Proapoptotic Bcl-2 inhibitor as host directed therapy for pulmonary tuberculosis

Proapoptotic Bcl-2 inhibitor as host directed therapy for pulmonary tuberculosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Proapoptotic Bcl-2 inhibitor as host directed therapy for pulmonary tuberculosis Sanjay Jain, Medha Singh, Mona Sarhan, Nerketa Damiba, Alok Singh, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4926508/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Mycobacterium tuberculosis establishes within host cells by inducing anti-apoptotic Bcl-2 family proteins, triggering necrosis, inflammation, and fibrosis. Here, we demonstrate that navitoclax, an orally bioavailable, small-molecule Bcl-2 inhibitor, significantly improves pulmonary tuberculosis (TB) treatments as a host-directed therapy. Addition of navitoclax to standard TB treatments at human equipotent dosing in mouse models of TB, inhibits Bcl-2 expression, leading to improved bacterial clearance, reduced tissue damage / fibrosis and decreased extrapulmonary bacterial dissemination. Using immunohistochemistry and flow cytometry, we show that navitoclax induces apoptosis in several immune cells, including CD68 + and CD11b + cells. Finally, positron emission tomography (PET) in live animals using novel, clinically translatable biomarkers for apoptosis ( 18 F-ICMT-11) and fibrosis ( 18 F-FAPI-74) demonstrates that navitoclax significantly increases apoptosis and reduces fibrosis in pulmonary tissues, which are confirmed using post-mortem studies. Our studies suggest that proapoptotic drugs such as navitoclax can improve pulmonary TB treatments, and should be evaluated in clinical trials. Health sciences/Pathogenesis/Infection Health sciences/Biomarkers/Predictive markers Health sciences/Diseases/Infectious diseases/Tuberculosis Health sciences/Medical research/Translational research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Despite being preventable and treatable, tuberculosis (TB) remains the second leading cause of mortality globally, with an estimated 1.3 million deaths and 10.6 million new cases due to TB, reported in 2022 1 . The number of new TB cases represents the highest incidence recorded since the World Health Organization (WHO) initiated global TB surveillance in 1995, surpassing the pre-pandemic baseline observed in 2019. Further, the burden of drug-resistant TB [DR-TB, including multidrug resistant (MDR)-TB strains resistant to first-line TB drugs rifampin and isoniazid] also increased and there were an estimated 410,000 new cases of rifampin resistant or MDR TB in 2022. The global community has established a goal to eradicate the TB epidemic by 2030, but achieving this objective necessitates urgent and innovative treatments. During TB infection, early-stage apoptosis is host protective which results in immune clearance of Mycobacterium tuberculosis -infected cells, activating both innate and adaptive immune response 2 , 3 . At later stages, bacteria benefit from inhibiting apoptosis and promoting uncontrolled necrosis, which facilitates infection dissemination and disease persistence 4 – 6 . Necrosis increases TB-associated morbidity as it causes tissue destruction, promotes fibrosis, and thereby reduces the penetration of antibiotics to the regions where they are needed most. The necrotic granuloma also provides a breeding ground for M. tuberculosis replication and can transform into cavities, leading to an increased likelihood of developing drug resistance, failing treatment, and disease transmission 7 . Necrotic tissues, often heal by fibrosis, leading to lung dysfunction long after treatment completion, which is increasing being recognized as post-TB lung disease 8 . Conversely, apoptosis is host-protective by eliminating infected cells without triggering excessive inflammation 9 – 12 . Therefore, there has been recent interest in developing host-directed therapies (HDTs) that promote apoptosis 13 , and which could shorten the duration of TB treatments when given in combination with antibiotic regimens. Unlike antibiotics, HDTs modulate host cell responses to improve overall outcomes 14 , 15 , and currently, there are no clinically approved HDTs for pulmonary TB. Importantly, since HDTs target host (mammalian) cells without direct antibiotic effects, they are likely to work against drug-susceptible, as well as MDR M. tuberculosis strains 16 . Here, we study navitoclax (ABT-263), an orally bioavailable, proapoptotic small molecule Bcl-2 inhibitor in clinical trials for cancer treatments, as an adjunctive HDT for pulmonary TB. Addition of navitoclax to the first-line, standard TB treatment (rifampin – R, isoniazid – H and pyrazinamide – Z, RHZ regimen) promotes pulmonary bacterial clearance and reduces lung damage in mouse models of TB, by inhibiting tissue Bcl-2 expression. Further, positron emission tomography (PET) in live animals with 18 F-ICMT-11, a clinically translatable imaging biomarker for apoptosis that targets activated caspase 3/7, demonstrated higher tissue apoptosis in navitoclax-treated animals, which was confirmed using post-mortem analysis (Bcl-2, Bid, Annexin V, caspase 3). Using immunohistochemistry and flow cytometry, we also demonstrate that navitoclax induces apoptosis in multiple cell types, including CD68 + immune cells. Finally, addition of navitoclax to the standard TB treatment significantly reduces pulmonary fibrosis in live animals, as measured by 18 F-FAPI-74 PET, a clinically translatable imaging biomarker for fibrosis ( Fig. S1 ), and confirmed on post-mortem analysis (soluble collagen levels and Masson’s trichrome stains). Extra-pulmonary bacterial dissemination was also decreased in animals receiving adjunctive navitoclax. RESULTS Co-administration of Rifampin does not affect Navitoclax levels in mice Studies have shown that co-administration of navitoclax with rifampin moderately decreases (40%) navitoclax plasma levels in patients but does not change the C max , half-life or its safety profile 17 . We measured navitoclax levels using mass spectrometry in M. tuberculosis -infected mice co-administrated with rifampin as part of the standard TB treatment. The median (interquartile range) for navitoclax plasma and lung levels were 28.50 (25.65–28.75) µg/mL and 5.76 (5.38–11.32) µg/g, respectively ( Fig. S2 ), and consistent with published navitoclax levels achieved in mice without co-administration of rifampin 18 , 19 . Navitoclax administration has no effect on platelet counts in mice Given that reversible thrombocytopenia is the only major side effect of navitoclax in human studies 20 , we measured the platelets in blood samples from M. tuberculosis -infected mice ( Fig. S3 ). The median platelet counts in untreated mice, and those receiving standard TB treatments, with and without navitoclax were 1.09 x 10 6 / µL, 1.05 x 10 6 / µL and 0.99 x 10 6 / µL, respectively. There were no significant differences in the median platelet counts in mice receiving standard TB treatments with and without navitoclax ( P = 0.43). Navitoclax reduces Bacterial burden and Lung pathology TB treatments were initiated three weeks after an aerosol infection with M. tuberculosis . While treatment with navitoclax alone did not have any antimicrobial effects, when combined with the standard TB treatment (RHZ + navitoclax), there was a significant reduction in the bacterial burden compared to the standard treatment alone (RHZ) ( P < 0.01) (Fig. 1 a). Addition of navitoclax also improved lung pathology (Fig. 1 b), with a significant decrease in the percentage of affected lung regions (Fig. 1 c). Navitoclax induces Lung tissue apoptosis by inhibiting Bcl-2 We have previously reported 18 F-ICMT-11 PET as a non-invasive approach to measure intralesional proapoptotic responses in situ in mice 21 . Dynamic PET was performed in live M. tuberculosis -infected mice within sealed biocontainment cells 22 , 23 (Fig. 2 , S4). 18 F-ICMT-11 PET area under the curve (AUC) was significantly higher in the lungs of animals treated with the standard TB treatment in addition to navitoclax versus those receiving the standard treatment alone ( P = 0.01) (Fig. 2 c). Pulmonary 18 F-ICMT-11 PET activity was lowest in the untreated animals. To delineate the mechanistic basis of navitoclax effects, we assessed the tissue levels of anti-apoptotic protein Bcl-2, which is inhibited by navitoclax, and Bid (proapoptotic protein) in whole lung lysates using Western blots. Bcl-2 protein level was significantly lower ( P = 0.03), and Bid level was significantly higher ( P = 0.01) in animals receiving adjunctive navitoclax versus standard TB treatment alone (Fig. 2 e-i). Similarly, apoptosis markers, Annexin V (Fig. 2 d, S5), and caspase 3 (Fig. 2 j) were significantly higher in mice receiving navitoclax plus standard TB treatment versus standard TB treatment alone ( P ≤ 0.01). Effects of Navitoclax on Immune cells in Lung tissues We investigated the cell types targeted by navitoclax in the lungs of M. tuberculosis -infected mice using high-dimensional flow cytometry ( Fig. S6 ). While the proportion of Figure 3. High-dimensional flow cytometry in lung tissues. Cell suspensions from lung tissues of M. tuberculosis -infected mice from the different treatment arms after exclusion of debris and doublets were analysed, two weeks after initiation of TB treatments. a-c , Distribution of immune cells (CD45 + ) in the different treatment arms is shown. d , Percentage of cells positive for intracellular expression of cleaved caspase 3 is shown. Five animals were used for each group. Data are represented as median ± interquartile range. Statistical comparisons were made using the Mann-Whitney U test. immune cells were similar in treatment groups with or without navitoclax, the addition of navitoclax led to a