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Multipla skleroza (MS) je hronična inflamacijska bolest centralnog nervnog sistema (CNS) koju kao i njen in vivo model eksperimentalni autoimunski encefalomijelitis (EAE) karakterišu infiltracija imunskih ćelija, aktivacija mikroglije i astrocita, demijelinizacija, oštećenje aksona, ali i remijelinizacija posredovana oligodendrocitnim progenitorskim ćelijama (OPĆ). Tokom neuroinflamacije, ATP ostvaruje pro-, a adenozin antiinflamacijsko dejstvo putem P2, odnosno P1 purinskih receptora. Aktivacija specifičnih purinskih receptora zavisi od koncentracije ATP, ADP i adenozina koju u vanćelijskom prostoru regulišu ektonukleotidaze. Najzastupljenije ektonukelotidaze u CNS su NTPDaza1/CD39, NTPDaza2 i eN/CD73. U MS/EAE uloga NTPDaza1/CD39 i eN/CD73 eksprimiranih na ćelijama imunskog sistema uglavnom je poznata, dok je uloga ovih enzima prisutnih na ćelijama CNS nedovoljno istražena. Budući da aktivirana mikroglija i astrociti imaju ključnu ulogu u toku neuroinflamacije glavni cilj ove disertacije bio je ispitivanje ekspresije glavnih ektonukleotidaza CNS na pomenutim ćelijama i procena njihovog inflamacijskog fenotipa, kao i ekspresije purinskih receptora u kičmenoj moždini pacova tokom EAE kao animalnog modela MS. S obzirom na ulogu OPĆ u remijelinizaciji tokom MS/EAE, dodatni cilj bio je ispitivanje uticaja proinflamacijskih faktora na vijabilnost i funkcionalnost OPĆ linije Oli-neu i ekspresiju eN/CD73 na tim ćelijama. Rezultati prikazani u ovoj disertaciji pokazali su da tokom EAE dolazi do fazno-specifičnih promena ekspresije svih ispitivanih komponenti purinskog signalnog sistema u kičmenoj moždini pacova. Uočeno povećanje ekspresije NTPDaza1/CD39 uzrokovano je aktivacijom mikroglije i infiltracijom monocita/makrofaga kao i drugih perifernih imunskih ćelija tokom EAE, a povezano je i sa tranzicijom mikroglije/makrofaga u pravcu antiinflamacijskog fenotipa, kao i indukcijom polarizacije astrocita u pravcu neuroprotektivnog fenotipa. U pogledu NTPDaza2, smanjenje ekspr, that is characterized like its in vivo model experimental autoimmune encephalomyelitis (EAE) by immune cell infiltration, microglia and astrocyte activation, demyelination, axonal damage, as well as remyelination guided by oligodendrocyte progenitor cells (OPC). During neuroinflammation, ATP acts pro-, while adenosine acts anti-inflammatory via P2 and P1 purine receptors, respectively. Activation of specific purine receptor depends on ATP, ADP and adenosine extracellular concentrations that are regulated by by ectonucleotidases. In the CNS most abundant ectonucleotidases are NTPDase1/CD39, NTPDase2 and eN/CD73. Role of NTPDase1/CD39 and eN/CD73 in the cells of immune system, unlike in the CNS, in MS/EAE is mostly well known,. Since activated microglia and astrocytes have a key role in the course of neuroinflammation, the main goal of this dissertation was to study expression of major ectonucleotidases in the CNS at these cells and to assess their inflammatory phenotype, likewise to analyze expression of purine receptors in the rat spinal cord during EAE as animal model of MS. Considering role of OPC in remyelination during MS/EAE, additional goal was to assess the effects of proinflammatory factors at viability and functionality of OPC Oli-neu cell line and their expression of eN/CD73. Results presented herein have demonstrated disease phase-specific changes of all analyzed components of purine signaling system in rat spinal cord during EAE. Upregulation of NTPDase1/CD39 during EAE arised as a consequence of microglial activation and infiltration of monocytes/macrophages and other perypheral immune cells and also was related to transition of microglia/macrophages towards anti-inflammatory phenotype, likewise to induction of astrocyte polarization towards neuroprotective phenotype. Regarding NTPDase2, mainly expressed at white matter astrocytes, observed downregulation resulted from decreased expression by these cells during EAE. Additionally, during EAE all analyzed

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 (tolDC) 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 exists, it remains unclear whether ROS modulation is a productive way for skewing T cells and DC towards 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.

