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AIM OF THE STUDY
The capacity of mycobacteria to modulate effector functions of professional phagocytes is related to the ability to express and produce virulence factors. The role of M. marinum ESX-5 in the modulation of pro-inflammatory cytokine secretion by human macrophages has been demonstrated (Abdallah et al., 2008).
The aim of this thesis was to evaluate the ability of several Mtb ESX-5 mutant strains to modulate the effector functions of human professional phagocytes. Three ESX-5 mutants (Fig. 8) were chosen for the studies: MtbeccD5ko and Mtbppe25-pe19, which inactivate the
ESX-5 secretion system and show an attenuated phenotype in the SCID mouse model (Bottai et al., 2012), and Mtbrv1794ko whose orthologue in M. marinum was demonstrated to modulate cytokine secretion by human macrophages (Abdallah et al., 2008).
First, the role of Mtb ESX-5 in modulating cytokine profiles was assessed. To this aim, human monocyte derived pro-inflammatory (M1) and regulatory (M2) macrophage subsets and MoDCs were infected with Mtbwt or various Mtb ESX-5 mutants and the pattern of the cytokines released from the infected cells was evaluated by flow cytometry using a multiplex bead assay. In parallel, the surface expression on MoDCs of activation markers, co-stimulatory molecules or molecules involved in antigen presentation to T cells was analysed. We observed that disruption of Mtb rv1794 gene resulted in increased secretion of IL-1 and IL-18 from Mtbrv1794ko infected M1 and M2. Both IL-1 and IL-18 are produced in inactive “pro-” form and their activation and secretion from macrophages is tightly controlled by a two-step mechanism that involves a family of cytosolic multiprotein complexes known as “inflammasomes”. Activation of inflammasome in macrophages triggers caspase-1 activation, and, in turn, maturation of IL-1 and IL-18 to their active, secreted forms. Thus, to investigate the possible mechanism(s) responsible of the augmented IL-1 and IL-18
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secretion by Mtbrv1794ko infected cells, in a second set of experiments, we have tried to dissect the molecular steps involved in inflammasome activation.
In order to evaluate whether the higher level of IL-1 in culture supernatants of macrophages infected with Mtbrv1794ko was due to a major induction of pro-IL1, we analysed both the mRNA expression of pro-IL-1 and the protein levels of pro- and active IL-1 in macrophages infected with Mtbwt, Mtbrv1794ko or Mtbrv1794ko-C.
Next we investigated the involvement of this enzyme in IL-1 production by human macrophages. To this aim, we assessed the effect of the Mtbrv1794ko strain on: i) pro-caspase-1 synthesis, by measuring mRNA expression levels, and ii) caspase-1 activation, by using a specific inhibitor of caspase-1. Furthermore, to investigate the ability of the Mtbrv1794ko strain to induce cell death and apoptosis in human macrophages, M1 were infected with Mtbwt, Mtbrv1794ko or Mtbrv1794ko-C and dead and apoptotic cells were assessed by flow cytometry.
Finally, experiments were designed to investigate whether two of the inflammasome triggering mechanisms, reported to be involved in Mtb infection (i.e. the efflux of K+ and the release of cathepsin B), might have a role in the observed increased IL-1 production/secretion by Mtbrv1794ko infected M1.
