Troglitazone Reduction of Intracellular Mycobacterium tuberculosis Survival Via Macrophage Autophagy Through LKB1-AMPKα Signaling

Abstract

Tuberculosis causes by Mycobacterium tuberculosis (Mtb) and results in primary morbidity and mortality worldwide. In 2022, approximately 10 million individuals contracted tuberculosis, and 1.3 million tuberculosis deaths occurred [1]. Although 85% of patients with tuberculosis can be successfully cured after a 6-month drug regimen, there remains a proportion of recrudescent cases [1]. Moreover, drug-resistant tuberculosis, particularly isoniazid (INH)– and rifampicin-resistant tuberculosis, poses a significant challenge to the current treatment regimen. Therefore, the development of more effective antituberculosis drugs and therapeutic regimens is necessary.

Studies in recent years have focused on a novel treatment known as host-directed therapy (HDT), which enhances host defense mechanisms or modulates the host innate and adaptive immune response to improve treatment outcomes by targeting host factors [2]. HDTs include regularly administered drugs for noninfectious diseases with good safety profiles, immunomodulatory compounds, biologics, nutritional products, and cell therapies [2]. In vitro and in vivo studies have shown that HDTs improve outcomes for various infectious diseases, including Mtb infection, by ameliorating host immunity and pathological damage, as Mtb has been shown to attenuate the host innate and adaptive response and exploit immunopathology to its benefit [3, 4]. HDTs are considered potential adjuncts to standard antimicrobial treatment for overcoming antimicrobial resistance. These reports suggest that HDT is an attractive strategy for antituberculosis therapy, particularly for drug-resistant tuberculosis.

HDTs exert their regulatory function on host immunity through multiple mechanisms, such as autophagy induction. Autophagy is an important intrinsic biological process, which recycles and degrades impaired cellular components and organelles to sustain cellular homeostasis during stress [5]. Two major host autophagy pathways can defend against intracellular pathogens: xenophagy and microtubule-associated protein 1 light chain 3 (LC3)-associated phagocytosis (LAP) [6]. Targeting pathogens to LAP is inhibitory in some cases but contributes to bacterial survival in others [7]. Previous studies indicate that LAP does not facilitate the inhibition of Mtb in mouse macrophages and that targeting Mtb to xenophagy is successful for its restriction [6]. However, Mtb subverts host autophagy through multiple mechanisms [4], and overcoming bacterial inhibition may be a successful approach for drug development.

Autophagy-associated HDTs have been shown to contribute to host elimination of Mtb infection [8, 9]. The induction of autophagy may enhance phagolysosome fusion, improve antigen presentation, and reduce inflammation [10]. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), has been extensively studied as an autophagy inducer [11] and facilitates the host elimination of Mtb [12]; however, rapamycin may lead to interstitial pneumonitis and may be metabolized by rifampicin-induced CYP3A4, which may hinder its clinical role as an antituberculosis HDT [10]. Although several autophagy inducers as HDT candidates may have been identified, only a few have entered clinical trials for tuberculosis [10]. Thus, more potential HDT candidates are required to identify more effective and safer antituberculosis drugs.

Troglitazone (Trog), a thiazolidinedione and a ligand of peroxisome proliferator–activated receptor γ (PPAR-γ) [13, 14], is a synthetic antidiabetic drug for type 2 diabetes, enhances the sensitivity of tissues to insulin, and decreases blood glucose levels through a mechanism closely associated with PPAR-γ activation [15]. Studies have shown that Trog induces autophagy in numerous tumor cells through a mechanism dependent on PPAR-γ activation [15, 16]. Moreover, other diabetic drugs, such as metformin [17], inhibit Mtb growth by inducing autophagy; however, whether Trog induces autophagy in macrophages and subsequently contributes to Mtb clearance remains largely unknown. In the present study, we examined the potential of Trog as an HDT candidate and useful adjunct combined with first-line antituberculosis drugs.

METHODS

All experimental procedures were permitted by the Animal Care and Use Committee of Shenzhen Third People's Hospital, and mice were maintained according to committee guidelines. Bone marrow–derived macrophages (BMDMs) was obtained from 8-week-old female mice. For detailed information on methods, see the Supplementary materials.