significant increase in apoptosis in several myeloid / macrophage lineage cells ( Fig. 3 ; P < 0.01). Next, we performed immunofluorescence in lung tissue from M. tuberculosis -infected mice undergoing standard TB treatments, with and without navitoclax, to identify the apoptotic effects of navitoclax in key immune cells. CD68 and CD11b are markers of myeloid / phagocytic cells critical in TB pathogenesis 24 – 26 , and consistent with the published studies, were localized within the TB lesions in all treatment arms. Importantly, the co-localization of cleaved caspase 3 (marker of apoptosis) was significantly higher in animals receiving adjunctive navitoclax (versus standard TB treatment alone) with both CD68 ( P = 0.03) (Fig. 4 ) and CD11b ( P = 0.01) ( Fig. S7 ) positive cells. The cumulative mean fluorescence intensity (MFI) for cleaved caspase 3, was significantly higher in animals receiving navitoclax plus standard TB treatment versus standard TB treatment alone ( Fig. S8 ). Addition of Navitoclax to standard TB treatment reduces Lung fibrosis In another set of experiments, TB treatments were initiated six weeks after an aerosol infection with M. tuberculosis ( Fig. S9 ), when pulmonary fibrosis is well established in this model 27 . The overall trends in pulmonary bacterial reductions were similar to the prior experiment. However, the addition of navitoclax to the standard TB treatment reduced extrapulmonary dissemination to the spleen, with complete abrogation of brain dissemination ( Table S1 ). Fibroblast activation protein (FAP) is a type II transmembrane serine protease, highly expressed in fibrotic tissues at the remodelling interface in lung tissues 28 . We synthesized 18 F-FAPI-74 with a radiolabeling yield of 15.4 ± 0.1% (non-decay corrected) and a radiochemical purity of 97.5 ± 0.1%. The specific activity was 101 GBq/µmol by HPLC. ( Fig. S10 ). Dynamic 18 F-FAPI-74 PET was performed in M. tuberculosis -infected mice within sealed biocontainment cells 22 , 23 , demonstrating a significantly lower pulmonary PET AUC lesion/blood in animals receiving adjunctive navitoclax versus standard TB treatment alone (Fig. 5 , S11). The anti-fibrotic effect of navitoclax was also confirmed using postmortem studies demonstrating significantly lower pulmonary fibrosis [Masson’s trichrome staining ( P < 0.01) and soluble collagen levels ( P = 0.02)] in animals receiving adjunctive navitoclax versus standard TB treatment alone (Fig. 6 ). DISCUSSION Current TB treatments comprise multidrug regimens, administered for 4–6 months, even for the treatment of uncomplicated pulmonary TB. Importantly, unlike other respiratory infections, many patients with TB have permanently damaged tissues with successful treatments only transitioning these TB patients from harboring a communicable infectious disease, to a syndrome of chronic pulmonary morbidity, commonly referred to as post-TB lung disease 29 , 30 . In one recent analysis of 6,225 pulmonary TB patients, abnormal lung function was noted in 46.7%, persistent respiratory symptoms in 41.0%, and radiologic abnormalities in 64.6% 30 . Although the precise mechanisms underlying post-TB lung disease remain poorly characterized, it is primarily mediated by M. tuberculosis -induced host-tissue damage (necrosis) and subsequent fibrosis 29 . Currently, there are no approved treatments to prevent post-TB lung disease. Therefore, there is significant interest in developing HDTs that can not only improve TB treatments 13 , 31 , 32 , but also maintain lung function and protect against post-TB lung disease. During the early stages of infection, M. tuberculosis evades apoptosis via induction of anti-apoptotic Bcl-2 family proteins, leading to necrosis, increased inflammation, and vascular disruptions, ultimately leading to fibrosis 9 , 33 . Therefore, the strategic targeting of apoptosis using HDTs presents a novel therapeutic approach to improve TB treatments. Among the orally bioavailable, proapoptotic small molecule Bcl-2 inhibitors, navitoclax and venetoclax are available for human use, with an excellent safety profile 34 . Venetoclax is a selective Bcl-2 inhibitor and approved by the U.S. FDA 35 , while navitoclax is in clinical trials. However, we choose navitoclax for these studies as it inhibits a wide spectrum of Bcl-2 family proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1) 36 , targets multiple host cells, including myofibroblasts, exerting anti-fibrotic effect by blocking Bcl-XL, which can treat established fibrosis in several different organs 34 , 37 , 38 , and due to its excellent safety profile. Co-administration of navitoclax with rifampin can moderately decrease navitoclax plasma levels 17 , but we demonstrate that this was not observed in our studies with M. tuberculosis -infected mice. Reversible thrombocytopenia is the only major side effect of navitoclax in human studies but daily dosing reduces thrombocytopenia risk to ~ 5% 20 , which is less than with several commonly approved antibiotics 39 . Even though daily navitoclax dosing was used in our studies, we performed platelet counts in M. tuberculosis -infected mice which were consistent with the reported platelet counts for untreated adult mice 40 – 42 , and were no different between treatment groups with and without navitoclax. We evaluated navitoclax at human equipotent dosing (325 mg/day) in combination with the first-line, standard TB treatment (RHZ), also administered at human equipotent dosing 37 , 43 . C3HeB/FeJ mice were utilized as they develop human-like TB lung pathology 3 , 7 , 27 , 44 and accurately predict the effectiveness of novel TB regimens that have subsequently been translated to the clinic 27 , 45 , 46 . While navitoclax did not show any antimicrobial effect on its own, when combined with the standard TB treatment, it significantly decreased the pulmonary bacterial burden and improved lung pathology. Of note, while most HDTs decrease bacterial burden only modestly (~ 1 log 10 , presumably targeting the ~ 1–2% persister population) 47 , 48 , even this modest decrease in bacterial burden results in a substantial decrease (~ 50%) in relapse 47 , 48 , with similar outcomes anticipated with navitoclax. M. tuberculosis can disseminate outside the lungs and cause extrapulmonary TB, including TB meningitis 49 , 50 . We observed that mice receiving adjunctive navitoclax had significantly decreased bacterial burden in the spleen and no bacterial dissemination to the brain. This is an interesting finding and is likely due to the proapoptotic effects of navitoclax, which can decrease extralesional bacterial dissemination, and highlight the potential role of navitoclax in preventing extrapulmonary dissemination and will be the subject of future investigation. Since molecular and cellular alterations occur earlier than structural changes, molecular imaging is a powerful tool that has augmented early diagnosis, monitoring and investigation of various diseases 51 . Tomographic molecular imaging can evaluate disease processes deep within the body, noninvasively and relatively rapidly 52 . Although already critical in the management of patients with cancer, molecular imaging has similar potential for infectious diseases to provide molecular characterization of infected lesions, changes with progression or treatments, identification of patient-specific cellular and metabolic abnormalities and holistic three-dimensional visualization, which are less prone to sampling errors 53 . Here, we utilized novel, clinically translatable molecular imaging tools to noninvasively assess navitoclax-induced pulmonary apoptosis ( 18 F-ICMT-11) and TB-associated fibrosis ( 18 F-FAPI-74) in live animals, which were confirmed using postmortem studies. In the future, we anticipate that these imaging approaches could be used to noninvasively characterize post TB-lung disease as well as evaluate novel HDTs in early clinical trials. Since navitoclax is known to affect multiple cell types, we performed flow cytometry and immunofluorescence to define the immune cell profile as well as the key immune cell types targeted by navitoclax in our studies. Although the pulmonary immune cell profiles remained similar in mice receiving standard TB treatments, with or without navitoclax, administration of navitoclax-induced apoptosis in several myeloid / macrophage-linage of immune cells. Additional studies utilizing immunofluorescence with CD11b, a pan myeloid marker and CD68, a marker for monocytes and macrophages 26 , 54 – 56 confirmed that navitoclax-induced apoptosis in these immune cells. Importantly, we provide mechanistic data that the effects of navitoclax are mediated by a decrease in anti-apoptotic protein Bcl2 and increased expression of proapoptotic protein Bid. Overall, these data suggest that navitoclax can improve pulmonary TB treatments by enhancing bacterial clearance and reducing tissue pathology, supporting its role as an HDT for pulmonary TB. METHODS All protocols were approved by the Johns Hopkins University Biosafety, Radiation Safety, and Animal Care and Use Committees (MO19M382). Animal infection and treatments Six-to-seven week-old female C3HeB/FeJ (Jackson Laboratory) mice were aerosol infected with frozen titrated bacterial stocks of M. tuberculosis H37Rv using the Middlebrook Inhalation Exposure System (Glas-Col) 44 . Animals were housed within the ABSL-3 facility with ad libitum access to food and water. Five mice were sacrificed using isoflurane (Henry Schein) overdose one day of infection to assess implantation and just prior to treatment initiation to assess the bacterial burden. At the start of treatments, animals were randomly allocated to receive standard TB treatment with or without navitoclax. Untreated animals served as controls, and in some studies, navitoclax was administered alone. All drugs were administered via oral gavage, five days per week, at human equipotent dosing: rifampin (10 mg/kg/day), isoniazid (10 mg/kg/day), pyrazinamide (150 mg/kg/day) and navitoclax (100 mg/kg/day; MedChemExpress) 37 . After animal euthanasia, whole organs were removed aseptically, homogenized in phosphate-buffered saline (PBS), and plated by serial dilution onto Middlebrook 7H11 agar plates, which were incubated at 37°C for three weeks before CFU were counted. Imaging Imaging was performed in live M. tuberculosis -infected mice within sealed biocontainment cells 22 , 23 using the nanoScan PET/CT (Mediso). 