic cells (cDCs), CCL19, and CCR7 in the CNS. Also, naringenin blocked the chemotaxis and antigen-presenting function of cDCs that resulted in reducing T-cell secreting cytokines (IFN-γ, IL-17, and IL-6) in the spleen. Importantly, naringenin blocked pathogenic T cells infiltrated into the CNS and attenuates passive EAE. Therefore, by blocking chemokine-mediated migration of DCs and pathogenic T cells into the CNS, naringenin attenuates EAE pathogenesis and might be a potential candidate for the treatment of autoimmune diseases, such as MS and other chronic T-cell mediated autoimmune diseases.     

Dietary supplementation with fructooligosaccharides attenuates allergic peritonitis in mice

Fructooligosaccharides (FOS) are a prebiotic supplement, which can enhance immunological responses in the host to activate mucosal immunity probably through regulation of gastrointestinal microflora. Nonetheless, the therapeutic potential of prebiotics on allergic pathologies has not been fully elucidated. Therefore, the purpose of this study was to evaluate the preventive and therapeutic effects of dietary supplementation with FOS on a murine model of allergic peritonitis induced by ovalbumin (OVA). Male C3H/HeN mice were intraperitoneally administrated with OVA (1 μg) bi-weekly (Day 0-42, total four times) and were fed a diet containing 0 or 2.5% FOS ad libitum (Day 7-43). At Day 43, mice were killed and several parameters were evaluated. As results, supplementation with FOS alleviated OVA-related peritoneal inflammation characterized by trafficking of polymorphonuclear leukocytes such as eosinophils and neutrophils in the peritoneal cavity. Also, FOS significantly suppressed the protein level of interleukin (IL)-5 and eotaxin in the peritoneal lavage fluid elicited by OVA. In addition, a FOS-supplemented diet significantly reduced the serum allergen specific-IgG(1) level, whereas it significantly increased total IgA levels in the cecal contents as compared with a control diet in the presence of OVA. These results suggest that dietary supplementation with FOS can prevent/ameliorate allergic peritoneal inflammation induced by OVA. The efficacy can at least partially be associated with the regulation of Ig class switching and inhibition of the local expression of IL-5 and eotaxin.     

实验性自身免疫性脑脊髓炎小鼠的视神经损害

目的:探讨实验性自身免疫性脑脊髓炎(EAE)的视神经损害以及将其作为视神经炎模型的可能性.方法:取MOG35-55多肽和完全弗氏佐剂制备成抗原乳剂免疫C57BL/6小鼠,并在腹腔内注射2次百日咳杆菌,建立EAE模型.在EAE疾病高峰时,观察小鼠视神经炎的发生率及病理学改变.结果:模型组12只小鼠中有10只从免疫后第12天开始陆续起病,约在第17～19天达到疾病高峰,发病率为83.3%;而对照组未出现任何神经功能受损的症状.HE染色结果显示模型组的视神经组织中有大量的炎症细胞浸润,动物视神经炎的发生率为83.3%,其炎症评分为2.1±0.8分,与对照组相比均有统计学意义.LFB染色可见模型组的视神经有明显的脱髓鞘改变,病灶内可见炎症细胞浸润;而对照组小鼠的视神经未见明显异常改变.结论:借助EAE建立视神经炎模型是可行的,这为深入探讨视神经炎的发病机制提供了理想的动物平台.     

Dietary zinc supplementation attenuates hyperglycemia in db/db mice

Although zinc (Zn) deficiency has been associated with insulin resistance, and altered Zn metabolism (e.g., hyperzincuria, low-normal plasma Zn concentrations) may be present in diabetes, the potential effects of Zn on modulation of insulin action in Type II diabetes have not been established. The objective of this study was to compare the effects of dietary Zn deficiency and Zn supplementation on glycemic control in db/db mice. Weanling db/db mice and lean littermate controls were fed Zn-deficient (3 ppm Zn; dbZD and InZD groups), Zn-adequate control (30 ppm Zn; dbC and InC groups) or Zn-supplemented (300 ppm Zn; dbZS and InZS groups) diets for 6 weeks. Mice were assessed for Zn status, serum and urinary indices of diabetes, and gastrocnemius insulin receptor concentration and tyrosine kinase activity. Fasting serum glucose concentrations were significantly lower in the dbZS group compared with the dbZD group (19.3 +/- 2.9 and 27.9 +/- 4.1 mM, respectively), whereas the dbC mice had an intermediate value. There was a negative correlation between femur Zn and serum glucose concentrations (r = -0.59 for lean mice, P = 0.007). The dbZS group had higher pancreatic Zn and lower circulating insulin concentrations than dbZC mice. Insulin-stimulated tyrosine kinase activity in gastrocnemius muscle was higher in the db/db genotype, and insulin receptor concentration was not altered. In summary, dietary Zn supplementation attenuated hyperglycemia and hyperinsulinemia in db/db mice, suggesting that the roles of Zn in pancreatic function and peripheral tissue glucose uptake need to be further investigated.     