RESULTS

Trog Reduction of Intracellular Mtb Survival in Macrophages

Colony-forming unit (CFU) counts was done to determine whether the antidiabetic drugs including Trog, rosiglitazone (Rosi), and pioglitazone (Piog) [18] could suppress intracellular Mtb survival in macrophages. As shown in Figure 1A, Mtb growth in THP-1 macrophages was significantly decreased after Trog treatment (50 µmol/L) for 24–72 hours; however, no difference was observed following Rosi and Piog treatment (Figure 1A). The antibacterial activity of Trog was not the result of cytotoxicity, as a cell viability assay indicated median inhibitory concentrations of 96.30 and 125.20 µmol/L, respectively, in Mtb-uninfected and -infected THP-1 macrophages, higher than the concentration used in the present study (Supplementary Figure 1A and 1B). Moreover, cell viability was essentially unchanged in Mtb-infected THP-1 macrophages preincubated with 100 µmol/L Rosi and 20 µmol/L Piog (Supplementary Figure  1C and 1D). The mycobactericidal activity of THP-1 macrophages was further enhanced by Trog treatment in a dose-dependent manner (Figure 1B); however, this trend was not observed following Rosi or Piog treatment (Supplementary Figure 2A and 2B).

Figure 1.

Troglitazone (Trog) reduces intracellular Mycobacterium tuberculosis (Mtb) survival in macrophages. A, THP-1 macrophages were preincubated with rosiglitazone (Rosi; 50 µmol/L), pioglitazone (Piog; 10 µmol/L), and Trog (50 µmol/L) and infected with Mtb H37Rv (multiplicity of infection [MOI], 10:1) for 4 hours. After washing 3 times with prewarmed phosphate-buffered saline, the cells were incubated with the drugs for another 24, 48, and 72 hours. Colony-forming units (CFUs) were enumerated at each time point. Abbreviation: DMSO, dimethyl sulfoxide. B, THP-1 macrophages were pretreated with Trog (25, 50, and 100 µmol/L) for 1 hour and infected with Mtb H37Rv (MOI, 10:1) for 4 hours. The infected cells were washed and treated as shown in A. C, Bone marrow–derived macrophages were pretreated with Trog (50 µmol/L) for 1 hour and then infected with Mtb H37Rv (MOI, 10:1) for 4 hours. The infected cells were washed and treated as shown in A. D, Mtb H37Rv, grown in 7H9-OADC liquid medium, were incubated with various doses of Trog (0–150 µmol/L) for the indicated durations. The growth rate of Mtb H37Rv was determined by measuring the optical density at 600 nm (OD₆₀₀) at each time point. Data are presented as means with SDs from 3 independent experiments. Statistical analysis was performed using 2-way analysis of variance, followed by Bonferroni test (A–C). *P < .05; **P < .01; ***P < .001; NS, not significant.

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Trog-treated BMDMs also increased mycobactericidal activity (Figure 1C). The results indicated that Trog reduced Mtb survival within macrophages. To determine whether the antibacterial effect of Trog resulted from the direct suppression of Mtb, a bacteriostatic assay was performed in vitro by adding Trog at the indicated concentrations (Figure 1D). Trog did not inhibit the growth of Mtb at a concentration of 50 µmol/L; however, a mild inhibitory effect on Mtb was observed at a concentration of 150 µmol/L, which also caused significant cytotoxicity (Figure 1D). Taken together, the results suggest that Trog may target macrophages and regulate their functions to exert an antibacterial effect.

Effect of Trog in Decreasing Mtb Survival in Macrophages by Improving Autophagy

Autophagy plays an important role in Mtb elimination by host macrophages [8]; however, Mtb could subvert this process [4]. Several studies have shown that Trog promotes autophagy in tumor cells [15, 16, 19, 20]. We hypothesize that Trog-mediated autophagy activation of macrophages decreases Mtb intracellular survival. As expected, Trog stimulation significantly improved autophagy within Mtb-infected and uninfected THP-1 macrophages, as increased lipid-bound LC3B-II transformed from LC3B-I and p62 was observed compared with the untreated control (Figure 2A and Supplementary Figure 3A and 3B). Trog treatment did not induce apoptosis, as similar levels of cleaved caspase 3– and annexin V–positive cells (Supplementary Figure 3A–3D) were observed. No difference in pyroptosis markers, including cleaved caspase 1 and cleaved gasdermin-D (GSDMD) (N-terminal GSDMD, N-GSDMD), (Supplementary Figure 3A and 3B) was observed following Trog treatment.

Figure 2.

