Fig 1.
TAG, CE, and free cholesterol levels in pulmonary tuberculous lesions of marmosets and rabbits.
Haematoxylin and eosin staining of lung tissue sections from M. tuberculosis-infected marmosets (A-B) and rabbits (C-D). In panels A and C, the boxes indicate the regions of granulomas that are shown at higher magnification in panels B and D. In necrotizing granulomas, the center of the lesion is occupied by caseum (C) (“cheese-like” material that becomes increasingly acellular as necrosis progresses), which is surrounded by a cellular region (CR) containing macrophages and lymphocytes. The inner layers of the cellular region are enriched in macrophages, including foam cells, epithelioid, and multinucleated giant cells; lymphocytes are predominantly found in the outermost cellular area [6, 7, 9]. In panels B and D, foam-cell rich regions are indicated by arrows. The presence of foam cells is demonstrated by the vacuole-rich areas resulting from loss of lipids during tissue preparation for haematoxylin and eosin staining. (E) Haematoxylin and eosin staining of the M. tuberculosis-infected rabbit lung tissue section used for confocal microscopy. The haematoxylin and eosin staining differs in intensity from the images in panels A through D because it was performed after fluorescence staining, to confirm the structure of the lesion. (F-G) Confocal microscopy images of tuberculous rabbit lung tissue sections stained with Nile red (red) and DAPI (blue). In Panel F, the box indicates the region of the granuloma that is shown at higher magnification in panel G. Lipid droplets stained with Nile Red, a dye widely used to visualize lipid droplet-laden cells both in vitro and in vivo [7, 71, 85, 105], are visible in the cellular region surrounding the caseum. A three-dimensional image of foam cells is provided in the S1 Video. (H-I) Measurements of TAG, CE, and free cholesterol levels. Areas of caseum and macrophage-rich cellular region of lung lesions, and regions of uninvolved lung were sampled by laser capture microdissection from 4–10 rabbits and 6–9 marmosets (1–2 samples per animal). Lipids were extracted and TAG, CE species, and free cholesterol quantified by LC-MS. All measurements are expressed as micrograms of lipid per gram of tissue (μg/g). The box plots show lower quartile, median, and upper quartile of the distribution. The whiskers represent the minimum and maximum values. Statistical significance (p < 0.025) was calculated by the Mann-Whitney and Wilcoxon signed-rank tests and the multiple-comparison Bonferroni correction. Statistical differences between lesional and uninvolved lung areas (+p < 0.025, ++p < 0.01 by the Mann-Whitney test), and between TAG and CE (*p <0.025, **p < 0.01, ***p < 0.001 by the Wilcoxon signed-rank test) are indicated. TAG: triglycerides, CE: cholesteryl esters, CHO: free cholesterol.
Fig 2.
TAG species profiles in pulmonary tuberculous lesions of rabbits, marmosets, and humans.
TAG species in microdissected areas of caseum and cellular regions of lesions, and uninvolved lung areas were quantified by LC-MS. In all graphs, each line represents one sample: uninvolved lung areas (light blue), cellular region (green), and caseum (orange). Marmosets: five lesional and three uninvolved lung areas (from four animals). Rabbits: six lesional and five uninvolved lung areas (from six animals). Humans: two lesional areas (one per patient) from two active tuberculosis cases, and one uninvolved lung area (from one of the two patients). One of the dominant species in this figure (TAG 52:2) was revealed by the electrospray ionization mass spectrometry analysis of human tuberculous caseum in a previous study [85].
Fig 3.
TAG and lipid droplet levels, and expression of TAG metabolism genes in macrophages infected with M. tuberculosis in vitro.
Human monocyte-derived macrophages (MDM) were infected with M. tuberculosis for 24 h or left uninfected. Infected cells were treated with either DMSO (vehicle control) or A922500 (DGAT inhibitor). (A) TAG measurement by mass spectrometry. Lipids were extracted from uninfected and infected cells, and TAG species quantified by LC-MS. (B) Lipid droplet content determination by imaging flow cytometry. Uninfected and infected macrophages were stained with Bodipy 493/503 and imaged by ImageStreamXMark II imaging flow cytometer at 60× magnification. Images were analyzed by IDEAS software and lipid droplet content was expressed as median Bodipy fluorescence intensity per cell (MFI) (the baseline measurements in uninfected cells reflect the scanty lipid droplet induction occurring during macrophage differentiation in vitro). (C) Effect of A922500 on lipid droplet content. Lipid droplet content of infected macrophages was determined as described. Results were expressed as ratio between inhibitor- and vehicle-treated cells. In A, B, and C, average and standard deviation of three donors are shown. (D) Gene expression analysis. RNA was isolated from uninfected and infected cells, and mRNAs enumerated by qPCR using gene-specific primers and molecular beacons (S5 Table). Gene expression was calculated using the 2 -ΔΔCt method and normalized to the housekeeping ACTB gene. Graphs show the medians of six donors, with each dot representing one donor. Statistical significance (*p < 0.05, **p < 0.01) was assessed by paired (panels A, B, and D) and one-sample (panel C) student t-tests. The comparisons in the paired tests are as indicated; the comparison in the one-sample student t-test was between treated and untreated cells. SREBF1 encodes SREBP-1c, a transcription factor that functions as master regulator of TAG biosynthesis [106]; LPIN1 encodes LIPIN1, a TAG biosynthetic enzyme [107] that also functions as transcriptional coactivator of SREBP-1c [108].
Fig 4.
