NRF2 Plays a Crucial Role in the Tolerogenic Effect of Ethyl Pyruvate on Dendritic Cells.

Ethyl pyruvate (EP) is a redox-active compound that has been previously shown to be effective in restraining immune hyperactivity in animal models of various autoimmune and chronic inflammatory diseases. Importantly, EP has also been proven to have a potent tolerogenic effect on dendritic cells (DCs). Here, the influence of EP on the signaling pathways in DCs relevant for their tolerogenicity, including anti-inflammatory NRF2 and pro-inflammatory NF-κB, was explored. Specifically, the effects of EP on DCs obtained by GM-CSF-directed differentiation of murine bone marrow precursor cells and matured under the influence of lipopolysaccharide (LPS) were examined via immunocytochemistry and RT-PCR. EP counteracted LPS-imposed morphological changes and down-regulated the LPS-induced expression of pro-inflammatory mediators in DCs. While it reduced the activation of NF-κB, EP potentiated NRF2 and downstream antioxidative molecules, thus implying the regulation of NRF2 signaling pathways as the major reason for the tolerizing effects of EP on DCs.

Complete Freund's adjuvant as a confounding factor in multiple sclerosis research.

Complete Freund's adjuvant (CFA) is used as a standard adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model in multiple sclerosis studies. Still, CFA induces glial activation and neuroinflammation on its own and provokes pain. In addition, as CFA contains Mycobacteria, an immune response against bacterial antigens is induced in parallel to the response against central nervous system antigens. Thus, CFA can be considered as a confounding factor in multiple sclerosis-related studies performed on EAE. Here, we discuss the effects of CFA in EAE in detail and present EAE variants induced in experimental animals without the use of CFA. We put forward CFA-free EAE variants as valuable tools for studying multiple sclerosis pathogenesis and therapeutic approaches.

Brain inflammation in experimental autoimmune encephalomyelitis induced in Dark Agouti rats with spinal cord homogenate.

We have recently characterized experimental autoimmune encephalomyelitis (EAE) induced in DA rats with spinal cord homogenate without complete Freund's adjuvant (CFA). The main advantage of this multiple sclerosis model is the lack of CFA-related confounding effects which represent the major obstacles in translating findings from EAE to multiple sclerosis. Here, antigen specificity of the cellular and humoral immune response directed against the central nervous system was explored. The reactivity of T and B cells to myelin basic protein, myelin oligodendrocyte glycoprotein, and ß-synuclein was detected. Having in mind that reactivity against ß-synuclein was previously associated with autoimmunity against the brain, the infiltration of immune cells into different brain compartments, i.e. pons, cerebellum, hippocampus, and cortex was determined. T cell infiltration was observed in all structures examined. This finding stimulated investigation of the effects of immunization on DA rat behavior using the elevated plus maze and the open field test. Rats recovered from EAE displayed increased anxiety-like behavior. These data support CFA-free EAE in DA rats as a useful model for multiple sclerosis research.

Ethyl pyruvate ameliorates acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) became a focus of intensive research due to its death toll during the Covid-19 pandemic. An uncontrolled and excessive inflammatory response mediated by proinflammatory molecules such as high mobility group box protein 1 (HMGB1), IL-6, and TNF mounts as a response to infection. In this study, ethyl pyruvate (EP), a known inhibitor of HMGB1, was tested in the model of murine ARDS induced in C57BL/6 mice by intranasal administration of polyinosinic:polycytidylic acid (poly(I:C)). Intraperitoneal administration of EP ameliorated the ARDS-related histopathological changes in the lungs of poly(I:C)-induced ARDS and decreased numbers of immune cells in the lungs, broncho-alveolar lavage fluid and draining lymph nodes (DLN). Specifically, fewer CD8+ T cells and less activated CD4+ T cells were observed in DLN. Consequently, the lungs of EP-treated animals had fewer damage-inflicting CD8+ cells and macrophages. Additionally, the expression and production of proinflammatory cytokines, IL-17, IFN-γ and IL-6 were downregulated in the lungs. The expression of chemokine CCL5 which recruits immune cells into the lungs was also reduced. Finally, EP downregulated the expression of HMGB1 in the lungs. Our results imply that EP should be further evaluated as a potential candidate for ARDS therapy.