18 F-ICMT-11 was synthesized using an acetal protected tosylate precursor 21 , 57 , while 18 F-FAPI-74 was synthesized as outlined ( Fig. S10 ) 58 , 59 . For anatomical co-registration, a CT was acquired following the PET. Four hours after oral administration of navitoclax, mice received 3.09 ± 0.88 MBq of 18 F-ICMT-11 via the tail vein and dynamic PET (45 min) was performed 15 min post-tracer injection. Dynamic PET (60 min) was performed immediately after an intravenous injection of 3.93 ± 0.86 MBq of 18 F-FAPI-74 via tail vein. Volumes of interest (VOIs) were drawn manually using the CT as reference using VivoQuant 4.0 (Invicro) and the PET signal quantified from the registered images 52 . Heatmap overlays were created using AMIRA 5.2.1 (Visage Imaging, Inc.) and AMIDE 1.0.6 (Andreas Loening). 18 F-ICMT-11 PET was represented as percent (%) injected dose (ID) per volume of tissue (mL) 21 . 18 F-FAPI-74 PET was represented as lesion to blood AUC ratio 58 , 59 , with blood signal obtained by placing a VOI in the left ventricle of the heart. Histopathology and Immunofluorescence Lungs were harvested after systemic perfusion with PBS, fixed in 4% paraformaldehyde. The lung lesions were identified on H&E stains and quantified using ImageJ (NIH). Masson’s trichrome stains were used to assess fibrosis and quantified using ImageJ (NIH) using Colour Deconvolution2 plugin enabled with Masson’s trichrome vector for analysis. Immunostaining was performed at the Johns Hopkins Oncology Tissue Services Core on formalin-fixed, paraffin-embedded sections using a Ventana Discovery Ultra autostainer (Roche Diagnostics). Primary antibody (anti‐CD68, 1:300 dilution, ab125212, Abcam; anti‐CD11b, 1:9000 dilution, ab133357, Abcam; anti‐cleaved caspase 3, 1:1000 dilution, 9661S, Cell Signalling Technology) was applied at 36˚C for 40 minutes. Primary antibodies were detected using an anti-rabbit HQ detection system (7017936001 and 7017812001, Roche Diagnostics) followed by OPAL 520 (FP1487001KT, Akoya Biosciences) diluted 1:150 in 1X Plus Amplification Diluent (FP1498, Akoya Biosciences). Image acquisition was performed using the Leica DM6B system (Leica). Plasma and lung homogenate assays Plasma and lung tissue homogenates were extracted and navitoclax levels were measured using mass spectrometry (Johns Hopkins Oncology core) four hours after receiving 100 mg/kg of oral navitoclax. For studies used to determine platelet levels, fresh blood was collected in EDTA tubes. Blood smears were fixed in cold methanol followed by Wright-Giemsa staining (ab245888) and counted at 100x magnification. The platelet count was obtained using methods described previously 40 – 42 . Soluble collagen was quantified in whole lung homogenates using fluorometric Soluble Collagen Quantification Assay Kit (Sigma, CS0006). Fluorescent intensity was measured at 465 nm (excitation 375 nm) and µg of soluble collagen was calculated using a standard curve. Western blot analysis was performed using a standardized protocol using primary antibodies specific to GAPDH (MA5-15738, Thermo Fisher, dilution 1:1,000), Bid (ab272880, Abcam, dilution 1:1,000), and Bcl-2 (ab182858, Abcam, dilution 1:1,000) and a goat, anti-Rabbit (ab97051, dilution 1:5,000) secondary antibody. The protein bands were visualized on the membranes using chemiluminescent substrates (Supersignal West Pico maximum sensitivity substrate, cat. no. 34580) and analysed using FIJI ImageJ. Caspase 3 activity was quantified four hours after oral administration of navitoclax in mice using the caspase 3 assay kit (Abcam, ab39383) according to the manufacturer’s protocol. caspase 3 activity was quantified as fold-increase relative to uninfected animals. Annexin V (Thermo Fisher Scientific, A13199) assays were performed using single-cell lung tissue suspensions analysed using the LSRII flow cytometer (BD) and Flowjo v10.8 software (BD). Flow cytometry Three weeks after an aerosol challenge with M. tuberculosis mice were randomly allocated to receive PBS, or isoniazid with or without navitoclax. All drugs were administered via oral gavage, five days per week, at human equipotent dosing: isoniazid (10 mg/kg/day), and navitoclax (100 mg/kg/day; MedChemExpress). Two weeks after treatment initiation, mice were sacrificed with isoflurane overdose, lungs were harvested and single-cell suspensions were prepared. Surface staining was performed by incubating samples with a master mix of surface antibodies ( Table S2 ). For caspase 3 staining, primary and secondary antibodies were added sequentially during permeabilization. Flow cytometry was conducted using the FACS ARIA II. The gating strategy adhered to guidelines from the American Thoracic Society ( Fig. S6 ). Initial steps involved removing debris, excluding doublets and dead cells, identifying immune cells (CD45+), and excluding lymphoid cells (CD3+, CD19+, CD19). Myeloid cells were further delineated based on CD11b and CD11c positivity. Statistical Analysis Data were analysed using Prism 10 Version 10.1.1 (GraphPad). Bacterial burden (CFU) are represented on a logarithmic scale (base 10) as mean ± SD and comparisons were made using a student t-test. All other data are represented as median ± IQR and comparisons were made using a Mann-Whitney U test. P values ≤ 0.05 were considered statistically significant. Declarations DATA AVAILABILITY All data are available in the main text or the supplementary materials. Source data are provided with this paper. Competing interests All authors declare that they have no competing interests. Author contributions M.S. and S.K.J. conceptualized and designed the studies. 18 F-ICMT-11 precursor was developed and provided by E.O.A. M.O.S. and L.S.C. developed and performed the radiotracer syntheses for 18 F-ICMT-11 and 18 F-FAPI-74. M.S., A.S. and A.A.O. performed mouse studies. N.N.L.D. performed the Western blots. O.J.N.-M. performed the PET/CT imaging and analyses. M.S. analyzed the data in the manuscript. A.V.-R., X.C., and F.D. performed flow cytometry. S.K.J. provided funding and supervised the project. M.S. and S.K.J. wrote the manuscript. All the authors reviewed and edited the manuscript. Acknowledgments We thank the Johns Hopkins University PET Center for assistance with 18 F-FAPI-74 synthesis. 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J Clin Invest 132 Ordonez AA et al (2016) Mouse model of pulmonary cavitary tuberculosis and expression of matrix metalloproteinase-9. Dis Model Mech 9:779–788 Xu J et al (2019) Contribution of pretomanid to novel regimens containing bedaquiline with either linezolid or moxifloxacin and pyrazinamide in murine models of tuberculosis. Antimicrobial agents and chemotherapy 63, 10.1128/aac . 00021 – 00019 Tasneen R et al (2011) Sterilizing Activity of Novel TMC207-and PA-824-Containing Regimens in a Murine Model of Tuberculosis. Antimicrob Agents Chemother 55:5485–5492 Ordonez AA et al (2018) Adjunct antibody administration with standard treatment reduces relapse rates in a murine tuberculosis model of necrotic granulomas. PLoS ONE 13:e0197474 Skerry C, Harper J, Klunk M, Bishai WR, Jain SK (2012) Adjunctive TNF inhibition with standard treatment enhances bacterial clearance in a murine model of necrotic TB granulomas. PLoS ONE 7:e39680 Jain SK et al (2018) Tuberculous meningitis: a roadmap for advancing basic and translational research. Nat Immunol 19:521–525 Be NA, Kim KS, Bishai WR, Jain SK (2009) Pathogenesis of central nervous system tuberculosis. Curr Mol Med 9:94–99 Higgins LJ, Pomper MG (2011) The evolution of imaging in cancer: current state and future challenges. Semin Oncol 38:3–15 Ordonez AA et al (2020) Dynamic imaging in patients with tuberculosis reveals heterogeneous drug exposures in pulmonary lesions. Nat Med 26:529–534 Ordonez AA et al (2021) Visualizing the dynamics of tuberculosis pathology using molecular imaging. J Clin Invest 131 Bentley JK et al (2013), Rhinovirus colocalizes with CD68- and CD11b-positive macrophages following experimental infection in humans. J Allergy Clin Immunol 132, 758–761 e753 Hong J-H et al (2008) Distribution of CD68-positive and CD11b-positive cells in the TRAMP-C1 tumors after high-dose in vivo irradiation. Cancer Res 68:5335–5335 Betjes MGH, Haks MC, Tuk CW, Beelen RH (1991) J. Monoclonal-Antibody Ebm11 (Anti-Cd68) Discriminates between Dendritic Cells and Macrophages after Short-Term Culture. Immunobiology 183:79–87 Fortt R, Smith G, Awais RO, Luthra SK, Aboagye EO (2012) Automated GMP synthesis of [(18)F]ICMT-11 for in vivo imaging of caspase-3 activity. Nucl Med Biol 39:1000–1005 Chen R et al (2023) Tumor-to-blood ratio for assessment of fibroblast activation protein receptor density in pancreatic cancer using [68Ga] Ga-FAPI-04. Eur J Nucl Med Mol Imaging 50:929–936 Glatting FM et al (2022) Subclass analysis of malignant, inflammatory and degenerative pathologies based on multiple timepoint FAPI-PET acquisitions using FAPI-02, FAPI-46 and FAPI-74. Cancers 14:5301 Additional Declarations There is NO Competing Interest. Supplementary Files 20240816SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4926508","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":343850754,"identity":"4af53f1f-a330-40a1-919d-c79b1bf68bc9","order_by":0,"name":"Sanjay