Anti-encephalitogenic mechanisms of ethyl pyruvate in experimental autoimmune encephalomyelitis

Multipla skleroza (MS) je hronična, inflamacijska, demijelinizirajuća bolest centralnog nervnog sistema (CNS-a). Autoimunski odgovor usmeren protiv CNS-a je bitan element patogeneze MS-e. Eksperimentalni autoimunski encefalomijelitis (EAE) predstavlja životinjski model MS-e pomoću kog se istražuju patogenetski mehanizmi ove bolesti. Glavne ćelije koje učestvuju u pokretanju autoimunskog odgovora usmerenog protiv CNS-a su antigen prezentujuće ćelije (APĆ) koje aktiviraju naivne CD4+ T-ćelije specifične za antigene CNS-a. Ove CD4+ T-ćelije se potom diferenciraju u efektorske Th1 (engl. T helper cells – Th ćelije) koje imaju sposobnost produkcije interferona γ (IFN-γ) i Th17 koje produkuju interleukin 17 (IL-17). Prolaskom kroz-krvno-moždanu barijeru, Th1 i Th17 ćelije dolaze u CNS gde ih reaktiviraju rezidentne APĆ, te one sva.ojim produktima privlače druge imunske ćelije u CNS, što sve dovodi do inflamacije koja vodi oštećenju tkiva CNS-a. Patogenezi bolesti doprinose i rezidentne ćelije CNS-a kao što su astrociti i mikroglija. Etil-piruvat (EP) je lipofilni estar pirogrožđane kiseline koji poseduje antioksidativna i antiinflamacijska svojstva. U ovoj studiji je ispitivan antiencefalitogeni efekat EP-a na tok EAE-a i ćelije ukjučene u patogenezu EAE-a. Takođe, ispitivan je i njegov in vitro i in vivo tolerogeni uticaj na dendritske ćelije (DĆ). Rezultati su pokazali da EP ostvaruje terapijsko dejstvo na EAE kada se daje pacovima svakodnevno, počev od pojave prvih kliničkih simptoma bolesti sve do njihovog inicijalnog oporavka. Svoj antiencefalitogeni efekat EP je ispoljio sprečavanjem infiltracije imunskih ćelija u CNS, inhibicijom produkcije IL-17 od strane CD4+ T-limfocita u kičmenoj moždini, čime je sprečio zapaljensku reakciju u CNS-u. EP je doveo i do redukcije broja reaktivnih makrofaga i ćelija mikroglije, kao i do inhibicije reaktivnosti astrocita. Takođe, sprečio je i oštećenje neurona. Jedan od mehanizama kojim je EP ostvario svoje dejstvo je inhibicija HMGB1 (grupa proteina visoke mobilnosti 1 (engl. High-mobility group box 1) u reaktivnim makrofagima/mikrogliji. Da EP ima i druge efekte na APĆ pokazali su rezultati na makrofagima, in vitro. EP je redukovao produkciju proinflamacijskih citokina od strane makrofaga i ekspresiju molekula bitnih za prezentaciju antigena na ovim ćelijama. Zatim, istraživanje je prošireno na DĆ kao profesionalne APĆ. Rezultati su pokazali da EP vrši tolerogeni uticaj na DĆ poreklom iz kostne srži miševa. Naime, tretman DĆ-a in vitro EP-om inhibira njihovu sposobnost efikasne prezentacije antigena, aktivacije T ćelija u alogenoj reakciji i produkcije proinflamacijskih citokina. Molekulski mehanizmi kojim EP ostvaruje svoje dejstvo na mišje DĆ, uključuju stimulaciju signalnog molekula nuklearnog faktora povezanog sa eritrocitima 2 (engl. Nuclear Factor Erythroid 2–related Factor 2-Nrf2) i enzima hem oksigenaze 1 (HO-1) i NQO1 (engl. NADPH-quinone oxidoreductase 1), kao i inhibiciju regulatornog proteina transkripcije nuklearnog faktora κB (NF-κB). EP-om tretirane DĆ ispoljavaju efekte in vivo inhibiranjem imunskog odgovora indukovanog kompletnim Frojndovim adjuvansom u miševa. Konačno, tolerogeno dejstvo EP-a pokazano je i na DĆ-ma diferenciranim iz monocita ljudi (engl. Monocyte derived Dendritic Cells - MoDĆ) dobrovoljnih davalaca krvi i pacijenata obolelih od MS-e. Iz rezultata ove doktorske disertacije proizilazi da je EP efikasno imunomodulacijsko jedinjenje koje inhibira EAE, kao i da je efikasno tolerogeno jedinjenje koje usmerava DĆ ka imunosupresivnom fenotipu. Samim tim, zaključuje se da EP ima potencijal da bude primenjen u terapiji MS-e. Primena EP bi mogla biti direktna ili u pripremi tolerogenih DĆ za ćelijsku