THE INFLUENCE OF HEAT SHOCK PROTEIN 70 AND GLYCOPROTEIN 96 ON MATURATION PROGRAM OF DECIDUAL CD1a+ DENDRITIC CELLS IN THE FIRST TRIMESTER OF PREGNANCY

Abstract

Cilj istraživanja: Bjelančevine toplinskog šoka su visoko očuvane i ubikvitarne molekule prisutne unutar stanice na majčino-fetalnom spoju. Tijekom implantacije dolazi do opsežne tkivne pregradnje, pri čemu se u izvanstanični prostor mogu otpustiti bjelančevine toplinskog šoka. Snažno imunogeno djelovanje ovih bjelančevina može postati okidač za aktivaciju pro-upalnog imunološkog odgovora te prijetnja održavanju trudnoće. Cilj ovog rada bio je utvrditi obrazac izražaja bjelančevina toplinskog šoka 70 i gp96 (od engl. glycoprotein 96) te njihovih receptora CD91 i TLR4 (od engl. Toll Like Receptor 4) na mjestu prodora trofoblasta u decidualno tkivo prvog tromjesečja normalne i patoloških trudnoća (anembrijska trudnoća i zadržani pobačaj) te tkiva posteljice na terminskoj trudnoći. Nadalje, ispitati mehanizme nadziranja izražaja bjelančevina toplinskog šoka (HSP70 i gp96) i njihovih receptora CD91 i TLR4 pod utjecajem PIBF-a (od engl. Progesterone Induced Blocking Factor) i pro-upalnih citokina (IL-15 i IL-2). Također, proučiti utjecaj HSP70 i gp96 na fenotip i funkciju decidualnih CD1a+ dendritičkih stanica in vitro. Materijali i metode: Raspodjela Hsc70, Hsp70, gp96, CD91 i TLR4 u decidualnom tkivu prvog tromjesečja normalne i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći uklopljenih u parafin ispitana je metodama imunohistologije i imunoflorescencije. Enzimatskom razgradnjom tkiva i centrifugiranjem na gradijentu gustoće izdvojene su decidualne mononuklearne stanice (DMS) i analiziran je utjecaj rastućih koncentracija PIBF-a, IL-15 i IL-2 na izražaj HSP70, gp96, CD91 i TLR4 protočnom citometrijom i RT-qPCR metodom. Vezanje HSP70 i gp96 za receptore CD91 i TLR4 na CD1a+ DS dokazano je testom specifičnog vezanja. Ispitan je utjecaj HSP70 i gp96 na fenotip, stupanj sazrijevanja, unutarstanično izražavanje citokina i kemokina u decidualnim CD1a+ DS protočnom citometrijom nakon 18-sati stimulacije in vitro. Rezultati: Hsc70, Hsp70, gp96, CD91 i TLR4 prisutni su u uzorcima decidualnog tkiva prvog tromjesečja zdrave i patoloških trudnoća te tkiva posteljice na terminskoj trudnoći i to posebno u žlijezdanom epitelu, endotelu kapilara, stanicama raspršenim u stromi decidue i stanicama trofoblasta. Pod utjecajem PIBF-a dolazi do smanjenja izražaja HSP70, gp96, CD91 i TLR4 na razini gena i bjelančevina u suspenziji DMS. Citokini, IL-2 i IL-15 smanjuju izražaj gRNK za HSP70, CD91 i TLR4, dok povećavaju izražaj gRNK za gp96. IL-2 također, smanjuje udio CD91+ i TLR4+ stanica u različitim staničnim subpopulacijama DMS-a. IL-15 povećava udio CD91+ stanica NK, ali smanjuje udio TLR4+ stanica NK, limfocita T, nezrelih i zrelih DS. HSP70 i gp96 specifično se vežu za CD91 i TLR4 na decidualnim CD1a+ DS. Pod utjecajem HSP70 i gp96 nezrele CD1a+ DS povećavaju izražaj CD83, HLA-DR, ko-stimulacijskih molekula (CD80 i CD86) te kemokina (CCL3 i CCL22) i citokina (INF- i IL-15). Zaključak: Bjelančevine toplinskog šoka (HSP70, gp96) i njihovi receptori (CD91 i TLR4) su prisutni na majčino-fetalnom spoju tijekom prvog tromjesečja zdrave i patoloških trudnoća te u tkivu posteljice na terminskoj trudnoći, što predstavlja molekulsku osnovu za njihovo međudjelovanje. Intenzitet izraženosti ovih molekula mogla bi biti posljedica složenih međuodnosa PIBF-a, IL-15 i IL-2. U pokusima in vitro, HSP70 i gp96 kao prirodni ligandi za CD91 i TLR4 potiču sazrijevanje i aktivaciju CD1a+ DS sa stvaranjem pretežno pro-upalnih medijatora, što može podržati štetan