Studies of effector functions in mycobacterium tuberculosis-infected macrophages with implications for host-directed therapies

Abstract

Tuberculosis infection remains a global health problem and the emergence of multidrugresistant TB (MDR-TB) adds further challenges in the battle to stop the spread of infection. Macrophages are the primary host cells to be infected with Mycobacterium tuberculosis (Mtb), and are therefore key cells in the initial control of TB infection. However, to survive and persist in activated macrophages, Mtb has developed strategies to counteract antimicrobial immunity. This thesis work aimed to explore macrophage responses in Mtbinfected cells, the nature of innate effector mechanisms and how these can be enhanced. For this purpose, we used an in vitro macrophage infection model as well as an organotypic lung tissue model system.

The thesis work is based on four papers, Study I and III, involved studies on the effects of the immunomodulatory compounds, vitamin D3 (VitD3) and phenylbutyrate (PBA), on the induction of antimicrobial peptides, primarily human cathelicidin LL-37, and the ability to kill intracellular Mtb. In Study I, we observed that Mtb can down-regulate the expression of LL-37 in infected macrophages, but this effect was effectively counteracted by treatment with VitD3 and/or PBA. PBA and/or VitD3 prevented intracellular Mtb growth via induction of LL-37 as well as activation of autophagy in Mtb-infected macrophages. In Study III, we further demonstrated that PBA+VitD3 enhanced intracellular Mtb killing of both MDR-TB and drug-susceptible strains and this effect was dependent on LL-37.

In Study IV, we explored the role of VitD3 in polarization of macrophages in comparison to activation with conventional stimuli such as GM-CSF (polarization of inflammatory M1 macrophages) or MCSF (polarization of anti-inflammatory M2 macrophages). The findings from Study IV, suggested that polarization with VitD3 enhanced the ability of Mtb-infected macrophages to control intracellular Mtb growth in comparison to M1 and M2 subsets. Enhanced Mtb growth control was associated with elevated levels of pro-inflammatory cytokines and LL-37, but reduced Mtb-induced expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase).

Study II, aimed to investigate the expression and function of matrix metalloproteinases (MMPs) in early TB granuloma formation using the organotypic lung tissue model that enabled studies on macrophage-Mtb interactions in a more physiological environment in tissue. In this study, we found that pre-treatment of macrophages with the global MMP inhibitor, marimastat, resulted in effective inhibition of TB granuloma formation, which was associated with reduced Mtb growth detected in the lung tissue model. Thus, reduced degradation of extracellular matrix proteins, could prevent Mtb multiplication and spread of the infection.

The main conclusion from this thesis work is that immunomodulatory compounds with the ability to boost or block innate effector mechanisms in Mtb-infected macrophages may be used as adjunct host-directed therapies that could support standard anti-TB drugs to enhance clinical recovery from TB.