Primary macrophages were isolated largely from peritoneal exudates of BALB/c mice. Other mice used included C3H/HeJ (Tlr4 mutant), C57BL/6J, and tlr2-knockout mice maintained in the Central Animal Facility, Indian Institute of Science (IISc). Both male and female mice were used in this study. Briefly, mice were injected immunoprecipitation with 1 ml of 8% Brewer thioglycollate. Mice were sacrificed after 4 days and peritoneal cells were harvested by lavage from peritoneal cavity with ice-cold PBS. The cells were cultured in DMEM (Gibco-Invitrogen/Thermo Fisher Scientific) containing 10% FBS (Gibco-Invitrogen/Thermo Fisher Scientific) for 24 h and adherent cells were used as peritoneal macrophages. Murine RAW264.7 macrophage cell line was obtained from an already-existing collection of Cell Repository at the National Center for Cell Sciences (NCCS), Pune, India. NIH3T3 and K562 cell lines were kind gifts from Prof. Anjali Karande (Dept. of Biochemistry, IISc) and Prof. Satees Raghavan (Dept. of Biochemistry, IISc). M. tuberculosis H37Rv ΔdosR was a kind gift from Dr. David Sherman, University of Washington, M. tuberculosis H37Rv ΔsecA was a kind gift from Dr. William Jacobs, Albert Einstein College Medicine, M. tuberculosis H37Rv ΔwhiB3 and M. tuberculosis H37Rv ΔwhiB3-comp were kind gifts from Dr. Amit Singh, IISc (61). All studies involving virulent mycobacterial strains were carried out at the BSL-3 facility at Centre for Infectious Disease Research (CIDR), IISc. Bacteria were grown to mid-log phase and used at 10 multiplicity of infection in all experiments unless mentioned otherwise.

Mycobacterium tuberculosis H37Rv was grown in Middlebrook 7H9 medium (Difco) containing 0.2% glycerol, 0.05% Tween 80, and 10% ADC. Cultures were grown at 37°C to log phase. Culture was washed with PBS and passed 10 times each, through 26, 29, and 31 gauge needles to make single-cell suspensions of Mtb H37Rv. BALB/c mice were infected with Mtb using a Madison chamber aerosol generation instrument calibrated to 100 CFU/mice and maintained in securely commissioned BSL3 facility. 28 days postinfection, mice were given 10 doses of Imatinib (66 mg/kg) (Calbiochem, 504595) immunoprecipitation (every 2 days) (n = 10). On day 56, mice were sacrificed, left lobe of the lung and whole spleen was homogenized in sterile PBS, serially diluted and plated on 7H11 agar containing OADC to quantify Mtb H37Rv CFU. Superior lung lobe was fixed in formalin, embedded in paraffin and stained with hematoxylin and eosin to assess pathology. Granuloma features were characterized and assigned different scores: with necrosis (Score = 5), without necrosis (Score = 2.5), with fibrosis (Score = 1). Total granuloma scores were calculated by multiplying the characterized feature score with the number of granuloma in each lung.

The following antibodies were utilized in this study: Cell Signaling Technology: BMPR2 (6979), p-Smad1/5/8 (9511), ID2 (3431), c-Abl (2862), KAT5 (12058), Flag (2368), pSTAT1 (9171), pSTAT3 (9131), and pSmad2/3 (8828). PeproTech Inc.: BMP2 (500-P195). Santa Cruz Biotechnology Inc.: BMP4 (sc-12721) and p-Tyrosine (PY99). Calbiochem: PCNA (NA-03). Abcam: TLR3 (ab62566), TICAM1 (ab13810), BMPR1a (ab38560), pPDGFR (ab5443), and p-c-KIT (ab5616). Sigma: iNOS (N7782) and β-ACTIN (A3854). Jackson Immuno Research: Goat anti-Rabbit HRP (111-035-045) and Mouse anti-Rabbit Light Chain specific HRP (211-032-171), DyLight 488 IgG Fraction Monoclonal Mouse Anti-Rabbit. Invitrogen: p-Threonine (PT-5H5). Tonbo Biosciences: PE Anti-Mouse F4/80 Antigen (BM8.1) (50-4801), APC Anti-Mouse Ly-6G (1A8) (20-1276). BD Biosciences: PE Anti-Mouse CD11c (HL3) (557401).