Jain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIie3OMQrCQBBA0VkWYhOxE7tcYToRJXcJAdNqEyzXJpUHiHgMG8uVAdMs8QDbmBsEbCwUDJsDOHaC+5uZYh4MgM/3k2nQ4rmQCEIBBGyill+RLqEI0G0cEm2pofXpOpiOSUGb02eC+oK0N1bODokSZc0hoJHCwEq0iZLDgkEiVbUdqXvy4hDQBmlY6J4IDkFtVhSa1JHzrs4Yj5XV8R5u4hRt1twe+Zzx2ES7kbonGfddI+VGzLv2+Xy+v+wN3h9DePvHAwsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9620-7070","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Sanjay","middleName":"","lastName":"Jain","suffix":""},{"id":343850755,"identity":"2e87e7aa-1159-46e9-8d0d-53acbeec457f","order_by":1,"name":"Medha Singh","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Medha","middleName":"","lastName":"Singh","suffix":""},{"id":343850756,"identity":"8b38edeb-9c1a-4d09-b61b-44f5b2bbb353","order_by":2,"name":"Mona Sarhan","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mona","middleName":"","lastName":"Sarhan","suffix":""},{"id":343850757,"identity":"c36f20fb-27f5-4907-9ba6-ab58387dc58a","order_by":3,"name":"Nerketa Damiba","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nerketa","middleName":"","lastName":"Damiba","suffix":""},{"id":343850758,"identity":"44096c20-9ca2-47b3-8c5f-148e6127ac95","order_by":4,"name":"Alok Singh","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Alok","middleName":"","lastName":"Singh","suffix":""},{"id":343850759,"identity":"dc8f9a34-bbaa-4836-8fb4-5070a9685d4d","order_by":5,"name":"Andres Villabona-Rueda","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Andres","middleName":"","lastName":"Villabona-Rueda","suffix":""},{"id":343850760,"identity":"32b04713-8990-4ce7-a98c-293919eaf9a6","order_by":6,"name":"Oscar Nino Meza","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"Nino","lastName":"Meza","suffix":""},{"id":343850761,"identity":"1e37b63d-6f00-4d55-8c95-a67b3bcd9ff1","order_by":7,"name":"Xueyi Chen","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xueyi","middleName":"","lastName":"Chen","suffix":""},{"id":343850762,"identity":"56d41e9e-163c-4a9c-8076-743607428641","order_by":8,"name":"Alvaro Ordonez","email":"","orcid":"https://orcid.org/0000-0002-8571-0655","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Alvaro","middleName":"","lastName":"Ordonez","suffix":""},{"id":343850763,"identity":"16a84850-3213-41b4-9bd2-c21efe49d5c4","order_by":9,"name":"Franco D'Alessio","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Franco","middleName":"","lastName":"D'Alessio","suffix":""},{"id":343850764,"identity":"b3527bd7-862f-4623-99ea-66953d832216","order_by":10,"name":"Eric Aboagye","email":"","orcid":"","institution":"Imperial College, London","correspondingAuthor":false,"prefix":"","firstName":"Eric","middleName":"","lastName":"Aboagye","suffix":""},{"id":343850765,"identity":"2cc53838-d5d3-49f5-99f1-bd511374bd98","order_by":11,"name":"Laurence Carroll","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Laurence","middleName":"","lastName":"Carroll","suffix":""}],"badges":[],"createdAt":"2024-08-16 17:40:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4926508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4926508/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-58190-x","type":"published","date":"2025-03-27T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63800683,"identity":"c993a94f-8a51-4c2a-926c-868bfc8758d2","added_by":"auto","created_at":"2024-09-02 13:10:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":332994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNavitoclax treatment in mouse model of pulmonary tuberculosis at human equipotent dosing. \u003c/strong\u003e\u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice were randomly allocated to receive standard TB treatment (R, rifampin; H, isoniazid; Z, pyrazinamide) with or without navitoclax at human equipotent dosing via oral gavage. \u003cstrong\u003ea\u003c/strong\u003e, Bacterial burden [colony-forming unit (CFU) per mL (log\u003csub\u003e10\u003c/sub\u003e) from whole lung] after three weeks of treatment (n = 4 mice/regimen). \u003cstrong\u003eb\u003c/strong\u003e, Hematoxylin \u0026amp; Eosin (H\u0026amp;E) stained lung sections of mice demonstrating lung pathology. \u003cstrong\u003ec, \u003c/strong\u003eH\u0026amp;E-stained lung tissue sections were used to quantify the percentage of affected lung tissue regions. For CFU, data are represented as mean ± standard deviation and statistical comparisons were made using the student t-test. For affected lung tissue regions, data are represented as median ± interquartile range. Statistical comparisons were made using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/7f93d11277bda6bb892a17fa.jpg"},{"id":63800678,"identity":"5e98e63e-b032-42d7-a42f-3ce6dba4b214","added_by":"auto","created_at":"2024-09-02 13:10:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":903609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoptosis imaging in live \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. tuberculosis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-infected mice.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Maximum intensity projection (MIP) and transverse \u003csup\u003e18\u003c/sup\u003eF-ICMT-11 PET/CT from representative \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected\u003cstrong\u003e \u003c/strong\u003emice from the different treatment arms, two weeks after initiation of TB treatments. Quantification of pulmonary \u003csup\u003e18\u003c/sup\u003eF-ICMT-11 PET signal as percent injected dose/mL (%ID/mL) (panel \u003cstrong\u003eb\u003c/strong\u003e) and heatmaps representing area under curve (AUC) (panel \u003cstrong\u003ec\u003c/strong\u003e) (n = 3-4 mice/group) are shown. \u003cstrong\u003ed\u003c/strong\u003e, Flow cytometry of single-cell suspensions to analyse the percentage of Annexin V positive cells (n = 3 animals per group; samples were acquired in duplicates for some groups). Levels of anti-apoptotic protein Bcl2 (panels \u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e), and proapoptotic protein Bid (panels \u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e) from lung tissue homogenates using GAPDH as an internal control (panel \u003cstrong\u003ei\u003c/strong\u003e) are shown (n = 4 animals per group).\u003cstrong\u003e j\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eLung tissue homogenate caspase 3 activity is shown (n = 4 animals per group). Data are represented as median ± interquartile range. Statistical comparisons were made using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/4b11805b3e991966134000e3.png"},{"id":63800680,"identity":"b7c68ddf-8346-44a6-bf02-f1356e5133c6","added_by":"auto","created_at":"2024-09-02 13:10:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh-dimensional flow cytometry in lung tissues. \u003c/strong\u003eCell suspensions from lung tissues of \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice\u003cstrong\u003e \u003c/strong\u003efrom the different treatment arms after exclusion of debris and doublets were analysed, two weeks after initiation of TB treatments. \u003cstrong\u003ea-c\u003c/strong\u003e, Distribution of immune cells (CD45\u003csup\u003e+\u003c/sup\u003e) in the different treatment arms is shown. \u003cstrong\u003ed\u003c/strong\u003e, Percentage of cells positive for intracellular expression of cleaved caspase 3 is shown. Five animals were used for each group. Data are represented as median ± interquartile range. Statistical comparisons were made using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/6a7fed65c677348d20dfbe68.png"},{"id":63800679,"identity":"01fd0b78-dd2d-49e8-b734-79b5a2a7962e","added_by":"auto","created_at":"2024-09-02 13:10:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1801334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry in lung tissues. \u003c/strong\u003eFixed lung tissues from \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice from the different treatment arms, two weeks after initiation of TB treatments (n = 4 sections; 2 sections per animal). \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eH\u0026amp;E-stained images and immunostained panels - cleaved caspase 3, CD68 and merged are shown (40x magnification). \u003cstrong\u003eb\u003c/strong\u003e, Pearson’s coefficient to quantify colocalization of cleaved caspase 3 and CD68 is shown. There is significantly higher colocalization of cleaved caspase 3 and CD68 in mice receiving navitoclax plus standard TB treatment versus those receiving standard treatment alone (\u003cem\u003eP\u003c/em\u003e = 0.03). Data are represented as median ± interquartile range. Statistical comparisons were made using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/f51c25983e6757e19a4cf913.png"},{"id":63800682,"identity":"65058b6a-0b80-45c5-8c56-5352a76e6444","added_by":"auto","created_at":"2024-09-02 13:10:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":571023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibrosis imaging in live \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. tuberculosis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-infected mice.