Troglitazone (Trog) decreases intracellular Mycobacterium tuberculosis (Mtb) survival in macrophages by improving autophagy. A, THP-1 macrophages were pretreated with Trog (50 µmol/L) for 1 hour and then infected with Mtb H37Rv (multiplicity of infection [MOI], 10:1) for the indicated times. Cell lysates were subjected to Western blot analysis using anti-microtubule-associated protein 1 light chain 3B (LC3B) and anti–β-actin antibodies. The relative band intensity (target protein/β-actin) is shown as a bar graph (with shades of black and gray corresponding to different treatment conditions, from left to right). Abbreviation: DMSO, dimethyl sulfoxide. B, monomeric red fluorescent protein (mRFP)–green fluorescent protein (GFP)–microtubule-associated protein 1 light chain 3B (LC3B) reporter THP-1 macrophages were treated and infected as shown in A. Cells were fixed and visualized by confocal microscopy. One representative image is shown (left; bars, 5 µm). The autophagosome (yellow) and autolysosome puncta (red) were calculated. Abbreviation: DAPI, 4',6-diamidino-2-phenylindole. C, THP-1 macrophages were treated and infected as shown in A, using GFP-overexpressing Mtb. Cells were fixed, incubated with anti-p62 and anti-lysosome-associated membrane glycoprotein 1 (LAMP1) primary and corresponding secondary antibodies, and then visualized by confocal microscopy. Left, Representative image (bars, 5 µm). Right, Percentage of colocalization of Mtb with p62 and LAMP1 in THP-1 macrophages. A total of 100 bacterial cells were counted. D, THP-1 macrophages were transfected with either autophagy protein 5 (ATG5)-specific small interfering RNA (siRNA; siATG5) or scrambled siRNA (siNC) for 72 hours. Macrophages were treated with Trog (50 µmol/L) for 24 hours. Left, Cell lysates were subjected to Western blot analysis using anti-ATG5, anti-LC3B, and anti–β-actin antibodies. THP-1 macrophages transfected with siATG5 and siNC for 72 hours were pretreated with Trog (50 µmol/L) for 1 hour and infected with Mtb H37Rv (MOI, 10:1) for 4 hours. Cells were washed with prewarmed phosphate-buffered saline (PBS) and reincubated with Trog for an additional 72 hours. Middle, THP-1 macrophages preincubated with Trog (50 µmol/L) were infected with Mtb H37Rv (MOI, 10:1) for 4 hours in the presence or absence of bafilomycin A₁ (BafA1; 100 nmol/L). After washing 3 times with prewarmed PBS, the cells were incubated with Trog for an additional 72 hours, in the presence or absence of BafA1. Right, Colony-forming units (CFUs) were enumerated. Data are presented means with SDs from 3 independent experiments. Statistical analysis was performed using 2-way analysis of variance (ANOVA) followed by Bonferroni test (A, D) or 1-way ANOVA followed by Tukey test (B) or unpaired t test (C) . *P < .05; **P < .01; ***P < .001; NS, not significant.

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Although the level of secreted interleukin 1β decreased following Trog treatment (Supplementary Figure 3E), similar to findings in a previous study [21], that may be the result of Trog’s anti-inflammatory effect. Moreover, Trog treatment did not affect ferroptosis as no differences were observed in GPX4 or lipid peroxide (malondialdehyde) (Supplementary Figure 3A, 3B, and 3F) levels. These results suggest that Trog-induced autophagy, but not cell death pathways, facilitates intracellular Mtb clearance in macrophages.

Ammonium chloride a lysosomal inhibitor that suppresses autophagic flux activation, as evidenced by reduced degradation of LC3B-II and p62. Trog-induced LC3B-II and p62 levels were further enhanced in the presence of ammonium chloride which suggests that autophagic flux activation occurred (Supplementary Figure 3G and 3H). Transmission electron microscopic results consistently showed increased autophagic vacuoles in Trog-pretreated and Mtb-infected macrophages (Supplementary Figure 3I and 3J). Rapamycin-treated macrophages were used as a positive control.

To further confirm Trog-induced autophagy activation, monomeric red fluorescent protein–green fluorescent protein–LC3 reporter THP-1 cells were used to visualize LC3 accumulation in the autophagosomes and autolysosomes. Confocal microscopy revealed elevated autophagosome and autolysosome punctate formation in Mtb-infected and uninfected THP-1 macrophages preincubated with Trog compared with their respective controls (Figure 2B and Supplementary Figure 3K–3M). In addition, Trog induced increased xenophagy, as evidenced by enhanced colocalization between bacteria, p62, and lysosome-associated membrane glycoprotein 1 (LAMP1) (Figure 2C). Trog had no effect on LAP, with no significant difference observed in colocalization between bacteria and p40^phox (Supplementary Figure 4A and 4B). The results indicate that the Trog-induced increase in autophagy flux inhibits intracellular Mtb survival.