Transient transfection of 0.5 × 10⁶ RAW264.7 macrophages with 5 μg of mentioned plasmid constructs was performed using polyethylenimine (PEI, Sigma-Aldrich). In case of experiments involving siRNA, RAW264.7 macrophages were transfected with carefully titrated concentration of 100 nM siRNA. Abl1, Twist1, non-targeting siRNA, and siGLO Lamin A/C were obtained from Dharmacon as siGENOME SMARTpool reagents, which contain a pool of four different double-stranded RNA oligonucleotides. Transfection efficiency was found to be 70% in all the experiments as determined by the number of siGLO Lamin A/C positive cells in a microscopic field using fluorescent microscope. Further, 36 h post-transfection, the cells were treated or infected as indicated and processed for analysis.

0.5 × 10⁶ RAW264.7 cells were transfected with specific promoter luciferase plasmids using PEI (Sigma-Aldrich). After 36 h of transfection, cells were infected for 12 h where indicated. Cells were harvested and lysed in reporter lysis buffer (Promega, USA) and luciferase activity was assayed using luciferase assay reagent (Promega, USA). The results were normalized for transfection efficiencies by assay of β-galactosidase activity. O-nitrophenol β-d-galactopyranoside (ONPG, Hi-Media, RM582) was utilized for the assay.

In all in vitro experiments, cells were treated with the given reagents 1 h prior to experimental treatments at the following concentrations: Imatinib (10 μM) (Gleevec), Alk5 inhibitor (10 nM) (Sigma, SB431542), Compound C (5 μM) (Calbiochem, 171260), Poly I:C (10 μg/ml) (Invivogen), Dibenziodolium chloride (10 μM) (DPI, Calbiochem, 300260), 1400W (Calbiochem, 100050), and IFN-γ (200 U/ml) (Biolegend). 2′,7′-Dichlorofluorescin diacetate (DCFDA, D6883) was purchased from Sigma. Recombinant PDGF-AB (100-00AB) and SCF (300-07) were purchased from PeproTech Inc. and used at concentrations of 10 and 100 ng/ml, respectively.

Microtome sections (5 μm) were obtained from 3.6% formaldehyde-fixed, decalcified, and paraffin-embedded tissues using Leica RM2245 microtome (Leica). These sections were first deparaffinized, subjected to antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0) for 10 min, treated with 1% H₂O₂ for 10 min, and blocked with 5% BSA (Sigma-Aldrich) for 1 h at room temperature. The tissue sections were further incubated with primary antibodies overnight. After incubation with anti-rabbit HRP-conjugated secondary antibody for 90 min, sections were stained with 0.05% diaminobenzidine (DAB, Sigma-Aldrich) in 0.03% H₂O₂ solution and counterstained with hematoxylin, dehydrated, and mounted. Stained tissue sections were imaged with Axio Scope A1 microscope (Zeiss) at indicated magnification. All experiments were performed with appropriate isotype-matched control antibodies and a minimum of three tissue sections were assessed.

The murine lung was rapidly frozen in liquid nitrogen in optimal cutting temperature (OCT) media (Jung, Leica). Cryosections of 10–15 μm were taken in Leica CM 1510 S or Leica CM 3050 S cryostat with the tissue embedded in OCT onto the glass slides and stored at −80°C.

Cryosections were thawed to room temperature and after blocking with 2% BSA containing saponin, the sections were stained for specific antibodies at 4°C overnight. The sections were incubated with DyLight 488-conjugated secondary antibody for 2 h and nuclei stained with DAPI. A coverslip was mounted on the section with glycerol as the medium. Confocal images were taken on Zeiss LSM 710 Meta confocal laser scanning microscope (Carl Zeiss AG) using a plan-Apochromat 40×/1.4 Oil DIC objective (Carl Zeiss AG) and images were analyzed using ZEN 2009 software.

