Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth

Abstract

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

Figure 1. High-content imaging assay development and categorization of screen hits.

(A) J774 cells in 96-well dishes were infected with GFP-expressing H37Rv at an MOI of 1∶1. Cells were then treated with rifampin at the indicated concentrations. Day 3 after infection, cells were fixed, stained with DAPI, and imaged for both DAPI and GFP. Images were analyzed using CellProfiler to determine the mean integrated GFP intensity across each well normalized to macrophage number. The graph represents one of four independent experiments with data represented as an average +/− SEM for each condition for 84 wells. (B) Of 133 unique hits in the screen, 41 were agonists or antagonists at G-protein coupled receptors, 16 were inhibitors or activators of ion channels, 15 were kinase activators or inhibitors, 5 were membrane transport modulators, 7 were anti-inflammatory agents, and 46 did not fall into one of the above major categories.

Figure 2. Selected screen hits have dose-dependent activity in J774 cells and bone-marrow derived macrophages.

Selected hit compounds were retested at varying doses in (A) J774 murine macrophages and (B) mouse bone marrow-derived macrophages (BMDM). Cells were infected with M. tuberculosis strain H37Rv at an MOI of 1∶1 and treated with each compound at the indicated doses (µM) after a 4 h phagocytosis period. At day 3 (J774) or day 5 (BMDM) after infection, cells were washed, lysed, and plated for CFU. Each column represents the mean and standard deviation of four biological replicates. Each experiment was repeated three times and a representative experiment is shown. With the exception of fluoxetine at 6.25 µM, all p-values were <0.05 for the comparison of each compound treatment condition with DMSO. p-values were calculated using the Mann Whitney U test.

Figure 3. Specific compounds enhance autophagy or inflammatory cytokine release.

(A) J774 murine macrophages were infected with H37Rv, then treated with the indicated compounds. Cells were harvested for Western-blot analysis of LC3 conversion from LC3-I to LC3-II as an indicator of autophagy. Data represents one of three independent experiments. Densitometry for LC3-II to LC3-I ratio for the shown Western blot was performed using ImageJ image analysis software. (B) Cells were infected with H37Rv, then treated with compound after a 4 hour phagocytosis. Supernatants were collected at 24 hours after infection and TNF-α concentration was determined by ELISA. Points represent average for 2 wells +/− standard deviation. Data represents one of two independent experiments.

Figure 4. Inhibitors of protein kinases impair mycobacterial growth in macrophages.

(A) J774 cells or (B) BMDM were infected with M. tuberculosis strain H37Rv at an MOI of 1∶1, and treated with each kinase inhibitor at the indicated concentrations (µM) after a 4 h phagocytosis period. At day 3 (J774) or day 5 (BMDM) after infection, cells were washed, lysed, and plated for CFU. Each column represents the mean and standard deviation of four biological replicates and each graph represents one of three independent experiments. For (A) all p<0.001 with the exception of GNF-2 at 2.5 µM, imatinib at 5 µM and gefitinib at 5 µM. For (B) inhibitors were used at the following concentrations: AKTi1/2 5 µM (p<0.03), H-89 5 µM (p<0.03), GNF-2 10 uM (not significant), imatinib 5 µM (p<0.03), gefitinib 5 µM (p<0.03), lapatinib 5 µM (p = <0.06). All p values were calculated using the Mann Whitney U test.

Figure 5. AKT and ABL are important for mycobacterial infection of macrophages.

(A) J774 cells were infected with H37Rv at an MOI of 1 or an MOI of 10. Cells were harvested two hours after infection, and lysates were probed for AKT activation using an antibody to phospho-serine at position 473. (B) J774 cells were infected with H37Rv at an MOI of 1 for 4 hours, then washed and treated with compound. Cells were harvested at 3 hours after treatment and lysates were probed for AKT activation using an antibody to phospho-serine at position 473. (C) akt1 and akt2 or akt1, akt2, and akt3 were silenced with siRNA in J774 cells. Cells were then infected with H37Rv at an MOI and 1, and infection was allowed to progress for 3 days. Cells were then lysed and plated for CFU. (D) abl1 was silenced in J774 macrophages using siRNA. Cells were then infected with H37Rv. After 4 hours of phagocytosis, wells were lysed and plated for CFU to determine uptake (day 0). Infection was allowed to progress in the remaining wells; day 3 after infection, cells were lysed and plated for CFU. Each experiment was repeated a minimum of three times, and a representative experiment is shown. Error bars are standard deviation, *p = 0.0195 by Mann Whitney U for (C) and (D).

Figure 6. EGFR is important for mycobacterial infection of macrophages and in a mouse model of infection.

(A) human primary PBMC derived macrophages were infected with a luciferase-expressing Erdman strain, then treated with EGFR-neutralizing antibodies (αEGFR) and/or gefitinib at 10 µM (gef) 4 h after the phagocytosis period. Antibodies and inhibitor was refreshed daily, and bacterial growth was determined by luminescence on day 5 after infection. The data is representative of two independent experiments. (B) J774 cells were infected with M. tuberculosis strain H37Rv. 4 hours after infection, cells were washed and gefitinib or DMSO was added. Cells were lysed at 15 minutes, 30 minutes, or 60 minutes after drug treatment and lysates were probed for phospho-p38 and total p38 by Western blot. The data is representative of three independent experiments. (C) J774 cells were infected with H37Rv-GFP. 4 hours after infection, cells were washed and DMSO or AMG548 was added at the indicated concentrations. Day 3 after infection, cells were washed and fixed with 4% paraformaldehyde. Plates were imaged and analyzed using our CellProfiler pipeline to determine integrated GFP intensity normalized to macrophage number. The data represent one of two independent experiments. (D) Mice were infected with M. tuberculosis strain Erdman via the aerosol route with ∼200 CFU and infection was allowed to progress for 7 days. Beginning day 8 after infection, mice were treated with DMSO or gefitinib 100 mg/kg by intraperitoneal injection daily for six days. Day 14 after infection, mice were sacrificed and lungs were plated for CFU. Data combined from three independent experiments is shown. ***p<0.001, *p = 0.034 by non-parametric Mann-Whitney U test. Error bars are standard deviation.