The role of SIRT1 in the process of Toxoplasma gondii infection of RAW 264.7 macrophages

Abstract

Toxoplasma gondii is an opportunistic pathogenic protozoan that can infect almost all kinds of warm-blooded animals, including humans. T. gondii can evade the host's immune response, a process known as immune evasion. Our main objective was to evaluate the role played by Sirtuin1 (SIRT1) [one of the sirtuins (SIRTs) that are a family of nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases (HDACs)] in the T. gondii infection of RAW264.7 macrophages. In this study, we evaluated and observed alterations in the activity, expression, and localization of SIRT1 and assessed its involvement in the CD154/IFN-γ (CD40 ligand/interferon gamma) killing pathway and in autophagy during T. gondii infection. The inhibition of SIRT1 in host cells effectively reduced the number of intracellular tachyzoites, and the mechanism behind this effect might be the upregulation of IRGM1 [murine ortholog of IRGM (immunity-related GTPase family M)] and the initiation of autophagy. To the best of our knowledge, our study is the first to prove that T. gondii infection upregulates SIRT1 in RAW264.7 cells and that the inhibition of SIRT1 reduces the number of intracellular tachyzoites. Moreover, the upregulation of IRGM1 and the activation of autophagy may contribute to the intracellular inhibition of T. gondii caused by SIRT1 inhibition.

Keywords: SIRT1; Toxoplasma gondii; autophagy; innate immunity; macrophages.

Figure 1

Changes in the expression, activity, location, and degradation of Sirtuin 1 (SIRT1) at different stages of Toxoplasma gondii infection. RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection. The NAD⁺/NADH (nicotinamide adenine dinucleotide) samples of the macrophages were extracted at 2, 18, and 24 h after infection of uninfected cells. The specific details of NAD⁺/NADH ratio measurement are illustrated in the NAD⁺/NADH ratio assay of the Experimental Methods section. The total protein of the host cells was extracted at 2, 18, and 24 h after infection, and the total SIRT1 level was detected by Western Blot. The confocal samples of the macrophages were extracted at 2, 36, and 45 h after infection of uninfected cells. (A) T. gondii infection leads to an increment in the NAD⁺/NADH ratio of host cells over time, indicating the upregulation of SIRT1 activity. The ratio of NAD⁺/NADH in murine macrophages did not increase significantly at 2 h of RH tachyzoite infection, but at 18 and 24 h, the ratio of NAD⁺/NADH increased significantly and increased with the prolongation of infection time. (B) T. gondii infection upregulated SIRT1 levels in host cells over time. (C) Fluorescent graphics showing the SIRT1 expression and lysosome activity of macrophages infected with RH tachyzoites for 2, 36, and 45 h. In the absence of T. gondii, SIRT1 in murine macrophages is predominantly expressed in the nucleus. With the prolongation of RH tachyzoite infection time, the expression of SIRT1 in host cells increased. Furthermore, the distribution of SIRT1 in murine macrophages also gradually shifted mainly from the nucleus to the cytoplasm, and gradually accumulated in the location of intracellular T. gondii. It also resulted in an upregulated activity of lysosomes, as seen by the red fluorescence. The intracellular T. gondii gradually accumulated in the lysosomes. These results were obtained from three independent experiments. The values shown are the means ± SEM from three independent experiments. SIRT1, green fluorescence; Lysotracker, red fluorescence; DAPI, blue fluorescence, PMT (photomultiplier tube), the light field with PMT as the detector and laser 488 nm as the light source. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2

Intracellular growth of RH tachyzoites with SRT1720 and EX527 in vitro. RH tachyzoites were harvested to infect RAW 264.7 mouse macrophages at an infection ratio of 5:1 (parasite/host cell) for 2 h, followed by the removal of excess parasites and stimulation with SRT1720 (5 μM) and EX527 (10 μM) for 24 h and further examination by staining with Wright's stain. RAW 264.7 cells stimulated with SRT1720 (5 μM) and EX527 (10 μM) for 2, 6, 12, or 18 h were collected and further examined by transmission electron microscopy (TEM) or lysed for Western Blot. We adjusted the protein concentration of each group of samples to be consistent and then carried out the experiment. Anti-SIRT1 antibody and anti-beta-Actin antibody were used at a 1:1,000 dilution. (A) T. gondii infection upregulated SIRT1 levels in host cells over time. Infected macrophages treated with SRT1720 exhibited an upregulation of SIRT1 at 2 and 18 h compared to that for the T. gondii infection group. Infected macrophages treated with EX527 showed a downregulation of SIRT1 at 2 and 18 h, compared to that for the T. gondii infection group. (B) A scattered distribution chart reveals the mean intracellular tachyzoite counts. Values represent actual numbers (scattered dots) and means (black lines) from three independent experiments; in each experiment, at least 200 infected cells were counted. The average number of T. gondii tachyzoites in macrophages stimulated by EX527 was significantly reduced compared to the control group. The average number of tachyzoites in the control group was about 10, while the average number of tachyzoites in the EX527 group dropped to about 5. T.g, T. gondii tachyzoites ***P < 0.001. The infected macrophages treated with SRT1720 had a much heavier intracellular tachyzoite burden than those incubated with EX527. (C) The number of T. gondii tachyzoites in the cell increased with the duration of the infection. At the same time, compared with the control group, the group with SRT1720 addition had more intracellular T. gondii tachyzoites. In contrast, the EX527 stimulation group had fewer intracellular T. gondii tachyzoites than the control group. These results were obtained from three independent experiments. The values shown are the means ± SEM from three independent experiments. RH, T. gondii RH strain. ***P < 0.001.