Autophagy protein 5 (ATG5) is important to autophagy initiation and processing as well as host defense against Mtb infection [22–24]. ATG5 knock-down macrophages were constructed to further demonstrate a role for Trog in autophagy activation and intracellular Mtb clearance. ATG5 knock-down decreased ATG5 expression (30%–45% reduction) (Figure 2D and Supplementary Figure 5A). Moreover, Trog treatment significantly enhanced autophagy in scrambled small interfering RNA (siRNA) (siNC)–transfected macrophages compared with that of its controls; however, the difference almost vanished in Trog-treated ATG5 knock-down macrophages with decreased LC3B-II (Figure 2D and Supplementary Figure 5A).

Similar results were obtained with confocal microscopy, as autophagosome and autolysosome puncta were decreased in Trog-treated, Mtb-infected, and uninfected ATG5 knock-down macrophages compared with their respective controls (Supplementary Figure 5B–5G). Similarly, Trog treatment significantly decreased intracellular Mtb survival in siNC-transfected macrophages, whereas the effect was significantly decreased in ATG5 knock-down macrophages (Figure 2D). We further enumerated CFUs in the presence of bafilomcin A₁, which prevents autophagy by suppressing lysosomal degradation, and found that the Trog-mediated mycobactericidal effect nearly disappeared (Figure 2D). Overall, our data suggest that Trog decreases intracellular Mtb survival in macrophages by promoting autophagy.

Trog-Mediated Autophagy and Mycobactericidal Activity involved in STRADA-LKB1-AMPKα Signaling

A previous study reported that Trog treatment promoted AMPKα phosphorylation (p-AMPKα) and autophagy in cancer cells [19]. Therefore, to determine the underlying mechanism of Trog-mediated autophagy, p-AMPKα levels were measured following Trog treatment. As shown in Supplementary Figure 6A and 6B, p-AMPKα was significantly increased in Trog-treated THP-1 macrophages. A similar trend was observed in LC3B-II levels, but no significant difference in AMPKα levels was observed (Supplementary Figure 6A and 6B). Moreover, Trog before incubation significantly improved p-AMPKα and LC3B-II levels in Mtb-infected macrophages, whereas AMPKα protein levels were not significantly changed (Figure 3A and 3B and Supplementary Figure 6C and 6D).

Figure 3.

Troglitazone (Trog)–mediated autophagy and mycobactericidal activity relate to 5'-AMP-activated protein kinase α (AMPKα) activation. A, As shown in Figure 2A, THP-1 macrophages treated with Trog and infected with Mycobacterium tuberculosis (Mtb) H37Rv were subjected to Western blot analysis using the indicated antibodies. Abbreviation: DMSO, dimethyl sulfoxide; LC3B, microtubule-associated protein 1 light chain 3B. B, Relative band intensity (target protein/β-actin) in A, shown as a bar graph (with shades of black and gray corresponding to different treatment conditions, from left to right). C, THP-1 macrophages were treated with Trog (50 µmol/L) for 24 hours. Cell lysates were subjected to Western blot analysis using the indicated antibodies. Abbreviation: Raptor, regulatory associated protein of mammalian target of rapamycin. D, Relative band intensity (target protein/β-actin) in C, shown as a bar graph (with shades of black and gray corresponding to different treatment conditions, from left to right). Data are presented as means with SDs from 3 independent experiments. Statistical analysis was performed using 2-way analysis of variance, followed by Bonferroni test (B, D) . *P < .05; **P < .01; ***P < .001.

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Similar results were observed in Mtb-uninfected and Mtb-infected BMDMs after Trog treatment (Supplementary Figure 6E–6H). AMPK can directly phosphorylate the mTORC1 subunit Raptor (regulatory associated protein of mTOR) and inhibit the mTORC1 pathway, which in turn suppresses the inhibitory effect of mTORC1 on serine/threonine-protein kinase ULK1 (ULK1), an important forward component of the autophagy pathway [25]. Moreover, AMPK can directly phosphorylate and activate ULK1 [25], which triggers autophagy. As expected, Trog treatment improved Raptor and ULK1 phosphorylation levels (Figure 3C and 3D).

A similar trend was observed in p-AMPKα and LC3B-II levels, whereas Raptor, ULK1, and AMPKα levels were relatively unchanged (Figure 3C and 3D). The results suggest that the mTORC1 pathway is inhibited, while AMPK activity and autophagy are induced. Although we observed increased p-AMPKα after Piog, but not Rosi, treatment, Piog-induced p-AMPKα levels were far lower compared with those following Trog treatment (Supplementary Figures 7 and 8). Moreover, we observed only a slight increase in LC3B-II levels following Piog, but not Rosi, treatment (Supplementary Figures 7 and 8). These results may explain the minimal mycobactericidal effect of Piog and Rosi in macrophages. The results indicate that Trog-induced p-AMPKα is involved in autophagy activation in macrophages.

