Mycobacterium tuberculosis Exploits Focal Adhesion Kinase to Induce Necrotic Cell Death and Inhibit Reactive Oxygen Species Production

Abstract

Tuberculosis is a deadly, contagious respiratory disease that is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb is adept at manipulating and evading host immunity by hijacking alveolar macrophages, the first line of defense against inhaled pathogens, by regulating the mode and timing of host cell death. It is established that Mtb infection actively blocks apoptosis and instead induces necrotic-like modes of cell death to promote disease progression. This survival strategy shields the bacteria from destruction by the immune system and antibiotics while allowing for the spread of bacteria at opportunistic times. As such, it is critical to understand how Mtb interacts with host macrophages to manipulate the mode of cell death. Herein, we demonstrate that Mtb infection triggers a time-dependent reduction in the expression of focal adhesion kinase (FAK) in human macrophages. Using pharmacological perturbations, we show that inhibition of FAK (FAKi) triggers an increase in a necrotic form of cell death during Mtb infection. In contrast, genetic overexpression of FAK (FAK⁺) completely blocked macrophage cell death during Mtb infection. Using specific inhibitors of necrotic cell death, we show that FAK-mediated cell death during Mtb infection occurs in a RIPK1-depedent, and to a lesser extent, RIPK3-MLKL-dependent mechanism. Consistent with these findings, FAKi results in uncontrolled replication of Mtb, whereas FAK⁺ reduces the intracellular survival of Mtb in macrophages. In addition, we demonstrate that enhanced control of intracellular Mtb replication by FAK⁺ macrophages is a result of increased production of antibacterial reactive oxygen species (ROS) as inhibitors of ROS production restored Mtb burden in FAK⁺ macrophages to same levels as in wild-type cells. Collectively, our data establishes FAK as an important host protective response during Mtb infection to block necrotic cell death and induce ROS production, which are required to restrict the survival of Mtb.

Keywords: Mycobacterium tuberculosis (Mtb); focal adhesion kinase (FAK); host-directed therapy; macrophage cell death; necroptosis; protein tyrosine kinase 2 (PTK2); reactive oxygen species.

Figure 1

Mtb infection downregulates FAK expression. (A) THP-1 macrophages were infected with Mtb at a multiplicity of infection (MOI) of 10. Cell lysates were prepared at the indicated time points post-infection, and total FAK protein levels were analyzed by western blot. (B) PTK2 expression levels in THP-1 macrophages infected with Mtb as in (A) were quantified using qRT-PCR at the indicated days post infection. (C) RAW 264.7 cells were infected Mtb as in (A), and cell lysates were prepared at the indicated time post infection. Blots shown in (A, B) are representative of three independent experiments. Densitometry analysis of the blots was performed using Image Quant LAS 4000 software with intensities normalized to GAPDH. Relative fold change of total FAK levels was expressed relative to uninfected cells. (D) Ptk2 expression levels in RAW 264.7 macrophages were measured as in (B). qRT-PCR data were analysed using the ΔΔCT method normalizing to (B) ACTB or (D) GAPDH as a reference gene, and fold change is expressed relative to uninfected cells. qRT-PCR data represent the mean of three replicates. ***p < 0.001.

Figure 2

Genetic overexpression and pharmacological inhibition of FAK. (A) Total FAK protein levels in cell lysates of THP-1 and THP-1 cells stably overexpressing FAK (THP-FAK⁺) were analyzed by western blot. (B) THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10 and cell lysates were prepared at indicated time points, and analysed by western blot. Blots shown in (A, B) are representative of three independent experiments. Densitometry analysis of the blots were performed using Image Quant LAS 4000 software with intensities normalized to GAPDH. Relative fold change of total FAK levels was expressed relative to THP-1 cells (A) and uninfected THP-FAK⁺ cells (B). (C, D) THP-1 macrophages were treated with varying concentrations of PF-573-228 for 5 days, and (C) representative phase-contrast microscopy (20x objective) images are shown along with (D) quantification of cell viability using the resazurin assay. (E, F) RAW 264.7 macrophages were treated with varying concentrations of PF-562-271 for 5 days, and (E) representative phase-contrast microscopy (20x objective) images are shown along with (F) quantification of cell viability using the resazurin assay. Images are representative of 3 visual fields for each inhibitor concentration. Relative viability is normalized to untreated control cells as 100%. Error bars represent the mean ± SD of three independent biological replicates. Scale bar, 100 μm.

