PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis-Infected Macrophages

Abstract

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis in cattle population across the world. Human beings are at equal risk of developing tuberculosis beside a wide range of M. bovis infections in animal species. Autophagic sequestration and degradation of intracellular pathogens is a major innate immune defense mechanism adopted by host cells for the control of intracellular infections. It has been reported previously that the catalytic subunit of protein phosphatase 2A (PP2Ac) is crucial for regulating AMP-activated protein kinase (AMPK)-mediated autophagic signaling pathways, yet its role in tuberculosis is still unclear. Here, we demonstrated that M. bovis infection increased PP2Ac expression in murine macrophages, while nilotinib a tyrosine kinase inhibitor (TKI) significantly suppressed PP2Ac expression. In addition, we observed that TKI-induced AMPK activation was dependent on PP2Ac regulation, indicating the contributory role of PP2Ac towards autophagy induction. Furthermore, we found that the activation of AMPK signaling is vital for the regulating autophagy during M. bovis infection. Finally, the transient inhibition of PP2Ac expression enhanced the inhibitory effect of TKI-nilotinib on intracellular survival and multiplication of M. bovis in macrophages by regulating the host's immune responses. Based on these observations, we suggest that PP2Ac should be exploited as a promising molecular target to intervene in host-pathogen interactions for the development of new therapeutic strategies towards the control of M. bovis infections in humans and animals.

Keywords: AMPK; Mycobacterium bovis (M. bovis), PP2Ac; TKI; autophagy; macrophages.

Figure 1

PP2Ac expression in BMDM cells infected with M. bovis. (A–C) BMDM cells were infected with M. bovis (multiplicity of infection (MOI) 5, 10, 20 and 40) for 24 h. (A,B) Cell lysates were used for PP2Ac protein expression by Western blot (WB) assay. (C) Total RNA was extracted and the expression of PP2Acα at mRNA level was determined by quantitative real-time polymerase chain reaction (qRT-PCR). (D,F) BMDM cells were infected with M. bovis (MOI 1:10) for indicated time periods. (D,E) Cell lysates were used for the expression of PP2Ac protein level by WB. β-actin was used as loading control. (F) mRNA level of PP2Acα was determined by qRT-PCR. Data represent the mean ± standard deviation (SD) from three independent experiments. (hpi stands for hours post infection) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

PP2Ac expression in RAW264.7 cells infected with M. bovis. (A–C) RAW264.7 cells were infected with M. bovis (MOI 5, 10, 20 and 40) for 24 h. (A,B) Cell lysates were used for PP2Ac protein expression by WB assay. (C) Total RNA was extracted and the expression PP2Acα at mRNA level was determined by qRT-PCR. (D,F) RAW264.7 cells were infected with M. bovis (MOI 1:10) for indicated time period. (D,E) Cell lysates were used for the expression of PP2Ac protein by WB. β-actin was used as loading control. (F) The mRNA level of PP2Acα was determined by qRT-PCR. Data represent the mean ± SD from three independent experiments. (MOI stands for multiplicity of infection, hpi stands for hours post infection) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Targeting PP2Ac via tyrosine kinase inhibitor (TKI)-nilotinib in M. bovis infected macrophages. (A–E) BMDM cells were pretreated with TKI-nilotinib (0 µM or 10 µM) (DMSO 0.1% used as sample diluent control) for 2 h and then infected with M. bovis (MOI 1:10) for indicated time periods. The expression level of (B) PP2Ac, (C) p-AMPK and (D) p-ULK1 were evaluated by using WB and normalized to β-actin. (E) The relative expression of PP2Acα at mRNA level was determined by using qRT-PCR. (F–J) RAW264.7 cells were treated with TKI-nilotinib (0 µM and 10 µM) followed by M. bovis (MOI 1:10) infection for indicated time periods. The expression levels of (F) PP2Ac, (G) p-AMPK and (H) p-ULK1 cells lysates were determined by WB and normalized to β-actin. (J) The relative expression of PP2Acα at mRNA level was determined by using qRT-PCR. Data represent the mean ± SD from three independent experiments. (hpi stands for hours post infection) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Upregulation of PP2Ac abrogates autophagy via modulating AMP-activated protein kinase (AMPK) activation during M. bovis infection. (A) BMDM cells were pretreated with okadaic acid (10 nM), TKI-nilotinib (10 µM) alone or okadaic acid + nilotinib followed by M. bovis (MOI 1:10) 24 h (DMSO 0.1% used as solvent control). The expression levels of (B) PP2Ac, (C) p-AMPK, (D) LC3-II, and (E) P62 were determined by WB and normalized against β-actin. Data represents the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments ) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Down-regulation of PP2Ac promotes autophagy via mediating AMPK activation during M. bovis infection. (A) BMDM cells were pretreated with forskolin (10 µM), TKI-nilotinib (10 µM) alone or forskolin + nilotinib followed by infection with M. bovis (MOI 1:10) 24 h (DMSO 0.1% used as solvent control). The expression levels of (B) PP2Ac, (C) p-AMPK, (D) LC3-II, and (E) P62 were determined by WB and normalized against β-actin. Data represents the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

AMPK agonist contributes the regulation of autophagy by TKI (nilotinib) in M. bovis-infected macrophages. (A) BMDM cells were treated with metformin (10 µM), TKI-nilotinib (10 µM), alone or metformin + TKI-nilotinib followed by infection with M. bovis (MOI 1:10) for 24 h (DMSO 0.1% used as solvent control). The expression levels of (B) p-AMPK, (C) LC3-II and (D) P62 were determined by using WB and normalized against GAPDH. Data represent the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

AMPK inhibitor restrict the effect of TKI (nilotinib) on the regulation of autophagy in M. bovis infected macrophages. (A) BMDM cells were treated with compound-c (10 µM), TKI-nilotinib (10 µM) alone or compound-c + TKI-nilotinib followed by infection with M. bovis (MOI 1:10) for 24 h (DMSO 0.1% used as solvent control). The expression levels of (B) p-AMPK, (C) LC3-II and (D) P62 were determined by WB and normalized to GAPDH. Data represent the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 8

PP2Ac promotes intracellular survival of M. bovis in murine macrophages. (A) PP2Ac protein level was determined by WB from BMDMs transfected with 50 µM SiRNA negative control and SiRNA PP2Ac for 36 h. (B) BMDMs were transfected with 50 µM SiRNA negative control and SiRNA PP2Ac and treated or untreated with TKI-nilotinib (10 µM) followed by infection with M. bovis for 24 h. The expression levels of (C) p-AMPK, (D) LC3-II and (E) P62 were determined by WB and normalized against β-actin. (F) The colocalization of LC3 with M. bovis was determined by confocal microscopy. (G) The colocalization % of LC3 was calculated by image-J software. (H) BMDMs were transfected with 50 µM SiRNA negative control and SiRNA PP2Ac and treated or untreated with nilotinib (10 µM) followed by infection with M. bovis for 24 h. A colony-forming unit (CFU) assay was performed for enumeration of total viable M. bovis bacilli. Data represent the mean ± SD from three independent experiments. (“−“refers the untreated group and “+” refers the treated group for the mentioned treatments ) (* p < 0.05, ** p < 0.01, *** p < 0.001).