The pathogenic M.tb laboratory strain H37Rv, M. bovis BCG and M. avium (MTCC1723) were grown in Middlebrook 7H9 broth supplemented with 10% OADC (Himedia), 0.5% (v/v) glycerol and 0.05% (v/v) Tween-80. The culture was checked for possible contamination using Ziehl-Neelsen (ZN) staining procedure. Bacteria were grown in 7H9 broth with gentle shaking to an OD₆₀₀ of 1.0 and harvested at 3,500 × g for 5 min. The cells were then resuspended in RPMI without antibiotics and dispersed by passage through a 100 U insulin syringe 10 times. The cells were counted following McFarland Nephelometer standards. MOIs were calculated based on these numbers and used for infection for 4 h at 37°C with 5% CO₂. The cells were washed 4 h post-infection thrice with PBS, followed by suspension in RPMI complete media with antibiotics and further incubated for the indicated time at 37°C with 5% CO₂ and processed as per the experiments described. All the experiments involving mycobacterial strains were performed in BSL3 facility following appropriate biosafety protocols approved by Institutional Biosafety committee (IBSC No: SB-N-98).

The infection assays and CFU enumeration assays were performed using either human monocytic cell line, THP-1, or PBMCs isolated from healthy volunteers.

The human monocytic cell line, THP-1 (ATCC) was maintained in RPMI 1640 medium supplemented with 10% (v/v) FBS and differentiated using 25 ng/mL PMA overnight followed by a resting period of 10–12 h. Subsequently, cholesterol treatment of differentiated THP-1 cells was carried out following the method described by Lu et al. with slight modifications. 250X (~7 mM) Cholesterol Lipid Concentrate (CLC) (GIBCO, Grand Island, NY, USA) was diluted in RPMI and then added to THP-1 cells at indicated time points and concentrations. In the case of infection, cells were pre-treated with cholesterol (80 μM) for 12 h followed by infection for 4 h as described. Small molecule M1 (Sigma) treatment was given post-infection till the rest of the assay.

Peripheral Blood Mononuclear Cells (PBMCs) isolation was performed as per the protocol described by Panda et al. (2012). Fifteen milliliter of peripheral blood was taken from clinically healthy human volunteers (N = 7) after taking prior written consents from subjects as per approved guidelines of Institutional Ethics Committee (IEC). These volunteers were profiled for their total serum cholesterol (TC), HDL, and LDL cholesterol from an NABL¹ (Govt. of India) accredited diagnostic lab. The PBMCs were isolated from heparinized blood by density gradient centrifugation method using Histopaque (Sigma). Briefly, the blood from each individual was diltuted twice with RPMI complete media and gently overlayed on Histopaque at 1:1 ratio followed by centrifugation at 100 × g for 30 min. The buffy coat layer containing PBMCs was gently aspirated and transferred into sterile centrifuge tubes. The cells were harvested and washed with RPMI and further cultured in complete RPMI media supplemented with antibiotics. The cell number was quantified and adjusted to 0.2 × 10⁶ cells/well in 24 well plates. The cells were cultured for 7 days at 37°C. The non-adherent cells were eliminated by washing with RPMI media and the adherent cells were used for infections.

The various experimental categories included (i) cholesterol untreated uninfected; (ii) cholesterol treated uninfected; (iii) Cholesterol untreated infected; (iv) cholesterol treated infected; (v) cholesterol untreated uninfected, M1 treatment (vi) cholesterol untreated infected, M1 treatment (vii) cholesterol treated uninfected, M1 treatment and (viii) cholesterol treated infected, M1 treatment.

For the CFU count, 0.2 × 10⁶ THP-1 cells were seeded per well in a 24-well plate and infected with mycobacteria as per the experiments described. At the end of the experiment, infected cells were washed five times with PBS to remove any residual extracellular bacilli. The cells were then lyzed with sterile water at 37°C for 10 min. The lysates were diluted and plated on 7H10 agar plates. The plates were incubated at 37°C with 5% CO₂ and 95% humidity for 2–3 weeks and the colonies were enumerated.