Increased regulatory activity of intestinal innate lymphoid cells type 3 (ILC3) prevents experimental autoimmune encephalomyelitis severity.

Experimental autoimmune encephalomyelitis (EAE) induced in inbred rodents, i.e., genetically identical animals kept under identical environmental conditions, shows variable clinical outcomes. We investigated such variations of EAE in Dark Agouti rats immunized with spinal cord homogenate and identified four groups: lethal, severe, moderate, and mild, at day 28 post immunization. Higher numbers of CD4+ T cells, helper T cells type 1 (Th1) and 17 (Th17) in particular, were detected in the spinal cord of the severe group in comparison with the moderate group. In addition, increased proportion of Th1 and Th17 cells, and heightened levels of interferon (IFN)-γ and interleukin (IL)-6 were detected in the small intestine lamina propria of the severe group. A selective agonist of free fatty acid receptor type 2 (Ffar2) applied orally in the inductive phase of EAE shifted the distribution of the disease outcomes towards milder forms. This effect was paralleled with potentiation of intestinal innate lymphoid cells type 3 (ILC3) regulatory properties, and diminished Th1 and Th17 cell response in the lymph nodes draining the site of immunization. Our results suggest that different clinical outcomes in DA rats are under determinative influence of intestinal ILC3 activity during the inductive phase of EAE.

ILC3: a case of conflicted identity.

Innate lymphoid cells type 3 (ILC3s) are the first line sentinels at the mucous tissues, where they contribute to the homeostatic immune response in a major way. Also, they have been increasingly appreciated as important modulators of chronic inflammatory and autoimmune responses, both locally and systemically. The proper identification of ILC3 is of utmost importance for meaningful studies on their role in immunity. Flow cytometry is the method of choice for the detection and characterization of ILC3. However, the analysis of ILC3-related papers shows inconsistency in ILC3 phenotypic definition, as different inclusion and exclusion markers are used for their identification. Here, we present these discrepancies in the phenotypic characterization of human and mouse ILC3s. We discuss the pros and cons of using various markers for ILC3 identification. Furthermore, we consider the possibilities for the efficient isolation and propagation of ILC3 from different organs and tissues for in-vitro and in-vivo studies. This paper calls upon uniformity in ILC3 definition, isolation, and propagation for the increased possibility of confluent interpretation of ILC3's role in immunity.

Phenethyl Ester of Gallic Acid Ameliorates Experimental Autoimmune Encephalomyelitis.

Gallic acid is a phenolic acid present in various plants, nuts, and fruits. It is well known for its anti-oxidative and anti-inflammatory properties. The phenethyl ester of gallic acid (PEGA) was synthesized with the aim of increasing the bioavailability of gallic acid, and thus its pharmacological potential. Here, the effects of PEGA on encephalitogenic cells were examined, and PEGA was found to modulate the inflammatory activities of T cells and macrophages/microglia. Specifically, PEGA reduced the release of interleukin (IL)-17 and interferon (IFN)-γ from T cells, as well as NO, and IL-6 from macrophages/microglia. Importantly, PEGA ameliorated experimental autoimmune encephalomyelitis, an animal model of chronic inflammatory disease of the central nervous system (CNS)-multiple sclerosis. Thus, PEGA is a potent anti-inflammatory compound with a perspective to be further explored in the context of CNS autoimmunity and other chronic inflammatory disorders.

Phenethyl ester of rosmarinic acid ameliorates experimental autoimmune encephalomyelitis.

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+ T cells purified from the lymph nodes. Also, PERA inhibited NO production in the CNS and the lymph nodes, as well as in macrophages and microglial cells. Finally, microglial ability to produce pro-inflammatory cytokines IL-6, and tumor necrosis factor (TNF) were also reduced by PERA. Our results clearly imply that PERA possesses anti-encephalitogenic properties. Thus, further studies on the relevance of the observed effects for the therapy of multiple sclerosis are warranted.

Ethyl pyruvate, a versatile protector in inflammation and autoimmunity.