For detection of miR27a, total RNA was isolated from the indicated cells/tissues using the TRI reagent. Quantitative real-time RT-PCR for miR27a (assay ID 00408) was performed using TaqMan miRNA assays (Life Technologies, 4324018) as per manufacturer’s instructions. U6 snRNA (assay ID 4427975) was used for normalization.

Chromatin immunoprecipitation assays were performed using a protocol provided by Upstate Biotechnology, USA, with certain modifications. Macrophages were fixed with 1.42% formaldehyde for 7 min at room temperature followed by inactivation of formaldehyde with addition of 125 mM glycine. Cells were lysed using 0.1%SDS lysis buffer (with 1% Triton X-100) and chromatin was sheared in Bioruptor (Diagenode) Sonicator (High Power, 70 cycles of 30 s pulse ON and 45 s pulse OFF). Chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using specific antibody or isotype IgG. Immunoprecipitated complexes were sequentially washed with Wash Buffer A [50 mM Tris–HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS, and protease/phosphatase inhibitors], Wash Buffer B [50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium deoxycholate and protease/phosphatase inhibitors], TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA], and eluted in elution buffer [1% SDS, 0.1 M NaHCO₃]. After treating the eluted samples with RNase A and Proteinase K, DNA was precipitated using phenol–chloroform–ethanol method. Purified DNA was analyzed by quantitative real-time RT-PCR. All values in the test samples were normalized to amplification of the specific gene in Input and IgG pull down and represented as fold change. All ChIP experiments were repeated at least three times and the primers utilized are listed in Table 1.

Cells were cultured in a 96-well plate, using triplicates for each condition and proper controls. Cell supernatants were collected and 50 μl was transferred to a new 96-well plate. Simultaneously, standard stock solution was diluted to obtain a standard curve. 50 μl of Sulfanilamide Solution was added to each sample, control well, and mixed well. It was incubated at room temperature for 10 min in the dark. Further, 50 μl of N-1-napthylethylenediamine dihydrochloride solution was added to each sample, control well, and mixed well. The plate was incubated at room temperature for 10 min in the dark. Absorbance was measured immediately at 540 nm.

Treated cells were loaded with the DCFDA dye (at a final concentration of 20 μM) and kept for 30 min in the dark, in a conventional incubator (37°C, 5% CO₂). Carboxy-H2DCFDA containing medium was discarded and cells were washed twice with PBS. ROS was assessed immediately by analyzing cells utilizing fluorescence plate reader at 529 nm.

Cells were washed with ice-cold PBS, scraped from the culture dish and collected by centrifugation. Cell pellets were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.25% Sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/ml of each aprotinin, leupeptin, pepstatin, 1 mM Na₃VO₄, 1 mM NaF). Whole cell lysate was collected by centrifuging lysed cells at 13,226 × g, 15 min at 4°C. Bradford’s method of protein estimation was used to assess protein concentration in each cell lysate. Equal protein from each sample was subjected to SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes (Millipore, USA) by the semidry (Bio-Rad, USA) method. Membranes were blocked with 5% non-fat dry milk powder (bovine, Sigma-Aldrich) in TBST [20 mM Tris–HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20] for 60 min. The blots were incubated overnight at 4°C with primary antibodies diluted in TBST with 5% BSA. After washing with TBST, blots were incubated with necessary secondary antibody conjugated to HRP (Jackson Immunoresearch, USA) diluted in 5% non-fat milk for 4 h. After further washes in TBST, the immunoblots were developed with ECL reagent (Perkin Elmer, USA). β-ACTIN was used as loading control.

