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. 2017 Oct 10:7:439.
doi: 10.3389/fcimb.2017.00439. eCollection 2017.

Small Molecule Mediated Restoration of Mitochondrial Function Augments Anti-Mycobacterial Activity of Human Macrophages Subjected to Cholesterol Induced Asymptomatic Dyslipidemia

Suman Asalla  1 , Krishnaveni Mohareer  1 , Sharmistha Banerjee  1
Affiliations

Small Molecule Mediated Restoration of Mitochondrial Function Augments Anti-Mycobacterial Activity of Human Macrophages Subjected to Cholesterol Induced Asymptomatic Dyslipidemia

Suman Asalla et al. Front Cell Infect Microbiol. .

Abstract

Mycobacterium tuberculosis (M.tb) infection manifests into tuberculosis (TB) in a small fraction of the infected population that comprises the TB susceptible group. Identifying the factors potentiating susceptibility to TB persistence is one of the prime agenda of TB control programs. Recently, WHO recognized diabetes as a risk factor for TB disease progression. The closely related pathological state of metabolic imbalance, dyslipidemia, is yet another emerging risk factor involving deregulation in host immune responses. While high cholesterol levels are clinically proven condition for perturbations in cardiac health, a significant fraction of population these days suffer from borderline risk cholesterol profiles. This apparently healthy population is susceptible to various health risks placing them in the "pre-disease" range. Our study focuses on determining the role of such asymptomatic dyslipidemia as a potential risk factor for susceptibility to TB persistence. Macrophages exposed to sub-pathological levels of cholesterol for chronic period, besides impaired release of TNF-α, could not clear intracellular pathogenic mycobacteria effectively as compared to the unexposed cells. These cells also allowed persistence of opportunistic mycobacterial infection by M. avium and M. bovis BCG, indicating highly compromised immune response. The cholesterol-treated macrophages developed a foamy phenotype with a significant increase in intracellular lipid-bodies prior to M.tb infection, potentially contributing to pre-disease state for tuberculosis infection. The foamy phenotype, known to support M.tb infection, increased several fold upon infection in these cells. Additionally, mitochondrial morphology and function were perturbed, more so during infection in cholesterol treated cells. Pharmacological supplementation with small molecule M1 that restored mitochondrial structural and functional integrity limited M.tb survival more effectively in cholesterol exposed macrophages. Mechanistically, M1 molecule promoted clearance of mycobacteria by reducing total cellular lipid content and restoring mitochondrial morphology and function to its steady state. We further supported our observations by infection assays in PBMC-derived macrophages from clinically healthy volunteers with borderline risk cholesterol profiles. With these observations, we propose that prolonged exposure to sub-pathological cholesterol can lead to asymptomatic susceptibility to M.tb persistence. Use of small molecules like M1 sets yet another strategy for host-directed therapy where re-functioning of mitochondria in cholesterol abused macrophages can improve M.tb clearance.

Keywords: Mycobacterium tuberculosis; cholesterol; dyslipidemia; infection; mitochondria; pre-disease; small molecule M1.