Ethyl pyruvate (EP) has potent influence on redox processes, cellular metabolism, and inflammation. It has been intensively studied in numerous animal models of systemic and organ-specific disorders whose pathogenesis involves a strong immune component. Here, basic chemical and biological properties of EP are discussed, with an emphasis on its redox and metabolic activity. Further, its influence on myeloid and T cells is considered, as well as on intracellular signaling beyond its effect on immune cells. Also, the effects of EP on animal models of chronic inflammatory and autoimmune disorders are presented. Finally, a possibility to apply EP as a treatment for such diseases in humans is discussed. Scientific papers cited in this review were identified using the PubMed search engine that relies on the MEDLINE database. The reference list covers the most important findings in the field in the past twenty years.

Ethyl Pyruvate Ameliorates Experimental Autoimmune Myocarditis.

Ethyl pyruvate (EP) has profound anti-inflammatory and immunomodulatory properties. Here, its effects were determined on experimental autoimmune myocarditis (EAM) induced in mice by heart-specific myosin-alpha heavy chain peptide immunization. EP was applied intraperitoneally, daily, starting with the immunization. Severity of EAM was determined by histological assessment of immune cell infiltrates into the heart. Cells were phenotypically characterized by flow cytometry. Concentration of cytokines in cell culture supernatants and sera was determined by ELISA. EP reduced the infiltration of immune cells into the heart and lessened heart inflammation. Smaller number of total immune cells, as well as of CD11b+ and CD11c+ cells were isolated from the hearts of EP-treated mice. A reduced number of antigen-presenting cells, detected by anti-CD11c, MHC class II and CD86 antibodies, as well as of T helper (Th)1 and Th17 cells, detected by anti-CD4, IFN-γ and IL-17 antibodies, was determined in mediastinal lymph nodes draining the heart, in parallel. In the spleen, only the number of CD11c+ cells were reduced, but not of the other examined populations, thus implying limited systemic effect of EP. Reduced production of IFN-γ and IL-17 by myosin-alpha heavy chain peptide-restimulated cells of the lymph nodes draining the site of immunization was observed in EP-treated mice. Our results clearly imply that EP restrains autoimmunity in EAM. Therapeutic application of EP in the treatment of myocarditis in humans should be addressed in the forthcoming studies.

Sepsis and multiple sclerosis: Causative links and outcomes.

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.

Modulation of Intestinal ILC3 for the Treatment of Type 1 Diabetes.

Gut-associated lymphoid tissue (GALT) is crucial for the maintenance of the intestinal homeostasis, but it is also the potential site of the activation of autoreactive cells and initiation/propagation of autoimmune diseases in the gut and in the distant organs. Type 3 innate lymphoid cells (ILC3) residing in the GALT integrate signals from food ingredients and gut microbiota metabolites in order to control local immunoreactivity. Notably, ILC3 secrete IL-17 and GM-CSF that activate immune cells in combating potentially pathogenic microorganisms. ILC3 also produce IL-22 that potentiates the strength and integrity of epithelial tight junctions, production of mucus and antimicrobial peptides thus enabling the proper function of the intestinal barrier. The newly discovered function of small intestine ILC3 is the secretion of IL-2 and the promotion of regulatory T cell (Treg) generation and function. Since the intestinal barrier dysfunction, together with the reduction in small intestine ILC3 and Treg numbers are associated with the pathogenesis of type 1 diabetes (T1D), the focus of this article is intestinal ILC3 modulation for the therapy of T1D. Of particular interest is free fatty acids receptor 2 (FFAR2), predominantly expressed on intestinal ILC3, that can be stimulated by available selective synthetic agonists. Thus, we propose that FFAR2-based interventions by boosting ILC3 beneficial functions may attenuate autoimmune response against pancreatic ß cells during T1D. Also, it is our opinion that treatments based on ILC3 stimulation by functional foods can be used as prophylaxis in individuals that are genetically predisposed to develop T1D.

ILC3, a Central Innate Immune Component of the Gut-Brain Axis in Multiple Sclerosis.

