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. 2020 Sep 24;117(41):25679–25689. doi: 10.1073/pnas.2005111117

Fig. 5.

Fig. 5.

Concerted 15-LO and sPLA2 activity is needed for formation of TLR4-activating ligands in synEVs. SynEVs composed of 30% AAPE and 70% POPC were prepared. (A) HEK293T cells expressing hTLR4 and hMD-2/CD14 or hTLR4 alone as well as firefly luciferase under NF-κB promotor and Renilla luciferase for normalization were stimulated with synEVs (10 μg/mL), hydrolyzed and oxidized synEVs, oxidized and hydrolyzed synEVs (10 μg/mL), or LPS (10 ng/mL) as a positive control for 24 h. Negative controls are transfected but unstimulated cells. A dual luciferase test for NF-κB activity was performed. (B, C, E, and H) WT macrophages (B, C, and H) and WT or TLR4 KO BMDMs (E) were stimulated for 6 h with indicated sPLA2 (1.9 μg/mL) or synovial fluid (SF) (14,7 μL), synEVs (10 μg/mL), oxidized synEVs (10 μg/mL), indicated oxidized and hydrolyzed synEVs (10 μg/mL) or LPS (2 ng/mL) as a positive control. Il1b, Il6, Il23a, and Socs2 mRNA levels were determined using qPCR. (D) RAW 264.7 cells were stimulated with synEVs, oxidized synEVs, oxidized and hydrolyzed sPLA2-IIA synEVs (40 μg/mL), or LPS (1 μg/mL) as a positive control for 6 h. TLR4/MD-2 dimerization using anti-MTS510-PE Ab was determined by flow cytometry. (F) WT or TLR4 KO BMDMs were stimulated for 16 h with synEVs (10 μg/mL), oxidized synEVs (10 μg/mL), oxidized and hydrolyzed sPLA2-IIA synEVs (10 μg/mL), or LPS (2 ng/mL) as a positive control. Il-6 in supernatants was measured by ELISA. (G) PLA2 activity was measured in the SF (ultracentrifuged and concentrated 3×) from RA and gout patients. (G) Mice (n = 4 to 5 per group) were injected with 150 and 90 μL K/BxN serum on days 0 and 2. On day 2 recombinant human sPLA2-II (9.5 μg) was injected. The development of arthritis was followed for 14 d. (A) Three indep. exps. (n = 6 or 9); t test (two-tailed; unequal var.); (B) three indep. exps. (n = 3); t test (two-tailed; paired); (C and E) three indep. exps. (n = 4); t test (two-tailed; paired or one-tailed; paired for KO). For D, additional an indep. exp. is shown in SI Appendix, Additional data. (F) Two indep. exps. (n = 6); t test (two-tailed; unequal var. or one-tailed; unequal var. for KO); (H) two indep. exps. (n = 2); t test (two-tailed; paired); (I) two-way ANOVA between WT+sPLA2 and TLR4KO+sPLA2 (n = 4 or 5); *P < 0.05, **P < 0.01.