Because Trog is a ligand of PPAR-γ, we hypothesized that Trog-triggered autophagy activation is dependent on the PPAR-γ pathway. Therefore, we constructed PPAR-γ knock-down THP-1 macrophages and found that the PPAR-γ expression was significantly decreased; however, no difference was observed in p-AMPK and LC3B-II levels between siNC and siPeroxisome proliferator-activated receptor gamma (PPAR-γ) macrophages in either the DMSO- the or Trog-treated group (Supplementary Figure 9A). Similarly, Trog treatment significantly inhibited the intracellular survival of Mtb in both siNC and siPPAR-γ macrophages (Supplementary Figure 9B). Our data suggest that Trog-mediated mycobactericidal activity is independent of PPAR-γ signaling.

Human tumor suppressor serine/threonine-protein kinase STK11 (LKB1), a master serine-threonine kinase, directly phosphorylates and activates AMPKα [26]. To determine whether Trog-induced AMPKα phosphorylation activation is associated with LKB1, LKB1 knock-down THP-1 macrophages were constructed, followed by Trog treatment. LKB1-specific siRNAs significantly decreased LKB1 expression in macrophages (Supplementary Figure 10 and Figure 4A). Moreover, p-AMPKα and LC3B-II were significantly reduced in LKB1 knock-down macrophages without Trog treatment (Supplementary Figure 10). Trog-stimulated p-AMPKα and LC3B-II were significantly enhanced in siNC-transfected macrophages, whereas the effect was reduced in LKB1 knock-down macrophages (Figure 4A).

Figure 4.

Troglitazone (Trog)–mediated autophagy and mycobactericidal activity are involved in serine/threonine-protein kinase STK11 (LKB1)-AMPKα signaling. A, THP-1 macrophages were transfected with either LKB1-specific small interfering RNA (siRNA) (siLKB1) or scrambled siRNA (siNC) for 72 hours. Macrophages were treated with Trog (50 µmol/L) for 24 hours. Cell lysates were subjected to Western blot analysis using anti-LKB1, anti–p-AMPK, anti-microtubule-associated protein 1 light chain 3B (LC3B), and anti–β-actin antibodies. Relative band intensity (target protein/β-actin) in A is shown as a bar graph (with shades of black and gray corresponding to different treatment conditions, from left to right). B, Monomeric red fluorescent protein (mRFP)–green fluorescent protein (GFP)–LC3B reporter THP-1 macrophages transfected with siLKB1 and siNC for 72 hours were pretreated with Trog (50 µmol/L) for 1 hour and then infected with Mycobacterium tuberculosis (Mtb) H37Rv for 4 hours. Cells were washed with prewarmed phosphate-buffered saline, reincubated with Trog for another 24 hours, fixed, and visualized with confocal microscopy. One representative image is shown (bars, 5 µm). Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; Rapa, rapamycin. C, Autophagosome (yellow) and autolysosome puncta (red) in B were calculated. D, THP-1 macrophages were transfected and pretreated as described in B, and colony-forming units were enumerated. Data are presented as means with SDs from 3 independent experiments. Statistical analysis was performed using 2-way analysis of variance (ANOVA) followed by Bonferroni test (A, D) or 1-way ANOVA followed by Tukey test (C). *P < .05; **P < .01; ***P < .001; NS, not significant.

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A similar conclusion was reached from confocal microscopy experiments (Supplementary Figure 11A–11C). Furthermore, Trog-induced autophagosome and autolysosome puncta formation were decreased in Mtb-infected LKB1 knock-down macrophages (Figure 4B and 4C). Similarly, Trog-mediated suppression of intracellular Mtb survival was significantly abolished in LKB1 knock-down macrophages (Figure 4D). These results demonstrated that Trog-mediated autophagy and suppression of intracellular Mtb survival involves LKB1-AMPKα signaling.

To identify potential Trog targets, we searched LKB1-interacting proteins that regulate LKB1 activity in the UNIPROT database (https://www.uniprot.org/), and each of the proteins was used for molecular docking experiments with Trog (Supplementary Table 1). Of these proteins, STRADA showed the highest binding capacity with Trog (Supplementary Table 1). STRADA is a pseudokinase that binds to adenosine triphosphate (ATP) and interacts with and activates LKB1 by promoting LKB1 phosphorylation (p-LKB1) [27, 28]. The molecular structure of Trog is similar to that of ATP, and molecular docking results showed that Trog can compete with ATP for binding to STRADA (Figure 5A). Therefore, similar to findings with ATP, we suspect that STRADA binding to Trog promotes STRADA interaction with LKB1 and then improves phosphorylation and activation of LKB1. Consistent with this hypothesis, Trog treatment enhanced p-LKB1 in a dose-dependent manner within Mtb-infected macrophages (Figure 5B and 5C). Moreover, Trog treatment enhanced the interaction of STRADA with LKB1 under similar conditions (Figure 5D). Together, these results suggest that Trog enhances the phosphorylation and activity of LKB1 by targeting STRADA, followed by activating AMPK and autophagy.