Gut immune cells have been increasingly appreciated as important players in the central nervous system (CNS) autoimmunity in animal models of multiple sclerosis (MS). Among the gut immune cells, innate lymphoid cell type 3 (ILC3) is of special interest in MS research, as they represent the innate cell counterpart of the major pathogenic cell population in MS, i.e. T helper (Th)17 cells. Importantly, these cells have been shown to stimulate regulatory T cells (Treg) and to counteract pathogenic Th17 cells in animal models of autoimmune diseases. Besides, they are also well known for their ability to stabilize the intestinal barrier and to shape the immune response to the gut microbiota. Thus, proper maintenance of the intestinal barrier and the establishment of the regulatory milieu in the gut performed by ILC3 may prevent activation of CNS antigen-specific Th17 cells by the molecular mimicry. Recent findings on the role of ILC3 in the gut-CNS axis and their relevance for MS pathogenesis will be discussed in this paper. Possibilities of ILC3 functional modulation for the benefit of MS patients will be addressed, as well.

Complete Freund's adjuvant-free experimental autoimmune encephalomyelitis in Dark Agouti rats is a valuable tool for multiple sclerosis 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.

Redox Regulation of Tolerogenic Dendritic Cells and Regulatory T Cells in the Pathogenesis and Therapy of Autoimmunity.

Significance: Autoimmune diseases are progressively affecting westernized societies, as the proportion of individuals suffering from autoimmunity is steadily increasing over the past decades. Understanding the role of reactive oxygen species (ROS) in modulation of the immune response in the pathogenesis of autoimmune disorders is of utmost importance. The focus of this review is the regulation of ROS production within tolerogenic dendritic cells (tolDCs) and regulatory T (Treg) cells that have the essential role in the prevention of autoimmune diseases and significant potency in their therapy. Recent Advances: It is now clear that ROS are extremely important for the proper function of both DC and T cells. Antigen processing/presentation and the ability of DC to activate T cells depend upon the ROS availability. Treg differentiation, suppressive function, and stability are profoundly influenced by ROS presence. Critical Issues: Although a plethora of results on the relation between ROS and immune cells exist, it remains unclear whether ROS modulation is a productive way for skewing T cells and DCs toward a tolerogenic phenotype. Also, the possibility of ROS modulation for enhancement of regulatory properties of DC and Treg during their preparation for use in cellular therapy has to be clarified. Future Directions: Studies of DC and T cell redox regulation should allow for the improvement of the therapy of autoimmune diseases. This could be achieved through the direct therapeutic application of ROS modulators in autoimmunity, or indirectly through ROS-dependent enhancement of tolDC and Treg preparation for cell-based immunotherapy. Antioxid. Redox Signal. 34, 364-382.

Benfotiamine Reduces Dendritic Cell Inflammatory Potency.

BACKGROUND: Benfotiamine is a synthetic liposoluble derivative of vitamin B1 that has been shown to have anti-inflammatory properties. OBJECTIVE: To study the effects of benfotiamine on dendritic cells. METHODS: Dendritic cells were obtained from murine bone marrow precursor cells in the presence of GM-CSF. Benfotiamine was applied to the cell culture during the process of bone marrow cell differentiation into dendritic cells. Dendritic cells were stimulated with lipopolysaccharide (LPS) and expression of MHC class II molecules and CD86 was determined by flow cytometry, while levels of tumor necrosis factor (TNF) and interleukin (IL)-1ß in cell culture supernatants were measured by ELISA. F-Actin, NF-κB and Nrf2 were visualized by immunofluorescent staining and microscopy. RESULTS: Benfotiamine potently reduced LPS-induced expression of MHC class II molecules and CD86, in addition to suppressing the release of pro-inflammatory cytokines TNF and IL-1ß. It also prevented LPS-imposed morphological changes of dendritic cells, i.e. enlargement and intensified protrusions. The effects were paralleled with the reduction of NF-κB translocation to the nucleus, but not of Nrf2 activation inhibition. CONCLUSION: Having in mind the importance of dendritic cells for the configuration of the immune response, our results imply that benfotiamine has the ability to regulate the immune response through inhibition of inflammatory properties of dendritic cells.

Upregulation of Tolerogenic Pathways by the Hydrogen Sulfide Donor GYY4137 and Impaired Expression of H2S-Producing Enzymes in Multiple Sclerosis.

