Table 1.
Baseline characteristics of the IBD patients anti TNF-α failure.
Table 2.
Baseline characteristics of the IBD patients naïve to TNF-α.
Fig 1.
Circulating exosomes isolation and characterization.
Exosomes were isolated from serum by Exoquick and quantified by Exocet. Panel A and B illustrate the mean concentration of exosomes in 12 blood donor (CTRL) and 17 UC patients, subdivided in anti- TNFα naïve (TNF-N) and anti-TNFα antagonist exposed (TNF-E). * p<0.05 compared to anti- TNFα naïve patients C) Exosomes were validated for the expression of exosomal markers by flow cytometry. Exosome-bound beads (white peak) were compared with beads alone (grey peak).
Fig 2.
Circulating exosomes express α4β7 integrin.
(A) Purified vesicles isolated from serum of blood donor (CTRL) and UC patients subdivided in anti- TNFα naïve (TNF-N) and anti-TNFα antagonist exposed (TNF-E) were captured on antibody-coated beads and analyzed by flow cytometry. Representative plots of the FACS analyses for CTRL(green line) and UC-derived exosomes (red line) are shown. Graphs reported the expression levels (percentage of exosome-bound beads compared with beads alone) of CD9 (B), CD3 (C), CD14 (D), α4β7 integrin (E) and MadCAM-1 (F) on exosome surface, * p<0.05 compared to anti- TNFα naïve patients.
Fig 3.
VDZ is bound to exosomes in UC patients.
(A)Serum levels of VDZ free and bound to exosomes were quantified by Promonitor-VDZ ELISA. (B)The expression of α4β7 integrin bound to VDZ was confirmed by native immunoblotting on exosomes lysate. (C)The sequestration of VDZ in exosomes was expressed as the percentage of VDZ bound to exosomes over the total level measured in serum. (D) Spearman correlation between the expression of α4β7 integrin and the amount of VDZ bound to 1010 exosomes isolated from 12 UC patients.
Fig 4.
Comparison of three different methods of exosome isolation.
Exosomes were isolated from a pool of patient’s sera by polymer precipitation (Exoquick), membrane-affinity (ExoEasy) and size-exclusion column (qEV2) methods. Vesicles were quantified by Exocet (A), validated for the expression of exosomal markers by immunoblotting (B) and analysed for size distribution by NTA (C). The levels of VDZ bound to exosomes were quantified by ELISA. Data are shown as mean ± SD. * p<0.05 compared to exosomes isolated by ExoEasy and qEV2; § p<0.05 compared to exosomes isolated by ExoQuick and qEV2.
Fig 5.
Exosomes compete with T cells for binding to VDZ.
VDZ bound to exosomes (A) or to CD4+ T cells (B) were evaluated by native immunoblotting. One representative blot is shown of three independent experiments. (C) CD4+ T cells were incubated with VDZ with or without exosomes and added to MAdCAM-1-coated plate. Representative microscope images of adherent cells stained with Hoechst dye are shown. Data are shown as mean ± SD. * p<0.05 compared to adherent cells treated with 5 ng/ml VDZ alone.