Figure 5.

Troglitazone (Trog) promotes the activation of serine/threonine-protein kinase STK11 (LKB1) by targeting STE20-related kinase adapter protein alpha (STRADA). A, The affinity between adenosine triphosphate (ATP)/Trog and STRADA was analyzed by molecular docking. B, C, THP-1 macrophages (B) or bone marrow–derived macrophages (C) were pretreated with Trog (50 or 100 µmol/L) for 1 hour and then infected with Mycobacterium tuberculosis (Mtb) H37Rv (multiplicity of infection [MOI], 10:1) for 24 hours. Cell lysates were subjected to Western blot analysis using anti–p-LKB1 and anti–β-actin antibodies. Relative band intensity (target protein/β-actin) is shown as bar graphs (with shades of black and gray corresponding to different treatment conditions, from left to right). D, THP-1 macrophages treated with Trog and infected with Mtb and shown in B were coimmunoprecipitated using anti-LKB1–specific antibody. Abbreviation: IP, immunoprecipitation. Data are presented as means with SDs from 3 independent experiments. Statistical analysis was performed using 1-way analysis of variance, followed by Tukey test (B, C). *P < .05; **P < .01; ***P < .001.

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Trog + INH Facilitation of Mtb Elimination in Vitro and in Vivo

CFU enumeration assay was performed to explore whether the combination of INH and Trog showed higher bactericidal activity in macrophages than INH treatment alone. As expected, this combination showed the highest inhibition of intracellular Mtb survival among THP-1 macrophages, BMDMs, and human monocyte–derived macrophages (hMDMs) compared with either INH or Trog treatment alone (Figure 6A–6C). Moreover, both Trog alone and the combination of INH with Trog significantly decreased INH-resistant clinical Mtb (C2) survival in THP-1 macrophages, BMDMs, and hMDMs but not in INH-treated macrophages (Figure 6A–6C).

Figure 6.

The combination of isoniazid (INH) with troglitazone (Trog) additively facilitates Mycobacterium tuberculosis (Mtb) elimination within macrophages. A, THP-1 macrophages preincubated with Trog for 1 hour were infected with Mtb H37Rv or INH-resistant Mtb (C2; multiplicity of infection [MOI], 10:1) for 4 hours. After washing 3 times with prewarmed phosphate-buffered saline (PBS), the infected THP-1 macrophages were incubated with Trog (50 µmol/L) in combination with INH (0.1 µg/mL) for an additional 24, 48, or 72 hours. Cells were lysed, and colony-forming units (CFUs) were enumerated. Abbreviation: DMSO, dimethyl sulfoxide. B, Bone marrow–derived macrophages (BMDMs) preincubated with Trog for 1 hour were infected with Mtb H37Rv or C2 (MOI, 10:1) for 4 hours. After washing 3 times with prewarmed PBS, the infected BMDMs were incubated with Trog (50 µmol/L) in combination with INH (0.1 µg/mL) for another 72 hours. Cells were lysed, and CFUs were enumerated. C, Human monocyte–derived macrophages (hMDMs) preincubated with Trog for 1 hour were infected with Mtb H37Rv or C2 (MOI, 10:1) for 4 hours. After washing 3 times with prewarmed PBS, the infected hMDMs were incubated with Trog (50 µmol/L) in combination with INH (0.1 µg/mL) for another 72 hours. Cells were lysed, and CFUs were enumerated. Data are presented as means with SDs from 3 independent experiments. Statistical analysis was performed using 2-way analysis of variance, followed by Bonferroni test (A–C). *P < .05; **P < .01; ***P < .001; NS, not significant.

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We also found that the mycobactericidal effect of Trog was relatively weak in hMDMs and BMDMs compared with THP-1 macrophages. This may be the result of the high basal autophagy in hMDMs compared with that in THP-1 macrophages (Supplementary Figure 12), which undermines Trog's mycobactericidal effect. In contrast, Trog induced relatively weak autophagy in BMDMs compared with that in THP-1 macrophages (Supplementary Figure 12). To further determine the combined effect between INH and Trog, the fractional inhibitory concentration was determined. As shown in Supplementary Figure 13 and Supplementary Table 2, the minimum inhibitory concentrations of Trog and INH alone were 80 µmol/L and 0.0375 µg/mL, respectively. The combined use of Trog with INH, the minimum inhibitory concentration of Trog and INH was 50 µmol/L and 0.01 µg/mL respectively. This produced fractional inhibitory concentration values of 0.892, which indicates an additive effect.