Intracellular mycobacterial survival was also evaluated using Alamar Blue assays as described earlier (Ganji et al., 2016).

MTT assay was performed as per Vemula et al. (2016). Briefly, at the end of the experiment, MTT dye (5 mg/mL) was added at 25 μL per 100 μL media for 2 h. The resulting formazan crystals were solubilized by 100 μL of DMSO. The absorbance was measured at 540 nm using a multimode reader (BioTek).

THP-1 cells were seeded in 6 well plates at a density of 2 × 10⁶ cells/well and cultured overnight in complete medium. Following cholesterol treatment for indicated time, the amount of total cellular cholesterol in macrophage cell lysate was measured using commercial enzyme-based cholesterol assay kit (Cat# STA-384 Cell Biolabs, Inc.) according to manufacturer's instructions.

Oil Red O staining was performed as per the protocol described in Ganji et al. (2016). The cells were washed with PBS, fixed with 10% formalin at room temperature for 30 min. The cells were then washed with sterile water and incubated with 60% isopropanol for 5 min, followed by staining with 60% Oil Red O Stain at 37°C for 5 min in the dark. The excess stain was washed thrice with sterile water. Intracellular lipid bodies were observed microscopically. The amount of lipid bodies were quantified by eluting the intracellular Oil Red O Stain using 100% isopropanol at room temperature for 10 min and measuring the intensity at 500 nm spectrophotometrically with 100% isopropanol as blank.

Mitochondrial membrane potential (MMP) was measured by JC-1 staining kit (Cat. no. CS0390, Sigma) following manufacturer's instructions. 0.2 × 10⁶ THP-1 cells were seeded in 24 well plates and cultured overnight in complete medium. At the end of the experiment, cells from the different experimental categories described earlier were assayed. Briefly, the cells were washed with PBS and stained with 2.5 μM of JC-1 in 1X JC-1 staining buffer. These cells were further incubated for 20 min at 37°C in a humidified atmosphere containing 5% CO_2. Further, the cells were washed with ice-cold 1X JC-1 staining buffer. The cells were then resuspended in 0.5 ml of the same buffer and analysis of the samples was performed using spectrofluorimetry (spectramax m5). JC-1 monomers were detected in the FL1 channel (green) and the aggregates were detected in FL2 channel (red), and the ratio of FL2/FL1 was calculated for determination of mitochondrial membrane potential.

ATP and ADP levels were measured in cells of different experimental categories as described earlier by using ADP/ATP Ratio Assay Kit (Bioluminescent, ab65313), based on the bioluminescent detection, following manufacturer's instructions.

THP-1 cells at a density of 0.2 × 10⁶ cells per well were seeded in 6 well plates and incubated overnight in complete medium. At the end of the experiment, cells from experimental categories, described in the text, were stained for 30 min with fluorogenic dye MitoSOX™ Red for mitochondrial ROS measurement following manufacturer's (Molecular Probes) instructions. Analysis of the samples was performed using spectrofluorimetry (spectramax m5).

2 × 10⁶ THP-1 cells were seeded in six-well plates containing 20-mm-diameter glass coverslips and cultured overnight. At the end of the experiment, cells from various experimental categories were washed with PBS and stained with 50 nM Mito-Tracker Red (M7512) in complete medium at 37°C for 30 min, washed again with PBS and finally fixed with 4% paraformaldehyde. The coverslips were then mounted in Vectashield (Vector Laboratories), and images were visualized in confocal laser scanning microscope (Leica, Germany) with 63 × oil immersion objective lens.