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naive astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell-astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca(2+)increase in astrocytes by promoting glial release of ATP. ATP propagated Ca(2+)elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca(2+)increase is specifically triggered by the autoreactive CD4(+)T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca(2+)signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell-astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.

Neuroinflamacija je urođeni akutni imunski proces u centralnom nervom sistemu, koji ima za cilj oporavak tkivne homeostaze nakon metaboličke, mehaničke i ishemijske povrede ili infekcije patogenima. Centralnu ulogu u neuroinflamaciji imaju rezidentne ćelije CNS, astrociti i mikroglija, i veliki broj reaktivnih, signalnih i trofičkih molekula, koje štite nervno tkivo od daljih oštećenja i pokreću proces rezolucije i oporavka. Neodgovarajuća ili nedovoljno izvedena rezolucija vodi ka hroničnoj neuronflamaciji i neurodegeneraciji. Dok je karakter akutne neuroinflamacije reaktivni i zaštitni, hronična forma dovodi do glioze i autoimunske reakcije i predstavlja osnovu za razvoj brojnih neuropatologija. Jedan od prvih fenomena u inicijalnoj fazi neuroinflamacije jeste aktivacija astrocita, koja podrazumjeva brojne morfološke i funkcionalne promjene. Te su promjene inicirane signalima opasnosti (DAMP), među kojima je jedan od najvažnijih ATP. Ovaj purinski nukleotid se oslobađa u vanćelijsku sredinu, gdje preko svojih P2 receptora, djeluje kao „nađi me“ signal za astrocite i mikrogliju. U vanćelijskoj sredini se ATP razgrađuje djelovanjem enzima ektonukleotidaza, koje sekvencijalno razgrađuju ATP do adenozina. Adenozin je takođe signalni molekul, koji djelujući na svoje P1 receptore, ostvaruje homeostatske i imunosupresivne uloge. Ektonukleotidaze svojom aktivnošću kontrolišu odnos koncentracija ATP/adenzina u vanćelijskom prostoru, a u tom smislu ključni enzim je ekto-5’-nukleotidaza (eN/CD73). Ovaj enzim katalizuje konverziju AMP u adenozin, ali i funkcioniše kao ćelijski adhezivni molekul za interakciju sa molekulima vanćelijskog matriksa. Brojna istraživanja su pokazala da u akutnoj ili hroničnoj neuroinflamaciji, naročito na reaktivnim astrocitima, dolazi do snažne ushodne regulacije Nt5e gena, a takođe i do posledičnih promjena na nivou količine proteina i aktivnosti eN/CD73..., with a physiological purpose of restoring the tissue homeostasis, endangered by a metabolic, ischemic and physical injury or pathogen invasion. Physiological significance of acute neuroinflammation is to protect the tissue from further damage and to support the recovery and restore the homeostasis. Central role in neuroinflammation is performed by resident glial cells, astrocytes, and microglia, which produce and liberate various reactive, signaling and trophic molecules, to restrict and protect the tissue from further damage and initiate the process of resolution and reparation. However, insufficient or inadequate resolution may lead to chronic neuroinflammation and neurodegeneration. While the nature of acute neuroinflammation is reactive and protective, chronic neuroinflammation leads to fibrosis and autoimmune reaction, which are the basis of many neuropathological conditions. One of the first events in neuroinflammation is activation of astrocytes, which comprises prominent morphological and functional changes of the cells. The changes are initiated by danger signals (DAMP), among which ATP has a particularly important role. Challenged or daying cells massively release the purine nucleotide into the extracellular space, where it acts upon its P2 purinoreceptors as a „find me“ signal for astrocytes and microglia. Extracellular ATP is degraded by ectonucleotidase enzyme chain, which sequentially hydrolyzes ATP to adenosine. Nucleoside adenosine is also a very strong signal, which acts upon P1 adenosine receptors, to impose its homeostatic and immunosuppressive actions. Ectonucleotidases control the ratio of ATP/adenosine in the extracellular milieu, thus determing the inflammatory state of the tissue. As the only enzyme which produces adenosine in the extracellular space and the rate-limiting enzyme of the cascade, ecto-5′-nucleotidase (eN/CD73) plays a critical role in this matter. Ecto-5′-nucleotidase/CD73 hydrolyzes conversion of AMP to adenosine, but it also

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 HMG, 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 E

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.

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.