We also evaluated the additive effect of Trog and INH in vivo. Mtb-infected mice were administered either INH or Trog alone or combined INH and Trog intragastrically. Similar results were observed as shown in the in vitro experiments (Figure 7A and 7B). Pathological lung damage was ameliorated in the Trog-treated group compared with the solvent-treated group in either Mtb H37Rv– or C2-infected mice (Figure 7C). The INH-treated group showed a significant amelioration of pathological lung damage, which was further enhanced in the Trog + INH–treated group in Mtb H37Rv-infected mice (Figure 7C). However, INH-treated and C2-infected mice showed severe pathological lung damage in the solvent-treated group, which was ameliorated in the Trog + INH–treated group (Figure 7C).

Figure 7.

The combination of isoniazid (INH) with troglitazone (Trog) additively facilitates Mycobacterium tuberculosis (Mtb) elimination in Mtb-infected mice. C57BL/6 mice were subjected to aerosol infection with approximately 200 colony-forming units (CFUs) of wild-type (WT) H37Rv or C2 per mouse. Drug administration began after 2 weeks of infection with continuous infusion for 2 weeks. The control group (negative control [NC]) was gavaged with 0.5% methylcellulose only, and the other groups were gavaged with 0.5% methylcellulose containing either Trog or INH alone or the combination of INH + Trog. Mice were euthanized at 1, 14, or 28 days. A, B, CFUs were enumerated in the lungs of the WT H37Rv-infected (A) and C2-infected (B) mice (n = 6). C, Mtb-infected lungs were stained with hematoxylin-eosin 28 days after infection. One representative image is shown (scale bars, 200 µm). D, In WT H37Rv-infected lungs, immunofluorescent staining with the indicated antibodies was performed 28 days after infection. One representative image is shown (scale bars, 200 or 100 µm), along with quantitative analysis of the mean fluorescence intensity (MFI) of microtubule-associated protein 1 light chain 3B (LC3B) in CD68⁺ cells in the lungs. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate. Data are presented as means with SDs (n = 6). Statistical analysis was performed using 1-way analysis of variance, followed by Tukey test (A, B, D). *P < .05; **P < .01; ***P < .001; NS, not significant.

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Moreover, the Trog-treated group showed an increase in the mean fluorescence intensity of LC3B in CD86⁺ macrophages, compared with that of the INH-treated and solvent-treated group, in both wild-type H37Rv-infected and C2-infected groups (Figure 7D and Supplementary Figure 14A and 14B). This trend was also observed in the Trog + INH–treated group (Figure 7D and Supplementary Figure 14A and 14B). These results suggest that Trog-triggered autophagy plays a major role in mycobactericidal activity in vivo. Taken together, our data indicate that Trog is a potential HDT candidate that may be used for antituberculosis therapy, alone or in combination with antituberculosis drugs.

DISCUSSION

Studies have shown that HDTs are a potentially effective strategy for treating tuberculosis, particularly drug-resistant tuberculosis [23, 29, 30]. Drugs with validated safety profiles have been screened and repurposed for antituberculosis treatment. The present study demonstrated that Trog may be repurposed as a potential HDT candidate for antituberculosis therapy. Trog, Rosi, and Piog are thiazolidinediones and are used for antidiabetic treatment [18]; however, our results demonstrated that only Trog exhibited antimycobacterial activity. Other studies demonstrated Piog-induced autophagy [31], which is consistent with our results showing (Supplementary Figures 7 and 8). However, Piog-stimulated autophagy activation was weak in THP-1 macrophages (Supplementary Figures 7 and 8), which may explain why Piog did not exert significant antibacterial effects. Moreover, it appears reasonable that Rosi did not show any antimycobacterial effect because several studies reported that it reduced autophagy [32]. Our results indicated that Rosi did not have an induction effect on autophagy (Supplementary Figure 8); thus, we focused on Trog for further studies.

Previous studies showed that Trog-treated cancer cells induce autophagy [19, 20]. In the present study, we demonstrated that Trog induced autophagy in macrophages. Autophagy is considered a double-edged sword in the context of tumorigenesis [33]; however, many studies indicate that it plays an important role in restricting the survival of invasive pathogens, including Mtb [8, 34]. Golovkine et al [35] demonstrated that autophagy suppressed Mtb survival during acute infection in mice. Therefore, many autophagy-based Food and Drug Administration–approved drugs have been repurposed for antituberculosis therapy, including bazedoxifene [23] and ibrutinib [29], which significantly inhibit intracellular Mtb growth in vitro and in vivo. Moreover, several antituberculosis drugs, including pyrazinamide and bedaquiline, also promote autophagy activation and contribute to intracellular Mtb clearance by targeting host factors, irrespective of their direct killing of Mtb [36, 37].