Extraction of RNA from experimental cells was performed using trizol method as per manufacturer's instructions (Invitrogen). Two microgram of total RNA from each experimental category was used for reverse transcription using iScript cDNA Synthesis Kit as per manufacturer's protocol (BioRad) using 2X SYBR green mix (iTaq™ Universal SYBR Green Supermix, BioRad). GAPDH gene was used as endogenous control. The primers used for qRT-PCR are tabulated in Table S1. The real-time PCR data analyses were carried out using the 2^−ΔΔCt method (Schmittgen and Livak, 2008).

2 × 10⁶ THP-1 cells were seeded in six-well plates and followed by cholesterol treatment and infection. Cell culture supernatant was collected and filtered through 0.22 μm syringe filter after infection and indicated time. For measurement of cytokine levels, BD OptEIA enzyme-linked immunosorbent assay (ELISA) kit was used following manufacturer's instructions. The absorbance was measured at 450 and 570 nm in multimode reader (BioTek). Each sample was assayed in triplicate.

The protocol was essentially followed as reported previously (Rajapakse et al., 2001) with minor modifications. Briefly, cells from each experimental category were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C and washed with PBS twice each for 45 min, then post-fixed in 1% aqueous Osmium Tetraoxide (OsO4) for 2 h. Subsequently, the cells were washed with deionized distilled water for four times each for 45 min, dehydrated in series of graded alcohols followed by infiltration and finally embedded in araldite 6005 resin or spur resin. The cells were then incubated at 80°C for 48 h for complete polymerization. Ultra thin (60 nm) sections were made with a glass knife on an ultramicrotome (Leica Ultra cut UCT-GA-D/E-1/00), followed by mounting on copper grids and stained with saturated aqueous Uranyl acetate (UA) and counterstained with Reynolds lead citrate (LC). Sections were viewed under TEM (JEOL from Japan). Images were acquired digitally.

Each experiment was carried out at the least three times (or more). The data are presented as mean ± SD (standard deviation). The statistical significance was assessed by t-test for the data distributed normally as inferred by normality test, for other data Mann-Whitney test was applied. This was performed using SigmaPlot version 11.0. A p-value of ≤ 0.05 was considered statistically significant.

With an aim to investigate the sequence of molecular events underlying the impact of long-term exposure (chronic) to sub-pathological levels of cholesterol on the clearance of intracellular pathogen M.tb by human macrophages, we first developed a suitable model for mycobacterial infection wherein human THP-1 monocytes, differentiated into macrophages using PMA, were infected with pathogenic mycobacteria H37Rv during exposure to different levels of cholesterol for 64 h (Figure 1). A systematic titration of cholesterol concentration, cholesterol exposure time and MOI of M.tb infection was exercised ensuring THP-1 remained viable during the course of experiments. The cells were treated with increasing concentrations of cholesterol (i.e., 40, 80, 160, and 250 μM cholesterol) for 24 h and evaluated for viability by using tetrazolium salt (MTT) metabolite assay. We observed that up to 80 μM of cholesterol, there was no effect on the viability of THP-1 while cholesterol concentration above 160 μM showed significant cell death (see Figure S1A). It was further observed that cells remained viable up to 72 h upon 80 μM cholesterol treatment, while 100 μM cholesterol treatment decreased the cell viability significantly (see Figure S1B). With these experiments, we concluded that 80 μM of cholesterol was the maximum concentration of cholesterol that could be tolerated by THP-1 cells for 48 h post-infection in our experimental conditions. Hence, treatment with 80 μM of cholesterol was subsequently used to represent borderline risk cholesterol (from now on referred to as sub-pathological cholesterol). After precise titrations of H37Rv infections, we observed that cholesterol treated THP-1 cells could withstand H37Rv infection at MOI 20 for 48 h (see Figure S1C), which was used for further studies. In summary, in our model, THP-1 cells were pre-treated with 80 μM cholesterol for 12 h followed by mycobacterial infection (MOI 20) for 4 h and were kept in sustained presence of 80 μM cholesterol for 48 h post-infection. The total exposure time of cholesterol in our experimental set-up was 64 h, after which assays were carried out, (Figure 1A). Since cells remained viable throughout the experimental period, these simulated conditions where cells exposed to “borderline risk cholesterol” were “asymptomatic and apparently healthy”.