Eksperimentalni autoimunski encefalomijelitis (EAE) je model multiple skleroze (MS), hronične inflamatorne bolesti centralnog nervnog sistema (CNS). Osnovne patogene populacije u EAE su efektorski T limfociti koji produkuju interferon-γ (Th1) ili interleukin-17 (Th17) i aktivirani makrofagi koji dovode do oštećenja CNS. Imajući u vidu da krastavac (Cucumis sativus) predstavlja bogat izvor sekundarnih metabolita kao što su polifenoli i kukurbitacini, u ovoj studiji je po prvi put ispitivano dejstvo različitih ekstrakata krastavca na encefalitogeni potencijal T limfocita, efektorske funkcije makrofaga i antigen-prezentujuću aktivnost dendritskih ćelija (DĆ). Rezultati su pokazali da ekstrakt lista krastavca (ELK) ostvaruje najpotentnije dejstvo, inhibirajući produkciju IFN-γ i IL-17 od strane encefalitogenih T limfocita, modulišući signalne puteve Nf-κB i p38 (MAPK). Pored toga, ELK je ostvario inhibitorno dejstvo na efektorske funkcije makrofaga i antigen-prezentujuću aktivnost DĆ u koncentracijama koje nisu pokazale citotoksičnost na embrione zebrice (Danio rerio) in vivo. Dalje, ELK je uspešno inhibirao encefalitogene T limfocite nakon in vivo primene u EAE. Kvantitativna i kvalitativna analiza ELK metodom masene spektrometrije potvrdila je prisustvo 37 različitih jedinjenja od kojih su kukurbitacini bili najzastupljenija jedinjenja. Shodno tome, ispitana su i imunomodulacijska dejstva kukurbitacina B i kukurbitacina E na glavnim imunskim ćelijama uključenim u patogenezu EAE. Ova ispitivanja su potvrdila dejstva ELK, sugerišući da su kukurbitacini, kao glavni konstituenti ovog ekstrakta, barem delimično odgovorni za ostvarena dejstva ELK. Rezultati ove doktorske disertacije ukazuju na to da ekstrakt lista krastavca i kukurbitacini ostvaruju snažne antiencefalitogene efekte u modelu EAE i ukazuju na važnost dodatnih istraživanja u cilju pronalaženja odgovarajućih tretmana autoimunosti CNS., Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). T lymphocytes, which produce interferon-γ (Th1) or interleukin-17 (Th17), and activated macrophages, that lead to CNS damage, are the major pathogenic cell populations in EAE. Having in mind that cucumber (Cucumis sativus) is a rich source of secondary metabolites such as polyphenols and cucurbitacins, in this study, for the first time, the effects of various cucumber extracts were investigated on encephalytogenic T lymphocytes, effector function of macrophages and antigen-presenting activities of dendritic cells (DC). The results show that cucumber leaf extract (CLE) expresses the most potent effect by inhibiting the production of IFN-γ and IL-17 in encephalytogenic T lymphocytes, through modulation of signaling pathways Nf-κB and p38 (MAPK). Additionally, CLE imposes an inhibitory effect on the effector functions of macrophages and antigen-presenting activity of DC in concentrations that do not show cytotoxicity in zebrafish (Danio rerio) in vivo. Also, CLE inhibits generation of encephalitogenic cells in vivo. Phytochemical analysis of CLE, characterized by mass spectrometry, confirmed the presence of 37 different compounds, among which cucurbitacins were the most abundant compounds. Hence, the immunomodulatory effects of cucurbitacin B and cucurbitacin E were also examined on the major immune cells involved in EAE pathogenesis. The obtained results confirm the effects of CLE, suggesting that cucurbitacins are, at least partially, responsible for its effects. The results of this doctoral dissertation indicate that the cucumber leaf extract and its major constituents cucurbitacins have anti-encephalitogenic effects in the EAE model, Moreover, they suggest the importance of additional research towards novel highly efficient CNS autoimmunity treatments.

