Investigation of the underlying molecular mechanisms of immune modulation by the contraceptive Medroxyprogesterone acetate (MPA) on immune responses to mycobacteria

Ehlers, Lizaan (2014-04)

Thesis (MScMedSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Background Individuals who are latently infected with Mycobacterium tuberculosis (M.tb) are able to quell the infection by balancing the innate and adaptive immune responses. Glucocorticoids (GCs) can affect this balance and can increase the risk of reactivation of TB. The three month injectable contraceptive medroxyprogesterone acetate (MPA) is widely used by women in developing countries, where TB is rife. MPA, unlike the two monthly contraceptive norethisterone enanthate (NET), possesses selective glucocorticoid activity, and could therefore alter immune responses to TB. Aims The aim of my investigation was to elucidate the immune modulatory effects of the synthetic progestins, MPA and NET, compared to the endogenous hormones, cortisol and progesterone, in Mycobacterium bovis Bacillus Calmette–Guérin (BCG) or anti-CD3 stimulated peripheral blood mononuclear cells (PBMC). I aim to determine the effects of MPA, NET, cortisol and progesterone on the receptor expression of glucocorticoid and various progesterone receptors. I investigate the effect of the above mentioned hormones on the downstream signalling cascades in the presence or absence of either BCG or anti-CD3. The overall immune modulation will be determined with regard to the cytokine production in PBMCs. Methods The presence of receptors for these steroid hormones in PBMCs was verified and BCG, anti-CD3 and hormone induced changes in receptor expression determined through RT-PCR. The impact of cortisol, MPA, NET and progesterone on BCG or anti-CD3 mediated activation of downstream signalling molecules were determined by Western blot as well as Luminex analysis. Results and Conclusion My results show that BCG and anti-CD3 mediated activation of the T cell receptor associated signalling molecules, Lck, ZAP-70, LAT was inhibited by the steroid hormones. Similarly several kinases including JNK, ERK and p38 and transcription factors including STAT3, STAT5 and CREB were differentially affected by the hormones. The inhibition of phosphorylation seen in the different signalling molecules indicated an inhibition of activation of downstream signalling cascades. To investigate the impact of the hormone induced changes in the signalling cascades on the expression of inflammatory and anti-inflammatory cytokines Luminex analysis was performed on the supernatant of the BCG and anti-CD3 stimulated PBMC cultures. Cortisol and MPA, but not NET and progesterone, significantly inhibited the secretion of IL-1α, IL-1β, IL-6, IL-10, TNF-α, IL-12 and IL-13. These results suggest that the immune suppressive effects of MPA are likely mediated through a combination of direct genomic GR action as well as through direct or indirect inhibition of several signalling molecules. The inhibition of the IFN-γ, IL-12, IL-1and IL-6 secretion by MPA could potentially increase the risk of susceptibility to TB in women using this contraceptive. Therefore the absence of glucocorticoid activity seen with NET could make this contraceptive a better choice for women in TB endemic areas.

AFRIKAANSE OPSOMMING: Agtergrond Individue wat latent met Mikobakterium tuberkulose (M.tb) geïnfekteer is, is in staat om die infeksie te onderdruk deur die ingebore en aanpasbare immuunrespons te balanseer. Glukokortikoïede (GCs) kan hierdie balans beïnvloed en kan die risiko van heraktivering van tuberkulose (TB) verhoog. Die drie maande inspuitbare voorbehoedmiddel medroksiprogestroon-asetaat (MPA) word algemeen gebruik deur vroue in ontwikkelende lande, waar TB volop is. MPA, in teenstelling met die twee maandelikse voorbehoedmiddel noretisteroon enantaat (NET), beskik selektiewe glukokortikoïed aktiwiteit, en kan dus die immuunrespons teenoor TB verander. Doelwitte Die doel van my studie was om die immuunregulerende effekte van die sintetiese progestiene, MPA en NET, toe te lig , in vergelyking met die endogene hormone, kortisol en progesteroon, in Mycobacterium bovis Bacillus Calmette - Guerin (BCG) of anti- CD3 gestimuleerde perifere bloed mononukleêre selle (PBMSe). Ek het beoog om die gevolge van MPA, NET, kortisol en progesteroon op die reseptor uitdrukking van glukokortikoïede en verskeie progesteroon reseptore te bepaal. Ek het ondersoek ingestel op die effek van die bogenoemde hormone op die sein transduksie in die teenwoordigheid of afwesigheid van óf BCG of anti-CD3. Die algehele immuun -modulasie sal bepaal word met betrekking tot die produksie van sitokiene in PBMSe . Metodes Die teenwoordigheid van reseptore vir die steroïedhormone in PBMSe is geverifieer en BCG en anti-CD3 en die veranderinge deurdie hormone in verband met die reseptor uitdrukking bepaal deur RT -PCR. Die impak van kortisol, MPA, NET en progesteroon op BCG of anti- CD3 aktivering van sein transduksie molekules is bepaal deur ‘Western blot’ asook Luminex analise. Resultate en gevolgtrekking My resultate toon dat BCG en anti-CD3 die aktivering van die T-sel reseptor wat verband hou met sein molekules , LCK , ZAP -70 , en LAT word geïnhibeer deur die steroïedhormone . Van die kinases insluitend JNK , ERK en p38 en transkripsie faktore, insluitend STAT3 , STAT5 en CREB is beïnvloed deur die hormone. Die inhibisie van fosforilering gesien in die verskillende sein molekules dui daarop aan dat 'n inhibisie van aktivering van sein transduksie. Die impak van die hormoon veroorsaak veranderinge in die sein transduksie met betrekking tot die uitdrukking van inflammatoriese en anti -inflammatoriese sitokiene Luminex analise is uitgevoer op die supernatant van die BCG en anti-CD3 gestimuleerde PBMS kulture. Kortisol en MPA, maar nie NET en progesteroon , het aansienlik die produksie van IL-1α , IL-1β , IL-6 , IL-10 , TNF-α , IL-12 en IL-13 geïnhibeer. Hierdie resultate dui daarop dat die immuunstelsel se onderdrukkende effekte van MPA is waarskynlik bemiddel deur 'n kombinasie van direkte genomiese GR interaksie sowel as deur direkte of indirekte inhibisie van verskeie sein molekules . Die inhibisie van die IFN-γ, IL-12, IL-1 en IL-6 sekresie deur MPA kan potensieel die risiko verhoog van vatbaarheid vir TB in vroue wat hierdie voorbehoedmiddel gebruik. Daarom oor die afwesigheid van glukokortikoïede aktiwiteit wat gesien is met NET, kan maak laat hierdie voorbehoedmiddel 'n beter keuse vir vroue in TB endemiese gebiede.

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