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Maximum intensity projection (MIP) and transverse \u003csup\u003e18\u003c/sup\u003eF-FAPI-74 PET/CT from representative \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected\u003cstrong\u003e \u003c/strong\u003emice from the different treatment arms, two weeks after initiation of TB treatments. Quantification of pulmonary \u003csup\u003e18 \u003c/sup\u003eF-FAPI-74 PET signal as lesion to blood ratio (panel \u003cstrong\u003eb\u003c/strong\u003e) and heatmaps representing area under the curve (AUC\u003csub\u003elesion/blood\u003c/sub\u003e) ratio (panel \u003cstrong\u003ec\u003c/strong\u003e) (n = 4 mice/group) are shown. Data are represented as median ± interquartile range. Statistical comparisons were made using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/1c706effccfa1200c8fe11c5.png"},{"id":63802319,"identity":"7ea244bc-a6ec-4570-90f1-90f0d6ac97f5","added_by":"auto","created_at":"2024-09-02 13:18:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1274677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePostmortem studies to quantify lung tissue fibrosis. \u003c/strong\u003eFixed lung tissues from \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice from the different treatment arms, four weeks after initiation of TB treatments (n = 6 sections; 2 sections per animal). \u003cstrong\u003ea\u003c/strong\u003e, Representative\u003cstrong\u003e \u003c/strong\u003eMasson’s trichrome stained sections and quantification (panel \u003cstrong\u003eb\u003c/strong\u003e). \u003cstrong\u003ec\u003c/strong\u003e, Soluble collagen was quantified in whole lung lysates (n = 3-4 animals/group). Data are represented as median ± interquartile range. Statistical comparisons were made using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/c6be41cf2601c52b9616a7b5.png"},{"id":79414675,"identity":"2f8a66c8-05bb-40d0-8275-3063f8d4f5b5","added_by":"auto","created_at":"2025-03-28 07:07:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5600561,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/2c202c7e-3cab-45d4-af26-ba6e43fbd03f.pdf"},{"id":63800684,"identity":"ecd418e0-1db7-4385-a9aa-4b2ebfefffe8","added_by":"auto","created_at":"2024-09-02 13:10:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6240456,"visible":true,"origin":"","legend":"","description":"","filename":"20240816SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4926508/v1/6bfb3be0bb1c3c2ec4799845.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Proapoptotic Bcl-2 inhibitor as host directed therapy for pulmonary tuberculosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDespite being preventable and treatable, tuberculosis (TB) remains the second leading cause of mortality globally, with an estimated 1.3\u0026nbsp;million deaths and 10.6\u0026nbsp;million new cases due to TB, reported in 2022\u003csup\u003e1\u003c/sup\u003e. The number of new TB cases represents the highest incidence recorded since the World Health Organization (WHO) initiated global TB surveillance in 1995, surpassing the pre-pandemic baseline observed in 2019. Further, the burden of drug-resistant TB [DR-TB, including multidrug resistant (MDR)-TB strains resistant to first-line TB drugs rifampin and isoniazid] also increased and there were an estimated 410,000 new cases of rifampin resistant or MDR TB in 2022. The global community has established a goal to eradicate the TB epidemic by 2030, but achieving this objective necessitates urgent and innovative treatments.\u003c/p\u003e \u003cp\u003eDuring TB infection, early-stage apoptosis is host protective which results in immune clearance of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e-infected cells, activating both innate and adaptive immune response\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. At later stages, bacteria benefit from inhibiting apoptosis and promoting uncontrolled necrosis, which facilitates infection dissemination and disease persistence\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Necrosis increases TB-associated morbidity as it causes tissue destruction, promotes fibrosis, and thereby reduces the penetration of antibiotics to the regions where they are needed most. The necrotic granuloma also provides a breeding ground for \u003cem\u003eM. tuberculosis\u003c/em\u003e replication and can transform into cavities, leading to an increased likelihood of developing drug resistance, failing treatment, and disease transmission\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Necrotic tissues, often heal by fibrosis, leading to lung dysfunction long after treatment completion, which is increasing being recognized as post-TB lung disease\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Conversely, apoptosis is host-protective by eliminating infected cells without triggering excessive inflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Therefore, there has been recent interest in developing host-directed therapies (HDTs) that promote apoptosis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and which could shorten the duration of TB treatments when given in combination with antibiotic regimens. Unlike antibiotics, HDTs modulate host cell responses to improve overall outcomes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and currently, there are no clinically approved HDTs for pulmonary TB. Importantly, since HDTs target host (mammalian) cells without direct antibiotic effects, they are likely to work against drug-susceptible, as well as MDR \u003cem\u003eM. tuberculosis\u003c/em\u003e strains\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we study navitoclax (ABT-263), an orally bioavailable, proapoptotic small molecule Bcl-2 inhibitor in clinical trials for cancer treatments, as an adjunctive HDT for pulmonary TB. Addition of navitoclax to the first-line, standard TB treatment (rifampin \u0026ndash; R, isoniazid \u0026ndash; H and pyrazinamide \u0026ndash; Z, RHZ regimen) promotes pulmonary bacterial clearance and reduces lung damage in mouse models of TB, by inhibiting tissue Bcl-2 expression. Further, positron emission tomography (PET) in \u003cem\u003elive\u003c/em\u003e animals with \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11, a clinically translatable imaging biomarker for apoptosis that targets activated caspase 3/7, demonstrated higher tissue apoptosis in navitoclax-treated animals, which was confirmed using post-mortem analysis (Bcl-2, Bid, Annexin V, caspase 3). Using immunohistochemistry and flow cytometry, we also demonstrate that navitoclax induces apoptosis in multiple cell types, including CD68\u0026thinsp;+\u0026thinsp;immune cells. Finally, addition of navitoclax to the standard TB treatment significantly reduces pulmonary fibrosis in \u003cem\u003elive\u003c/em\u003e animals, as measured by \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74 PET, a clinically translatable imaging biomarker for fibrosis (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), and confirmed on post-mortem analysis (soluble collagen levels and Masson\u0026rsquo;s trichrome stains). Extra-pulmonary bacterial dissemination was also decreased in animals receiving adjunctive navitoclax.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCo-administration of Rifampin does not affect Navitoclax levels in mice\u003c/h2\u003e \u003cp\u003eStudies have shown that co-administration of navitoclax with rifampin moderately decreases (40%) navitoclax plasma levels in patients but does not change the C\u003csub\u003emax\u003c/sub\u003e, half-life or its safety profile\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We measured navitoclax levels using mass spectrometry in \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice co-administrated with rifampin as part of the standard TB treatment. The median (interquartile range) for navitoclax plasma and lung levels were 28.50 (25.65\u0026ndash;28.75) \u0026micro;g/mL and 5.76 (5.38\u0026ndash;11.32) \u0026micro;g/g, respectively (\u003cb\u003eFig. S2\u003c/b\u003e), and consistent with published navitoclax levels achieved in mice without co-administration of rifampin\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNavitoclax administration has no effect on platelet counts in mice\u003c/h2\u003e \u003cp\u003eGiven that reversible thrombocytopenia is the only major side effect of navitoclax in human studies\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, we measured the platelets in blood samples from \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice (\u003cb\u003eFig. S3\u003c/b\u003e). The median platelet counts in untreated mice, and those receiving standard TB treatments, with and without navitoclax were 1.09 x 10\u003csup\u003e6\u003c/sup\u003e / \u0026micro;L, 1.05 x 10\u003csup\u003e6\u003c/sup\u003e / \u0026micro;L and 0.99 x 10\u003csup\u003e6\u003c/sup\u003e / \u0026micro;L, respectively. There were no significant differences in the median platelet counts in mice receiving standard TB treatments with and without navitoclax (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.43).