Figures and data
Central role of TWIK2 in triggering ATP-induced AMφ training of bacterial killing.
(A). Diagram of the ATP-induced AMφ training model in vivo. WT or KO mice were exposed to ATP (1mM, i.n.) and AMφ were isolated on day 7 to determine acquisition of training. For in vitro training, BMDMs or RAW cells were trained with or without ATP (1mM) and recovered for 7 days. (B). AMφ subjected to training as described in A were incubated with live PA-GFP (MOI:50-100), PA killing was evaluated by decreased GFP fluorescence. (C). BMDMs subjected to in vitro training (1mM ATP for 7 days as described in A) were incubated with live PA-GFP (MOI:50-100) and PA killing activity were determined. (D). Survival of control or ATP pretrained mice after receiving 40mL of 5*106 c.f.u. of P. aeruginosa intranasally instilled, n = 5. (E) Comparison of bactericidal activity of AMφ trained with various DAMPs: ATP, histone or NAD. ATP showed the most dramatic response. ∗, p<0.05, ∗∗∗, p<0.001 (n = 3).
Assessment of TWIK2 PM and phagosome translocation.
A. Images of RAW cells expressing TWIK2-GFP post-ATP challenge (1mM, PM stained in red). White arrowheads show PM translocated TWIK2. Bar, 5µm. B. Western blot analysis of ATP trained or untrained macrophage lysate after ultracentrifuge fractionation. Membrane and non-membranous fractions were compared to assess the relative abundance of TWIK2 and the Na-K ATPase with and without ATP training. C. Ratiometric fluorescence intensity analysis of TWIK2-GFP signal within 1 micrometer of the plasmalemma to the corresponding TWIk2-GFP cytoplasmic intensity, highlighting enhanced and long-term membrane association after ATP treatment. D. Duration of PM-TWIK2 untreated or ATP or LPS treated cells. *, P<0.05, **, P<0.01, ***, P<0.001, compared with control (n = 3).
PM-TWIK2 re-internalizes into phagosomes upon phagocytosis.
A. Fluorescence images of TWIK2-GFP expressing RAW 264.7 cells phagocytosing zymosan (red) trained with or without ATP. B. Quantification of A. We observed significantly greater phagosome-associated TWIK2 in ATP-trained cells vs. controls. Arrowheads point to phagosomes with TWIK2-GFP. ∗, p<0.01 compared with untreated control (n = 3). C, D, Representative images (C) and quantification (D) of K+ enrichment (indicated by a specific K+ dye, ION K+, green) in phagosomes of control and ATP-trained BMDMs, ∗∗∗, p < 0.001.
ATP training regulates transcriptional profile and metabolic reprogramming of Mφ.
A. Normalized PA killing ability of BMDMs trained with or without ATP in the presence of metabolic inhibitors 2DG or protease inhibitors. B. RNA isolated from BMDMs trained with or without ATP was mapped to the Mus musculus genome and subsequently sorted into GO terms in Galaxy. Shown are the top 10 GO categories overrepresented in ATP trained BMDMs. C. Basal glycolytic and mitochondrial metabolism in BMDMs trained with or without ATP as exhibited by Seahorse assay. D, E. Western blot (D) and quantification (E) of NLRP3 assembly in BMDMs trained with or without 1mM ATP and challenged with 1.0 MoI PA for 2 hours. These cells were lysed with DSP crosslinker and assessed for NLRP3 oligomerization, n=3.
ATP training rescues lung immunosuppression caused by pneumonia.
A. Diagram of the double-infection model of primary pneumonia (1° PN) with 1×106 E. coli and secondary pneumonia (2° PN) with 1×106 GFP-PA. B. Colony-forming units (CFU) per ml of BAL with 1° or 2° pneumonia (both induced by E. coli). ∗, p < 0.05, n = 3. C. Bacterial killing ability in WT or designated KO mice trained with or without ATP in the double exposure model.
