Figure 1.
PMNs treated with MSU crystals release NETs.
PMNs from control subjects treated with MSU crystals for different time points. NETs are assessed by immunofluorescence microscopy after co-staining with DAPI (blue) and MPO (green). Control represents untreated PMNs incubated for 180 min. Scale bars, 30 µm. Original magnification 400x. One out of three independent experiments is shown. B) Percentage of NET releasing cells presented in panel A. Data are representative of three independent experiments and presented as mean ± SD. * P<0.05; ns = non significant compared to control cells.
Figure 2.
PI3K signaling and endosomal acidification is involved in NET release after treatment with MSU crystals.
A) Treatment with 3-MA or bafilomycin A prevents the release of NETs from PMNs from control subjects treated with MSU crystals for 180 min. NETs are determined by co-staining with DAPI (blue) and MPO (green) and visualized by immunofluorescence microscopy. Scale bars, 30 µm. Original magnification 400x. One out of four independent experiments is shown. B) The effect of treatment with 3-MA, LY294002, bafilomycin A or PP1 in the percentage of NET releasing PMNs incubated with MSU crystals. Data are representative of four independent experiments and presented as mean ± SD. * P<0.05; ns = non significant compared to MSU treated cells.
Figure 3.
Induction of autophagy in PMNs treated with MSU crystals.
A) Induction of autophagy in control PMNs treated with MSU crystals for 20 min and 60 min, as assessed by immunofluorescence microscopy of endogenous LC3B. LC3B-positive autophagosome formation was blocked by 3-MA. DNA is labeled with DAPI (blue). LC3B was stained with polyclonal anti-LC3B Ab (green). Scale bars, 5 µm. Original magnification 1000x. One out of three independent experiments is shown. B) Induction of LC3B-I to LC3B –II conversion in PMNs treated with MSU crystals for 45 min, as assessed by immunoblotting. Attenuation of LC3B conversion in PMNs treated with 3-MA. C) Measurement of integrated optical density (IOD) of bands presented in B, expressed as LC3B-II to LC3B-I ratio ± SD. Representative data from four independent experiments are shown in B and C. * P<0.05 compared to MSU treated cells.
Figure 4.
NET release from synovial cells and peripheral PMNs from patients with gout.
A) NET formation by synovial cells, peripheral PMNs from patients with gout and from control PMNs treated with synovial fluid or serum from patients with gout, as assessed by immunofluorescense microscopy after co-staining with DAPI (blue) and MPO (green). Inhibitory effect of treatment with 3-MA on the release of NETs from synovial cells. Scale bars, 30 µm. Original magnification 400x. One out of six independent experiments is shown. B) The effect of treatment with 3-MA or bafilomycin A in the percentage of NET releasing cells presented in panel A. Data are representative of six independent experiments and presented as mean ± SD. ‡ P<0.05 compared to control, * P<0.05.
Figure 5.
Implication of IL-1β in the induction of NET release from PMNs treated with synovial fluid or serum from patients with gout.
A) The inhibitory effect of treatment with anakinra on NET release from control PMNs treated with centrifuged or not synovial fluid from patients with gout, as assessed by immunofluorescense microscopy after co-staining with DAPI (blue) and MPO (green). Treatment with recombinant IL-1β induces NET release. Scale bars, 30 µm. Original magnification 400x. One out of six independent experiments is shown. B) The inhibitory effect of anakinra on the percentage of NET releasing PMNs presented in panel A and in PMNs treated with serum from patients with gout. Incubation with anti-IL-1β mAb reduced the percentage of NET releasing PMNs treated with centrifuged synovial fluid. Data are representative of six independent experiments and presented as mean ± SD. ‡ P<0.05 compared to control, * P<0.05.
Figure 6.
Localization of HMGB-1 on extracellular fibrous DNA structures.
A) Expression of HMGB-1 in extracellular DNA structures released from PMNs derived from control subjects treated with MSU crystals or synovial fluid and synovial cells from patients with gout, as assessed by immunofluorescense microscopy. DNA is labeled with DAPI (blue). HMGB-1 is stained with anti-HMGB-1 mAb (green). Control represents untreated PMNs. One representative out of four independent experiments is shown. Scale bars, 5 µm. Original magnification 1000x. B) Expression of HMGB-1 in NETs released from control PMNs stimulated with MSU crystals or synovial fluid from patients with gout, as assessed by immunoblotting of NET-derived proteins. The inhibitory effect of bafilomycin A is shown. One out of three independent experiments is shown.