Extracellular vesicles are considered to be an inflammatory factor in several acute and chronic inflammatory diseases. Researchers from Kyoto Prefectural University demonstrate that exosomes from macrophages (Mφ) infected with live Escherichia coli induced secretion of proinflammatory factors by uninfected Mφ. Inflammatory responses induced by exosomes derived from Mφ infected with heat-inactivated E. coli or lipopolysaccharide were significantly weaker than those elicited by outer membrane vesicles (OMVs) released from live E. coli. Proteome analysis of exosomes from Mφ infected with live or heat-inactivated E. coli revealed that E. coli proteins OmpA, GroL1, DegP, CirA, and FepA are candidate triggers of exosome-mediated inflammatory responses. OMVs from a cirA-deleted strain suppressed exosome-mediated inflammatory responses by uninfected Mφ. The C terminus of the CirA protein (residues 158 to 633), which was relayed from E. coli-derived OMV to Mφ-derived exosomes, promoted exosome-mediated inflammatory responses by uninfected Mφ. These results suggest an alternative mechanism by which extracellular vesicles from E. coli OMV-elicited Mφ transmit proinflammatory responses to uninfected Mφ.
Characterization of E. coli-derived outer membrane vesicles (OMVs)
(a) OMVs from the culture medium of E. coli K-12 strain during 24 h were isolated by ultracentrifugation. (b) The growth of E. coli was assessed by measuring at a wavelength of 600 nm (OD600) and is shown by the line graph. The number of OMV proteins is shown in the bar graph (n = 1). The experiment was repeated with at least three independent biological replicates. (c) OMVs from 1.2 mL of culture medium were prepared by ultracentrifugation. The protein expression of OmpA, an OMV marker, was detected by Western blotting. (d) OMVs were visualized with a transmission electron microscope. (e) After RAW264.7 cells were incubated with or without Ec-med, Ec-sup, or OMVs for 9 h, cells were incubated with fresh medium for 1 h. After we changed fresh medium again, cells were incubated for 9 h. The protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in whole-cell lysates was measured by Western blotting. β-Actin was used as loading control. (f) After RAW264.7 cells (recipient cells; 5 × 105 cells) were incubated with each exosome from donor cells (5 × 105 cells) for 24 h, the protein expression of iNOS and COX-2 in whole-cell lysates was measured by Western blotting. β-Actin was used as loading control. The protein levels of iNOS, COX-2, and β-actin were quantitatively analyzed by using CS Analyzer 3.0 as image analysis software. Relative intensity was expressed as the mean ± SD (n = 3) of at least three independent biological replicates. One-way ANOVA and Tukey’s test were used for statistical analysis; *, P < 0.05; ***, P < 0.005; ****, P < 0.001.