Multipla skleroza je hronična, inflamacijska, neurodegenerativna, demijelinizirajuća bolest centralnog nervnog sistema (CNS). Etiologija multiple skleroze još uvek nije poznata, ali je sve više podataka koji pokazuju ulogu crevne mikrobiote u patogenezi bolesti. Više podataka o značaju crevne mikrobiote u autoimunosti CNS dobijeno je istraživanjima na eksperimentalnom autoimunskom encefalomijelitisu (EAE), životinjskom modelu multiple skleroze. U EAE-u se autoimunski odgovor protiv CNS-a pokreće u limfnim čvorovima koji dreniraju mesto imunizacije, gde se CD4+ T ćelije specifične za antigene CNS-a aktiviraju i diferenciraju u Th (engl. T helper) 1 ćelije koje produkuju IFN-γ i Th17 ćelije koje produkuju IL-17. Kada dospeju u CNS ove Th ćelije bivaju reaktivirane od strane lokalnih antigen-prezentujućih ćelija. Nakon reaktivacije, encefalitogene Th ćelije pokreću i održavaju inflamaciju unutar CNS-a. Albino oksford (AO) pacovi su veoma otporni na indukciju EAE-a. S druge strane, Dark Agouti (DA) pacovi razvijaju EAE čak i nakon blage imunizacije. U dosadašnjim istraživanjima je pokazano da se AO pacovi u odnosu na DA pacove razlikuju po slaboj aktivaciji Th1 i Th17 ćelija u relevantnim limfnim čvorovima u odgovoru na encefalitogenu imunizaciju. Takođe, pokazano je da relativno mali broj encefalitogenih ćelija infiltrira CNS AO pacova. Do sada nije vršeno ispitivanje uloge GALT-a i mikrobiote creva u otpornosti pacova soja AO na indukciju EAE-a. Sve je više istraživanja koja pokazuju da mikroorganizmi creva imaju važnu ulogu u regulaciji imunskog odgovora kroz interakciju sa limfnim tkivom creva (GALT, engl. gut-associated lymphoid tissue). U istraživanjima na EAE-u je pokazano da određeni mikroorganizmi creva pospešuju tok bolesti, dok drugi ostvaruju terapeutske efekte modulacijom odnosa Th1 i Th17 encefalitogenih ćelija i regulatornih T ćelija (Treg). Poznato je i da encefalitogene CD4+ T ćelije migriraju u GALT, uključujući mezenterične limfne čvorove i Pejerove p, Multiple sclerosis is a chronic inflammatory neurodegenerative demyelinating disease of the central nervous system (CNS). Etiology of multiple sclerosis is still unknown, but data are showing that gut microbiota plays an important role in the pathogenesis of this disease. Information about the significance of gut microbiota in CNS autoimmunity has been dominantly obtained in studies in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The autoimmune response against CNS starts in lymph nodes draining the site of immunization in EAE. There, CD4+ T cells are being activated and differentiated into Th1 (helper T cells) and Th17 cells that produce IFN-γ and IL-17, respectively. When Th cells arrive in the CNS, they are reactivated by local antigen-presenting cells. After the reactivation, these encephalitogenic T cells initiate and propagate CNS inflammation. Albino Oxford (AO) rats are highly resistant to EAE induction. On the other hand, Dark Agouti (DA) rats develop EAE even after the mild immunization. In our earlier studies it was shown that AO rats had less activated Th1 and Th17 cells in the relevant lymph nodes in response to encephalitogenic immunization, unlike DA rats. Also, it was shown that relatively small number of encephalitogenic cells infiltrated CNS in AO rats. Up until now, the studies of role of GALT and gut microbiota in resistance of AO rats in EAE induction have not been conducted. It has been increasingly appreciated that gut microbiota plays an important role in the regulation of the immune response through interaction with the cells of gut-associated lymphoid tissue (GALT). In EAE studies specific gut microorganisms have been proposed to promote the disease, while others have been shown to have therapeutic effects by modulating ratio and activity of encefalitogenic Th1 and Th17 cells and regulatory T cells (Treg). Encefalitogenic cells can also migrate to GALT, including mesenteric lymph nodes and Peyer’s

Albino Oxford (AO) rats are extremely resistant to induction of experimental autoimmune encephalomyelitis (EAE). EAE is an animal model of multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS), with established autoimmune pathogenesis. The autoimmune response against the antigens of the CNS is initiated in the peripheral lymphoid tissues after immunization of AO rats with CNS antigens. Subsequently, limited infiltration of the CNS occurs, yet without clinical sequels. It has recently become increasingly appreciated that gut-associated lymphoid tissues (GALT) and gut microbiota play an important role in regulation and propagation of encephalitogenic immune response. Therefore, modulation of AO gut microbiota by antibiotics was performed in this study. The treatment altered composition of gut microbiota in AO rats and led to a reduction in the proportion of regulatory T cells in Peyer's patches, mesenteric lymph nodes, and in lymph nodes draining the site of immunization. Upregulation of interferon-. and interleukin (IL)-17 production was observed in the draining lymph nodes. The treatment led to clinically manifested EAE in AO rats with more numerous infiltrates and higher production of IL-17 observed in the CNS. Importantly, transfer of AO gut microbiota into EAE-prone Dark Agouti rats ameliorated the disease. These results clearly imply that gut microbiota is an important factor in AO rat resistance to EAE and that gut microbiota transfer is an efficacious way to treat CNS autoimmunity. These findings also support the idea that gut microbiota modulation has a potential as a future treatment of multiple sclerosis.