Dietary supplementation of N-acetylcysteine enhances early inflammatory responses during cutaneous wound healing in protein malnourished mice

Prolonged wound healing is a complication that contributes to the morbidity and mortality of protein malnutrition (PM). The molecular mechanisms that underlie impaired wound healing in PM may begin in the early inflammatory stage of the process. We hypothesized that the impaired wound healing observed in PM occurs as a consequence of excessive reactive oxygen species (ROS) production that impairs the wound healing process by depressing nuclear factor kappa B (NFkappaB) activation and the subsequent synthesis and release of proinflammatory cytokines that are critical mediators of the inflammatory response. In this study, we showed that the time to wound closure was significantly prolonged in PM mice. During the early wound healing in PM, inhibitory kappa B alpha (IkappaBalpha), interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) expression and neutrophil infiltration were significantly decreased in PM mice. The role of excess ROS in PM was demonstrated by using transgenic mice with overexpression of copper zinc superoxide dismutase and with dietary supplementation of N-acetylcysteine (NAC). Both interventions improved the extent of wound closure in PM mice. Moreover, NAC supplementation in PM mice restored the expression of IkappaBalpha, IL-1beta and TNF-alpha and infiltration of neutrophils to levels observed in control animals. These findings support the notion that wound healing defects in PM may result from dysregulation of ROS-mediated and NFkappaB-regulated signaling pathways.     

Corticotropin-releasing hormone contributes to the peripheral inflammatory response in experimental autoimmune encephalomyelitis

Peripheral corticotropin-releasing hormone (CRH) is thought to have proinflammatory effects. We used the model of experimental autoimmune encephalomyelitis (EAE) to study the role of CRH in an immune-mediated disease. We showed that CRH-deficient mice are resistant to EAE, with a decrease in clinical score as well as decreased cellular infiltration in the CNS. Furthermore, Ag-specific responses of primed T cells as well as anti-CD3/anti-CD28 TCR costimulation were decreased in crh(-/-) mice with decreased production of Th1 cytokines and increased production of Th2 cytokines. Wild-type mice treated in vivo with a CRH antagonist showed a decrease in IFN-gamma production by primed T cells in vitro. This effect of CRH is independent of its ability to increase corticosterone production, because adrenalectomized wild-type mice had similar disease course and severity as control mice. We found that IkappaBalpha phosphorylation induced by TCR cross-linking was decreased in crh(-/-) T cells. We conclude that peripheral CRH exerts a proinflammatory effect in EAE with a selective increase in Th1-type responses. These findings have implications for the treatment of Th1-mediated diseases such as multiple sclerosis.     

Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice

Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8(-/-) mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC(50) = 0.4 nM). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis.     

Fatal neurological disease in scrapie-infected mice induced for experimental autoimmune encephalomyelitis

During the years or decades of prion disease incubation, at-risk individuals are certain to encounter diverse pathological insults, such as viral and bacterial infections, autoimmune diseases, or inflammatory processes. Whether prion disease incubation time and clinical signs or otherwise the pathology of intercurrent diseases can be affected by the coinfection process is unknown. To investigate this possibility, mice infected with the scrapie agent at both high and low titers were subsequently induced for experimental autoimmune encephalomyelitis, an immune system-mediated model of central nervous system (CNS) inflammation. We show here that co-induced mice died from a progressive neurological disease long before control mice succumbed to classical scrapie. To investigate the mechanism of the co-induced syndrome, we evaluated biochemical and pathological markers of both diseases. Brain and spleen PrP(Sc) levels in the dying co-induced mice were comparable to those observed in asymptomatic scrapie-infected animals, suggesting that co-induced disease is not an accelerated form of scrapie. In contrast, inflammatory markers, such as demyelination, immune cell infiltrates, and gliosis, were markedly increased in co-induced mouse spinal cords. Activated astrocytes were especially elevated in the medulla oblongata. Furthermore, PrP(sc) depositions were found in demyelinated white matter areas in co-induced mouse spinal cords, suggesting the presence of activated infected immune cells that infiltrate into the CNS to facilitate the process of prion neuroinvasion. We hypothesize that inflammatory processes affecting the CNS may have severe clinical implications in subjects incubating prion diseases.     