Cells were treated as indicated, harvested by centrifugation, and gently resuspended in ice-cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After incubation on ice for 15 min, cell membranes were disrupted with 10% NP-40 and the nuclear pellets were recovered by centrifugation at 13,226 × g for 15 min at 4°C. The supernatants from this step were used as cytosolic extracts. Nuclear pellets were lysed with ice-cold Buffer C (20 mM HEPES pH7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) and nuclear extracts were collected after centrifugation at 13,226 × g for 20 min at 4°C.

Immunoprecipitation (IP) assays were performed using a protocol provided by Millipore, USA, with certain modifications. Briefly, macrophages were gently lysed in ice-cold RIPA buffer on an orbital shaker. Whole cell lysate was collected by centrifuging lysed cells at 13,226 × g, 10 min at 4°C. Cell lysate was pre-cleared with Protein A (Bangalore Genei, 62211018005A) beads blocked with 2% BSA. Bradford’s method of protein estimation was used to assess protein concentration in each sample. Cell lysates containing equal protein were incubated with respective antibody or IgG at 4°C for 6 h on the orbital shaker. The immunocomplexes were captured using Protein A agarose at 4°C for 4 h. The beads were harvested, washed (with ice-cold RIPA buffer) and boiled in 5× Laemmli buffer for 10 min. The samples were separated by SDS-PAGE and further subjected for immunoblotting.

BALB/c mice (n = 16) were infected with mid-log phage Mtb H37Rv, as mentioned above. On day 56, mice (n = 8) were injected with 10 mg/kg Poly I:C (intravenously), 12 h prior to sacrifice. RNA was isolated from the lungs and spleen of these mice and assessed for TLR3 stimulated signaling. PBS (n = 3), Poly I:C (n = 8) injected and Mtb H37Rv (n = 8) infected mice were used as controls.

The full length 3′UTR of Ticam1 was PCR amplified and cloned into a pmirGLO vector using the restriction enzyme pair SacI and XbaI. The miR27a binding site in the 3′UTR of Ticam1, was mutated by nucleotide replacements through site-directed mutagenesis using the inverse PCR method. The forward and reverse primers were perfectly complementary and comprised the desired mutations flanked by 10–15 nucleotides on each side (Table 1). The newly synthesized plasmid was separated from the parental plasmid by digesting the reaction with the restriction enzyme DpnI followed by transformation and screening for mutants.

Macrophages were treated or infected as indicated and total RNA was isolated using TRI reagent (T9424, Sigma-Aldrich, USA). For RT-PCR, 1 μg of total RNA was converted into cDNA using First Strand cDNA synthesis kit (M3682, Promega). Quantitative real-time RT-PCR was performed with SYBR Green PCR mixture (F416, Thermo Fisher Scientific). All the experiments were repeated at least three times, independently, to ensure reproducibility of the results. Gapdh was used as internal control. The primers used for quantitative real-time RT-PCR are summarized in Table 1.

Levels of significance for comparison between samples were determined by the Student’s t-test distribution or one-way ANOVA followed by Tukey’s multiple comparisons. The data in the graphs are expressed as the mean ± SE for the values from at least three or more independent experiments and P values < 0.05 were defined as significant. GraphPad Prism 5.0 software (GraphPad Software) was used for all the statistical analysis.

