Fig 1: a-Synuclein in CNS was drained to the macrophage and activated it in the dCLN through meningeal lymphatics. A Representative fluorescence images of dCLNs of C57 mice injected (i.c.m) with vehicle or AF647-a-synuclein (red). Scale bar, 200 µm. B Number of macrophages containing AF647-a-synuclein in dCLNs of C57 mice injected (i.c.m) with vehicle or AF647-a-synuclein was evaluated by flow cytometry. The macrophages were labelled by CD11b-FITC and CD45-PE-Cy7. C Quantitative analysis of the number of macrophages containing AF647-a-synuclein in dCLNs of C57 mice. D Representative fluorescence images of dCLNs in WT and A53T mice. a-Synuclein in dCLN was immunolabelled by MJFR-1 (red). Scale bar, 200 µm. E Macrophages in dCLN were sorted by FACS. The macrophages were labelled by CD11b-FITC and CD45-PE-Cy7. F Quantitative analysis of a-synuclein oligomer in macrophages of dCLNs and total dCLNs using MSD. N = 3 independent experiments in each group. G Quantitative analysis mRNA levels of Il1b, Il6 and Tnf in BMDMs treated with vehicle control, 100 nM a-syn monomer, or 100 nM a-syn oligomer using qPCR. N = 3 independent experiments in each group. H Quantitative analysis of the levels of IL-1ß, IL6 and TNF-a using ELISA, released by BMDMs treated with vehicle control, a-syn monomer, or a-syn oligomer. N = 3 independent sample pools in each group. Values are means ± S.E.M, t test (C, F), one-way ANOVA test (G, H). *, P < 0.05; **, P < 0.01; ****, P < 0.0001
Fig 2: BMDCs autophagy was reduced after overexpression of PI3K. The overexpression plasmids (oe-NC or oe-PI3K) were first transfected, and BMDCs were treated with OmpA (10 µg/mL) for 24 h. (A) PI3K, AKT and mTOR expressions were monitored using qRT-PCR. (B) Western blot detection of PI3K/mTOR pathway related proteins, Beclin1, P62, and LC3II/I expressions. (C) Flow cytometry detection of BMDCs apoptosis. (D) The expression of BMDCs mature markers MHC-II + CD11c, CD80 + CD11c, and CD86 + CD11c were tested by flow cytometry. E. IL-18, NLRP3, IL-1ß, and TNF-a contents were assessed through ELISA. *p < .05 versus OmpA+oe-NC. BMDC, bone marrow-derived dendritic cell; IL, interleukin; PI3K/AKT, protein kinase B; mTOR, mammalian target of rapamycin; TNF, tumor necrosis factor.
Fig 3: OmpA promoted the maturation and inflammation of BMDCs by affecting autophagy. BMDCs were pretreated with chloroquine for 30 min, and BMDCs were treated with OmpA (10 µg/mL) for 24 h. (A) IF was utilized to assess LC3 level. Scale bar = 25 µm (×400). (B) Western blot detection of Beclin1, P62, and LC3II/I expressions. (C) The levels of MHC-II + CD11c, CD80 + CD11c, CD86 + CD11c, the mature markers of BMDCs, were monitored by flow cytometry. (D) Flow cytometry assessment of BMDCs apoptosis. (E) IL-18, NLRP3, IL-1ß, and TNF-a contents were tested using ELISA. *p < .05 versus Control, # p < .05 versus OmpA. BMDC, bone marrow-derived dendritic cell; LC, light chain; IL, interleukin; TNF, tumor necrosis factor.
Fig 4: Peripheral inflammation in A53T mice. A Quantitative analysis of mRNA levels of Il1b, Il6 and Tnf in dCLNs of WT and A53T mice using qPCR. N = 3 independent animals in each group. B Western blot to assess the level of IL-1ß, IL-6 and TNF-a in dCLNs of WT and A53T mice. C Quantitative analysis of the protein level of IL-1ß, IL6 and TNF-a in dCLNs of WT and A53T mice, N = 3 independent in each group (3 samples were pooled from 6 animals). D Representative fluorescence images of dCLNs from WT and A53T mice immunolabeled by CD11b (red or green) with IL-1ß (green), IL-6 (red), or TNF-a (red). Scale bar, 100 µm. E Quantitative analysis of IL-1ß, IL6 and TNF-a positive area in dCLNs of WT and A53T mice. N = 5 independent animals in WT and A53T. F Quantitative analysis of the levels of IL-1ß, IL6 and TNF-a using ELISA, in plasma of WT and A53T mice. N = 4 independent animals in WT group and N = 5 independent animals in A53T group. Values are means ± S.E.M, t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001
Fig 5: HA ameliorates colitis induced by DSS. (A) Experimental design. (B) Percentage change in body weight after DSS treatment. (C) Disease activity index on day 7. (D,E) Representative images and length of the colon. (F,G) Representative image of H&E staining of colon and pathological score. The right lower panels (20×) are images enlarged from the low magnification panels (2.5×). (H) Relative mRNA expression of inflammatory factors in the colon. (I–M) Concentrations of IL4, IL6, TNFa, IL17, and IL10 in serum. Data are expressed as mean ± SE, n = 8–10, * p < 0.05, ** p < 0.01.
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