Tenascin-C (TnC) je glikoprotein prisutan u vanćelijskom matriksu (VĆM) različitih tkiva kičmenjaka tokom razvića, gde je uključen u regulaciju ćelijskog rasta, migracije i adhezije preko aktivacije različitih unutarćelijskih signalnih puteva. Ekspresija TnC je značajno smanjenja u adultnom organizmu, međutim ostaje prisutna samo u određenim perifernim tkivima, kao i u delovima centralnog nervnog sistema koji zadržavaju visok nivo plastičnosti, kao što je mali mozak, gde je uočena njegova uloga u modulaciji sinaptičkih funkcija. Obogaćena sredina (OS) oblikuje mnoge aspekte ponašanja i stoga može da posluži kao pristup za proučavanje neuronalne plastičnosti u adultnom organizmu. Izlaganje OS dovodi do brojnih promena na molekularnom i ćelijskom nivou u različitim regionima mozga uključujući mali mozak. Cilj ove studije je bio da se ispita uloga TnC u modulaciji strukturne plastičnosti malog mozga indukovane OS praćenjem distribucije perineuronalnih mreža (PNM), promene u veličini i gustini ekscitatornih i inhibitornih presinaptičkih završetaka, kao i aktivnosti glavnih enzima koji vrše razgradnju VĆM, MMP-2 i MMP-9. Takođe, ova studija je imala za cilj da ispita kako nedostatak TnC interaguje sa spoljašnjom sredinom u oblikovanju različitih domena ponašanja povezanih sa funkcijom malog mozga. U te svrhe, miševi sa nedostatkom TnC (TnC-/-) ili MMP-9 (MMP-9-/-) i odgovarajući kontrolni miševi starosti 3 nedelje su izlagani standardnim uslovima gajenja (SS) ili OS 4 ili 8 nedelja. Ova studija je pokazala da izlaganje OS u toku 8 nedelja dovodi do smanjenja intenziteta bojenja na PNM, kao i smanjenja u veličini GABAergičkih i povećanja broja i veličine glutamatergičkih sinaptičkih završetaka u kontrolnim miševima. Nasuprot tome, TnC-/- miševi su pokazali smanjen intenzitet bojenja na PNM u poređenju sa kontrolnim životinjama gajenim u SS, dok njihovo izlaganje OS nije dovelo da smanjenja, već do blagog povećanja intenziteta PNM..., Tenascin-C (TnC) is a glycoprotein present in the extracellular matrix (ECM) of a variety of vertebrate tissues during development, where it plays multiple roles in cell growth, migration and adhesion by activating diverse intracellular signaling pathways. Expression of TnC is markedly downregulated in the adulthood, persisting in a few peripheral structures and in central nervous system areas known to retain high degree of plasticity, such as the cerebellum, where it is involved in the modulation of synaptic functions. Enriched environment (EE) shapes many aspects of behavior and may, therefore, serve as a paradigm to study neuronal plasticity in the adult. Exposure to EE leads to numerous changes at the molecular and cellular levels, which target various brain regions including the cerebellum. The aim of present study was to examine the role of TnC in the modulation of cerebellar structural plasticity induced by the exposure to EE by following the appearance of perineuronal nets (PNN), changes in size and density of excitatory and inhibitory synaptic terminals, and the activity of major ECM degrading enzymes, MMP-2 and MMP-9. Furthermore, the present study aimed to examine how TnC deficiency interacts with the environment in shaping different behavioral domains associated with the cerebellum. To this end, 3-week-old mice lacking TnC (TnC-/-) or MMP-9 (MMP-9-/-) and corresponding wild-type mice were exposed to standard conditions (SC) or an enriched environment (EE) for 4 or 8 weeks. The present study shows that 8 weeks of exposure to EE leads to reduced staining of PNN, reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-/- mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, while exposure to EE did not further reduce, but even slightly increased PNN staining...

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 NFKB 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. (C) 2017 Elsevier Ltd. All rights reserved.