Using this model, the efficacy of these macrophages to eliminate intracellular H37Rv was then evaluated. THP-1 cells were infected with H37Rv in the presence and absence of cholesterol and live intracellular H37Rv were scored through CFU. We observed significantly high CFU counts from THP-1 treated with cholesterol, showing 1790 ± 192.0 CFU as against 991.7 ± 94.35 CFU seen from untreated THP-1 (Figure 1B). Thus, there was an increase in live persistent bacteria by 80.5% in cholesterol-treated cells. Since some of the earlier cell culture-based studies have implicated the role of cholesterol in supporting entry of intracellular pathogens like Brucella and M. bovis into macrophages (Gatfield and Pieters, 2000; Naroeni and Porte, 2002; Viswanathan et al., 2015), we monitored mycobacterial CFU in treated and untreated THP-1 cells at 0 h post-infection (see Figure S1D). The results showed that there was an insignificant difference in mycobacterial CFU at 0 h in both treated (1,788 ± 411.5 CFU) and untreated cells (1,705 ± 271CFU) clearly showing that cholesterol concentration and exposure time in our model did not affect mycobacterial entry. This suggested that intracellular events post-mycobacterial entry may have supported mycobacterial survival in the cholesterol exposed THP-1 cells.

Having observed that 64 h of sub-pathological cholesterol exposure impaired clearance of pathogenic mycobacteria by otherwise viable macrophages (Figure 1B), we checked the levels of intracellular cholesterol and lipid bodies in these cells. Recent studies have pointed that, macrophages with accumulated lipid bodies (foamy macrophages) contribute to tissue pathology and exhibit limited protection against persistent bacteria (Russell et al., 2009; Daniel et al., 2011). At the same time, pathogen-induced dysregulation of host lipid synthesis and requisitioning have been shown to impair protective immunity against TB in a mouse model (Martens et al., 2008). While such observations have been made with high cholesterol treatments, we observe that even chronic exposure to sub-pathological cholesterol levels resulted in the accumulation of cholesterol in macrophages. Intracellular lipid and total cellular cholesterol concentrations were evaluated as described in methods. Both microscopic examination (Figure 1C) and spectrophotometric quantification (Figure 1D) showed that cholesterol pre-treatment for 12 h in THP-1 cells resulted in an increase of intracellular lipid bodies by 32.7% and 90.5% when sustained for next 48 h (please refer Figure 3B), giving these cells a sustained “foamy” phenotype during the course of the experiment.

We then investigated the potential of these cells to release TNF-α, a potent pyrogenic cytokine that stimulates the acute phase of the immune response and is also one of the first cytokines to be released in response to a pathogen (Beutler, 1999). TNF-α plays a key role both in initial and chronic phase of M.tb infection (Lin et al., 2007). As reported earlier, we observed an induction of TNF-α secretion in response to H37Rv infection (1.48-fold increase as compared to unifected, untreated). However, it was observed that both treated and untreated THP-1 cells released a similar amount of TNF-α (Treated 417.7 ± 37.17 and untreated 454.5 ± 22.92 pg/mL). But upon H37Rv infection, the treated cells failed to release adequate levels of TNF-α (276.5 ± 19.94 pg/mL) as compared to untreated infected cells (675.6 ± 87.86 pg/mL) (Figure 1E). Taken together, these experiments showed that while cholesterol exposed cells were viable, they failed to respond appropriately to mycobacterial infection.