The aim of this study was to examine the in vitro effects of the slow-releasing H2S donor GYY4137 on the immune cells involved in the pathogenesis of the central nervous system (CNS) autoimmune disease, multiple sclerosis (MS). GYY4137 specifically potentiated TGF-ß expression and production in dendritic cells and significantly reduced IFN-γ and IL-17 production in the lymph node and spinal cord T cells obtained from mice immunized with CNS antigens. Both the proportion of FoxP3+ regulatory CD4+ T cells in the lymph node cells, and the percentage of IL-17+ CD4+ T cells in the spinal cord cells were reduced upon culturing with GYY4137. Interestingly, the peripheral blood mononuclear cells obtained from the MS patients had a lower expression of the H2S-producing enzyme, 3-mercaptopyruvate-sulfurtransferase (MPST), in comparison to those obtained from healthy donors. A significant inverse correlation between the expression of MPST and several pro-inflammatory factors was also observed. Further studies on the relevance of the observed results for the pathogenesis and therapy of MS are warranted.

MIF and insulin: Lifetime companions from common genesis to common pathogenesis.

Pro-inflammatory nature of macrophage migration inhibitory factor (MIF) has been generally related to the propagation of inflammatory and autoimmune diseases. But this molecule possesses many other peculiar functions, unrelated to the immune system, among which is its supportive role in the post-translational modifications of insulin. In this way MIF enables proper insulin conformation within the pancreatic beta cell and its full activity. The inherent or acquired changes in MIF expression might therefore lead to different insulin processing and initiation of autoimmunity. The relation between MIF and insulin does not stop at this point; these two molecules continue to interact during pathological states characterized by inflammation and insulin resistance. In this context, MIF indirectly and negatively influences insulin action by boosting inflammatory environment and disabling target cells to respond to insulin. On the other side, insulin might interfere with MIF action as well, acting as an anti-inflammatory mediator. Therefore, the proper interaction between MIF and insulin is crucial for maintaining homeostasis, while anti-inflammatory therapies based on the systemic MIF blockage may disturb this balance. This review covers MIF-insulin relationship in the physiological and pathological conditions and discusses the approaches for MIF inhibition and their net effect specifically considering possible impact on insulin misfolding and the possible misinterpretation of previous results due to the discovery of MIF functional homolog D-dopachrome tautomerase.

Ethyl Pyruvate Induces Tolerogenic Dendritic Cells.

Dendritic cells (DC) are professional antigen presenting cells that have a key role in shaping the immune response. Tolerogenic DC (tolDC) have immuno-regulatory properties and they are a promising prospective therapy for multiple sclerosis and other autoimmune diseases. Ethyl pyruvate (EP) is a redox analog of dimethyl fumarate (Tecfidera), a drug for multiple sclerosis treatment. We have recently shown that EP ameliorates experimental autoimmune encephalomyelitis, a multiple sclerosis murine model. Here, we expanded our study to its tolerogenic effects on DC. Phenotypic analysis has shown that DC obtained from mice or humans reduce expression of molecules required for T cell activation such as CD86, CD83, and HLA-DR under the influence of EP, while CD11c expression and viability of DC are not affected. Furthermore, EP-treated DC restrain proliferation and modulate cytokine production of allogeneic lymphocytes. These results demonstrate that EP has the ability to direct DC toward tolDC.

Comparison of dendritic cells obtained from autoimmunty-prone and resistant rats.

Dendritic cells (DC) are responsible for the initiation and shaping of the adaptive immune response and are in the focus of autoimmunity research. We were interested in comparison of DC obtained from autoimmunity-prone Dark Agouti (DA) rats and autoimmunity-resistant Albino Oxford (AO) rats. DC were generated from bone marrow precursors and matured (mDC) by lipopolysaccharide. Tolerogenic DC (tolDC) obtained by vitamin D3 treatment were studied in parallel. Profile of cytokine production was different in AO and DA mDC and tolDC. Expression of MHC class II molecules and CD86 were higher in DA DC, while vitamin D3 reduced their expression in dendritic cells of both strains. Allogeneic proliferation of CD4+ T cells was reduced by AO tolDC, but not with DA tolDC in comparison to respective mDC. Finally, expression of various genes identified as differentially expressed in human mDC and tolDC was also analyzed in AO and DA DC. Again, AO and DA DC differed in the expression of the analyzed genes. To conclude, AO and DA DC differ in production of cytokines, expression of antigen presentation-related molecules and in regulation of CD4+ T proliferation. The difference is valuable for understanding the divergence of the strains in their susceptibility to autoimmunity.