Evaluation of micro RNA expression profiles during BCG infection in the presence and absence of endogenous and synthetic steroids and possible implications on the host immune response to the pathogen

Thiart, Leani (2014-04)

Thesis (MScMedSc)--Stellenbosch University, 2014.


ENGLISH ABSTRACT: Individuals latently infected with Mycobacterium tuberculosis (M.tb) contain the infection without showing signs of illness. Steroid hormones such as glucocorticoids (GCs) however can lead to reactivation of TB. Currently two different injectable contraceptives are available in South Africa, medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET). MPA is able to bind to and activate the glucocorticoid receptor (GR) and possesses selective GC activity, a pharmacological characteristic absent in NET. The aim of this study was to investigate the immune modulatory effects of the two contraceptives MPA and NET on immune responses to mycobacteria in vitro and in vivo. Human peripheral blood mononuclear cells (PBMCs) were infected with BCG (M. bovis Bacille Calmette-Guérin) and treated with MPA, NET, progesterone or cortisol and cytokine expression was measured in order to determine whether the synthetic progestins mimic endogenous progesterone or the GC cortisol. MPA, but not NET suppressed the expression of IFN-γ, IL-1α, IL-1β, IL-2, IL-12p40 and IL-13 similarly to cortisol. Furthermore expression of miRNAs, small double stranded RNA molecules that bind to complementary sequences in mRNAs of target genes and cause their degradation, was determined under the different experimental conditions. The expression of several miRNAs including miR-30c, miR-222, miR-454 and miR-331-3p were differentially influenced by MPA, cortisol and/or NET in PBMCs stimulated with BCG. For example, BCG induced the expression of miR-454 (p=0.003) which was then inhibited to basal levels by cortisol (p=0.008), MPA (p=0.002) and NET (p=0.002). These results demonstrate that steroid hormones including the contraceptives MPA and NET can modulate immune responses to mycobacteria at the miRNA level. To determine the effect of MPA and NET on BCG-induced expression of miRNAs in vivo a mouse model was used. C57BL/6 mice were injected weekly with either MPA or NET using a dose equivalent to humans and then infected with BCG. Mice treated with MPA had a higher spleen bacterial load 21 days after infection compared to both NET treated and control mice (p=0.023). In whole blood, MPA and NET treatment suppressed the BCG-induced production of miR-100 and miR-509-3p to basal levels. In contrast to NET, MPA induced expression of miR-99a expression independent of BCG infection. In the lung, the site of disease, a total number of 22 out of 377 miRNAs tested were differentially expressed 21 days after infection. The results of this study indicate that both synthetic progestins altered the immune response to BCG at the level of cytokine expression as well as the miRNA level. MPA was found to mimic cortisol by inhibiting secretion of inflammatory cytokines whereas NET appeared to have more progestogenic properties. Each of the steroid hormones was observed to induce a characteristic miRNA expression profile, both in vitro as well as in vivo, although not identical. These results highlight that the two contraceptives – although used interchangeably by women in developing countries - are pharmacologically unique and differentially modulate immune responses to mycobacteria. These data support the urgent need for further research into the link between hormonal contraceptives and susceptibility to infectious diseases.

AFRIKAANSE OPSOMMING: Individue wat latent met Mycobacterium tuberculosis ( M.tb ) geïnfekteer is, onderdruk die infeksie en wys geen simptome van die siekte nie. Steroïed hormone soos glukokortikoïede (GKe) kan egter tot die heraktivering van TB lei. Daar is tans twee verskillende inspuitbare voorbehoedmiddels beskikbaar in Suid-Afrika, medroksiprogesteroon-asetaat (MPA) en noretisteroon enantaat (NET). MPA is staat om aan die glukokortikoïed reseptor (GR) te bind en dit te aktiveer. MPA beskik ook selektiewe GK aktiwiteit, 'n farmakologiese eienskap wat afwesig is in NET. Die doel van hierdie studie was om die immuun-regulerende effekte van die twee voorbehoedmiddels, MPA en NET, op immuunresponse teen mikobakterieë in vitro en in vivo te ondersoek. Menslike perifêre bloed mononukleêre selle (PBMSe) is met BCG geïnfekteer en met MPA, NET, progesteroon of kortisol behandel. Sitokien uitdrukking was gemeet om vas te stel of die sintetiese progestiene, endogene progesteroon of die GK kortisol naboots. MPA, maar nie NET, onderdruk die produksie van IFN-γ, IL- 1α, IL- 1β, IL- 2, IL- 12p40 en IL- 13 soortgelyk aan kortisol. Verder is uitdrukking van miRNAs, klein dubbelstring RNS molekules wat aan komplimentêre volgorde in mRNAs van teiken gene bind en wat hul degradering veroorsaak, bepaal in elk van die verskillende eksperimentele toestande. Die uitdrukking van verskeie miRNAs insluitende miR-30c, miR-222, miR-454 en miR-331-3p is differensieël beïnvloed deur MPA, kortisol en/of NET in PBMSe wat met BCG gestimuleer is. Byvoorbeeld, BCG veroorsaak die uitdrukking van miR-454 wat dan geïnhibeer word tot agtergrondvlakke deur kortisol, MPA en NET. Hierdie resultate toon dat steroïed hormone, insluitend die voorbehoedmiddels MPA en NET, die immuunrespons teen mikobakterieë op miRNA-vlak affekteer. Om die effek van MPA en NET op BCG-geïnduseerde uitdrukking van miRNAs in vivo te bepaal, is ʼn muismodel gebruik. C57BL/6 muise is weekliks met 'n dosis van MPA of NET, ekwivalent aan dosisse gebruik in die mens, ingespuit en met BCG geïnfekteer. Muise wat met MPA behandel is, het 'n hoër bakteriële lading in die milt 21 dae na infeksie in vergelyking met NET-behandelde muise en kontrole muise. In hul bloed, onderdruk MPA en NET behandeling die BCG-geïnduseerde produksie van miR-100 en miR-509-3p tot basale vlakke. In teenstelling met NET, induseer MPA die uitdrukking van miR-99a onafhanklik van BCG infeksie. In die long is 'n totaal van 23 miRNAs differensieël uitgedruk 21 dae na die infeksie. Die resultate van hierdie studie dui daarop dat beide sintetiese progestien die immuunrespons teen BCG verander op sitokien sowel as miRNA vlak. MPA boots hoofsaaklik kortisol na deur inhibering van sitokien-produksie terwyl NET meer progesterone eienskappe het. Op miRNA vlak veroorsaak elk van die steroïed hormone 'n kenmerkende miRNA uitdrukkingsprofiel, beide in vitro asook in vivo. Hierdie resultate beklemtoon dat die twee voorbehoedmiddels - alhoewel hulle afwisselend gebruik word deur vroue in ontwikkelende lande - farmakologies uniek is en differensieël die immuunrespons reguleer teen Mycobacterium. Hierdie data ondersteun die dringende behoefte aan verdere navorsing in verband met hormonale voorbehoedmiddels en vatbaarheid vir aansteeklike siektes.

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