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. 2013;8(3):e59276.
doi: 10.1371/journal.pone.0059276. Epub 2013 Mar 14.

Outer membrane vesicles derived from Escherichia coli up-regulate expression of endothelial cell adhesion molecules in vitro and in vivo

Ji Hyun Kim  1 , Yae Jin Yoon, Jaewook Lee, Eun-Jeong Choi, Namwoo Yi, Kyong-Su Park, Jaesung Park, Jan Lötvall, Yoon-Keun Kim, Yong Song Gho
Affiliations

Outer membrane vesicles derived from Escherichia coli up-regulate expression of endothelial cell adhesion molecules in vitro and in vivo

Ji Hyun Kim et al. PLoS One. 2013.

Abstract

Escherichia coli, as one of the gut microbiota, can evoke severe inflammatory diseases including peritonitis and sepsis. Gram-negative bacteria including E. coli constitutively release nano-sized outer membrane vesicles (OMVs). Although E. coli OMVs can induce the inflammatory responses without live bacteria, the effect of E. coli OMVs in vivo on endothelial cell function has not been previously elucidated. In this study, we show that bacteria-free OMVs increased the expression of endothelial intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1, and enhanced the leukocyte binding on human microvascular endothelial cells in vitro. Inhibition of NF-κB and TLR4 reduced the expression of cell adhesion molecules in vitro. OMVs given intraperitoneally to the mice induced ICAM-1 expression and neutrophil sequestration in the lung endothelium, and the effects were reduced in ICAM-1(-/-) and TLR4(-/-) mice. When compared to free lipopolysaccharide, OMVs were more potent in inducing both ICAM-1 expression as well as leukocyte adhesion in vitro, and ICAM-1 expression and neutrophil sequestration in the lungs in vivo. This study shows that OMVs potently up-regulate functional cell adhesion molecules via NF-κB- and TLR4-dependent pathways, and that OMVs are more potent than free lipopolysaccharide.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Up-regulation of endothelial cell adhesion molecule expression by E. coli OMVs in vitro. (A
–C) HMVECs were treated as indicated in the figures for 12 hours in 5% FBS/EBM, and the expression of ICAM-1, E-selectin, and VCAM-1 was measured by Western blot of whole cell lysates (10 µg). A. The dose-dependent effect of E. coli OMVs (in total protein) compared with TNF-α (10 ng/mL). B. The effect of a protein synthesis inhibitor, cycloheximide. HMVECs were pre-treated with cycloheximide (1 µg/mL) for 30 minutes, followed by treatment with E. coli OMVs (10 ng/mL in total protein) and cycloheximide (1 µg/mL) for 12 hours. C. The effect of several signaling inhibitors. HMVECs were treated with E. coli OMVs (10 ng/mL in total protein) and the signaling inhibitors (PD98059 for ERK, 20 µM; SB203580 for p38 MAPK, 10 µM; SP600125 for JNK, 10 µM; BAY11-7082 for NF-κB, 1 µM). D. The time course of IκB phosphorylation (p-IκB) by treating E. coli OMVs (10 ng/mL in total protein) on HMVECs. In A–D, the numbers under each blot indicate ratios calculated by dividing the densitometry values for ICAM-1, E-selectin, VCAM-1, IκB, or p-IκB by those for actin. (E–G) HMVECs were treated with PBS, E. coli OMVs (10 ng/mL in total protein), or TNF-α (10 ng/mL) for 12 hours in 5% FBS/EBM, followed by adding CMFDA-labeled U937 cells and incubating for 45 minutes. E. Representative visualization of adherent cells under fluorescence microscopy. F. The number of adherent cells per field (n = 8). G. The inhibitory effect of BAY11-7082 (1 µM) on CMFDA-labeled U937 cell adhesion to OMV-treated HMVECs (n = 8). *P<0.05; ***P<0.001. Results are represented as means ± SD.
Figure 2
Figure 2. Role of LPS in inducing endothelial cell adhesion molecules by E. coli OMVs in vitro.
HMVECs were treated as indicated in the figures for 12 hours in 5% FBS/EBM, and the expression of ICAM-1, E-selectin, and VCAM-1 was measured by Western blot of whole cell lysates (10 µg). A. The effect of various TLR agonists (HKLM (1×107 cells/mL) for TLR2; ultrapure LPS from E. coli K-12 (100 ng/mL) for TLR4 (InvivoGen); flagellin (100 ng/mL) for TLR5) or E. coli OMVs (10 ng/mL in total protein). B. The effect of OMVs (10 ng/mL in total protein) derived from E. coli K-12 W3110 wild-type or ΔmsbB mutant (contain inactive LPS). (C-E) HMVECs were treated with E. coli OMVs (10 ng/mL in total protein), LPS (75 ng/mL) purified from the E. coli strain isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice , or TNF-α (10 ng/mL). The effects of polymyxin B (1 µg/mL) and TLR4 antagonist (R. sphaeroides LPS, 10 µg/mL) are shown in C and D, respectively. E. The effect of absence or presence of FBS (5%) or CD14 (1 µg/mL) in EBM. The numbers under each blot indicate ratios calculated by dividing the densitometry values for ICAM-1, E-selectin, or VCAM-1 by those for actin.
