In vitro model of Mycobacterium tuberculosis (Mtb): Impact of donor BCG vaccination

• Author / Creator

  Bhavanam,Sudha

• Tuberculosis (TB) is a chronic pulmonary disease caused by Mycobacterium tuberculosis (Mtb). Mtb spreads by aerosol and infects through the airways. Mtb is phagocytosed by macrophages in the lung, and is able to replicate inside these cells. The initial infection of Mtb with macrophage results in the recruitment of other innate and adaptive immune cells resulting in the formation of a granuloma, the hallmark of Mtb infection. Maintenance of the granuloma is thought to be depended on the host immune system. A Bacille Calmette-Guérin (BCG) vaccine was developed in 1920 to prevent Mtb infection and is still the only available vaccine. The efiicacy of this vaccine is variable in terms of protection against pulmonary TB infection. This thesis focuses on the impact of BCG vaccination history of donor human peripheral blood mononuclear cells (PBMCs) on the interaction between the host immune cells and Mtb. This work is the first study to investigate BCG vaccination history as a potential confounding variable when evaluating the immune responses in an in vitro PBMC model of early Mtb infection using the attenuated strain Mtb H37Ra. After validating an in vitro PBMC-collagen matrix culture model of Mtb infection, I characterized the immune responses to Mtb infection in PBMCs from 10 donors. Aggregates of PBMCs were initially observed on day 3 and the size of aggregates continued to increase until day 8 post-infection when macrophages and T cell subsets were identified to be present. The levels of cytokines secreted by PBMCs infected with Mtb peaked on day 3 and decreased on days 5 and 8. I then stratified the data by donor prior BCG vaccination history. Mycobacterial load was significantly lower in infected PBMCs from BCG vaccinated donors compared to unvaccinated donors. The proinflammatory cytokines secreted by T helper 1 cells (Th1): INF-γ, TNF-α, IL-6, IL-4, and IL-17 in the supernatants were higher in BCG vaccinated donors compared to BCG unvaccinated donors. In contrast, the secretion of one anti-inflammatory cytokine secreted by T helper 2 cells (Th2) as well as Tr1 cells: IL-10, was significantly lower in Mtb-infected PBMCs from BCG vaccinated donors compared to BCG unvaccinated donors. These results demonstrated that prior BCG vaccination led to a greater Th1 than a potential Th2 immune response and there by increased the suppression of Mtb growth. Tregs are a subset of T lymphocytes whose role is to suppress the immune response to infection. The role of Tregs in the suppression of Mtb growth was studied in donor PBMCs with or without Tregs (depleted by antibody- coated magnetic bead separation). This study is the first report to investigate the role of Tregs in an early in vitro PBMC model of Mtb H37Ra infection cultured for 8 days. Cell aggregates were unexpectedly smaller in PBMCs without Tregs compared to PBMCs with Tregs at day 8 post-infection, suggesting that Tregs promoted rather than impaired the formation of cell aggregates. The number of Mtb colony forming units (CFUs) increased in PBMCs without Tregs compared to PBMCs with Tregs at days 3, 5 and 8, suggesting that Tregs may play a role in suppressing the growth of Mtb. Levels of IL-17 and proinflammatory Th1 cytokines IFN-γ (at days 3 and 5), TNF-α and IL-6 (at day 3) were lower in PBMCs without Tregs compared to PBMCs with Tregs. In contrast, anti-inflammatory Th2 cytokines IL-10 and IL-4 were higher at day 3 in PBMCs depleted of Tregs compared to PBMCs with Tregs demonstrating that depletion of Tregs led to an unexpected decrease in Th1 to a potential Th2 cytokine response. This study highlights the possibility that BCG vaccination may confound results that utilize human PBMCs to study Mtb infection. The unexpected finding that depletion of Tregs increased Mtb growth and decreased Th1 immune responses to infection suggests further investigation is needed to understand the role of Tregs in early Mtb infection.

• Subjects / Keywords

  • Immunology
  • Mycobacterium Tuberculosis
  • Regulatory T cells

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3HD7P83M

• License

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