imunoterapiju. Buduće predkliničke i kliničke studije bi trebalo da budu usmerene na istraživanje mogućnosti primene EP u terapiji MS-e, ali i drugih autoimunskih i hroničnih inflamacijskih bolesti. Ključne reči: eksperimentalni autoimunski encefalomijelitis, multipla skleroza, etil-piruvat, tolerogene dendritske ćelije Naučna oblast: Biologija Uža naučna oblast: Imunologija UDK broj: Anti-encephalitogenic mechanisms of ethyl pyruvate in experimental autoimmune encephalomyelitis Abstract Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) with proposed autoimmune pathogenesis. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenetic mechanisms of the disease. Antigen presenting cells (APC) are major cells that are involved in the initiation of the autoimmune response against the CNS by activating CNS-specific naive CD4+ T cells. Consequently, these CD4+ T cells differentiate into effector T helper cells 1 (Th1) that produce interferon γ (IFN-γ) and Th17 cells that produce interleukin 17 (IL-17). Passing through the blood brain barrier, Th1 and Th17 cells arrive in the CNS where they become reactivated by the resident APC. Afterwards, other immune cells infiltrate the CNS, thus causing inflammation and tissue damage. The resident cells of the CNS, such as astrocytes and microglia, also contribute to the disease pathogenesis. Ethyl pyruvate (EP) is a lipophilic ester of pyruvic acid that possesses anti-oxidative and anti-inflammatory properties. The anti-encephalitogenic effect of EP in EAE and on cells of the CNS involved in the disease pathogenesis, were investigated in this study. Also, its in vitro and in vivo tolerogenic effect on dendritic cells (DC) was studied. Results showed that EP had a therapeutic effect on EAE when applied to the rats once a day, starting from the first clinical symptoms until their initial recovery. EP prevented immune cells infiltration into the CNS and inhibited T cell production of IL-17 in the spinal cord. Thus, EP restrained the inflammatory reaction in the CNS and therefore exerted its anti-encephalitogenic effect. Furthermore, treatment with EP led to the reduction of macrophages and microglia cell number, inhibition of astrocyte activity, as well as neuron destruction. Inhibition of HMGB1 (High-Mobility Group Box 1) molecule in activated macrophages/microglia was one of the mechanisms of the EP effects in the CNS. Moreover, in vitro treatment of stimulated macrophages with EP showed that EP had also an impact on APC. Treatment with EP led to reduced production of pro-inflammatory cytokines by macrophages as well as to downregulation of the expression of molecules relevant for antigen presentation. Furthermore, EP had a tolerogenic effect on the major APC, i.e. DC. Mice bone marrow derived DC were investigated. In vitro treatment of DC with EP inhibited their ability to efficiently present antigens, to activate T cells in allogeneic reaction, and to produce pro-inflammatory cytokines. Molecular mechanisms involved in the tolerogenic effects of EP on mice DC included stimulation of nuclear factor erythroid 2–related factor 2 (Nrf2) signaling, increase of heme oxygenase-1 (HO1) and NADPH-quinone oxidoreductase 1 (NQO1) expression, and inhibition of nuclear factor κB (NF-κB) transcription factor activation. Also, EP-treated DC inhibited immune response in vivo induced with complete Freund’s adjuvant in mice. Finally, the tolerogenic effects of EP were demonstrated in human monocyte derived DC (MoDC) obtained from healthy individuals and MS patients. Results of this doctoral thesis show that EP ameliorates EAE and that is also an effective tolerogenic agent that shifts DC towards immune-suppressing phenotype. Thereby, EP has the potential to be applied in MS therapy. Its application could be direct or through generation of tolerogenic DC as a mean of cell-based immunotherapy. Future preclinical and clinical studies should be focused towards investigating the possible application of EP in MS therapy, as well as in other autoimmune and chronic inflammatory diseases.Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) with proposed autoimmune pathogenesis. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenetic mechanisms of the disease. Antigen presenting cells (APC) are major cells that are involved in the initiation of the autoimmune response against the CNS by activating CNS-specific naive CD4+ T cells. Consequently, these CD4+ T cells differentiate into effector T helper cells 1 (Th1) that produce interferon γ (IFN-γ) and Th17 cells that produce interleukin 17 (IL-17). Passing through the blood brain barrier, Th1 and Th17 cells arrive in the CNS where they become reactivated by the resident APC. Afterwards, other immune cells infiltrate the CNS, thus causing inflammation and tissue damage. The resident cells of the CNS, such as astrocytes and microglia, also contribute to the disease pathogenesis. Ethyl pyruvate (EP) is a lipophilic ester of pyruvic acid that possesses anti-oxidative and anti-inflammatory properties. The anti-encephalitogenic effect of EP in EAE and on cells of the CNS involved in the disease pathogenesis, were investigated in this study. Also, its in vitro and in vivo tolerogenic effect on dendritic cells (DC) was studied. Results showed that EP had a therapeutic effect on EAE when applied to the rats once a day, starting from the first clinical symptoms until their initial recovery. EP prevented immune cells infiltration into the CNS and inhibited T cell production of IL-17 in the spinal cord. Thus, EP restrained the inflammatory reaction in the CNS and therefore exerted its anti-encephalitogenic effect. Furthermore, treatment with EP led to the reduction of macrophages and microglia cell number, inhibition of astrocyte activity, as well as neuron destruction. Inhibition of HMGB1 (High-Mobility Group Box 1) molecule in activated macrophages/microglia was one of the mechanisms of the EP effects in the CNS. Moreover, in vitro treatment of stimulated macrophages with EP showed that EP had also an impact on APC. Treatment with EP led to reduced production of pro-inflammatory cytokines by macrophages as well as to downregulation of the expression of molecules relevant for antigen presentation. Furthermore, EP had a tolerogenic effect on the major APC, i.e. DC. Mice bone marrow derived DC were investigated. In vitro treatment of DC with EP inhibited their ability to efficiently present antigens, to activate T cells in allogeneic reaction, and to produce pro-inflammatory cytokines. Molecular mechanisms involved in the tolerogenic effects of EP on mice DC included stimulation of nuclear factor erythroid 2–related factor 2 (Nrf2) signaling, increase of heme oxygenase-1 (HO1) and NADPH-quinone oxidoreductase 1 (NQO1) expression, and inhibition of nuclear factor κB (NF-κB) transcription factor activation. Also, EP-treated DC inhibited immune response in vivo induced with complete Freund’s adjuvant in mice. Finally, the tolerogenic effects of EP were demonstrated in human monocyte derived DC (MoDC) obtained from healthy individuals and MS patients. Results of this doctoral thesis show that EP ameliorates EAE and that is also an effective tolerogenic agent that shifts DC towards immune-suppressing phenotype. Thereby, EP has the potential to be applied in MS therapy. Its application could be direct or through generation of tolerogenic DC as a mean of cell-based immunotherapy. Future preclinical and clinical studies should be focused towards investigating the possible application of EP in MS therapy, as well as in other autoimmune and chronic inflammatory diseases.