imunološki odgovor i nepovoljan ishod trudnoćeObjectives: Heat shock proteins are highly conserved and ubiquitous molecules present in the cells at the maternal-fetal interface. During the extensive tissue remodelling which is followed by the release of HSPs into the extracellular space. Strongly immunogenic activity of these proteins might bias the immune response towards the Th1 pattern and could cause undesirable pregnancy termination. The aim of the study was to evaluate the expression of HSP70 family members (Hsc70 and Hsp70), glycoprotein 96 (gp96) and their receptors CD91 and Toll like receptor 4 (TLR4) was investigated at maternal-fetal interface of the first trimester normal and pathological pregnancies (blighted ovum and missed abortion) and placental tissue at term. The mecchanisam of regulation of HSP70, gp96, CD91 and TLR4 expression was analysed by increasing concentration of Progesterone Induced Blocking Factor (PIBF), interleukin (IL)-15 and IL-2. The influence of HSP70 and gp96 on phenotype and maturation process of decidual CD1a+ dendritic cells (DC) was evaluated in vitro. Material and methods: Immunohistological and immunofluorescent labelling were performed on paraffin-embedded decidual sections of normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta to detect gp96, Hsc70, Hsp70, CD91 and TLR4. The samples of normal first trimester pregnancy decidua were enzymatically digested and gradient density centrifuged to obtain the suspension of decidual mononuclear cells (DMC). DMC were stimulated with increasing concentration of PIBF, IL-15 and IL-2 and the expression of HSP70, gp96, CD91 and TLR4 was analysed by flow cytometry and RT-qPCR. The specific binding of HSP70 and gp96 was asset for receptors CD91 and TLR4 on CD1a+ DC. Flow cytometry was used to evaluate the influences of HSP70 and gp96 on the phenotype, the maturation process and the intracellular expression of cytokines and chemokines in decidual CD1a+ DC. Results: Hsc70, Hsp70, gp96, CD91 and TLR4 are present in decidual tissue of normal and pathological pregnancies first trimester (blighted ovum and missed abortion) and term placenta and they were found in glandular cells, endothelial cells, cells randomly distributed in stroma and trophoblast cells. PIBF down-regulated HSP70, gp96, CD91 and TLR4 at gene and proteins levels in DMCs. Cytokines, IL-2 and IL-15 decreased mRNA for HSP70, CD91 and TLR4, while increased mRNA for gp96. IL-2 also decreased the frequency of CD91+ and TLR4+ cells in different subpopulations of DMC. IL-15 treatment increased the frequency of CD91+ NK cells, but decreased the frequency of TLR4 expressing NK cells, T cells, immature and mature DC. HSP70 and gp96 efficiently binds CD91 and TLR4 receptors on decidual CD1a+ DC. In the presence of HSP70 and gp96 immature CD1a+ DC assume a more mature phenotype due to increased expression of CD83, HLA-DR, co-stimulatory CD80 and CD86 molecules, chemokines (CCL22 and CCL3) and cytokines (IFN-γ and IL-15). Conclusion: The presence of heat shock proteins (HSP70 and gp96) and their receptors CD91 and TLR4 at the maternal-fetal interface during normal and pathological first trimester pregnancies (blighted ovum and missed abortion) and term placenta provides a molecular basis for their interaction. The expression intensity of these molecules could be influenced by the interplay among PIBF, IL-15 and IL-2. HSP70 and gp96, as natural ligands for CD91 and TLR4 receptors induce maturation process of CD1a+ DC toward the expression of predominantly pro-inflammatory mediators, which could support a harmful immune response and adverse pregnancy outcome