At the outset, we asked if BMP signaling is mycobacteria responsive. We examined the expression of hallmarks of BMP signaling during mycobacterial infection and found transcript levels of Bmp2, Bmp4, Bmpr2, and Id2 were consistently upregulated upon infection of mouse peritoneal macrophages with Mtb H37Ra as well as virulent Mtb H37Rv (Figure 1A). There was no change in the transcript levels of BMPR1a, emphasizing that phosphorylation of BMPR1a suffices the activation of BMP signaling. BMPR2, BMP2, BMP4, p-Smad1/5/8, and ID2 were also assessed at the protein level (Figure 1B). No significant differences were observed among the transcript or protein abundances of Mtb H37Ra and Mtb H37Rv-infected macrophages with respect to BMP signaling (Figure S1A in Supplementary Material). Further, it was found that activated BMP signaling was a delayed event (Figure 1C; Figure S1B in Supplementary Material). We established that mycobacteria-activated BMP signaling required canonical receptors BMPR2 and BMPR1a, as overexpressed CA BMPR1a spurred an activation of BMP signaling, while a dominant-negative form of BMPR2 failed to activate the cascade even during infection (Figure 1D). The same was also assessed utilizing a BMP responsive element (BRE) tagged to luciferase (Figure 1E). Among pattern recognition receptors (PRRs), TLR2 is known to play important roles in arbitrating the outcome of mycobacterial infections (26). TLR2 was observed to be indispensable for the ensuing activation of BMP signaling (Figure 1F). In order to assess the possibility of TGF-β receptors regulating BMP signaling, we evaluated the activation of BMP and TGF-β cognate response regulators p-Smad2/3 in the presence of ALK5 (a Type I TGF-β receptor kinase) inhibitor and found that BMP signaling operates independent of the TGF-β clade (Figures 1G,H). Thus, we established TLR2-dependent activation of BMP signaling during Mtb infection.

Earlier evidences suggested that c-Abl tunes BMP signaling through the direct phosphorylation of the BMP receptor BMPR1a in osteoblasts and thus activates downstream events (14, 15). In this respect, we found that c-Abl interacted poorly with BMPR1a during mycobacterial infection (Figure S1C in Supplementary Material). Further, although its threonine phosphorylation was robustly induced upon Mtb infection, its tyrosine phosphorylation status remained relatively unchanged (Figure S1D in Supplementary Material). These findings excluded a role for c-Abl in activating BMP signaling through its phosphorylation of BMPR1a. We next utilized Imatinib, a small-molecule inhibitor targeted against c-Abl, to negate the role of c-Abl in activating mycobacteria-dependent BMP signaling. Yet, when assessed in vitro at the protein level (Figure 2A), remarkably, Imatinib treatment curbed Mtb activated BMP signaling.

In addition to inhibiting Abl tyrosine kinases, Imatinib targets two other receptor tyrosine kinases, namely, c-KIT and platelet-derived growth factor receptor (PDGFR) with high affinity, albeit to varying degrees. Consistent reports suggest that the 50% inhibitory concentration (IC₅₀) values of Imatinib for c-Abl are approximately 10-fold lower than those for PDGFR and c-KIT (27–29), suggesting that Abl kinases are the foremost targets of Imatinib. In this context, we demonstrated a titration of Imatinib concentrations and assessed the phosphorylation of c-Abl, PDGFR and c-KIT in macrophages, NIH3T3, and K562 cells. We found that Imatinib demonstrated a substantial inhibition of c-Abl at a concentration of 5 μM and significantly reduced c-Abl phosphorylation at 10 μM (Figure S1E in Supplementary Material), while PDGF (Figure S1F in Supplementary Material) and c-Kit (Figure S1G in Supplementary Material) phosphorylation was inhibited at higher concentrations of Imatinib.

In order to assess this in vivo, we generated a mouse model of established TB utilizing Mtb H37Rv. Groups of mice were infected with Mtb H37Rv for 28 days, followed by treatment with vehicle or Imatinib at 28 days postinfection, for a total duration of 56 days (Figure 2B). In line with the in vitro data, lung IHC (Figure 2C) and transcript levels of hallmarks of BMP signaling in spleen (Figure 2D) of Mtb-infected mice demonstrated abrogated levels upon Imatinib treatment. Immunofluorescence studies focusing on the cell types expressing BMP2 revealed that macrophages predominantly display activated BMP signaling (appearance of yellow coloration through the co-localization of green BMP2 and red F4/80 signal), as do CD11c⁺ cells (mostly dendritic cells) to a lesser extent. Ly6G⁺ (neutrophils) cells however, displayed a poor activation of BMP signaling upon mycobacterial infection (Figure 2E). c-Abl overexpression sufficed the activation of BMP signaling, while a kinase dead (KD) form of c-Abl failed to bring the effect, highlighting that kinase activity of c-Abl was essential for the initiation of Mtb-driven BMP signaling (Figure S2A in Supplementary Material). While cellular c-Abl levels were unaffected during mycobacterial infection (Figure 2F), we observed its redistribution in the cytosolic and nuclear compartments. Upon infection, c-Abl was found to translocate increasingly to the nucleus (Figure 2G). This event was TLR2 driven (Figure 2H) and independent of TLR4 (Figure S2B in Supplementary Material). Thus, c-Abl was observed to regulate BMP signaling in macrophages, as in osteoblasts, yet the events leading to such an activation were unclear in the present scenario.

