Environmental influences on innate and adaptive immune responses against Mycobacterium tuberculosis

Loebenberg, Laurianne (2011-03)

Thesis (PhD)--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: The evaluation of the immune responses in peripheral blood and at the site of disease of people with differential outcomes following M.tb exposure will lead to the discovery of host biomarkers that will increase our understanding of the protective and non-protective immune responses against the bacterium. The main study consisted of a number of pilot studies and the objectives of the studies were: (1) To determine the background and stimulated whole blood cytokine profiles of children and adults of the community; (2) to establish biomarker profiles in whole blood of children with different M.tb infection phenotypes; (3) to investigate the anti-mycobacterial whole blood immune responses in HIV infected and uninfected children; (4) and to investigate the role of the innate immune system during human tuberculosis disease. The study designs were as follow: (1) Adults and children were enrolled in order to determine cytokine profiles in the community. Whole blood was stimulated with BCG and ESAT-6 or left unstimulated. Eighteen cytokines were measured in supernatants of each condition. Progression to active tuberculosis in the years after study participation was assessed by searching for patient entries in the tuberculosis register. (2) Children with known tuberculosis exposure in their households and with M.tb infection as assessed through interferon-ã release assays, children with exposure but no infection and a control group with no exposure and no infection were investigated. Whole blood was stimulated in QuantiFeron tubes overnight and 21 cytokines were measured in antigen stimulated and unstimulated supernatants by multiplex cytokine arrays. (3) HIV infected and uninfected children were enrolled in a hospital based study. Whole blood interferon-ã responses against specific mycobacterial antigens were investigated in a diluted 7 day whole blood assay and compared to QuantiFeron supernatants from the same participants. (4) Tuberculosis diseased adults were enrolled before the onset of treatment and innate and adaptive cell populations were investigated upon start of treatment and at treatment end. In addition, pleural effusion fluid was collected from tuberculosis and cancer patients and innate cell populations further investigated. The studies were performed in Cape Town, South Africa and included Tygerberg Academic Hospital and the surrounding neighbourhoods of Ravensmead, Uitsig and Elsies River. The main findings of the studies included: (1) We showed age related cytokine differences in our study community. Tuberculosis progressors had significantly higher levels of IL-10 in the unstimulated sample several years before the onset of tuberculosis disease. (2) Cytokines that distinguished best between children with tuberculosis infection and no infections were all cytokines that correlated with interferon-ã (interferon-ã was used to make the classification of M.tb infected and uninfected). Higher IL-1â and lower IL-17 levels in children with tuberculosis exposure without subsequent M.tb infection compared to children with no exposure were shown. (3) HIV infected children showed better responses after 7 day whole blood antigen stimulation compared to the overnight stimulation in QuantiFeron tubes. TB10.4 stimulation in HIV infected TST positive children gave higher interferon-ã responses than ESAT-6 and CFP-10. (4) The presence of myeloid derived suppressor cells is shown during tuberculosis disease circulating in peripheral blood. Upon treatment a decrease in the population is observed. No differences were seen in the myeloid derived suppressor cell frequencies between tuberculosis and cancer patients, however significantly lower frequencies were seen in healthy controls. The immune response against M.tb is complex and interactions between the different cell types are essential to control and fight infection and disease. In this thesis we presented new biomarkers that play important roles during different stages of M.tb pathogenesis from exposure to infection and even during disease. These may shed light on mechanisms of protection against M.tb, relevant to development of tuberculosis diagnostics and vaccine strategies. Combinations of multiple biomarkers including cytokines and chemokines and cell subsets are required to characterize biosignatures relevant to the diagnosis of tuberculosis infection and disease.