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNavitoclax reduces Bacterial burden and Lung pathology\u003c/h2\u003e \u003cp\u003eTB treatments were initiated three weeks after an aerosol infection with \u003cem\u003eM. tuberculosis\u003c/em\u003e. While treatment with navitoclax alone did not have any antimicrobial effects, when combined with the standard TB treatment (RHZ\u0026thinsp;+\u0026thinsp;navitoclax), there was a significant reduction in the bacterial burden compared to the standard treatment alone (RHZ) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Addition of navitoclax also improved lung pathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), with a significant decrease in the percentage of affected lung regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNavitoclax induces Lung tissue apoptosis by inhibiting Bcl-2\u003c/h2\u003e \u003cp\u003eWe have previously reported \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11 PET as a non-invasive approach to measure intralesional proapoptotic responses \u003cem\u003ein situ\u003c/em\u003e in mice\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Dynamic PET was performed in live \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice within sealed biocontainment cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, S4). \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11 PET area under the curve (AUC) was significantly higher in the lungs of animals treated with the standard TB treatment in addition to navitoclax versus those receiving the standard treatment alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Pulmonary \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11 PET activity was lowest in the untreated animals. To delineate the mechanistic basis of navitoclax effects, we assessed the tissue levels of anti-apoptotic protein Bcl-2, which is inhibited by navitoclax, and Bid (proapoptotic protein) in whole lung lysates using Western blots. Bcl-2 protein level was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03), and Bid level was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) in animals receiving adjunctive navitoclax versus standard TB treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-i). Similarly, apoptosis markers, Annexin V (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, S5), and caspase 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej) were significantly higher in mice receiving navitoclax plus standard TB treatment versus standard TB treatment alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Navitoclax on Immune cells in Lung tissues\u003c/h2\u003e \u003cp\u003e We investigated the cell types targeted by navitoclax in the lungs of \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice using high-dimensional flow cytometry (\u003cb\u003eFig. S6\u003c/b\u003e). While the proportion of\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3. High-dimensional flow cytometry in lung tissues.\u003c/b\u003e Cell suspensions from lung tissues of \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice from the different treatment arms after exclusion of debris and doublets were analysed, two weeks after initiation of TB treatments. \u003cb\u003ea-c\u003c/b\u003e, Distribution of immune cells (CD45\u003csup\u003e+\u003c/sup\u003e) in the different treatment arms is shown. \u003cb\u003ed\u003c/b\u003e, Percentage of cells positive for intracellular expression of cleaved caspase 3 is shown. Five animals were used for each group. Data are represented as median\u0026thinsp;\u0026plusmn;\u0026thinsp;interquartile range. Statistical comparisons were made using the Mann-Whitney U test.\u003c/p\u003e \u003cp\u003eimmune cells were similar in treatment groups with or without navitoclax, the addition of navitoclax led to a significant increase in apoptosis in several myeloid / macrophage lineage cells (\u003cb\u003eFig.\u0026nbsp;3\u003c/b\u003e; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eNext, we performed immunofluorescence in lung tissue from \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice undergoing standard TB treatments, with and without navitoclax, to identify the apoptotic effects of navitoclax in key immune cells. CD68 and CD11b are markers of myeloid / phagocytic cells critical in TB pathogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and consistent with the published studies, were localized within the TB lesions in all treatment arms. Importantly, the co-localization of cleaved caspase 3 (marker of apoptosis) was significantly higher in animals receiving adjunctive navitoclax (versus standard TB treatment alone) with both CD68 (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.03) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and CD11b (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) (\u003cb\u003eFig. S7\u003c/b\u003e) positive cells. The cumulative mean fluorescence intensity (MFI) for cleaved caspase 3, was significantly higher in animals receiving navitoclax plus standard TB treatment versus standard TB treatment alone (\u003cb\u003eFig. S8\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAddition of Navitoclax to standard TB treatment reduces Lung fibrosis\u003c/h2\u003e \u003cp\u003eIn another set of experiments, TB treatments were initiated six weeks after an aerosol infection with \u003cem\u003eM. tuberculosis\u003c/em\u003e (\u003cb\u003eFig. S9\u003c/b\u003e), when pulmonary fibrosis is well established in this model\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The overall trends in pulmonary bacterial reductions were similar to the prior experiment. However, the addition of navitoclax to the standard TB treatment reduced extrapulmonary dissemination to the spleen, with complete abrogation of brain dissemination (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFibroblast activation protein (FAP) is a type II transmembrane serine protease, highly expressed in fibrotic tissues at the remodelling interface in lung tissues\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We synthesized \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74 with a radiolabeling yield of 15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% (non-decay corrected) and a radiochemical purity of 97.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%. The specific activity was 101 GBq/\u0026micro;mol by HPLC. (\u003cb\u003eFig. S10\u003c/b\u003e). Dynamic \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74 PET was performed in \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice within sealed biocontainment cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, demonstrating a significantly lower pulmonary PET AUC\u003csub\u003elesion/blood\u003c/sub\u003e in animals receiving adjunctive navitoclax versus standard TB treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, S11). The anti-fibrotic effect of navitoclax was also confirmed using postmortem studies demonstrating significantly lower pulmonary fibrosis [Masson\u0026rsquo;s trichrome staining (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and soluble collagen levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02)] in animals receiving adjunctive navitoclax versus standard TB treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCurrent TB treatments comprise multidrug regimens, administered for 4\u0026ndash;6 months, even for the treatment of uncomplicated pulmonary TB. Importantly, unlike other respiratory infections, many patients with TB have permanently damaged tissues with successful treatments only transitioning these TB patients from harboring a communicable infectious disease, to a syndrome of chronic pulmonary morbidity, commonly referred to as post-TB lung disease\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In one recent analysis of 6,225 pulmonary TB patients, abnormal lung function was noted in 46.7%, persistent respiratory symptoms in 41.0%, and radiologic abnormalities in 64.6%\u003csup\u003e30\u003c/sup\u003e. Although the precise mechanisms underlying post-TB lung disease remain poorly characterized, it is primarily mediated by \u003cem\u003eM. tuberculosis\u003c/em\u003e-induced host-tissue damage (necrosis) and subsequent fibrosis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Currently, there are no approved treatments to prevent post-TB lung disease. Therefore, there is significant interest in developing HDTs that can not only improve TB treatments\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, but also maintain lung function and protect against post-TB lung disease.\u003c/p\u003e \u003cp\u003eDuring the early stages of infection, \u003cem\u003eM. tuberculosis\u003c/em\u003e evades apoptosis via induction of anti-apoptotic Bcl-2 family proteins, leading to necrosis, increased inflammation, and vascular disruptions, ultimately leading to fibrosis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Therefore, the strategic targeting of apoptosis using HDTs presents a novel therapeutic approach to improve TB treatments. Among the orally bioavailable, proapoptotic small molecule Bcl-2 inhibitors, navitoclax and venetoclax are available for human use, with an excellent safety profile\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Venetoclax is a selective Bcl-2 inhibitor and approved by the U.S. FDA\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, while navitoclax is in clinical trials. However, we choose navitoclax for these studies as it inhibits a wide spectrum of Bcl-2 family proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, targets multiple host cells, including myofibroblasts, exerting anti-fibrotic effect by blocking Bcl-XL, which can treat established fibrosis in several different organs\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and due to its excellent safety profile. Co-administration of navitoclax with rifampin can moderately decrease navitoclax plasma levels\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, but we demonstrate that this was not observed in our studies with \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice. Reversible thrombocytopenia is the only major side effect of navitoclax in human studies but daily dosing reduces thrombocytopenia risk to ~\u0026thinsp;5%\u003csup\u003e20\u003c/sup\u003e, which is less than with several commonly approved antibiotics\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Even though daily navitoclax dosing was used in our studies, we performed platelet counts in \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice which were consistent with the reported platelet counts for untreated adult mice\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, and were no different between treatment groups with and without navitoclax.