Having observed a compromised protective response by cholesterol treated infected cells, we hypothesized that such microenvironment could make macrophages susceptible to persistent infections even by opportunistic mycobacteria. To test the same, we infected treated and untreated THP-1 cells with non-tuberculous pathogenic mycobacteria (NTM) M. avium and attenuated mycobacteria, M. bovis BCG. We observed that as compared to untreated (1,550 ± 28.87 CFU), there was an increase of 80.64% of persistent M. avium (2,800 ± 435.9 CFU) in cholesterol treated THP-1 cells (Figure 1F), while for M. bovis BCG it was observed to be increased by 81.2% (untreated 495.0 ± 45.37 CFU and treated 897.0 ± 13 CFU) (Figure 1G). The increase in persistent bacteria inside cholesterol treated THP-1 cells indicated that these cells are relatively immune-compromised and provide a conducive environment to these opportunistic mycobacteria for their growth and survival. Collectively, with these experiments, we conclude chronic exposure of macrophages to sub-pathological cholesterol creates a conducive environment in the form of foamy phenotype, setting a “pre-disease” state in the cells supporting M.tb persistence. We further investigated the molecular events underlying the same using this model.

Some of the earlier assays with exogenous treatment of cholesterol suggested mitochondrial dysfunction resulting in apoptotic cell death (Lu et al., 2011). While most of these studies advocated that it leads to activation of apoptosis, our model clearly has taken conditions where cell death is minimal upon exogenous treatment of cholesterol.

To check if sub-pathological cholesterol can increase the risk of mitochondrial malfunction upon infection, we first studied alterations in mitochondrial structure microscopically. For this, mitochondrial morphology was monitored using fluorescent staining and transmission electron microscopy (TEM). Fluorescent staining of mitochondria from untreated and treated THP-1 cells, with or without infection, using MitotrackerRed showed distinct morphological differences (Figure 2A). In the control cells, the majority of mitochondria were observed to be rod-shaped, elongated, and forming interconnected structures, while infected cells showed predominantly nod-shaped, ovoid and solitary mitochondria, indicating stress morphology (Figure 2A, upper panel, columns i and ii). Such stress morphology could be observed in cholesterol exposed THP-1 cells even before infection, which augmented several fold upon infection where solitary spheres and rods of various sizes were predominantly seen (Figure 2A, lower panel, column i and ii). These observations were also supported by TEM imaging (Figure 2B). Transmission electron micrographs clearly show that mitochondrial morphology in control cells was predominantly elongated rod-shaped and interlinked, while the same in either cholesterol treated or infected cells were predominantly ovoid nod-shaped and solitary (Figure 2B, lower panel column i and upper panel column ii, respectively). Infection of cholesterol treated THP-1 cells resulted in a more noticeable change in mitochondrial morphology where mitochondria appeared swollen and spherical (Figure 2B, lower panel column ii). Additionally, we also looked for associated changes in nuclear morphology using confocal microscopy, where the images were focused on nuclei (Figure S5). We did not observe any changes in nuclear morphology. With these visual evidences, we could conclude that exposure to sub-pathological levels of cholesterol leads to changes in mitochondrial morphology, very similar to that caused by M.tb infection alone. This indicated that cholesterol exposed cells are already under stress, which gets several fold amplified upon infection as evident from swollen mitochondrial morphology.

To assess the mitochondrial structure in relation to changes in the expression of genes involved in mitochondrial dynamics, mRNA levels of mitochondrial fusion and fission genes mfn-1, mfn-2, drp-1, and mff in these macrophages were measured through qRT-PCR (Figures 2C–F). The levels of mfn-1 and mfn-2 were significantly decreased by ~2-fold in cholesterol treated infected cells, whereas mfn-2, but not mfn-1, was downregulated by ~2-fold during infection in untreated cells (Figures 2C,D). The levels of drp-1 did not change significantly in any condition (Figure 2E). However, levels of mff were observed to be significantly reduced by ~2-fold in cholesterol treated infected cells as compared to cholesterol treatment alone (Figure 2F). Overall, the changes in the expression levels of mitochondrial fission and fusion genes support the variations in the mitochondrial structural dynamics as observed microscopically.