The nuclear translocation of c-Abl during mycobacterial infection excluded a conventional role for c-Abl at the cytoplasmic membrane in phosphorylating receptor BMPR1a (Figure 2). c-Abl possesses a less examined DNA binding domain that was demonstrated to bind and recruit transcriptional factors such as p53 (30). We revisited the possibility of c-Abl regulating mycobacteria responsive BMP ligands, BMP2 and BMP4. A prediction of potential transcription factor binding sites using MatInspector (Genomatix) identified c-Abl binding sites in the 2 kb region of the promoters of BMP2 and BMP4 (Figure S2C in Supplementary Material). Further, a recent study demonstrated that lysine acetyl transferase KAT5, also known as Tip60, is a c-Abl partner (31). Here, Kaidi and Jackson reported c-Abl facilitated tyr44 phosphorylation of KAT5, thus rendering it active. We found an increased association of c-Abl with endogenous KAT5 during Mtb infection (Figure 3A). In addition, KAT5 levels were unperturbed during infection but its phosphorylated tyrosines (pY) were significantly increased. Further, inhibition of kinase activity of c-Abl brought about by Imatinib also resulted in a loss of phosphorylated tyrosine of KAT5 (Figure 3B) during infection. Interestingly, RAW264.7 macrophages transfected with c-Abl KD elucidate reduced c-Abl-KAT5 interaction (Figure 3C) during infection. When KAT5YF (Tyr44 was mutated to a non-phosphorylatable phenylalanine) was overexpressed in macrophages, followed by Mtb infection, BMP signaling failed to be activated (Figure 3D), thus underscoring the importance of c-Abl phosphorylated KAT5 in driving BMP signaling during Mtb infection. We observed increased H4K5 acetylation at the promoters of BMP2 and BMP4 upon Mtb infection, while overexpression of c-Abl KD or KAT5YF failed to bring about a similar effect (Figure 3E), suggesting an important role for KAT5-mediated H4K5 acetylation and subsequent activation of BMP2 and BMP4 promoters. Yet, c-Abl does not possess the ability for transcriptional activation. The promoters of BMP2/4 also indicated multiple TWIST1 binding sites (Figure S2C in Supplementary Material). TWIST1 has been demonstrated to play varied roles in the regulation of BMP signaling (32–34). We observed elevated levels of TWIST1 upon Mtb infection (Figure 4A). However, TWIST1 overexpression failed to simulate the activation of BMP signaling (Figure 4B), suggesting the necessity of mycobacteria triggered molecular machinery in orchestrating its activation. In support of these hypotheses, we observed an increased association of c-Abl-KAT5-TWIST1 during Mtb infection (Figure 4C). In line with these observations, we also confirmed the increased recruitment of TWIST1 to BMP2/4 promoters in the course of infection, which was hampered in the presence of c-Abl KD (Figure 4D). In addition, there was an increased recruitment of KAT5 to the promoters of BMP2 and BMP4 at their TWIST1 binding sites (Figure 4E), which was lost upon transfection of c-Abl KD in RAW264.7 macrophages. siRNA-mediated depletion of c-Abl or TWIST1 failed to trigger Mtb activated BMP signaling (Figures 4F–H), thus establishing a conclusive role for TWIST1 in conjunction with c-Abl-KAT5 in coordinating the regulation of BMP pathway.

