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

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 (18F-ICMT-11) and fibrosis (18F-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.


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 rst-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 bene t from inhibiting apoptosis and promoting uncontrolled necrosis, which facilitates infection dissemination and disease persistence [4][5][6] .Necrosis increases TB-associated morbidity as it causes tissue destruction, promotes brosis, 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 brosis, 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 in ammation [9][10][11][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 rst-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 con rmed using post-mortem analysis (Bcl-2, Bid, Annexin V, caspase 3).Using immunohistochemistry and ow 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 signi cantly reduces pulmonary brosis in live animals, as measured by 18 F-FAPI-74 PET, a clinically translatable imaging biomarker for brosis (Fig. S1), and con rmed on post-mortem analysis (soluble collagen levels and Masson's trichrome stains).Extra-pulmonary bacterial dissemination was also decreased in animals receiving adjunctive navitoclax.

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 pro le 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 signi cant 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 signi cant reduction in the bacterial burden compared to the standard treatment alone (RHZ) (P < 0.01) (Fig. 1a).Addition of navitoclax also improved lung pathology (Fig. 1b), with a signi cant decrease in the percentage of affected lung regions (Fig. 1c).

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). 18F-ICMT-11 PET area under the curve (AUC) was signi cantly 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. 2c).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 signi cantly lower (P = 0.03), and Bid level was signi cantly higher (P = 0.01) in animals receiving adjunctive navitoclax versus standard TB treatment alone (Fig. 2e-i).Similarly, apoptosis markers, Annexin V (Fig. 2d, S5), and caspase 3 (Fig. 2j) were signi cantly 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 ow cytometry (Fig. S6).While the proportion of immune cells were similar in treatment groups with or without navitoclax, the addition of navitoclax led to a signi cant increase in apoptosis in several myeloid / macrophage lineage cells (Fig. 3; P < 0.01).
Next, we performed immuno uorescence 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.

Addition of Navitoclax to standard TB treatment reduces Lung brosis
In another set of experiments, TB treatments were initiated six weeks after an aerosol infection with M. tuberculosis (Fig. S9), when pulmonary brosis 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).

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 brosis 29 .Currently, there are no approved treatments to prevent post-TB lung disease.Therefore, there is signi cant 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 in ammation, and vascular disruptions, ultimately leading to brosis 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 pro le 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 myo broblasts, exerting anti-brotic effect by blocking Bcl-XL, which can treat established brosis in several different organs 34,37,38 , and due to its excellent safety pro le.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][41][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 rst-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 signi cantly 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 signi cantly decreased bacterial burden in the spleen and no bacterial dissemination to the brain.This is an interesting nding 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, identi cation of patient-speci c 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 navitoclaxinduced pulmonary apoptosis ( 18 F-ICMT-11) and TB-associated brosis ( 18 F-FAPI-74) in live animals, which were con rmed 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 ow cytometry and immuno uorescence to de ne the immune cell pro le as well as the key immune cell types targeted by navitoclax in our studies.Although the pulmonary immune cell pro les 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 immuno uorescence with CD11b, a pan myeloid marker and CD68, a marker for monocytes and macrophages 26,54-56 con rmed 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 sacri ced using iso urane (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, ve 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, Soluble collagen was quanti ed in whole lung homogenates using uorometric Soluble Collagen Quanti cation 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.
Caspase 3 activity was quanti ed 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 quanti ed as fold-increase relative to uninfected animals.Annexin V (Thermo Fisher Scienti c, A13199) assays were performed using single-cell lung tissue suspensions analysed using the LSRII ow 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, ve 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 sacri ced with iso urane 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 signi cant.

Figure 3 .
Figure 3. High-dimensional ow 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.

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