Role of TNFR in lipid droplet formation in M. tuberculosis-infected macrophages.
MDM were infected with mCherry-expressing M. tuberculosis and treated either with R-7050 (TNFR chemical inhibitor) or DMSO (vehicle control). When TNFR neutralizing antibodies were used, MDM were either pre-treated with the antibodies or left untreated, and then infected. Uninfected samples were included as controls. Lipid droplet content was measured as described in Fig 3. (A) Representative images of uninfected and infected macrophages obtained by imaging flow cytometry (60× magnification) and lipid droplet content determination; lipid droplets are fluorescent green signals and bacteria are orange signals. Each bar of the graph represents the average and standard deviation of median Bodipy fluorescence intensity per cell obtained from three donors. Effect of R-7050 (B) or TNFR neutralizing antibodies (C) on lipid droplet content of infected macrophages. Results are expressed as the ratio between lipid droplet content of treated and untreated cells. Graphs show the median of six (B) and ten (C) donors (each dot represents one donor). Statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) was assessed by paired (panel A) and one-sample (panels B and C) student t-tests. The comparisons in the paired tests are as indicated; the comparison in the one-sample student t-test was between treated and untreated cells. UN: uninfected, INF: infected, TNFR1 AB: TNFR1 neutralizing antibodies, TNFR2 AB: TNFR2 neutralizing antibodies.
Fig 5.
Role of TNFR-mediated pathways in lipid droplet formation in M. tuberculosis-infected macrophages.
(A) Effect of pathway inhibitors on lipid droplet content of infected macrophages. The scheme at the top of the figure shows the intracellular signaling pathways activated by TNFR1 and TNFR2. Infected MDM were treated either with chemical inhibitors or vehicle; lipid droplet content was measured and results expressed as described in Fig 4. Statistical significance (*p < 0.05) of differences between treated and untreated cells was assessed by one-sample student t-test. Rapamycin: mTORC1 inhibitor, Z-IETD-FMK: caspase 8 inhibitor, Z-DEVD-FMK: caspase 3 inhibitor, QNZ: NF-κB inhibitor, SB203580: p38 inhibitor, JNK-IN-8: JNK inhibitor, GDC-0994: ERK inhibitor. (B) Transcriptomic analysis of human lung tuberculous granulomas. The differential expression between sample classes was determined for 182 Pathway Interaction Database pathways by coincident extreme ranks in numerical observations. 77 pathways were identified at a cutoff false discovery rate of 0.05; the p values for these were plotted onto the x-axis. To represent effect size, pathway gene sets with fewer genes were given greater bar height than were larger sets that yielded similar p values. Four of the pathways discussed in the text are highlighted in orange.
Fig 6.
Effects of TNFR neutralizing antibodies on mTORC1 and caspase activity.
MDM were treated with antibodies against TNFR1 or TNFR2 prior to infection. After 24 h of infection, whole cell lysates and RNA were obtained. (A) Analysis of phosphorylation state of mTORC1 and abundance of pro-caspase 8 by western blot. p-mTOR, band reacting with an antibody specific for mTOR phosphorylated at Ser-2448; mTOR, band reacting with an antibody recognizing total mTOR protein. Western blot bands were quantified by using ImageJ software. Numbers below each band indicate the intensity ratio of the test band relative to β-actin (loading control). Full-length blots are presented in S8 Fig. (B) Quantification of the western blot data in panel A. The graph shows the effect of TNFR antibody treatment (treated vs untreated) on the ratios of p-mTOR/mTOR and procaspase 8 abundance. The average and standard deviation for three donors is shown. (C) Gene expression analysis. Methods are described in the legend to Fig 3. The graph shows the median of five donors, with each dot representing one donor. Statistical significance (*p < 0.05, **p < 0.01) was assessed by one-sample (panel B) and paired (panel C) student t-tests. The comparisons in the paired tests are as indicated; the comparison in the one-sample student t-test was between treated and untreated cells. [It is noted that the decrease of 50% in the ratio of p-mTOR/mTOR observed with the anti-TNFR1 antibody treatment did not reach statistical significance (p = 0.09)]. Mtb: M. tuberculosis, TNFR1 AB: TNFR1 neutralizing antibodies, TNFR2 AB: TNFR2 neutralizing antibodies.
Fig 7.
Key nodes of the signaling network involved in TAG accumulation in tuberculous macrophages.
Infection of macrophages with M. tuberculosis induces production of TNFα, which has both autocrine and paracrine effects. TNFα binding to its surface receptors (TNFR1 and TNFR2) triggers the activation of intracellular caspase and mTORC1 pathways. Both mTORC1 and caspases directly interact with autophagy effectors to inhibit autophagic flux [42, 52, 109]. So does M. tuberculosis infection [110]. In addition, mTORC1-dependent block of autophagy can also activate the caspase cascade through accumulation of p62, which binds and activates caspase 8 [111]. When autophagy is blocked, lipid droplets are not degraded and accumulate in the cytoplasm. Our observation that treatment with TNFR neutralizing antibodies relieves the inhibition of autophagy by M. tuberculosis infection (S6 Fig) further supports inhibition of autophagy as a driver of lipid droplet accumulation in tuberculous macrophages. In addition, caspase activation also leads to mitochondrial dysfunction [43], which results in accumulation of lipid droplets due to reduced fatty acid utilization [39]. Effects on TAG biosynthesis may also be involved, since mTORC1 induces expression of SREBF1 [112–114] and caspases activate SREBPs [40, 41]. Arrowhead: positive regulation. LD: lipid droplet.