Fig 2: Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell death. (A) NB4 cells were pre-treated with 5 μg/mL STO609 and then treated with 1 μM ATRA for 72 h. Granulocytic differentiation was determined by cytofluorometric evaluation of CD11c expression. Left panel, representative flow cytometry histograms are shown; right panel, the median fluorescence intensity (MFI) of CD11c was scored for each sample. (B) NB4 cells were pre-treated with 44 μM 2APB and then treated with 1 μM ATRA for 72 h and granulocytic differentiation was determined. Results correspond to the means ± SD of three independent experiments realized in triplicate. p value * < 0.05, p value *** < 0.001.

Fig 3: Essential function and molecular mechanism of SIRT1 in modulating the host immune response to T. gondii infection. In macrophages, myeloid-specific SIRT1 increases autophagy activity during T. gondii infection, thereby eliminating intracellular T. gondii by inducing the colocalisation of parasitophorous vacuoles with autophagosomes/lysosomes. As a key regulatory mechanism of autophagy, SIRT1 attenuates the acetylation of the transcription factor FoxO1 and its PI3K/AKT-dependent phosphorylation (Thr24) in response to T. gondii, thus regulating FoxO1 transactivation activity by preventing its nuclear exclusion. Moreover, SIRT1 promotes FoxO3a deacetylation and its CaMKK2/AMPK-dependent phosphorylation (Ser413), leading to nuclear accumulation and the transactivation of FoxO3a. Pharmacological activation of SIRT1 by RES induces antiprotozoal autophagy by regulating FoxO1 and FoxO3a activity via the PI3K/AKT and CaMKK2/AMPK signalling pathways, respectively.

Fig 4: CaMKKß/AMPK activation plays an essential role in endogenous ?-3 PUFA-induced antiparasitic responses against Toxoplasma infection in BMDMs. (A,B) BMDMs from WT and Fat-1 transgenic mice were infected with T. gondii RH strain (MOI = 1) for the indicated time periods. (A) Immunoblot analysis of phosphorylated forms of AMPKa, ACCa/ß, CAMKKß, LKB1, and ß-tubulin. A representative gel from three independent experiments with similar results is shown. (left), Densitometry analysis (right) (B) Immunoblot analysis of phosphorylated forms of AMPKa, CAMKKß, LKB1, and ß-tubulin. A representative gel from three independent experiments with similar results is shown. (top), Densitometry analysis (bottom) (C,D) BMDMs from Fat-1 transgenic mice were incubated with increasing concentrations of the AMPK inhibitor Compound C (5, 10, or 25 µM) or the CaMKK2 inhibitor STO-609 (5 or 10 µM), and then infected with T. gondii RH strain (MOI = 1) for 18 h. SAG1 mRNA expression was evaluated by Real-Time Quantitative PCR analysis. (E,F) BMDMs transduced for 36 h with adenovirus expressing GFP (Ad-GFP), WT LKB1 (Ad-LKB1 WT), or LKB1 D194A mutant (Ad-LKB1 D194A), at a MOI of 10, were infected with T. gondii RH strain (MOI = 1) for 2 h, and then incubated with DHA (10 µM) for 18 h. SAG1 mRNA expression was evaluated by RT-qPCR analysis. Data are representative of three independent experiments and are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). NS, not significant; U, untreated; SC, solvent control.

Fig 5: CaMKK2-dependent AMPK signalling is crucial for the activation of SIRT1-mediated antiparasitic responses against Toxoplasma infection in primary murine macrophages. (A,B) BMDMs were stimulated with RES (10 µM) for the various time periods, and then immunoblot analysis was assessed to evaluate the phosphorylation of AMPKa (for A) or CaMKK2 and LKB1 (for B). (C,D) BMDMs were pretreated with increasing concentrations of CompC (5, 10, or 25 µM, for C) or STO (1, 5, or 10 µM, for D) for 45 min, then infected with T. gondii RH strain (MOI = 1) for 18 h in the presence or absence of RES. Sag1 mRNA expression was examined by qPCR analysis. (E) BMDMs transduced with Ad-GFP, Ad-LKB1 WT, or Ad-LKB1 D194A for 36 h (MOI of 10), respectively, were infected with a T. gondii RH strain (MOI = 1) for 2 h, then further incubated with RES (10 µM) for 22 h. Sag1 mRNA expression was evaluated by qPCR analysis. (F) BMDMs from mSirt1+/+ and mSirt1-/- mice were infected with T. gondii for various time periods. Cell lysates were subjected to immunoblot analysis using antibodies against p-AMPKa (Thr172), AMPKa, and ß-tubulin. (G,H) BMDMs from mSirt1+/+ and mSirt1-/- mice were transduced for 36 h with adenovirus-expressing GFP (Ad-GFP, for G,H), constitutively active AMPK (Ad-CA, for G), or WT LKB1 (Ad-LKB1 WT, for H), at a MOI of 10, and each cell then was infected with a T. gondii RH strain (MOI = 1) for 18 h. The mRNA expression of the Sag1 gene was evaluated by real-time qPCR analysis. Data are representative of three independent experiments and are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). Tg, Toxoplasma gondii; RES, resveratrol; CompC, Compound C; STO, STO-609; adenovirus-expressing GFP, Ad-GFP; adenovirus-expressing WT LKB1, Ad-LKB1 WT; adenovirus-expressing LKB1 D194A mutant, Ad-LKB1 D194A; ns, not significant.