Genska ekspresija matriks metaloproteinaze-9 (MMP-9) je pojačana u alveolarnim makrofagima (AM) HIV-1 pozitivnih pušača obolelih od emfizema pluća. Na osnovu podataka koji ukazuju da supernatant bronhoalveolarnog lavata u plućima HIV-1 pozitivnih pušača sadrži povišene koncentracije inflamatornih citokina u odnosu na ovu tečnost u plućima HIV-1 negativnih pušača, postavili smo hipotezu da povišene koncentracije inflamatornih citokina u populaciji HIV-1 pozitivnih pušača utiču na povišenje koncentracije metaloproteinaza, na taj način dovodeći do ranog razvoja emfizema pluća. Putem multicitokinske analize, analizirali smo supernatante bronhoalveolarnog lavata 5 različitih fenotipskih grupa: HIV-1 negativnih nepušača, HIV-1 negativnih pušača bez poremećaja disajnih puteva, HIV-1 negativnih pušača sa ranim emfizemom pluća, HIV-1 pozitivnih nepušača i HIV-1 pozitivnih pušača sa ranim emfizemom pluća. Pored pojačane ekspresije inflamatornih citokina u plućima pušača zaraženih virusom HIV-1, povišene vrednosti citokina koji indukuju Th-17, pre svega interleukina IL-23, predstavljaju zajedničku karakteristiku HIV-1 pozitivnih nepušača, HIV-1 pozitivnih pušača sa ranim emfizemom pluća i HIV-1 negativnih pušača sa ranim emfizemom pluća. Relativna ekspresija IL-23 gena u AM HIV-1 pozitivnih osoba je povišena u odnosu na AM HIV-1 negativnih ispitanika, i značajno je povišena u odnosu na AM HIV-1 pozitivnih pušača sa ranim emfizemom pluća. Akutna infekcija HIV-1 virusom in vitro dovodi do pojačane ekspresije IL-23 u HIV-1 negativnim AM. Stimulacija kultura AM IL-23 dovodi do pojačane ekspresije MMP-9 gena i pojačane koncentracije MMP-9. S obzirom na to da AM zdravih osoba ne eksprimiraju IL-23 receptor (IL-23R), činjenice da kulture makrofaga prečišćenih adherencijom sadrže izvestan procenat T limfocita i činjenice da dodavanje T limfocita ovim kulturama pojačava produkciju MMP-9, efekat IL-23 na pojačano lučenje MMP-9 može biti indirektan i zahteva učešće T limfocita u ovom pro, Matrix metalloproteinase-9 (MMP-9) is up-regulated in alveolar macrophages (AM) of HIV1+ smokers, who develop emphysema in an accelerated time frame compared to HIV1- smokers. The epithelial lining fluid (ELF) of HIV1+ smokers has increased levels of inflammatory cytokines compared to their HIV1- counterparts. To assess the hypothesis that up-regulation of lung cytokines may be functionally related to the increased concentrations of MMP-9, arrays were used to evaluate relative cytokine protein levels in the ELF obtained from 5 groups of individuals: HIV1‾ healthy nonsmokers, HIV1‾ healthy smokers, HIV1‾ smokers with early emphysema, HIV1 + nonsmokers, and HIV1 + smokers with early emphysema. While several proinflammatory cytokines were increased in smoking and HIV1+ phenotypes, increased levels of Th17-related cytokines such as IL-23 and IL-17 represented a common finding in HIV1- smokers with early emphysema as well as in HIV1+ individuals independently of their smoking history. We further hypothesized that IL-23 is up-regulated in the setting of HIV1 infection and that this pro-inflammatory cytokine may be directly correlated to increased concentrations of MMP-9 in the ELF of HIV1+ smokers with early emphysema. Relative gene expression of AM IL-23 is significantly increased in HIV1+ individuals, with a trend toward even greater expression in HIV1+ smokers with early emphysema. Infection with HIV1 laboratory strain JRFL in vitro induced IL-23 expression in normal AM. With the knowledge that AM cultured by the adherence method contain a small number of other cells, including lymphocytes, we hypothesized that in an AM/lymphocyte co-culture system IL-23 would up-regulate MMP-9. Stimulation of AM/lymphocyte co-cultures with IL-23 in vitro induced increased MMP-9 gene expression and protein. While AM obtained from healthy individuals do not express IL-23 receptors, T lymphocytes express IL-23R and act as an intermediary to up-regulate AM MMP-9...

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.