Mycolates of Mycobacterium tuberculosis modulate the flow of cholesterol for bacillary proliferation in murine macrophages

Abstract

The differentiation of macrophages into lipid-filled foam cells is a hallmark of the lung granuloma that forms in patients with active tuberculosis (TB). Mycolic acids (MAs), the abundant lipid virulence factors in the cell wall of Mycobacterium tuberculosis (Mtb), can induce this foam phenotype possibly as a way to perturb host cell lipid homeostasis to support the infection. It is not exactly clear how MAs allow differentiation of foam cells during Mtb infection. Here we investigated how chemically synthetic MAs, each with a defined stereochemistry similar to natural Mtb-associated mycolates, influence cell foamy phenotype and mycobacterial proliferation in murine host macrophages. Using light and laser-scanning-confocal microscopy, we assessed the influence of MA structure first on the induction of granuloma cell types, second on intracellular cholesterol accumulation, and finally on mycobacterial growth. While methoxy-MAs (mMAs) effected multi-vacuolar giant cell formation, keto-MAs (kMAs) induced abundant intracellular lipid droplets that were packed with esterified cholesterol. Macrophages from mice treated with kMA were permissive to mycobacterial growth, whereas cells from mMA treatment were not. This suggests a separate yet key involvement of oxygenated MAs in manipulating host cell lipid homeostasis to establish the state of TB.

Keywords: confocal microscopy; foam cell; infection; lipid droplets; liver X receptor; mycolic acid; tuberculosis.

Fig. 1.

MA structures. The biochemical structures of the synthetic MAs are given for examples of the αMA (JR1080), mMA (JR1046), and kMA (GK324) classes containing cis-cyclopropanation. Numbers in brackets represent carbon chain lengths and wiggly line indicates a mixture of stereoisomers at that position.

Fig. 2.

MAs of the methoxy oxygenation class promote the formation of multi-vacuolar foam cells. The proportion of vacuole-rich macrophages was determined by light microscope images of May-Grünwald-Giemsa-stained cells on cytospins and by laser-scanning-confocal microscopy (mean ± SEM). A: Percentage of foam cells in PEC fraction from the control (PBS and Lipo), αMA, mMA, and kMA treatments. (Shapiro-Wilk: W = 0.801, **P < 0.01; Kruskal-Wallis: H = 12.100, *P < 0.05, df = 4; n = 3 independent experiments; ns, not significant). B: Light microscope images of cytospins showing vacuolar foam cells in PEC fraction. Images were taken at 100× oil magnification. Arrows indicate MGCs. Scale bar: 10 μm. C: Induction of enlarged V+ cells is shown for control (PBS and Lipo) and the various MA-treated mouse peritoneal macrophages over time, as measured by laser-scanning-confocal microscopy (GLM: Wald chi-square = 753.924, ***P < 0.001, df = 14; n = 5 per time point; ns, not significant). D, E: Laser scanning confocal microscopy images showing enlarged V+ cells for PBS, αMA, and mMA treatments. Arrows indicate MGCs. Stacked images, 63× oil objective. Scale bar: 20 μm. E: Zoomed images from the mMA treatment in (D).

Fig. 3.

MAs of the keto oxygenation class induce foam cells rich in cholesterol-laden LDs. The induction of LDs is shown for peritoneal macrophages from control (PBS and Lipo) and αMA-, mMA-, and kMA-treated mice (mean ± SEM). A: LD accumulation in murine macrophages harvested at specified time points following the control and various MA treatments (GLM: Wald chi-square = 353.662, P < 0.001, df = 14; n = 5 per time point). B: Relative induction of LDs over time as compared with PBS (broken line). C: Laser-scanning-confocal microscopy images depicting variation in LD induction for PBS, αMA, and kMA treatments. Arrows indicate LD-filled cells. D: Cellular cholesterol content of variously treated cells. Bars represent total cholesterol, subdivided in the amount of free and esterified cholesterol for each of the treatments (micrograms per 10⁶ cells). Upper panel, fraction percentages of free and esterified cholesterol are shown in the bars (n = 12 mice; Shapiro-Wilk: W = 0.813, P < 0.05; Kruskal-Wallis: H = 24.726, P < 0.001, df = 3). Lower panel, the ratio of esterified-to-free cholesterol is given for the various treatments (Kruskal-Wallis: H = 26.554, P < 0.001, df = 3). E: kMA-induced LDs in peritoneal macrophages as identified by the neutral lipid probe, Bodipy^® 493/503. C, E: Stacked images, 63× oil objective. Scale bar: 20 μm. Significant P values were ranked as *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

Fig. 4.

MA-induced LD accumulation in macrophages promotes BCG proliferation. Following in vivo treatment with MA, peritoneal macrophages were infected ex vivo with BCG-dsRed (MOI 1, broken line) and cultured until timed interval measurements were taken by laser-scanning-confocal microscopy (mean ± SEM). A: Laser-scanning-confocal microscopy images (zoomed) showing macrophages from mock or BCG infections. The presence of a BCG-dsRed bacillus can be clearly distinguished. Scale bar: 2.5 μm. B: Upper panel, LD accumulation in peritoneal macrophages over time (GLM: Wald chi-square = 636.496, P < 0.001, df = 17; n = 5 per time point). Lower panel, proliferation of BCG bacilli over time (GLM: Wald chi-square = 250.303, P < 0.001, df = 17; n = 5 per time point). On the x-axis, “Mix” denotes a natural purified MA extract consisting of ∼53% αMA, ∼38% mMA, and ∼9% kMA. C, D: Laser-scanning-confocal microscopy images depicting a clear difference in the presence of BCG-dsRed bacilli in peritoneal macrophages from mice treated with either kMA or mMA. Stacked images, 63× oil objective. Scale bar: 20 μm. Arrows indicate BCG-dsRed bacilli. Significant P values were ranked as *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant.

Fig. 5.

Mycobacterial growth in LD-accumulating macrophages from LXR-deficient mice. A: Macrophages from WT and LXR-deficient mice were cultured for 5 days and the intracellular LD prevalence analyzed by laser-scanning-confocal microscopy. KO-cells contained ∼10-fold more cellular LDs in comparison to WT-cells, though no significant change in the number of LDs per 100 cells was observed over time (GLM: Wald chi-square = 99.093, P < 0.001, df = 5; n = 4 per time point; sequential Sidak pairwise comparisons P > 0.05). Laser-scanning-confocal microscopy images depicting clear morphological differences in LD prevalence between WT and KO macrophages. Scale bar: 20 μm. B: Cholesteryl esters accounted for 88% of total cholesterol in LXR-deficient cells (left panel) that also contained significantly elevated ratios of intracellular esterified-to-free cholesterol (right panel; Mann-Whitney: U = 144.000, P < 0.001, df = 1; n = 12 mice). C: Macrophages from WT and KO mice were infected ex vivo for 6 h with BCG-dsRed (MOI 1, broken line) and cultured for 5 days. Mycobacterial growth was measured by laser-scanning-confocal microscopy at specified time points, and was significantly increased in LXR-deficient cells over time (GLM: Wald chi-square = 576.689, P < 0.001, df = 17; n = 6 per time point; sequential Sidak pairwise comparisons P < 0.05). Data represent mean ± SEM. Significant P values were ranked as *P < 0.05 and ***P < 0.001; ns, not significant.