With the clear suggestion of changes in mitochondrial structure, we further studied the changes in mitochondrial function. MMP in untreated and cholesterol treated THP-1 cells, with and without infections were measured using fluorometric assays (Figure 2G). It was observed that while either infection or cholesterol alone did not significantly affect MMP, infection of cholesterol-treated cells caused a consistent (20.07%) reduction in MMP. Since the change in MMP should reflect in ATP production, in addition to this, mitochondrial function was assayed by measuring ATP/ADP ratio. ATP/ADP ratio in untreated infected cells was 15.65%, while that in treated infected cells was 21.08%, suggesting cumulative damage to mitochondria upon cholesterol treatment and infection together (Figure 2H). With these experiments, we registered a consistent change in mitochondrial functional parameters. Such subtle and consistent changes in mitochondria have been reported to have huge physiological consequences (Buckman and Reynolds, 2001) and should not be considered inconsequential. MitoROS production, upon infection of both untreated and treated cells, however, remained unaltered (please see Figure S2A). Further, as control experiments, to check if cholesterol concentrations <80 μM could cause alterations in mitochondrial structure and function, 20 μM and 40 μM cholesterol treatments were used to assay for their impact on both mitochondrial structure and function (Figures S2B,C) and clearance of mycobacteria (Figure S2D). We do not observe any significant change in mitochondrial morphology or function, neither any differences in mycobacterial clearance upon treatment with 20 and 40 μM cholesterol as compared to the untreated.

These results implicated that, prolonged exposure to sub-pathological cholesterol levels created an amicable environment for M.tb in otherwise apparently healthy macrophages, by compromising mitochondrial structure and function, which significantly decreases the anti-mycobacterial activity of infected cells, thereby promoting mycobacterial persistence.

We next attempted to restore the mitochondrial activity using a commercially available, pharmacological small molecule, M1, which has been reported to restore mitochondrial health in various systems upon different stresses (Wang et al., 2012). We standardized the effective concentration of M1 in our experimental conditions, keeping the cells viable (please see Figure S3A). THP-1 cells, untreated and cholesterol treated, with and without H37Rv infections, were treated with 30 μM of M1 post-infection for 48 h. All the mitochondrial parameters, viz; morphology, expression of genes responsible for mitochondrial dynamics, MMP and ATP/ADP ratio were reanalyzed in the presence of M1. We observed that mitochondrial morphology was substantially restored upon M1 treatment of both untreated and treated infected cells (Figure 2A lower panel column iii and Figure 2B lower panel column iii, respectively). M1 also restored mfn1and mfn2 levels in cholesterol treated infected cells, but not in untreated infected cells (Figures 2C,D respectively). We, for the first time, observed that M1 restored levels of the fission gene mff in cholesterol treated infected cells (Figure 2F). The restoration of the expression levels of these genes, supported by re-establishment of morphological features, suggested that M1 probably reinstated the balance between fission and fusion events, and this equilibrium maintained the steady state mitochondrial dynamics, more efficiently in cholesterol-treated cells. We also observed that consequent restoration of MMP (Figure 2G) and ATP/ADP ratio (Figure 2H) by M1 during infection of cholesterol exposed cells.

We next postulated if restoring mitochondrial structure and function in cholesterol exposed THP-1 by M1 will potentiate these cells for mycobacterial clearance. Having noted that M1 treatment rescued mitochondrial function in cholesterol treated THP-1, we checked the lipid bodies and intracellular cholesterol levels in these cells. In compliance with other studies, we observed that H37Rv infection alone resulted in the increase in lipid bodies by 53.77% (Figure 3A upper panel column ii and Figure 3B). This impact was more pronounced in cholesterol treated H37Rv infected cells which showed an increase in intracellular lipids by 76.2% as compared to infection in untreated cells (Figure 3A lower panel column ii and Figure 3B).

