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Rosmarinic acid is a polyphenolic compound, abundantly present in herbs of the Lamiaceae family. The aim of the study was to evaluate the immunomodulatory properties of a recently developed phenethyl ester derivative of rosmarinic acid (PERA), with enhanced ability of diffusion through biological membranes, in an animal model of the central nervous system (CNS) autoimmunity. To this end, experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis was used. Daily subcutaneous administration of PERA (30 mg/kg) from day 7 to day 22 after immunization successfully ameliorated EAE induced in Dark Agouti rats, shortening the disease duration and reducing maximal, cumulative and mean clinical score. PERA efficiently reduced production of major encephalitogenic cytokines, interferon (IFN)-γ and interleukin (IL)-17, in immune cells from the CNS or the lymph nodes draining the site of immunization of EAE rats, as well as in CD4

Sepsis is a life-threatening condition characterized by an acute cytokine storm followed by prolonged dysfunction of the immune system in the survivors. Post-septic lymphopenia and functional deficits of the remaining immune cells lead to increased susceptibility to secondary infections and other morbid conditions causing late death in the patients. This state of post-septic immunoparalysis may also influence disorders stemming from inappropriate or overactive immune responses, such as autoimmune and immunoinflammatory diseases, including multiple sclerosis. In addition, ongoing autoimmunity likely influences the susceptibility to and outcome of sepsis. This review article addresses the bidirectional relationship between sepsis and multiple sclerosis, with a focus on the immunologic mechanisms of the interaction and potential directions for future studies.

Experimental autoimmune encephalomyelitis (EAE) is classically induced with complete Freund's adjuvant (CFA). The immune response against CFA has a confounding influence on the translational capacity of EAE as a multiple sclerosis model. Here, we compare clinical, cellular and molecular properties between syngeneic spinal cord homogenate (SCH)- and SCH + CFA-immunized Dark Agouti rats. EAE signs were observed earlier and the cumulative clinical score was higher without CFA. Also, a higher number of immune cells infiltrates in the spinal cords was noticed at the peak of EAE without CFA. High spinal cord abundance of CD8+CD11bc+MHC class II+ cells was detected in SCH-immunized rats. Myelin basic protein -specific response can be elicited in the cells from the lymph nodes draining the site of SCH immunization. This CFA-free EAE is a reliable multiple sclerosis model.

C57BL/6, BALB/c and NOD mice are among the most frequently used strains in autoimmunity research. NOD mice spontaneously develop type 1 diabetes (T1D) and they are prone to induction of experimental autoimmune encephalomyelitis (EAE). Both diseases can be routinely induced in C57BL/6 mice, but not in BALB/c mice. Also, C57BL/6 mice are generally considered T helper (Th)1-biased and BALB/c Th2-biased mice. Having in mind increasingly appreciated role of gut associated lymphoid tissue (GALT) cells in autoimmunity, especially in relation to gut Th17 and regulatory T (Treg) cells, our aim was to determine if there are differences in proportion of CD4 + T cell populations in mesenteric lymph nodes and Peyer's p atches of these mouse strains. Lower proportion of Treg was observed in NOD PP, Th2 cells dominated in BALB/c mice in mesenteric lymph nodes (MLN) and Peyer's patches (PP), while Th1 cells prevailed in C57BL/6 MLN. Intradermal immunization of mice with complete Freund's adjuvant resulted in significant difference in Th cell distribution in GALT of NOD mice. Differences were less pronounced in C57BL/6 mice, while GALT of BALB/c mice was almost unresponsive to the immunization. The observed strain- and tissue-dependent changes in Treg proportion after the immunization was probably a consequence of different CCR2 or CCR6-related migration patterns and/or in situ Treg proliferation. In conclusion, NOD, a highly autoimmunity-prone mouse strain, exhibits more profound GALT-related immune response upon immunization compared to the strains that are less prone to autoimmunity. Immunology Letters (2017), 190: 282-288