\u003c/p\u003e \u003cp\u003eWe evaluated navitoclax at human equipotent dosing (325 mg/day) in combination with the first-line, standard TB treatment (RHZ), also administered at human equipotent dosing\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. C3HeB/FeJ mice were utilized as they develop human-like TB lung pathology\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and accurately predict the effectiveness of novel TB regimens that have subsequently been translated to the clinic\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. While navitoclax did not show any antimicrobial effect on its own, when combined with the standard TB treatment, it significantly decreased the pulmonary bacterial burden and improved lung pathology. Of note, while most HDTs decrease bacterial burden only modestly (~\u0026thinsp;1 log\u003csub\u003e10\u003c/sub\u003e, presumably targeting the ~\u0026thinsp;1\u0026ndash;2% persister population)\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, even this modest decrease in bacterial burden results in a substantial decrease (~\u0026thinsp;50%) in relapse\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, with similar outcomes anticipated with navitoclax. \u003cem\u003eM. tuberculosis\u003c/em\u003e can disseminate outside the lungs and cause extrapulmonary TB, including TB meningitis\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. We observed that mice receiving adjunctive navitoclax had significantly decreased bacterial burden in the spleen and no bacterial dissemination to the brain. This is an interesting finding and is likely due to the proapoptotic effects of navitoclax, which can decrease extralesional bacterial dissemination, and highlight the potential role of navitoclax in preventing extrapulmonary dissemination and will be the subject of future investigation.\u003c/p\u003e \u003cp\u003eSince molecular and cellular alterations occur earlier than structural changes, molecular imaging is a powerful tool that has augmented early diagnosis, monitoring and investigation of various diseases\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Tomographic molecular imaging can evaluate disease processes deep within the body, noninvasively and relatively rapidly\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Although already critical in the management of patients with cancer, molecular imaging has similar potential for infectious diseases to provide molecular characterization of infected lesions, changes with progression or treatments, identification of patient-specific cellular and metabolic abnormalities and holistic three-dimensional visualization, which are less prone to sampling errors\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Here, we utilized novel, clinically translatable molecular imaging tools to noninvasively assess navitoclax-induced pulmonary apoptosis (\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11) and TB-associated fibrosis (\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74) in \u003cem\u003elive\u003c/em\u003e animals, which were confirmed using postmortem studies. In the future, we anticipate that these imaging approaches could be used to noninvasively characterize post TB-lung disease as well as evaluate novel HDTs in early clinical trials.\u003c/p\u003e \u003cp\u003eSince navitoclax is known to affect multiple cell types, we performed flow cytometry and immunofluorescence to define the immune cell profile as well as the key immune cell types targeted by navitoclax in our studies. Although the pulmonary immune cell profiles remained similar in mice receiving standard TB treatments, with or without navitoclax, administration of navitoclax-induced apoptosis in several myeloid / macrophage-linage of immune cells. Additional studies utilizing immunofluorescence with CD11b, a pan myeloid marker and CD68, a marker for monocytes and macrophages\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e confirmed that navitoclax-induced apoptosis in these immune cells. Importantly, we provide mechanistic data that the effects of navitoclax are mediated by a decrease in anti-apoptotic protein Bcl2 and increased expression of proapoptotic protein Bid. Overall, these data suggest that navitoclax can improve pulmonary TB treatments by enhancing bacterial clearance and reducing tissue pathology, supporting its role as an HDT for pulmonary TB.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eAll protocols were approved by the Johns Hopkins University Biosafety, Radiation Safety, and Animal Care and Use Committees (MO19M382).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimal infection and treatments\u003c/h2\u003e \u003cp\u003eSix-to-seven week-old female C3HeB/FeJ (Jackson Laboratory) mice were aerosol infected with frozen titrated bacterial stocks of \u003cem\u003eM. tuberculosis\u003c/em\u003e H37Rv using the Middlebrook Inhalation Exposure System (Glas-Col)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Animals were housed within the ABSL-3 facility with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. Five mice were sacrificed using isoflurane (Henry Schein) overdose one day of infection to assess implantation and just prior to treatment initiation to assess the bacterial burden. At the start of treatments, animals were randomly allocated to receive standard TB treatment with or without navitoclax. Untreated animals served as controls, and in some studies, navitoclax was administered alone. All drugs were administered via oral gavage, five days per week, at human equipotent dosing: rifampin (10 mg/kg/day), isoniazid (10 mg/kg/day), pyrazinamide (150 mg/kg/day) and navitoclax (100 mg/kg/day; MedChemExpress)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. After animal euthanasia, whole organs were removed aseptically, homogenized in phosphate-buffered saline (PBS), and plated by serial dilution onto Middlebrook 7H11 agar plates, which were incubated at 37\u0026deg;C for three weeks before CFU were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImaging\u003c/h2\u003e \u003cp\u003eImaging was performed in live \u003cem\u003eM. tuberculosis\u003c/em\u003e-infected mice within sealed biocontainment cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e using the nanoScan PET/CT (Mediso). \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11 was synthesized using an acetal protected tosylate precursor\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, while \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74 was synthesized as outlined (\u003cb\u003eFig. S10\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. For anatomical co-registration, a CT was acquired following the PET. Four hours after oral administration of navitoclax, mice received 3.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 MBq of \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11 via the tail vein and dynamic PET (45 min) was performed 15 min post-tracer injection. Dynamic PET (60 min) was performed immediately after an intravenous injection of 3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 MBq of \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74 via tail vein. Volumes of interest (VOIs) were drawn manually using the CT as reference using VivoQuant 4.0 (Invicro) and the PET signal quantified from the registered images\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Heatmap overlays were created using AMIRA 5.2.1 (Visage Imaging, Inc.) and AMIDE 1.0.6 (Andreas Loening). \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11 PET was represented as percent (%) injected dose (ID) per volume of tissue (mL)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74 PET was represented as lesion to blood AUC ratio\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, with blood signal obtained by placing a VOI in the left ventricle of the heart.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology and Immunofluorescence\u003c/h2\u003e \u003cp\u003eLungs were harvested after systemic perfusion with PBS, fixed in 4% paraformaldehyde. The lung lesions were identified on H\u0026amp;E stains and quantified using ImageJ (NIH). Masson\u0026rsquo;s trichrome stains were used to assess fibrosis and quantified using ImageJ (NIH) using Colour Deconvolution2 plugin enabled with Masson\u0026rsquo;s trichrome vector for analysis.\u003c/p\u003e \u003cp\u003eImmunostaining was performed at the Johns Hopkins Oncology Tissue Services Core on formalin-fixed, paraffin-embedded sections using a Ventana Discovery Ultra autostainer (Roche Diagnostics). Primary antibody (anti‐CD68, 1:300 dilution, ab125212, Abcam; anti‐CD11b, 1:9000 dilution, ab133357, Abcam; anti‐cleaved caspase 3, 1:1000 dilution, 9661S, Cell Signalling Technology) was applied at 36˚C for 40 minutes. Primary antibodies were detected using an anti-rabbit HQ detection system (7017936001 and 7017812001, Roche Diagnostics) followed by OPAL 520 (FP1487001KT, Akoya Biosciences) diluted 1:150 in 1X Plus Amplification Diluent (FP1498, Akoya Biosciences). Image acquisition was performed using the Leica DM6B system (Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlasma and lung homogenate assays\u003c/h2\u003e \u003cp\u003ePlasma and lung tissue homogenates were extracted and navitoclax levels were measured using mass spectrometry (Johns Hopkins Oncology core) four hours after receiving 100 mg/kg of oral navitoclax. For studies used to determine platelet levels, fresh blood was collected in EDTA tubes. Blood smears were fixed in cold methanol followed by Wright-Giemsa staining (ab245888) and counted at 100x magnification. The platelet count was obtained using methods described previously\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSoluble collagen was quantified in whole lung homogenates using fluorometric Soluble Collagen Quantification Assay Kit (Sigma, CS0006). Fluorescent intensity was measured at 465 nm (excitation 375 nm) and \u0026micro;g of soluble collagen was calculated using a standard curve.