Figure 3

FAK expression alters cytokine production by macrophages infected with Mtb. THP-1, THP-FAKi and THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10 and cell culture supernatants were collected at 24 hours post-infection. Production of (A) TNF-α (B) IL-1β (C) IL-10 were measured in cell culture supernatants using a human ELISA assay kit. Error bars represent the mean ± SD of three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

FAK is required to block cell death of Mtb-infected macrophages. (A) THP-1, THP-FAKi and THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10, after which changes in adherence (viability) were monitored by RTCA as indicated by the Cell Index (CI). CI was measured continuously every hour for 120 hours post-infection. The data represent the mean of three independent biological replicates. (B) THP-1, THP-FAKi, THP-FAK⁺ macrophages, (C) mBMDMs, and (D) RAW 264.7 cells were infected with Mtb at an MOI of 10 and cell viability was quantified using Cell Titer-Glo (measured as Relative Luminescence Units, RLU). Data in (C, D) represent 4 days post-infection. (E) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10, and at the indicated time points post-infection, cells were stained with Fixable Viability Stain (FVS780) dye and analyzed using flow cytometry to measure the percentage of viable cells. Error bars represent the mean ± SD of three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

FAK controls necroptotic cell death in macrophages during Mtb infection. (A) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10, and cell lysates were prepared at the indicated days post-infection (d.p.i.). Total and cleaved caspase 3, and total FAK protein levels were analyzed by western blotting. Non-infected THP-1 cell lysates were used as a negative control for caspase 3 activation, while lysates containing cleaved caspase-3 (#9663, Cell Signaling Technologies) were used as a positive control. β-tubulin was used as loading control. (B) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected as in (A) and at the indicated days post-infection, cells were stained with annexin V and FVS780. Stained cells were analyzed by flow cytometry to quantify the percentage of healthy, apoptotic, and necrotic cells as described in the Methods. (C) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected with Mtb at an MOI of 10, and lactate dehydrogenase (LDH) released in culture supernatants were assessed using the CYQUANT LDH kit at indicated days post-infection. The amount of LDH in the supernatant is proportional to the measured absorbance at 490 nm. (D) THP-1, THP-FAKi and THP-FAK⁺ macrophages were mock treated or pre-treated with 1 µg/ml LPS for 4 h, followed by treatment with 5 µM nigericin for 24 h Macrophage viability was assessed using the Cell Titre-Glo assay. (E–G) THP-1, THP-FAKi, and/or THP-FAK⁺ macrophages were mock treated or pre-treated with (E) necrostatin-1s (Nec-1s, 10 µM), (F) GSK-872 (5 µM), or (G) necrosulfonamide (NSA, 10 µM) for 24 h Cells were then infected with Mtb at an MOI of 10 for 6 days, and cell viability was assessed using the Cell Titer-Glo assay. Error bars represent the mean ± SD of three independent biological replicates. *p < 0.05, ***p < 0.001, ns, non-significant.

Figure 6

Expression of FAK controls Mtb survival. (A) Mtb was treated with the indicated concentrations of FAK inhibitors (in the absence of macrophages), PF-573-228 and PF-562-271, for 5 days, after which Mtb growth was assessed using the resazurin viability assay. Relative viability was normalized to 100% using untreated controls. (B) THP-1, THP-FAKi, and THP-FAK⁺ macrophages, or (C) RAW 264.7 and RAW 264.7-FAKi macrophages were infected with Mtb-luciferase at an MOI of 10 for 6 days. Infected macrophages were lysed at the indicated time points post-infection and the resultant luminescence signal (RLU) was measured as a proxy for the relative number of viable of Mtb. (D, E) THP-1 macrophages were mock treated or pre-treated with (D) 30 µM necrostatin-1 or (E) 5 µM GSK-872 for 24 h, and subsequently infected as in (B). Infected macrophages were lysed at the indicated time points post-infection, and the resultant luminescence signal (RLU) was measured as a proxy for the relative number of viable of Mtb. Error bars represent the mean ± SD of three independent biological replicates. **p < 0.01, ***p < 0.001.

Figure 7

Expression of FAK induces production of ROS to control Mtb infection. (A) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were infected with Mtb-tdTomato (red signal) for 6 days, after which cells were stained with DCFH-DA (5 μM, green signal) for 30 minutes at 37°C. Representative Bright Field and fluorescence images of uninfected and Mtb-infected (red) macrophages (green, ROS), are shown using a 20x objective. Scale bar, 100 μm. (B) THP-1 macrophages were infected and stained with DCFH-DA as in (A) and the amount of ROS production was quantified at the indicated time points post-infection using a fluorescence plate reader at 485 nm/535 nm. (C) RAW 264.7 macrophages were infected and stained as in (A) and the amount of ROS production was quantified at day 6 post-infection. (D) THP-1 and THP-FAK⁺ macrophage were pre-treated with 10 mM N-acetyl-cysteine (NAC) or 25 µM GSK2795039 for 24 h and then infected with Mtb-luciferase at an MOI of 10. Infected macrophages were lysed at day 6 post-infection and the resultant luminescence signal was measured as a proxy for the relative number of viable of Mtb. RLU signals were normalised to mock treated THP-1 cells as 100% and depicted as % Mtb survival. (E) THP-1, THP-FAKi, and THP-FAK⁺ macrophages were mock treated or pre-treated with 30 µM necrostatin-1 for 24 h, after which cells were infected with Mtb at MOI of 10. At indicated time points post-infection, infected macrophages were stained with DCFH-DA as in (A) and fluorescence signals for ROS production was measured. Error bars represent the mean ± SD of three independent biological replicates. *p < 0.05, ***p < 0.001.