Cucumber (Cucumis sativus) fruit has been used in cuisine worldwide, while its leaves are rich in immunomodulatory compounds. Cucumber leaf extract (CLE) was characterized by the predominance of triterpenoids cucurbitacins and significant levels of phenolics. Effects of CLE on CD4 + T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system were investigated in our study. CLE potently inhibited production of major pathogenic Th cytokines: interferon-gamma and interleukin-17, as well as of nitric oxide and reactive oxygen species in macrophages. Antigen-presenting activity of macrophages and dendritic cells was also affected by CLE. The effects of CLE were co-incident with modulation of NFκB and p38 MAPK signaling. Concentrations of CLE used in vitro did not show toxic effects on zebrafish embryos. Moreover, CLE inhibited generation of encephalitogenic cells in vivo. These results demonstrate that CLE deserve further investigation on its anti-encephalitogenic therapeutic properties. Journal of Functional Foods (2017), 37: 249-262

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system. The aim of this work was to study the probiotic effect of γ-aminobutyric acid (GABA)-producer Lactobacillus brevis BGZLS10-17 on experimental autoimmune encephalomyelitis (EAE), an experimental animal MS model. Clinical EAE symptoms were monitored in Dark Agouti (DA) rats treated with L. brevis BGZLS10-17 strain, or supernatant obtained from 48 h culture of L. brevis BGZLS10-17 cultivated in growth medium with or without GABA precursor monosodium glutamate (MSG). The results revealed that oral administration of L. brevis BGZLS10-17 alleviates the symptoms of EAE in DA rats. Namely, treatment with BGZLS10-17 and the supernatant of the strain cultivated in medium with MSG delayed the onset, shortened the duration, and reduced the intensity of the disease in the period when the EAE symptoms in controls were most pronounced. The probiotic treated animals were completely recovered after forty days, unlike the control animals. The results indicate that supplementation with live strain or with supernatant containing GABA produced by L. brevis BGZLS10-17 could alleviate the EAE symptoms. However, the use of L. brevis BGZLS10-17 in functional food as probiotic for autoimmune diseases should be tested in clinical trials. [Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173019 and Grant no. 173035]

Cucurbitacin E (CucE) is a highly oxidized steroid consisting of a tetracyclic triterpene. It is a member of a Cucurbitacin family of biomolecules that are predominantly found in Cucurbitaceae plants. CucE has already been identified as a potent anti-inflammatory compound. Here, its effects on CD4(+) T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system, were investigated. Production of major pathogenic Th cell cytokines: interferon-gamma and interleukin-17 were inhibited under the influence of CucE. The effects of CucE on CD4(+) T cells were mediated through the modulation of aryl hydrocarbon receptor, STAT3, NFκB, p38 MAPK, and miR-146 signaling. Further, production of nitric oxide and reactive oxygen species, as well as phagocytic ability, were inhibited in macrophages treated with CucE. These results imply that CucE possesses powerful antiencephalitogenic activity.

Gut microbiota and gut-associated lymphoid tissue have been increasingly appreciated as important players in pathogenesis of various autoimmune diseases, including multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that can be induced with an injection of spinal cord homogenate emulsified in complete Freund’s adjuvant in Dark Agouti (DA) rats, but not in Albino Oxford (AO) rats. In this study, mesenteric lymph nodes (MLN), Peyer’s patches (PP) and gut microbiota were analysed in these two rat strains. There was higher proportion of CD4+ T cells and regulatory T cells in non-immunised DA rats in comparison to AO rats. Also, DA rat MLN and PP cells were higher producers of pro-inflammatory cytokines interferon-γ and interleukin-17. Finally, microbial analyses showed that uncultivated species of Turicibacter and Atopostipes genus were exclusively present in AO rats, in faeces and intestinal tissue, respectively. Thus, it is clear that in comparison of an EAE-susceptible with an EAE-resistant strain of rats, various discrepancies at the level of gut associated lymphoid tissue, as well as at the level of gut microbiota can be observed. Future studies should determine if the differences have functional significance for EAE pathogenesis.