Figure 3

Intracellular growth of RH tachyzoites inside mouse macrophages with interferon gamma (IFN-γ) or CD154 in vitro and Sirtuin 1 (SIRT1) regulation-infected macrophages stimulated with IFN-γ and CD154 at different time points. RH tachyzoites were harvested to infect RAW 264.7 mouse macrophages at an infection ratio of 5:1 (parasite/host cell) for 2 h, followed by the removal of excess parasites and incubation with IFN-γ (1 μg/ml) or CD154 (200 ng/ml) for an extra 24 or 12 h before observation under light microscopy or transmission electron microscopy (TEM). RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection, followed by incubation with IFN-γ (1 μg/ml) or CD154 (200 ng/ml) for 2, 18, or 24 h before collection of the cell lysates. We adjusted the protein concentration of each group of samples to be consistent and then carried out the experiment. Anti-SIRT1 antibody and anti-beta-Actin antibody were used at a 1:1,000 dilution. (A) A scattered distribution chart showing the mean intracellular tachyzoite counts. Values represent actual numbers (scattered dots) and means (black line) from three independent experiments; in each case, at least 200 infected cells were counted. The interference of IFN-γ or CD154 reduced the average number of intracellular tachyzoites from 10 to about 3 at 24 h after the infection of Toxoplasma gondii. (B) Observation under TEM showed that the infected macrophages had much heavier intracellular tachyzoite burdens than infected macrophages incubated with IFN-γ and CD154 at 12 h after infection. The arrows indicate the tachyzoites inside host cells. (C) Infected macrophages treated with IFN-γ and CD154 showed a downregulation of SIRT1 2, 18, and 24 h compared to the T. gondii infection group. Among them, the expression level of SIRT1 exhibited its greatest decrement at 24 h. These results were obtained from three independent experiments. RH, T. gondii RH strain. **P < 0.01 and ***P < 0.001.

Figure 4

Intracellular growth of RH tachyzoites inside mouse macrophages with regulation by Sirtuin 1 (SIRT1) and interferon gamma (IFN-γ) or CD154 at different time points. RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection, followed by incubation with IFN-γ (1 μg/ml), CD154 (200 ng/ml), IFN-γ (1 μg/ml) and SRT1720 (5 μM), IFN-γ (1 μg/ml) and EX527 (10 μM), CD154 (200 ng/ml) and SRT1720 (5 μM), or CD154 (200 ng/ml) and EX527 (10 μM) for 24 h. Before observation under light microscopy. (A) Observation under light microscopy pointed out that the infected macrophages had much heavier intracellular tachyzoite burdens than infected macrophages incubated with IFN-γ at 24 h after infection. SRT1720 neutralized the intracellular tachyzoites' downward trend caused by IFN-γ, while EX527 enhanced the downtrend. (B) Observation under a light microscope revealed that the infected macrophages had much heavier intracellular tachyzoite burdens than infected macrophages incubated with CD154 at 24 h after infection. SRT1720 neutralized the intracellular tachyzoites' downward trend caused by IFN-γ, while EX527 enhanced the downtrend. (C) A scattered distribution chart showing the means of the intracellular tachyzoite counts. Values represent actual numbers (scattered dots) and means (black line) from three independent experiments; in each case, at least 200 infected cells were counted. ***P < 0.001.

Figure 5

Murine ortholog of immunity-related GTPase family M (IRGM1) regulation by Sirtuin 1 (SIRT1) with or without interferon gamma (IFN-γ) or CD154 at different time points. RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection, followed by incubation with IFN-γ (1 μg/ml), CD154 (200 ng/ml), SRT1720 (5 μM), EX527 (10 μM), IFN-γ (1 μg/ml) and SRT1720 (5 μM), or IFN-γ (1 μg/ml) and EX527 (10 μM) for 2, 18, or 24 h before the collection of the cell lysates. We adjusted the protein concentration of each group of samples to be consistent and then carried out the experiment. Anti-IRGM1 antibody and anti-beta-Actin antibody were used at a 1:1,000 dilution. (A) The expression of IRGM1 in the Toxoplasma gondii infection group increased at 2 h but decreased at 18 and 24 h compared to that for the control group. Infected macrophages treated with IFN-γ displayed an upregulation of IRGM1 at 2, 18, and 24 h compared to that of the infection-only group. (B) Infected macrophages treated with CD154 showed no significant changes of IRGM1 compared to that for the infection-only group. (C) Infected macrophages treated with RH tachyzoites showed an upregulation of IRGM at 2 h and a downregulation at 18 or 24 h compared to that of the control group. Meanwhile, IRGM1 production was found to be decreased under SRT1720 stimulation and increased with the existence of EX527 at 2 h. However, both SRT1720 and EX527 failed to interfere with the IRGM1 level at 18 and 24 h. (D) When combining SIRT1 interference and IFN-γ stimulation, infected macrophages treated with IFN-γ and SRT720 showed a downregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. Meanwhile, infected macrophages treated with IFN-γ and EX527 showed an upregulation of IRGM at 2, 18, and 24 h compared to that of the IFN-γ group. These results were obtained from three independent experiments. RH, T. gondii RH strain. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6