We attempted to decipher the signaling events resulting in reduced survival of mycobacteria upon treatment with Imatinib. ROS and Reactive Nitrogen Intermediates (RNI) are recognized as important players mediating host protection during mycobacterial infection (35, 36). In this respect, we found Mtb stimulated ROS levels were unaffected in the presence of Imatinib or Compound C (Figure 5A), DPI treated cells were used as a control. Compound C (or Dorsomorphin) is an ALK2 (ActR1a), ALK3 (BMPR1a), and ALK6 (BMPR1B) inhibitor known to affect BMP-mediated Smad1/5/8 phosphorylation (37), while DPI (Diphenyleneiodonium) inhibits the production of ROS through the inhibition of NAD(P)H oxidase. However, cellular levels of nitrite were enhanced upon pretreatment of cells with both these inhibitors (Figure 5B), N-(3-(aminomethyl)benzyl)acetamidine (1400W) is a selective inhibitor for iNOS and was used as a control. This was also corroborated by elevated iNOS in the presence of Imatinib and Compound C (Figure 5C, left). Further, macrophages transfected with siRNA targeted against Abl1 recapitulated increased iNOS levels (Figure 5C, right). A study of the regulators of iNOS revealed that IFN-γ stimulated STAT1 and STAT3 phosphorylation are abrogated in response to mycobacterial infection (38), although there exist contrary reports. We found that Mtb requires activated BMP signaling to prevent the phosphorylation of STAT1/3 (Figure 5D). Mycobacteria regulated abrogation of STAT1/3 phosphorylation was also dependent on c-Abl, as c-Abl overexpression exhibited similar effects (Figure 5E). Further, Poly I:C induced activation of STAT1/3 was also inhibited by transient overexpression of c-ABL WT, yet this effect was not observed when c-Abl KD was overexpressed (Figure 5F). Taken together, we propose that Mtb employ c-Abl activated BMP signaling to maintain a check on nitric oxide induced upon infection in murine macrophages.

The Mtb granuloma is a defining yet complex structure, and there remains much to be discovered regarding forces orchestrating this phenomenon (39). In an in vivo established TB model (Figure 2B), characteristic solid lesions were observed in greater numbers on the interstitium of infected mice lungs as compared to that in Mtb-infected mice treated with Imatinib (Figure 6A). Gross histopathology of the lungs revealed merely degenerating neutrophil containing foci in Imatinib treated mice (Figure 6A, lower panel), while untreated mice demonstrated active granulomatous structures with foam cells (Figure 6A, upper panel). Treatment with Imatinib resolved mycobacterial granuloma, as was evident from the granuloma scores (Figure 6B). Histopathology and scoring was also performed for PBS injected mice at the end of 56 days (Figures 6A,B). Mtb H37Rv infected mice displayed a discernable splenomegaly as compared to Imatinib treated mice (Figure 6C). Day 1 and Day 28 CFUs were assessed in the lungs of Mtb infected mice (Figures 6D,E). Lung weights (Figure 6F) and splenic weights (Figure 6G) of Imatinib treated mice were significantly lower than that of untreated mice, as were the reduced mycobacterial CFUs with Imatinib at Day 56 (Figures 6H,I). Our findings underscore an important role for c-Abl in the success of established mycobacterial infections.