Gut microbiota dysbiosis has been considered the essential element in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Antibiotics were administered orally to Dark Agouti (DA) rats early in their life with the aim of perturbing gut microbiota and investigating the effects of such intervention on the course of EAE. As a result, the diversity of the gut microbiota was reduced under the influence of antibiotics. Mainly, Firmicutes and Actinobacteria were replaced by Proteobacteria and Bacteroidetes, while decreased proportions of Clostridia and Bacilli classes were accompanied by an increase in Gamma-Proteobacteria in antibiotic-treated animals. Interestingly, a notable decrease in the Helicobacteraceae, Spirochaetaceae and Turicibacteriaceae was scored in antibiotic-treated groups. Also, levels of short chain fatty acids were reduced in the faeces of antibiotic-treated rats. Consequently, aggravation of EAE, paralleled with stronger immune response in lymph nodes draining the site of immunization, and increased inflammation within the CNS, were observed in antibiotic-treated DA rats. Thus, the alteration of gut microbiota leads to an escalation of CNS-directed autoimmunity in DA rats. The results of this study indicate that antibiotic use in early life may have subsequent unfavourable effects on the regulation of the immune system.

MicroRNAs (miR) are small non-coding RNAs involved in the immune response regulation. miR-155 has been attributed a major pro-inflammatory role in the pathogenesis of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Here, a role of miR-155 in re-activation of encephalitogenic CD4(+) T cells was investigated. Dark Agouti rats were immunized with myelin basic protein (MBP) emulsified in complete Freund's adjuvant. CD4(+) T cells were purified from draining lymph node cells (DLNC) obtained in the inductive phase and from spinal cord immune cells (SCIC) isolated at the peak of EAE. CD4(+) T cells obtained from SCIC (i.e., in vivo re-activated cells) had markedly higher expression of miR-155 in comparison to those purified from DLNC (not re-activated). Likewise, in vitro re-activation of DLNC with MBP led to increase in miR-155 expression. Further, DLNC and DLNC CD4(+) T cells were transfected with an inhibitor of miR-155 during in vitro re-activation. As a result, expression of important CD4(+) T cell effector cytokines IFN-gamma and IL-17, but not of regulatory cytokines IL-10 and TGF-beta, was reduced. These results imply that miR-155 supports re-activation of encephalitogenic CD4+ T cells. Our results contribute to a view that miR-155 might be a valuable target in multiple sclerosis therapy. (C) 2015 Elsevier Masson SAS. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173035, 175038, 173013]

Ethyl pyruvate is a redox analogue of dimethyl fumarate (Tecfidera), a drug for multiple sclerosis treatment. We have recently shown that ethyl pyruvate ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. It affects encephalitogenic T cells and macrophages in vitro, as well as in lymph nodes draining the site of encephalitogenic immunization and within the central nervous system (CNS). Here, in vivo effects of ethyl pyruvate on EAE are thoroughly investigated in the CNS and within the gut associated lymphoid tissue. Ethyl pyruvate reduced infiltrates within the CNS and number of activated macrophages/microglia (ED1(+)/Iba1(+)) and proliferating astrocytes (GFAP(+)). Furthermore, it reduced expression of HMGB1 in activated macrophages/microglia. It also reduced number of activated T cells and antigen-presenting cells and expression of Th1/Th17-related molecules in mesenteric lymph nodes and Peyer's patches. These results contribute to our understanding of anti-encephalitogenic effects of ethyl pyruvate as they provide evidence of its effects within the CNS and imply that these effects are related to reduction of inflammatory immune response in gut associated lymphoid tissue. Biomedicine & Pharmacotherapy (2017), 96: 78-85