Autophagy regulation by Sirtuin 1 (SIRT1) and interferon gamma (IFN-γ) or CD154 at different time points. RAW 264.7 macrophages were either infected with RH tachyzoites at a ratio of 5:1 (parasite/host cell) for 2 h or left in the culture plate without infection, followed by incubation with SRT1720 (5 μM), EX527 (10 μM), or rapamycin (100 nM) for 2, 18, or 24 h before collection of the cell lysates or followed by incubation with rapamycin (100 nM) for 24 h for light microscopy or 12 h before transmission electron microscopy (TEM). We adjusted the protein concentration of each group of samples to be consistent and then carried out the experiment. Anti-LC3 I/LC3 II antibody, anti-phospho-mTOR (mechanistic target of rapamycin kinase) antibody, anti-mTOR, and anti-beta-Actin antibody were used at a 1:1,000 dilution. (A) The LC3 II/LC3 I ratio in the Toxoplasma gondii infection group increased over time compared to that for the negative control. Infected macrophages treated with SRT1720 showed a downregulation of LC3II/LC3 I at 2, 18, and 24 h compared to that for the T. gondii infection group. Infected macrophages treated with EX527 showed an upregulation of LC3II/LC3 I at 2, 18, and 24 h compared to that for the T. gondii infection group. (B) Infected macrophages treated with rapamycin showed an upregulation of LC3 II/LC3 I ratio at 2 and 18 h compared to that for the T. gondii infection group. (C) A scattered distribution chart showing the means of the intracellular tachyzoite counts. Values represent actual numbers (scattered dots) and means (black line) from two independent experiments; in each case, at least 200 infected cells were counted. (D) Observation under the light microscope showed that the infected macrophages had much heavier intracellular tachyzoite burdens than infected macrophages incubated with rapamycin at 24 h after infection. (E) Observation under TEM revealed that the infected macrophages had much heavier intracellular tachyzoite burdens than infected macrophages incubated with rapamycin at 12 h after infection. At the same time, host cells showed a large number of autophagosomes after being incubated with rapamycin for 12 h. These results were obtained from three independent experiments. Autophagosomes are multilayered structures containing cytoplasmic organelles. The arrows indicate the tachyzoites inside or outside host cells. The box indicates the autophagosome inside host cells. The values shown are the means ± SEM from three independent experiments. The arrows indicate the tachyzoites inside host cells. The box indicates the autophagosome inside host cells. (F) When infected by RH tachyzoites at two different time points, the mouse macrophages displayed an increased phosphorylation level of mTOR at 2 h and a decrease at 18 h. Infected macrophages treated with rapamycin showed a decrease in the mechanistic target of rapamycin kinase (mTOR) phosphorylation level at 2 and 18 h. At the same time, infected macrophages treated with rapamycin showed a downregulation of SIRT1 at 2 and 18 h compared to that of the infection-only group. Infected macrophages treated with SRT1720 or EX527 showed no significant changes of mTOR or phosphorylation level of mTOR compared to that of the infection-only group. p-mTOR, phosphorylated mTOR. t-mTOR, total mTOR. (G) The LC3 II/LC3 I ratio in the T. gondii infection group increased over time compared to that of the control group. Infected macrophages treated with IFN-γ showed an upregulation of LC3II/LC3 I at 2, 18, and 24 h compared to that for the T. gondii infection group. Infected macrophages treated with CD154 showed an upregulation of LC3II/LC3 I at 2 h and 18 h, but not at 24 h compared to the T. gondii infection group. These results were obtained from three independent experiments. RH, T. gondii RH strain. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 7

Toxoplasma gondii can be eliminated in infected cells by inhibiting the Sirtuin1 (SIRT1) protein. After T. gondii invades host cells, it causes the upregulation of the nicotinamide adenine dinucleotide (NAD⁺/NADH) ratio in host cells, which further leads to the upregulation of SIRT1. However, SIRT1 inhibits parasitic clearance via the immunity-related GTPase family M (IRGM) pathway and the activation of autophagy. Interferon gamma (IFN-γ) stimulation can clear intracellular T. gondii by simultaneously upregulating IRGM and activating autophagy. CD154 stimulation can clear intracellular T. gondii by activating autophagy. These two methods of eliminating intracellular T. gondii are assisted by inhibiting SIRT1. Directly using rapamycin to inhibit the mechanistic target of rapamycin kinase (mTOR) and initiate autophagy is also a way to eliminate intracellular T. gondii, which is also accompanied by SIRT1 inhibition.