Besides restoring mitochondrial structure and function, when cholesterol exposed infected cells were treated by M1, it resulted in a decrease in the intracellular lipid bodies by 58% (Figure 3A lower panel column ii and iii and Figure 3B), as well as total intracellular cholesterol levels by 15% (Figure 3C). M1 treatment could not reduce either lipid bodies or cholesterol significantly in untreated infected cells (Figure 3A upper panel column ii and iii, Figures 3B,C). Finally, the impact of M1 on mycobacterial clearance was scored. Pharmacological intervention by M1 reduced the intracellular H37Rv burden of cholesterol treated infected cells by 46.16% and of untreated infected cells by 27.87%. This indicated that the efficacy of M1 in clearance of mycobacterial infection was higher in cholesterol treated macrophages (Figure 3D). It is to be noted that M1 treatment of cholesterol untreated and treated cells in the absence of infection had insignificant impact on intracellular lipid and MMP (Figures S3B–D). It is also to be noted that M1 had no impact on the in-vitro growth of H37Rv cultures (please see Figure S4).

From the above experiments, we could infer that sub-pathological long term exposure to cholesterol impairs macrophages from adequately responding to mycobacterial infection and prevents its clearance. The small molecule M1 could rescue cholesterol exposed infected macrophages and helped in improving mycobacterial clearance. To validate these cell line based observations in primary cells, we used PBMC derived macrophages from human volunteers with either normal (referred to as NC) or borderline risk (referred to as BLC) cholesterol profiles (Table S2). Mycobacterial clearance was assayed by CFU enumeration from PBMC derived macrophages subjected to different conditions as in the chronic cell culture model (Figure 4). Similar to observations from the cell culture model, we found that intracellular clearance of mycobacteria by cholesterol treated PBMC derived macrophages from NC (139.8 ± 22.84) was significantly reduced (46.3%) as compared to untreated cells (64.76 ± 9.29) (Figure 4A). We then compared the clearance of mycobacteria by PBMC derived macrophages from NC and BLC. As expected, PBMC derived macrophages from BLC (108.7 ± 20.21) showed reduced clearance (61.3%) of mycobacteria as compared to those from NC (64.76 ± 9.29) (Figure 4B). Further, there was no difference in clearance of mycobacteria from PBMC derived macrophages of BLC either cholesterol treated (119.9 ± 17.84) or untreated (108.7 ± 20.21) (Figure 4C). Also, there was no significant difference in the clearance of mycobacteria from cholesterol treated PBMC derived macrophages from either NC (139.8 ± 22.84) or BLC (119.9 ± 17.84) (Figure 4D). These experiments showed that exogenously treated PBMC derived macrophages from NC behaved similar to those of untreated BLC confirming that PBMC derived macrophages from BLC had reduced efficiency of mycobacterial clearance.

M1 treatment did not improve the clearance of mycobacteria significantly from PBMC derived macrophages from either NC or BLC. However, when we compared improvement of clearance of mycobacteria between untreated NC and BLC, we found that while M1 could improve clearance in NC by 12.9%, the same in BLC was 25.6% (Figure 4E). On the other hand, impact of M1 on mycobacterial clearance was more efficient in cholesterol treated PBMC derived macrophages from both NC and BLC (Figure 4F) reiterating that M1 is more effective in improving clearance from cholesterol exposed macrophages. These results supported that chronic exposure to borderline risk cholesterol created a favorable environment for M.tb persistence.

In summary, we concluded that prolonged exposure to sub-pathological levels of cholesterol leads to increase in intracellular lipid bodies, alteration in mitochondrial structure and function that supported persistence of H37Rv and opportunistic pathogens. Pharmacological intervention by M1 reversed changes in cholesterol treated infected macrophages, thereby increasing the clearance of persistent mycobacteria (Figure 5).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.