Several instances report the cross-regulation among PRRs (40–42). We observed that Poly I:C triggered increase of Type I interferons (IFN-α and IFN-β) was inhibited substantially upon transient overexpression of c-Abl, and further, there was a rescue in their levels upon overexpression of c-Abl KD during Mtb infection (Figure 7A). Poly I:C mimics the effects of naturally occurring dsRNA and majority of its pleiotropic effects have been attributed to recognition by TLR3. In an in vivo model of TLR3-triggered events as depicted (Figure 7B), intravenous injection of Poly I:C resulted in a robust upregulation of IFN-α and IFN-β in lungs (Figure 7C) and spleens (Figure S3A in Supplementary Material) of these mice, while mice with established TB injected Poly I:C 12 h prior to sacrifice, manifested a fairly dampened IFN-α and IFN-β response. Further, we observed that this abrogation of TLR3 stimulated events was dependent on BMP signaling, as these events failed to unfold upon inhibition of BMP signaling using Compound C (Figure 7D). We found that type I interferon response was also elevated in mycobacteria-infected mice treated with Imatinib for 28 days, suggesting that treatment with Imatinib potentiated immune responses (Figure 7E; Figure S3B in Supplementary Material). In addition, Poly I:C induced TLR3 adaptor TICAM1 was downregulated during Mtb infection and this inhibition was lost if cells were pretreated with Compound C and Imatinib (Figure 7F) or siRNA directed against c-Abl (Figure 7G). Collectively, these results suggest that Mtb infection strongly inhibits Poly I:C induced inflammatory signature.

Tuberculosis infection is known to result in a deregulation of several host miRNA that are fundamental posttranscriptional regulators of gene expression. In this context, we observed Smad1/5 binding elements at the promoter of miR27a when assessed by MatInspector (Genomatix) (Figure 8A). These findings prompted us to assess the levels of miR27a during Mtb infection. miR27a was upregulated in vitro (Figure 8B) as well as in vivo in the lungs and spleen (Figure 8C) of Mtb infected mice. This upregulation was obstructed in vivo upon Imatinib treatment (Figure 8C) and in vitro upon pretreatment of macrophages with Compound C (Figure 8D). We validated our findings of induced miR27a through the overexpression of c-Abl WT and c-Abl KD during Mtb infection (Figure 8E). In addition, overexpression of c-Abl WT in the presence of Compound C failed to affect the expression of miR27a (Figure 8E), thus revealing a linear axis comprising c-Abl-BMP signaling which regulates miR27a. siRNA targeted against c-Abl prevented the Mtb induced upregulation of miR27a (Figure 8F), further underscoring the importance of the said sequence of events in directing the escalation of miR27a. Systematic bioinformatic analysis (Target Scan, miRanda, and miRWalk) identified target sites located at the residues spanning from 2,725–2,731 in the 3′UTR of TICAM1 as critical for its interaction with miR27a (Figure 8G). Enforced expression of miR27a inhibitor failed to bring about Mtb mediated negative regulation of TICAM1, while its mimic was sufficient to abrogate Poly I:C induced TICAM1 (Figure 8H). In addition, Mtb infection or transfection with miR27a mimic markedly reduced WT TICAM1 3′UTR luciferase activity. However, the reduction was not significant when mutant (for miR27a binding) TICAM1 3′UTR construct was utilized (Figure 8I). Altogether, Mtb responsive miR27a targeted Poly I:C induced signaling through the abrogation of TICAM1.

For many years, mycobacteria have exploited macrophages as their principle niche, which is also evident in this case of activated BMP pathway. To understand the mechanism of Mtb mediated modulation of BMP signaling, we employed Mtb mutants, namely, MtbΔwhiB3, MtbΔdosR, and MtbΔsecA that have been implicated in a global modulation of Mtb architecture. We observed that MtbΔdosR and MtbΔwhiB3 failed to activate BMP signaling (Figure 9A) as well as miR27a (Figure 9B). SecA2 was not seen to play a role in these events. Yet, unsurprisingly, iNOS was elevated in all three Mtb mutants (Figure 9A). Further, we chose to focus on the role for DosR and WhiB3 in mycobacterial pathogenesis and found that macrophages infected with MtbΔdosR and MtbΔwhiB3 also cross-regulated the TLR3 clade induced by Poly I:C (Figure 9C) including their effect on Type I interferons, Ifn-α and Ifn-β (Figure 9D). Altogether, our findings reveal novel roles for Mtb WhiB3 and DosR in regulating specific host signaling cascades (Figure 9E).