AFRIKAANSE OPSOMMING: Deur die immuunreaksie te ondersoek, in heelbloed en in die setel van infeksie, in mense met verskillende uitkomste van M.tb blootstelling sal lei tot die ontdekking van biologiese merkers en sal bydra tot ons begrip van die beskermde en nie-beskermde immuunreaksies teen die bakterium. Die hoofstudie het bestaan uit ‘n aantal loodsstudies en die doel van die studies was: (1) Om die sitokienprofiele in gestimuleerde heelbloed, asook agtergrond waardes, van kinders en volwassenes te bepaal, in die gemeenskap; (2) om die profiele van biologiese merkers in heelbloed van kinders met verskillende M.tb infeksie fenotipes te bepaal; (3) om die anti-mykobakteriële immuunreaksies in heelbloed by MIV geïnfekteerde en nie-geïnfekteerde kinders te bepaal; (4) om ondersoek in te stel na die doel van die aangebore immuunsisteem tydens tuberkulose siekte. Die studie ontwerpe was soos volg: (1) Volwassenes en kinders het deelgeneem aan die ondersoek van sitokienprofiele in die gemeenskap. Heelbloed is gestimuleer met BCG en ESAT-6 of is ongestimuleerd gelaat. Agtien sitokiene is gemeet in die bo-stand verkry van elke kondisie. Mense wat aktiewe tuberkulose siekte in die jare na die studie ontwikkel het, is geïdentifiseer deur die pastiëntinligting in die tuberkulose-register. (2) Kinders met gedokumenteerde huishoudelike tuberkulose blootstelling en met M.tb infeksie, soos bepaal deur vrygelate interferon-ã toetse, kinders met blootstelling maar geen infeksie en ‘n kontrole groep met geen blootstelling en geen infeksie, is ondersoek. Heelbloed is gestimuleer in die QuantiFeron buise oornag en 21 sitokiene is gemeet in die antigeen gestimuleerde en ongestimuleerde bostande deur die multiplex sitokienpaneel. (3) MIV geïnfekteerde en nie-geïnfekteerde kinders het deelgeneem aan ‘n hospitaal baseerde studie. Heelbloed interferon-ã reaksies teen spesifieke mykobakteriële antigene is bestudeer in ‘n verdunde 7 dag heelbloed toets en vergelyk met die QuantiFeron bostande van dieselfde deelnemers. (4) Siek tuberkulose volwassenes wat nie op behandeling is nie, het deelgeneem. Die aangebore en verworwe selpopulasies is bepaal aan die begin van behandeling asook voor die einde van behandeling. Verder is pluralevog van tuberkulose en kanker pastiënte bestudeer vir aangebore selpopulasies. Die studies is uitgevoer in Kaapstad, Suid-Afrika en sluit in Tygerberg Akademiese Hospitaal en die gemeenskappe van Ravensmead, Uitsig en Elsiesrivier. Die hoofbevindinge van die studies sluit in: (1) Ons het gewys dat daar ouderdomsverwante sitokien verskille in die studie gemeenskap is. Mense wat tuberkulose siekte ontwikkel het, het beduidende hoër vlakke van IL-10 in die ongestimuleerde monsters getoon ‘n paar jaar voor die begin van die siekte. (2) Sitokiene wat die beste onderskeiding gewys het tussen infeksie en geen infeksie was sitokiene wat ook korrelasie getoon het met interferon-ã (interferon-ã is gebruik om die klassifikasie te maak van M.tb infeksie of geen infeksie). Hoër IL-1â en laer IL-17 vlakke in kinders met tuberkulose blootstelling en sonder M.tb infeksie, is gewys wanneer dit vergelyk is met kinders sonder blootsteling. (3) MIV geïfekteerde kinders het beter reaksies getoon na 7 dag heelbloed antigeen stimulasie as met die oornag stimulasie in QuantiFeron buise. TB10.4 stimulasie in MIV geïnfekteerde TST positiewe kinders het hoër interferon-ã reaksies getoon as na stimulasie met ESAT-6 en CFP-10. (4) Die teenwoordigheid van miloïed afgeleide onderdrukkende selle in heelbloed, is getoon tydens tuberkulose siekte. Na behandeling is ‘n afname in die populasie gesien. Geen verskille is gesien in die aantal miloïed afgeleide onderdrukkende selle tussen tuberkulose en kanker pastiënte nie, alhoewel beduidende laer getalle is waargeneem in gesonde kontrole deelnemers. Die immuunreaksie teen M.tb is kompleks en interaksies tussen die verskillende seltipes is belangrik om infeksie en siekte te kontroleer en te beveg. In die tesis het ons nuwe biologiese merkers geïdentifiseer wat belangrike funksies het, tydens die verskillende stadiums van M.tb patogenesiteit, van blootstelling tot infeksie asook tydens siekte. Dit kan gebruik word as biologiese merkers betrokke by die immuunreaksie teen M.tb en sal bydra tot die diagnose van tuberkulose infeksie en siekte.

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