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Figures

Figure 1
Figure 1
Chronic exposure of THP-1 cells to sub-pathological levels of cholesterol impaired them from clearing intracellular Mycobacteria. (A) Schematic representation of the cell culture based model used in the study (B) Measurement of the clearance of intracellular pathogenic mycobacteria H37Rv by CFU counts in untreated and treated cells. (C) Oil Red O staining of intracellular lipid bodies in untreated and treated cells 12 h post cholesterol (80 μM) treatment before infection. The bright field images for each category are representative of at the least five fields (scale bar 10 μm). (D) Lipid bodies as visualized in (C) were quantified on microtiter plate spectroscopically after Oil Red O staining. (E) TNF-α released by untreated and cholesterol treated, with (MOI 20) or without H37Rv infection as measured by ELISA. Clearance of (F) non-tuberculous mycobacteria M. avium or (G) attenuated mycobacteria M. bovis BCG by untreated or treated THP-1 cells as evaluated by CFU. Each experiment was carried out at the least three times. Statistical analyses were performed as mentioned in methods. Error bars represent ± SD. *P ≤ 0.05; **P < 0.005.
Figure 2
Figure 2
Mycobacterial infection in cholesterol treated THP-1 cells altered mitochondrial structure and function, which was rescued by small-molecule M1. (A) Confocal microscopy showing distribution and shape of mitochondria stained by mito-tracker red (seen in red fluorescence) against a background of DAPI stained cells from each category. The images are representative of at the least five fields (scale bar 10 μm). The inset in each image is the zoomed image showing the gross mitochondrial shape. (B) Transmission electron micrographs depicting changes in the mitochondrial morphology from cells from each category. Arrows indicate representative mitochondria. The bar represents 0.2 μm scale. qRT-PCR-based changes in mRNA expression levels of mitochondrial fusion genes mfn-1 (C) and mfn-2 (D) as well as mitochondrial fusion genes drp-1 (E) and mff (F) in each category. Fold change in transcript levels were calculated as against untreated and uninfected THP-1 cells. The transcript levels were quantified by relative 2−ΔΔCt normalized against endogenous GAPDH. (G) Measurement of Mitochondrial Membrane Potential in each category as described in methods. The data is expressed as the ratio of FL2 to FL1. (H) Graphical representation of intracellular ATP/ADP ratios in each category using bioluminescence assay as described in methods. Each experiment was carried out at the least three times. Statistical analyses were performed as mentioned in methods. Error bars represent ± SD. *P ≤ 0.05; **P < 0.005.
Figure 3
Figure 3
Pharmacological intervention of M1 improves mycobacterial clearance by cholesterol treated THP-1 cells. (A) Representative bright field microscopy image showing the accumulation of lipid bodies by Oil Red O staining inside the untreated or cholesterol treated H37Rv infected cells upon 48 h post-infection, with or without M1 treatment. The white bar indicates 10 μm scale. Images were taken at 100 × magnification. (B) Bar graph representing the fold change in the accumulation of Oil Red O Stain as measured at Abs500 nm in different categories. Percentage changes were calculated with respect to the values of the untreated uninfected THP-1 cell. (C) Colorimetric assay quantifying total cellular cholesterol in each category. (D) Measurement of H37Rv clearance by CFU counts upon M1 treatment of untreated and cholesterol treated infected THP-1 as described. Each experiment was carried out at the least three times. Statistical analyses were performed as mentioned in methods. Error bars represent ± SD. *P ≤ 0.05; **P < 0.005.
Figure 4
Figure 4
PBMC derived macrophages from subjects with borderline risk cholesterol profiles showed reduced clearance of intracellular mycobacteria. PBMC derived macrophages were isolated from human volunteers with either normal (NC) or borderline risk (BLC) cholesterol profiles, were either untreated or cholesterol treated and then infected with M.tb H37Rv as discussed in methods. The clearance of mycobacteria by PBMC derived macrophages from the two categories was evaluated by CFU enumeration 48 h post-infection. Bar plots showing CFU enumeration from (A) Cholesterol untreated and treated PBMC derived macrophages of NC; (B) Cholesterol untreated PBMC derived macrophages of NC and BLC; (C) Cholesterol Untreated and treated PBMC derived macrophages of BLC; (D) Cholesterol treated PBMC derived macrophages of NC and BLC; (E) Cholesterol untreated PBMC derived macrophages of NC and BLC with or without M1 treatment and (F) Cholesterol treated PBMC derived macrophages of NC and BLC with or without M1 treatment. Each experiment was carried out at the least three times NC (n = 4) and BLC (n = 3). Statistical analyses were performed as mentioned in methods. Error bars represent ± SD. *P ≤ 0.05; **P < 0.005.
Figure 5
Figure 5
Schematic representation of mechanism involved in M1 mediated restoration of mitochondrial function and anti-mycobacterial activity of THP-1 cells in the pre-disease model mimicking borderline risk cholesterol exposure. Prolonged exposure to sub-pathological levels of cholesterol leads to increase in intracellular lipid bodies, alteration in mitochondrial structure and function that support persistence of M.tb. Pharmacological intervention by M1 reversed changes in cholesterol treated infected macrophages, in terms of restoring structure and function of mitochondria and reducing intracellular lipid, thereby increasing the clearance of intracellular mycobacteria.
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