\u003c/p\u003e \u003cp\u003eWestern blot analysis was performed using a standardized protocol using primary antibodies specific to GAPDH (MA5-15738, Thermo Fisher, dilution 1:1,000), Bid (ab272880, Abcam, dilution 1:1,000), and Bcl-2 (ab182858, Abcam, dilution 1:1,000) and a goat, anti-Rabbit (ab97051, dilution 1:5,000) secondary antibody. The protein bands were visualized on the membranes using chemiluminescent substrates (Supersignal West Pico maximum sensitivity substrate, cat. no. 34580) and analysed using FIJI ImageJ.\u003c/p\u003e \u003cp\u003eCaspase 3 activity was quantified four hours after oral administration of navitoclax in mice using the caspase 3 assay kit (Abcam, ab39383) according to the manufacturer\u0026rsquo;s protocol. caspase 3 activity was quantified as fold-increase relative to uninfected animals.\u003c/p\u003e \u003cp\u003eAnnexin V (Thermo Fisher Scientific, A13199) assays were performed using single-cell lung tissue suspensions analysed using the LSRII flow cytometer (BD) and Flowjo v10.8 software (BD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eThree weeks after an aerosol challenge with \u003cem\u003eM. tuberculosis\u003c/em\u003e mice were randomly allocated to receive PBS, or isoniazid with or without navitoclax. All drugs were administered via oral gavage, five days per week, at human equipotent dosing: isoniazid (10 mg/kg/day), and navitoclax (100 mg/kg/day; MedChemExpress). Two weeks after treatment initiation, mice were sacrificed with isoflurane overdose, lungs were harvested and single-cell suspensions were prepared. Surface staining was performed by incubating samples with a master mix of surface antibodies (\u003cb\u003eTable S2\u003c/b\u003e). For caspase 3 staining, primary and secondary antibodies were added sequentially during permeabilization. Flow cytometry was conducted using the FACS ARIA II. The gating strategy adhered to guidelines from the American Thoracic Society (\u003cb\u003eFig. S6\u003c/b\u003e). Initial steps involved removing debris, excluding doublets and dead cells, identifying immune cells (CD45+), and excluding lymphoid cells (CD3+, CD19+, CD19). Myeloid cells were further delineated based on CD11b and CD11c positivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were analysed using Prism 10 Version 10.1.1 (GraphPad). Bacterial burden (CFU) are represented on a logarithmic scale (base 10) as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and comparisons were made using a student t-test. All other data are represented as median\u0026thinsp;\u0026plusmn;\u0026thinsp;IQR and comparisons were made using a Mann-Whitney U test. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eAll data are available in the main text or the supplementary materials. Source data are provided with this paper.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAll authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eM.S. and S.K.J. conceptualized and designed the studies. \u003csup\u003e18\u003c/sup\u003eF-ICMT-11 precursor was developed and provided by E.O.A. M.O.S. and L.S.C. developed and performed the radiotracer syntheses for \u003csup\u003e18\u003c/sup\u003eF-ICMT-11 and \u003csup\u003e18\u003c/sup\u003eF-FAPI-74. M.S., A.S. and A.A.O. performed mouse studies. N.N.L.D. performed the Western blots. O.J.N.-M. performed the PET/CT imaging and analyses. M.S. analyzed the data in the manuscript. A.V.-R., X.C., and F.D. performed flow cytometry. S.K.J. provided funding and supervised the project. M.S. and S.K.J. wrote the manuscript. All the authors reviewed and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank the Johns Hopkins University PET Center for assistance with \u003csup\u003e18\u003c/sup\u003eF-FAPI-74 synthesis. This work was funded by the National Institutes of Health [R01-AI153349, R01-AI145435-A1, R56-AI179012-A1, S10-OD030381-A1, and P30-AI168436].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO (2023) Global Tuberculosis Report. World Health Organization, Geneva, Switzerland\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik ZA, Iyer SS, Kusner DJ (2001) Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages. 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Platelets 30:698\u0026ndash;707\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeters LL et al (2002) Large-scale, high-throughput screening for coagulation and hematologic phenotypes in mice. Physiol Genomics 11:185\u0026ndash;193\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJirouskova M, Shet AS, Johnson GJ (2007) A guide to murine platelet structure, function, assays, and genetic alterations. J Thromb Haemost 5:661\u0026ndash;669\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuiz-Bedoya CA et al (2022) High-dose rifampin improves bactericidal activity without increased intracerebral inflammation in animal models of tuberculous meningitis. J Clin Invest 132\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrdonez AA et al (2016) Mouse model of pulmonary cavitary tuberculosis and expression of matrix metalloproteinase-9. Dis Model Mech 9:779\u0026ndash;788\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J et al (2019) Contribution of pretomanid to novel regimens containing bedaquiline with either linezolid or moxifloxacin and pyrazinamide in murine models of tuberculosis. \u003cem\u003eAntimicrobial agents and chemotherapy\u003c/em\u003e 63, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aac\u003c/span\u003e\u003cspan address=\"10.1128/aac\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 00021\u0026thinsp;\u0026ndash;\u0026thinsp;00019\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTasneen R et al (2011) Sterilizing Activity of Novel TMC207-and PA-824-Containing Regimens in a Murine Model of Tuberculosis. 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J Allergy Clin Immunol 132, 758\u0026ndash;761 e753\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong J-H et al (2008) Distribution of CD68-positive and CD11b-positive cells in the TRAMP-C1 tumors after high-dose in vivo irradiation. Cancer Res 68:5335\u0026ndash;5335\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBetjes MGH, Haks MC, Tuk CW, Beelen RH (1991) J. Monoclonal-Antibody Ebm11 (Anti-Cd68) Discriminates between Dendritic Cells and Macrophages after Short-Term Culture. Immunobiology 183:79\u0026ndash;87\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFortt R, Smith G, Awais RO, Luthra SK, Aboagye EO (2012) Automated GMP synthesis of [(18)F]ICMT-11 for in vivo imaging of caspase-3 activity. Nucl Med Biol 39:1000\u0026ndash;1005\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen R et al (2023) Tumor-to-blood ratio for assessment of fibroblast activation protein receptor density in pancreatic cancer using [68Ga] Ga-FAPI-04. Eur J Nucl Med Mol Imaging 50:929\u0026ndash;936\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlatting FM et al (2022) Subclass analysis of malignant, inflammatory and degenerative pathologies based on multiple timepoint FAPI-PET acquisitions using FAPI-02, FAPI-46 and FAPI-74. Cancers 14:5301\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4926508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4926508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e establishes within host cells by inducing anti-apoptotic Bcl-2 family proteins, triggering necrosis, inflammation, and fibrosis. Here, we demonstrate that navitoclax, an orally bioavailable, small-molecule Bcl-2 inhibitor, significantly improves pulmonary tuberculosis (TB) treatments as a host-directed therapy. Addition of navitoclax to standard TB treatments at human equipotent dosing in mouse models of TB, inhibits Bcl-2 expression, leading to improved bacterial clearance, reduced tissue damage / fibrosis and decreased extrapulmonary bacterial dissemination. Using immunohistochemistry and flow cytometry, we show that navitoclax induces apoptosis in several immune cells, including CD68\u0026thinsp;+\u0026thinsp;and CD11b\u0026thinsp;+\u0026thinsp;cells. Finally, positron emission tomography (PET) in \u003cem\u003elive\u003c/em\u003e animals using novel, clinically translatable biomarkers for apoptosis (\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-ICMT-11) and fibrosis (\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eF-FAPI-74) demonstrates that navitoclax significantly increases apoptosis and reduces fibrosis in pulmonary tissues, which are confirmed using post-mortem studies. Our studies suggest that proapoptotic drugs such as navitoclax can improve pulmonary TB treatments, and should be evaluated in clinical trials.\u003c/p\u003e","manuscriptTitle":"Proapoptotic Bcl-2 inhibitor as host directed therapy for pulmonary tuberculosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-02 13:10:48","doi":"10.21203/rs.3.rs-4926508/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0008d362-3c8f-4fae-964f-3855b07a054d","owner":[],"postedDate":"September 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":36440561,"name":"Health sciences/Pathogenesis/Infection"},{"id":36440562,"name":"Health sciences/Biomarkers/Predictive markers"},{"id":36440563,"name":"Health sciences/Diseases/Infectious diseases/Tuberculosis"},{"id":36440564,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2025-03-28T07:07:25+00:00","versionOfRecord":{"articleIdentity":"rs-4926508","link":"https://doi.org/10.1038/s41467-025-58190-x","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-03-27 04:00:00","publishedOnDateReadable":"March 27th, 2025"},"versionCreatedAt":"2024-09-02 13:10:48","video":"","vorDoi":"10.1038/s41467-025-58190-x","vorDoiUrl":"https://doi.org/10.1038/s41467-025-58190-x","workflowStages":[]},"version":"v1","identity":"rs-4926508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4926508","identity":"rs-4926508","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}