Fig. 6: MEVsCa reduce inflammation via the SMAD7 pathway.
From: Immune modulation by complement receptor 3-dependent human monocyte TGF-β1-transporting vesicles
a No change in IL6 transcription occurs in whole-blood ex vivo infection with C. albicans, also after blocking TGF-β1 (data are presented as mean values + /− SD, p = 0.9947, unpaired two-tailed t test, n = 3 donors). b TGF-β1 on MEVsCa binds to TGF-βRII on monocytes. No such interaction was observed with MEVs. Cells were incubated with EVs for 30 min, and TGF-β1/TGF-βRII complexes detected by PLA using CLSM. PLA staining– red: TGF-β1/TGF-βRII complexes, blue: DNA. Bars: 10 µm. c MEVsCa and d TGF-β1 adhere to C. albicans and its hyphae. Monocytes were infected with opsonized C. albicans for 1 h, and immunogold-labeled TGF-β1 visualized by SEM. e Phosphorylated SMAD2/3 and SMAD7 increase in whole blood infected with C. albicans ex vivo (1 h), but not when TGF-β1 was neutralized. Cells were lysed and intracellular proteins were for total and phosphorylated SMAD2/3 and SMAD7 detected by Western blot. (see uncropped WB in Supplementary Fig. 7d) The experiments in b, c, d, e are representatives of each n = 3 experiments. f Illumina-based RNA-seq reveals 6363 significantly upregulated genes in C. albicans-infected monocytes (1 h) compared with control monocytes. g RNA-seq analysis shows upregulation of SMAD2, NFKBIA, and SMAD7. For f, g RNA from four different donors were pooled before sequencing. h TGF-β1 inhibits IL-1β synthesis in renal fibrosis. TGF-β1 binds to TGF-βRII, and induces the phosphorylation of SMAD2/3. Phosphorylated SMAD2/3 in combination with SMAD4 upregulate SMAD7, which in turn upregulates IκBα. IκBα inhibits the IL-1β-positive feedback loop by inhibiting phosphorylation of NFκB54. i Little upregulation of IL1B expression is observed in whole blood infected with C. albicans ex vivo, but strong upregulation of IL1B occurs when TGF-β1 or TGF-βRI was blocked during ex vivo infection (data are presented as mean values + /− SD, p = 0.0222, p = 0.0005, unpaired two-tailed t test, n = 3 donors). j IL1b expression is not upregulated in whole blood from wild-type mice (C57BL/6) infected with C. albicans, but in blood from CR3-deficient (CD11b KO) mice (B6.129S4-Itgamtm1Myd/J) (data are presented as mean values + /− SD, p = 0.3785, p = 0.0015, unpaired two-tailed t test, n = 4 donors). After 4 h whole blood infection, cells were lysed, and RNA was isolated and subjected to comparative qPCR.
