A study of the molecular mechanism of progestin-induced regulation of IL-12 and IL-10 and implications for HIV pathogenesis

Louw, Renate (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)), designed to mimic the actions of the endogenous hormone progesterone (Prog), are extensively used by women as contraceptives and in hormone replacement therapy (HRT). A number of reports have indicated that these synthetic progestins affect immune function in the female genital tract thereby increasing the risk of acquiring sexual transmitted infections. Despite these findings, very little is known about their mechanism of action at the cellular level, in particular their steroid receptor-mediated effects on cytokine gene expression. In the first part of this thesis, the effect of Prog, MPA and NET-A on the expression of the endogenous pro-inflammatory cytokine gene, interleukin (IL)-12p40, and anti-inflammatory cytokine gene, IL-10, was investigated in a human ectocervical epithelial cell line, Ect1/E6E7. Quantitative realtime PCR (qPCR) showed that all three ligands significantly upregulated the tumor necrosis factor alpha (TNF )-induced IL-12p40 gene expression, while IL-10 gene expression was downregulated. Moreover, by reducing the glucocorticoid receptor (GR) levels with siRNA, these effects were shown to be mediated by the GR. A more detailed investigation into the molecular mechanism of the progestogen-induced upregulation of IL-12p40 gene expression, using chromatin immunoprecipitation (ChIP), siRNA, co-immunoprecipitation and re-ChIP analyses, showed that the progestogen-bound GR is recruited to the CCAAT enhancer binding protein (C/EBP)- regulatory element of the IL-12p40 promoter, most likely via an interaction with the transcription factor C/EBP . Similar experiments for the progestogen-induced downregulation of IL-10 gene expression showed that the progestogen-bound GR is recruited to the signal transducer and activator of transcription (STAT)-3 regulatory element of the IL-10 promoter, most likely via an interaction with the transcription factor STAT-3. The second part of this study elucidated the influence of the HIV-1 accessory viral protein R (Vpr) on progestogen-induced regulation of IL-12p40, IL-12p35 and IL-10 in the Ect1/E6E7 cell line. Results showed that in these cells, the overexpression of Vpr significantly modulated the effects of Prog, MPA and NET-A on the mRNA expression of IL- 12p40 and IL-10, while only the NET-A effect was modulated on IL-12p35. Moreover, reducing the GR protein levels by siRNA suggested that the GR is required by Vpr to mediate its effects. Taken together, these results show that Prog, MPA and NET-A promote the pro-inflammatory milieu in the ectocervical environment, and that during HIV-1 infections, this milieu is modulated. Furthermore, the results suggest that the use of MPA or NET in vivo may cause chronic inflammation of the ectocervical environment, which may have important implications for ectocervical immune function, and hence susceptibility to infections such as HIV-1.

AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), ontwerp om die funksies van die natuurlike hormone progesteroon (Prog) na te boots, word wêreldwyd deur vroue as voorbehoedmiddels sowel as vir hormoon vervangingsterapie (HVT) gebruik. Daar is verskeie aanduidings dat hierdie sintetiese progestiene die immuunfunksie in die vroulike geslagskanaal kan beïnvloed en ook die moontlike vatbaarheid van seksueel oordraagbare infeksies kan verhoog. Ten spyte hiervan, is baie min bekend oor hulle meganisme van werking op ‘n molekulêre vlak, veral in die besonder hul effek op sitokinien geenuitdrukking. Die effek van Prog, MPA en NET-A op die geenuitdrukking van ’n endogene pro-inflammatoriese sitokinien, interleukin (IL)-12, en ’n anti-inflammatoriese sitokinien, IL-10, asook die onderliggend meganisme van werking, in ’n menslike ektoservikale sellyn, Ect1/E6E7, is in die eerste deel van hierdie studie ondersoek. Kwantitatiewe “realtime” polimerisasie ketting reaksie (PKR) het getoon dat al drie die ligande die tumor nekrosis faktor alfa (TNF- )-geïnduseerde IL-12p40 geenuitdrukking opreguleer en IL-10 geenuitdrukking onderdruk. Verder is gevind dat induksie van IL-12p40 en inhibisie van IL-10 deur Prog, MPA en NET-A deur die glukokortikoïed reseptor (GR) gedryf word, aangesien volledige opheffing van die effekte op hierdie sitokinien gene waargeneem is wanneer die GR proteïen vlakke deur middel van kort inmengende ribonukleïensuur (siRNS) verminder is. 'n Meer beskrywende ondersoek in die molekulêre meganisme is uitgevoer deur gebruik te maak van chromatien immunopresipitasie (ChIP), siRNS, mede-immunopresipitasie en her-ChIP analises. Hierdie resultate het voorgestel dat die progestogeen (Prog en die sintetiese progestiene)-gebonde GR tot die CCAAT verbeterende bindings protein (C/EBP)- regulatoriese element van die IL-12p40 promotor betrek word en dat die transkripsie faktor C/EBP benodig word om transkripsie van die IL-12p40 geen te aktiveer. Met betrekking tot IL-10, het die resultate voorgestel dat die progestogeen-gebonde GR tot die sein transduksie en aktiveerder van transkripsie (STAT)-3 regulatoriese element van die IL-10 promotor betrek word en dat die transkripsie faktor STAT-3 benodig word om transkripsie van die IL-10 geen te onderdruk. Die tweede deel van die studie het die invloed van die MIV-1 aksesorale virale proteïen R (Vpr) op sitokinien geenuitdrukking, spesifiek die progestogeen-geïnduseerde regulering van IL-12p40, IL-10 en IL-12p35, in die Ect1/E6E7 sellyn ondersoek. Resultate het getoon dat ooruitdrukking van Vpr in hierdie sellyn die effekte van Prog, MPA en NET-A op die mRNS uitdrukking van IL-12p40 en IL-10, en slegs die NET-A effek op IL-12p35, aansienlik moduleer. Vermindering van die GR proteïen vlakke deur middel van siRNS het getoon dat Vpr die GR benodig om hierdie veranderinge mee te bring. In samevatting, die resultate van hierdie proefskrif stel voor dat Prog, MPA en NET-A die pro-inflammatoriese milieu in die ektoservikale omgewing bevorder, en dat hierdie milieu gedurende MIV-1 infeksies verander. Verder, die resultate van hierdie studie impliseer dat die gebruik van MPA en NET in vivo nadelige lokale immuunonderdrukkende effekte mag hê wat kan lei tot kroniese inflammasie van die ektoservikale omgewing en ‘n moontlike verhoging in die vatbaarheid van infeksies soos MIV-1.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79822
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