Fig. 2

Effect of the PM exposure of monocytes on: (I) Mitochondrial respiration. (a) The Cell Mito Stress Test profile presenting OCR values for monocytes in control condition and after NIST or LAP treatment (data are presented from one representative experiment). Additionally, OCR values for: (b) basal respiration, (c) proton leak, (d) ATP production, (e) maximal respiration, (f) spare respiratory capacity and (g) non-mitochondrial respiration in control cells and monocytes treated with NIST or LAP (100 µg/mL) are presented separately, as median ± interquartile range from 4 independent experiments. All measurements were performed in triplicates. (II) Caspase-3–dependent pyroptotic pathway. (a) Activation of Apaf-1 and (e) GSDME after 15 min of monocyte exposure to NIST or LAP (100 µg/mL) was evaluated by Western blot analysis. GAPDH was used to confirm the equal protein loading. (b) In the case of Caspase-9 and Caspase-3, their activation was examined by flow cytometry using Caspase-9 (active) Staining Kit and PE-conjugated mouse anti-human active Caspase-3 monoclonal antibodies. Data are presented as median ± interquartile range from 4 independent experiments. (c, d) Dot plots (LEHD-fmk – FITC vs. PE and Caspase-3 – PE vs. FITC) show cells positive for Caspase-9 and Caspase-3 in unstimulated control and cells stimulated with NIST or LAP (100 µg/mL) for 15 min. As a positive control, cells stimulated with 100 ng/mL of LPS from Salmonella abortus equi, 200 µM TBHP or 10 µM CCCP, were used simultaneously. Monocytes pre-incubated for 1 h with 1.5 mM Mito-TEMPO prior to PM exposure were used as a negative control. (III) The canonical inflammasome pathway. (a) Activation of NRLP3, including after 1 h pre-incubation with1.5 mM Mito-Tempo (b) or 10 µM MCC950 (c) and GSDMD (g) was evaluated by Western blot analysis after 15 min of monocyte exposure to NIST or LAP (100 µg/mL). GAPDH was used to confirm the equal protein loading. (d) Specific inhibition of NLRP3. MCC950 was used to assess the effect of NLRP3 inactivation on monocyte Annexin V binding. (e) In the case of Caspase-1, its activation was examined by flow cytometry using Caspase-1 (active) Staining Kit. Data are presented as median ± interquartile range from 4 independent experiments. (f) Dot plots (FAM-yvad-fmk vs. PE) show cells positive for Caspase-1 in unstimulated control and cells stimulated with NIST or LAP (100 µg/mL) for 15 min. (h) Additionally, the level of IL-1β producing monocytes was evaluated by flow cytometry after cell staining with a PE-conjugated mouse anti-human IL-1β monoclonal antibodies. Human monocytes were cultured with or without NIST or LAP for 15 min. Data are presented as a percentage of IL-1β positive cells (median ± interquartile range from 3 independent experiments). (i) Dot plots (IL-1β – PE vs. FITC) show cells positive for IL-1β in unstimulated control and cells stimulated with NIST or LAP (100 µg/mL) for 15 min. As a positive control, cells stimulated with 100 ng/mL of LPS from Salmonella abortus equi and 400 U/mL of human recombinant IFN-γ, 200 µM TBHP or 10 µM CCCP, were used simultaneously. Monocytes pre-incubated for 1 h with 1.5 mM Mito-TEMPO or 50 µM Ac-yvad-cmk prior to PM exposure were used, as a negative control. Statistically significant differences were estimated at p < 0.05, p < 0.01, p < 0.001, p < 0.0001, ns – not significant