Antigen-induced suppression of mitogen responses and resistance to experimental autoimmune encephalomyelitis

Random-bred Hartley and inbred Strain 2 and Strain 13 guinea pigs were inoculated for acute experimental autoimmune encephalomyelitis (EAE). Sixty-six percent (69/103) of the Hartleys developed signs of EAE while the remaining 34% (34/103) were resistant. No Strain 2 and all Strain 13 guinea pigs developed EAE. Histologic examination of nervous tissue revealed that susceptible Hartleys and Strain 13 and Strain 2 animals had lesions characteristic of EAE. Tissue from resistant Hartleys showed fewer and less severe changes. Lymphocyte-transformation assays with EAE-inducing and noninducing antigens and T-cell mitogens revealed three different sets of responses in vitro: (i) lymphocytes from all animals responded to mitogens; (ii) lymphocytes from susceptible animals responded to EAE-inducing antigens; and (iii) lymphocytes from resistant Hartleys were suppressed from responding to the mitogens solely by EAE-inducing antigens. Plasmas from all EAE-sensitized animals had equivalent anti-myelin basic proteins (MBP) antibody titers and skin tests of EAE-inoculated Hartleys were all positive for MBP sensitization. Therefore, resistance and reduced histologic changes characteristic of EAE correlated with a disease-specific antigen-induced suppression of lymphocyte responses to T-cell mitogens. This suggests that clinical resistance to EAE in Hartley guinea pigs is mediated by an immunologic suppressor mechanism.     

Suppression of experimental autoimmune encephalomyelitis by dermatan sulfate

The effect of dermatan sulfate (DS) on the treatment of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined. DS, a sulfated glycosaminoglycan, has been reported to exhibit anticoagulant and fibrinolytic activities. DS treatment (50 mg/kg/day) facilitates recovery from the clinical manifestations of EAE. In this study, the fibrinolytic activity was higher in DS-treated rats than in saline-treated rats. Although the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in DS-treated rats compared to that in saline-treated rats, perivascular fibrin deposition was markedly suppressed in DS-treated rats. These findings suggest that DS would act as an effective therapeutic agent for EAE by preventing fibrin deposition.     

Hypercalcemia produced by parathyroid hormone suppresses experimental autoimmune encephalomyelitis in female but not male mice

Besides its role in regulating serum levels of calcium and phosphorus, 1alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) has potent effects on the immune system and suppresses disease in several animal models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. While the amount of 1,25-(OH)2D3 needed to prevent EAE is dependent on the gender of the mouse and amount of calcium available in the diet, the minimum levels of 1,25-(OH)2D3 sufficient to prevent disease cause hypercalcemia. To test if hypercalcemia independent of high levels of 1,25-(OH)2D3 can suppress EAE, we used a 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase) knockout mouse strain. Because these 1alpha-hydroxylase knockout mice lack the parathyroid hormone (PTH)-regulated enzyme that synthesizes 1,25-(OH)2D3, hypercalcemia from increased bone turnover was created by continuous administration of PTH without changing the circulating levels of 1,25-(OH)2D3. This PTH-mediated hypercalcemia generated after EAE induction prevented disease in female mice but not male mice. When hypercalcemia was prevented by diet manipulation, PTH administration no longer prevented EAE. We conclude that hypercalcemia is able to prevent EAE after disease induction in female mice.     

Impaired autoimmune T helper 17 cell responses following DNA vaccination against rat experimental autoimmune encephalomyelitis

Background

We have previously shown that vaccination with DNA encoding the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)_91–108 (pMOG) suppresses MOG_91–108-induced rat Experimental Autoimmune Encephalomyelitis (EAE), a model for human Multiple Sclerosis (MS). The suppressive effect of pMOG is dependent on inclusion of CpG DNA in the plasmid backbone and is associated with early induction of Interferon (IFN)-β.

