@phdthesis{
author = "Stanisavljević, Suzana",
year = "2018",
abstract = "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 ploče gde se mogu diferencirati u Treg. Smatra se da značajnu ulogu u ovoj rediferencijaciji imaju crevne bakterije i njihovi produkti. Zbog toga je važno utvrditi koje bakterije u crevima i na koji način ostvaruju ulogu u patogenezi multiple skleroze, kako bi se modulacijom mikrobiote creva omogućila prevencija i tretman bolesti. Nedavna uporedna istraživanja sastava mikrobiote creva osoba obolelih od multiple skleroze i zdravih osoba pokazuju kod pacijenata povećanu ili smanjenu zastupljenost pojedinih grupa bakterija, uključujući Clostridia klaster XIV i IV, Bacteroides fragilis, i rodova Pseudomonas, Mycoplana, Haemophilus, Dorea i Blautia. Međutim, rezultati dosadašnjih istraživanja nisu dovoljni da bi bilo razjašnjeno na koji način mikrobiota creva doprinosi patogenezi multiple skleroze. Mikrobiota creva i GALT se smatraju važnim učesnicima u pokretanju, propagaciji, ali i u prevenciji autoimunoskih procesa u CNS-u. Sastav mikrobiote creva se razlikuje kod pacijenata obolelih od multiple skleroze i zdravih osoba. U skladu sa tim, soj pacova podložan EAE-u, DA, i soj koji je otporan na indukciju EAE-a, AO, pokazali su razlike u sastavu mikrobiote creva u ovom istraživanju. Konkretno, Turicibacter sp. i članovi Lachnospiraceae familije su identifikovani kao mogući promoteri otpornosti na indukciju EAE-a ili oporavka od EAE-a. Takođe je pokazano da se ćelijski sastav mezenteričnih limfnih čvorova, kao i ćelijski sastav Pejerovih ploča razlikuje između AO i DA pacova, s tim da DA pacovi imaju više CD4+ ćelija, ali i više Treg-a. Pored toga, ćelije GALT-a AO soja pacova otpornog na EAE su produkovale manje IFN-γ i IL-17 u poređenju sa DA sojem pacova koji je podložan indukciji EAE-a. Produkcija IL-10 kao glavnog imunoregulatornog citokina u crevima koji ispoljava imunosupresivne efekte, razlikovala se između ova dva soja pacova. Antibiotski tretman je narušio otpornost AO pacova prema indukciji EAE-a, što se ogledalo u pojavi blagih kliničkih simptoma, povećanom broju infiltrata u kičmenoj moždini i povišenom nivou IL-17 u CNS-u. Primena antibiotika je dovela i do promena u drenirajućim limfnim čvorovima. U njima je primećen povećan broj ćelija sa povećanom zastupljenošću CD4+ T ćelija, koje su, produkovale više proinflamacijskih citokina. Analiza zastupljenosti Treg je pokazala da se procenat Treg razlikuje između sojeva samo u Pejerovim pločama neimunizovanih životinja, dok se nakon imunizacije smanjuje udeo Treg u drenirajućim limfnim čvorovima, mezenteričnim limfnim čvorovima i Pejerovim pločama kod životinja tretiranih antibioticima. Sastav mikrobiote creva AO pacova se znatno promenio nakon četiri nedelje primene antibiotika, nakon čega su se promene izgubile. Transferom mikrobiote creva AO pacova u DA pacove došlo je do ublažavanja simptoma EAE-a kod DA pacova. U CNS-u dolazi do smanjenja koncentracije IL-17 kod tako tretiranih DA životinja. Uočena je povećana celularnost u drenirajućim limfnim čvorovima, kao i veća zastupljenost Treg u drenirajućim i mezenteričnim limfnim čvorovima. Transfer mikrobiote je doveo i do značajne promene diverziteta crevne mikrobiote tretiranih DA pacova. Promene se naročito zapažaju u diverzitetu mlečnokiselinskih bakterija. Rezultati ove doktrorske disertacije jasno pokazuju da crevna mikrobiota ima bitnu ulogu u patogenezi autoimunskog procesa prisutnog u CNS-u i da bi njena modulacija mogla biti novi pristup u terapiji multiple skleroze., 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 patches, where they can be re-differentiated into
Treg. It is considered that intrinsic bacteria and their products play an important role in this re-differentiation. That is why it has become important to determine which bacteria and in what ways are implicated in multiple sclerosis pathogenesis, to prevent and treat the disease by modulating gut microbiota. Recent comparative studies of gut microbiota content of multiple sclerosis patients and healthy individuals have shown increased presence of some groups of bacteria in patients, including Clostridia clusters XIV and IV, Bacteroides fragilis, and Pseudomonas, Mycoplana, Haemophilus, Dorea, Blautia. However, the results of previous studies cannot clarify what are the mechanisms of contribution of gut microbiota to pathogenesis of multiple sclerosis.
Gut microbiota and GALT are considered to be important players in initiation, propagation, but also in prevention of CNS autoimmunity. Gut microbiota content differs among multiple sclerosis patients and healthy subjects. In accordance to that, EAE-resistant rat strain and EAE-prone rat strain have different gut microbiota content in our experiments. Specifically, Turicibacter sp. and members of Lachnospiraceae family are identified as possible promoters of EAE resistance or EAE recovery. Besides, it was demonstrated that mesenteric lymph nodes and Peyer’s patches differ between AO and DA rats. DA rats have higher proportion of CD4+ cells and more Tregs. Further, GALT cells of EAE-resistant rat strain produced less IFN-γ and IL-17 in comparison to EAE-prone rat strain. Production of IL-10, major immunoregulatory cytokine, was also different among these rat strains.
Antibiotic treatment disturbed EAE resistance in AO rats. It was observed in the form of mild clinical symptoms, higher number of spinal cord infiltrates and a higher concentration of IL-17 in the CNS. Antibiotic effects were seen in lymph nodes draining the site of immunization; they had higher cellularity, a higher proportion of CD4+ cells, and higher production of proinflammatory cytokines. Treg analysis showed that there was a difference in Peyer’s patches of unimmunized rats. After the immunization, less Treg was observed in draining lymph nodes, mesenteric lymph nodes and Peyer’s patches in antibiotic treated rats. Gut microbiota content of AO rats was significantly changed after four weeks of antibiotic treatment.
Gut microbiota transfer from AO rats into DA rats resulted in milder EAE symptoms in DA rats and lower concentration of IL-17 in CNS. Increased proportion of
Treg was observed in draining lymph nodes in the treated animals. Gut microbiota transfer led to more significant changes in gut microbiota diversity of treated rats, especially in lactic acid bacteria.
Results of this doctoral thesis clearly demonstrate important role of gut microbiota in the pathogenesis of the CNS autoimmunity. They also imply that modulation of gut microbiota could be used as a novel therapeutic approach in multiple sclerosis.",
publisher = "Belgrade: Universiy of Belgrade, Faculty of Biology",
journal = "University of Belgrade, Faculty of Biology",
title = "Uloga mikrobiote i limfnog tkiva creva u otpornosti pacova soja Albino Oksford na indukciju eksperimentalnog autoimunskog encefalomijelitisa, Role of gut microbiota and gut-associated lymphoid tissue in resistance of Albino Oxford rats to experimental autoimmune encephalomyelitis induction",
pages = "1-134",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_3105"
}