Multiple sclerosis is a chronic inflammatory disease of the central nervous system (CNS). It is widely accepted that autoimmune response against the antigens of the CNS is the essential pathogenic force in the disease. It has recently become increasingly appreciated that activated encephalitogenic cells tend to migrate towards gut associated lymphoid tissues (GALT) and that interrupted balance between regulatory and inflammatory immunity within the GALT might have decisive role in the initiation and propagation of the CNS autoimmunity. Gut microbiota composition and function has the major impact on the balance in the GALT. Thus, our aim was to perform analyses of gut microbiota in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Albino Oxford (AO) rats that are highly resistant to EAE induction and Dark Agouti (DA) rats that develop EAE after mild immunization were compared for gut microbiota composition in different phases after EAE induction. Microbial analyses of the genus Lactobacillus and related lactic acid bacteria showed higher diversity of Lactobacillus spp. in EAE-resistant AO rats, while some members of Firmicutes and Proteobacteria (Undibacterium oligocarboniphilum) were detected only in faeces of DA rats at the peak of the disease (between 13 and 16 days after induction). Interestingly, Turicibacter sp. that was found exclusively in non-immunized AO, but not in DA rats in our previous study was detected in DA rats that remained healthy 16 days after induction. Similar observation was obtained for the members of Lachnospiraceae. As dominant presence of the members of Lachnospiraceae family in gut microbial community has been linked with mild symptoms of various diseases, it is tempting to assume that Turicibacter sp. and Lachnospiraceae contribute to the prevention of EAE development and the alleviation of the disease symptoms. Further, production of a typical regulatory cytokine interleukin-10 was compared in GALT cells of AO and DA rats, and higher production was observed in DA rats. Our data contribute to the idea that gut microbiota and GALT considerably influence multiple sclerosis pathogenesis.

Ethyl pyruvate (EP) has been increasingly appreciated as an anti-inflammatory and neuroprotective agent with potent pharmacological properties relevant for treatment of various CNS disorders. Microglial cells seem to be particularly sensitive to its effects. In this study, microglial cells were exposed to EP for relatively short periods (10-120 min) and inflammatory properties of the cells were determined after 24 h of cultivation. Application of EP in the short-term periods inhibited production of interleukin-6, tumor necrosis factor and nitric oxide in microglial cells. At the same time, the effects on cell viability, reactive oxygen species generation and expression of F4/80 and CD40 of microglial cells were minor. NFkB activation was not affected by EP in the cells during the short exposures, thus implying that the observed effect of EP on cytokine and nitric oxide generation was performed in NFkB independent way. Importantly, effects of the short term EP treatment on microglial cells were detected by a real time cell analysis, as well. The observed ability of EP to affect microglial cell function after relatively short time of exposure is relevant for its therapeutic potential against inflammatory disorders of the CNS. (C) 2015 Elsevier Masson SAS. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}OI173035, OI173013]

Dimethyl fumarate (DMF), a new drug for multiple sclerosis (MS) treatment, acts against neuroinflammation via mechanisms that are triggered by adduct formation with thiol redox switches. Ethyl pyruvate (EP), an off-the-shelf agent, appears to be a redox analog of DMF, but its immunomodulatory properties have not been put into the context of MS therapy. In this article, we examined and compared the effects of EP and DMF on MS-relevant activity/functions of T cells, macrophages, microglia, and astrocytes. EP efficiently suppressed the release of MS signature cytokines, IFN-gamma and IL-17, from human PBMCs. Furthermore, the production of these cytokines was notably decreased in encephalitogenic T cells after in vivo application of EP to rats. Production of two other proinflammatory cytokines, IL-6 and TNF, and NO was suppressed by EP in macrophages and microglia. Reactive oxygen species production in macrophages, microglia activation, and the development of Ag-presenting phenotype in microglia and macrophages were constrained by EP. The release of IL-6 was reduced in astrocytes. Finally, EP inhibited the activation of transcription factor NF-kappa B in microglia and astrocytes. Most of these effects were also found for DMF, implying that EP and DMF share common targets and mechanisms of action. Importantly, EP had in vivo impact on experimental autoimmune encephalomyelitis, an animal model of MS. Treatment with EP resulted in delay and shortening of the first relapse, and lower clinical scores, whereas the second attack was annihilated. Further studies on the possibility to use EP as an MS therapeutic are warranted. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}OI173035, OI175038, OI173014, OI173013]