Principal Findings

In this study we examined the mechanisms underlying pMOG-induced protection. We found that in the DNA vaccinated cohort proinflammatory Interleukin (IL)-17 and IL-21 responses were dramatically reduced compared to in the control group, but that the expression of Foxp3 and Tumor Growth Factor (TGF)-β1, which are associated with regulatory T cells, was not enhanced. Moreover, genes associated with Type I IFNs were upregulated. To delineate the role of IFN-β in the protective mechanism we employed short interfering RNA (siRNA) to IFN-β in the DNA vaccine. SiRNA to IFN-β completely abrogated the protective effects of the vaccine, demonstrating that a local early elaboration of IFN-β is important for EAE protection. IL-17 responses comparable to those in control rats developed in rats injected with the IFN-β-silencing DNA vaccine.

Conclusions

We herein demonstrate that DNA vaccination protects from proinflammatory Th17 cell responses during induction of EAE. The mechanism involves IFN-β as IL-17 responses are rescued by silencing of IFN-β during DNA vaccination.     

Resistance to myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis by death receptor 6-deficient mice

Genetic disruption of death receptor 6 (DR6) results in enhanced CD4+ T cell expansion, Th2 differentiation, and humoral responses after stimulation. However, the in vivo consequences of DR6 targeting (DR6-/-) during the initiation and progression of inflammatory autoimmune disease are unclear. Using a myelin oligodendrocyte glycoprotein (MOG(35-55))-induced model of experimental autoimmune encephalomyelitis, DR6-/- mice were found to be highly resistant to both the onset and the progression of CNS disease compared with wild-type (WT) littermates. DR6-/- mice exhibited fewer inflammatory foci along with minimal demyelination and perivascular cuffing of inflammatory cells. Consistent with these observations, mononuclear cell infiltration, including CD4+ T cells and macrophages, in the spinal cord of DR6-/- mice was dramatically reduced. Furthermore, CD4+ T cells from DR6-/- mice exhibited profoundly reduced cell surface expression of VLA-4 before and after stimulation. Compared with WT mice, DR6-/- mice exhibited significantly increased autoantigen-induced T cell proliferative responses along with greater numbers of IL-4-producing and similar or slightly higher numbers of IFN-gamma-producing CD4+ T cells. DR6-/- CD4+ T cells secreted higher levels of the Th2 cytokine, IL-4, and similar levels of the Th1 cytokine, IFN-gamma, compared with WT cells. Taken together, our data demonstrate that DR6 plays an important role in regulating leukocyte infiltration and function in the induction and progression of experimental autoimmune encephalomyelitis.     

Inhibition of complement alternative pathway suppresses experimental autoimmune anterior uveitis by modulating T cell responses

The objective of the current study was to delineate the pathway of complement activation that is crucial for the induction of experimental autoimmune anterior uveitis (EAAU). We studied the development of EAAU in melanin-associated antigen (MAA)-sensitized Lewis rats treated with antibody against C4 or factor B. Control animals received isotype IgG control. Antibody against C4 had no effect on EAAU, and all of the animals developed EAAU similar to those injected with control IgG. In contrast, EAAU was completely inhibited in all MAA-sensitized Lewis rats injected with factor B antibody. Treatment with anti-factor B antibody resulted in suppression of ocular complement activation. Adoptive transfer of T lymphocytes harvested from draining lymph nodes of donor animals treated with anti-factor B did not transfer EAAU to naïve syngenic rats. Anti-factor B antibody inhibited the ability of MAA-specific CD4⁺ T cells to proliferate (in vitro) in response to MAA in a dose-dependent manner. Level of TNF-α and IFN-γ decreased in the presence of anti-factor B. Collectively, our results provide the novel finding that complement activation via the alternative pathway contributes to intraocular inflammation in EAAU, and anti-factor B-mediated inhibition of EAAU is due to diminished antigen-specific CD4⁺ T cell responses to MAA. Our findings explain the interactions between the complement system and T cells that are critical for the induction of EAAU and may lead to the development of therapy for idiopathic anterior uveitis based on selective blockade of the alternative pathway.