Similarly, our results showed that Trog treatment results in enhanced autophagy, flux as evidenced by increased lipid-bound LC3B-II as well as autophagosome and autolysosome puncta formation in both Mtb-infected macrophages and lungs. Moreover, Trog-mediated autophagy notably reduced Mtb survival in vitro and in vivo. Our findings and those of others suggest that targeting autophagy by repurposed drugs is an effective strategy to screen potential drugs for antituberculosis therapy.

AMPK contains a catalytic (AMPKα) and 2 regulatory (AMPKβ and AMPKγ) subunits, which act as energy sensors in all eukaryotes and represent an important therapeutic target in patients with diabetes and cancer [26]. Moreover, liver kinase B1 (LKB1), an upstream activator of AMPK, directly phosphorylates and activates AMPK [26, 38]. STRADA binds to ATP and activates LKB1 by promoting LKB1 phosphorylation [27, 28]. AMPK activation enhances autophagy, which is closely associated with ULK1 activity and the regulation of autophagy-related gene expression [25, 39]. The LKB1-AMPK pathway plays an important role in autophagy modulation [40, 41]. Thus, targeting AMPK activation followed by autophagy induction by HDTs, such as metformin, is a promising antimycobacterial treatment approach [8, 42].

Yan et al [19] found that Trog-induced autophagy is associated with AMPK signaling in porcine aortic endothelial cells. Similarly, we demonstrated that Trog-mediated autophagy activation and subsequent suppression of Mtb survival within macrophages involves STRADA-LKB1-AMPKα signaling. Trog treatment significantly promoted an interaction between STRADA and LKB1 and LKB1 phosphorylation, which activated AMPKα activation during Mtb infection. LKB1 knock-down decreased this effect and autophagy induction. These results suggest that the Trog-triggered mycobactericidal effect occurred primarily through autophagy induction.

HDTs as adjuncts combined with antituberculosis drugs represent an attractive strategy to treat tuberculosis, particularly drug-resistant tuberculosis, by enhancing the elimination of Mtb infection and decreasing immunopathology [43]. One study reported that dual mTORC1/mTORC2 inhibition by CC214-2 significantly enhanced the antimycobacterial effect of the first-line RHZE regimen in an Mtb-infected mouse model [44]. Imatinib, an anticancer drug repurposed as an HDT for tuberculosis treatment, decreases the Mtb bacterial burden and lung pathology. It also facilitates the killing of rifampicin-resistant strains [43, 45]. Moreover, a synergistic antibacterial effect was observed when imatinib was combined with first-line drugs, such as rifampicin or rifabutin [45]. Similarly, our data indicate that Trog treatment decreased the intracellular survival of INH-resistant Mtb. Trog + INH exhibited the highest antimycobacterial effect compared with either Trog or INH alone in vitro and in vivo.

One disadvantage to Trog and INH is hepatotoxicity, which is why Trog has been withdrawn from the market. Therefore, it is necessary to evaluate liver toxicity in Mtb-infected mice treated with the combination INH and Trog, as this additive effect could also cause liver toxicity. Although the present study is preclinical, we need to evaluate the liver toxicity of Trog.

In summary, we found that Trog treatment reduced Mtb survival in vitro and in vivo. Trog exerts its antibacterial activity through STRADA-LKB1-AMPKα signaling–mediated autophagy. Moreover, Trog treatment decreases the intracellular survival of INH-resistant Mtb, whereas the combination of Trog and INH shows additive antibacterial effects against Mtb H37Rv. Antidiabetic Trog may be repurposed as a potential HDT candidate and a useful adjunct combined with first-line antituberculosis drugs.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Note

Financial support. This work was supported by the National Natural Science Foundation of China (grants 82170009 and 82102403), the National Key Research and Development Plan (grant 2021YFA1300902), the Guangdong Science Fund for Distinguished Young Scholars (grant 0620220214), the State Key Laboratory of Respiratory Diseases Open Project (grant SKLRD-OP-202324), the Shenzhen Scientific and Technological Foundation (grants KCXFZ20211020163545004, RCJC20221008092726022, JCYJ20220530163216036, and JCYJ20220818095610021), the Sanming Project of Medicine in Shenzhen (grant SZZYSM202311009), and the Guangxi Scientific and Technological Foundation (grant AA22096027).

References

Author notes