Figure 3
Figure 3. Up-regulation of ICAM-1 expression and leukocyte adhesion on pulmonary endothelium by E. coli OMVs. (A-C)
C57BL/6 wild-type mice were intraperitoneally administered with PBS or E. coli OMVs (15 µg in total protein/mouse; n  =  5). A. The number of neutrophils in BAL fluid after OMVs injection. (B and C) Six hours after the administration, the lungs were harvested. B. Immunohistochemistry with confocal microscopy of ICAM-1 (green), endothelial cell marker CD31 (red), and nuclei (blue) in the lungs (scale bars, 100 µm). White arrows indicate ICAM-1 positive endothelial cells. C. Hematoxylin and eosin staining of the lungs (scale bars, 100 µm). Black arrows indicate leukocytes on the pulmonary endothelium. (D and E) C57BL/6 wild-type and ICAM-1-/- mice were intraperitoneally administered with PBS or E. coli OMVs (15 µg in total protein/mouse). Six hours after the administration, the lungs were harvested (n  =  3). D. Immunohistochemistry with confocal microscopy of a neutrophil marker NIMP-R14 (green) and nuclei (blue) in the lungs (scale bars, 50 µm). E. The number of neutrophils per field. *P<0.05; **P<0.01; ***P<0.001. Results are represented as means ± SEM.
Figure 4
Figure 4. Role of TLR4 in endothelial ICAM-1 expression and pulmonary neutrophil infiltration by E. coli OMVs
. C57BL/6 wild-type and TLR4-/- mice were intraperitoneally administered with PBS or E. coli OMVs (15 µg in total protein/mouse). Three hours after the administration, the lungs were harvested (n  =  3). A. Immunohistochemistry with confocal microscopy of ICAM-1 (green), endothelial cell marker CD31 (red), and nuclei (blue) in the lungs (scale bars, 50 µm). White arrows indicate ICAM-1 positive endothelial cells. B. The quantitative analysis of ICAM-1/CD31 co-localization. C. The number of neutrophils per field was determined as described in Figure 3D and 3E. **P<0.01; ***P<0.001. Results are represented as means ± SEM.
Figure 5
Figure 5. Comparison of E. coli OMVs and LPS in ICAM-1 expression and leukocyte adhesion on endothelium. (A
–C) HMVECs were treated with OMVs or equivalent amount of purified E. coli LPS for 12 hours in 5% FBS/EBM. One hundred nanograms of E. coli OMVs in total protein harbored 75 ng of LPS. A. The dose-dependent effect of E. coli OMVs and purified E. coli LPS on endothelial ICAM-1 expression. B. The effect of different dosage of purified E. coli LPS treated with E. coli K-12 W3110 ΔmsbB mutant OMVs (10 ng/mL in total protein) on HMVECs. E. coli K-12 W3110 wild-type OMVs (10 ng/mL in total protein) were used as positive control. In A–B, the numbers under each blot indicate ratios calculated by dividing the densitometry values for ICAM-1 by those for actin. C. Representative still photographs of adherent cells under flow. HMVECs were treated with PBS, E. coli OMVs (10 ng/mL in total protein), or equivalent amount of purified E. coli LPS (7.5 ng/mL) for 12 hours in 5% FBS/EBM, followed by continuous infusion of CMFDA-labeled U937 cells (1×106 cells/mL) under flow (physiological shear stress, 2 dyn/cm2). The cellular fluorescence was recorded using fluorescence microscopy for 2 minutes, which is shown in the Supporting Information Movie S1. (D–F) C57BL/6 wild-type mice were intraperitoneally administered with PBS, E. coli OMVs (15 µg in total protein/mouse), or equivalent amount of purified E. coli LPS (11.25 µg/mouse). Six hours after the administration, the lungs were harvested (n  =  5). D. Immunohistochemistry with confocal microscopy of ICAM-1 (green), endothelial cell marker CD31 (red), and nuclei (blue) in the lungs (scale bars, 100 µm). White arrows indicate ICAM-1 positive endothelial cells. E. Immunohistochemistry with confocal microscopy of a neutrophil marker NIMP-R14 (green) and nuclei (blue) in the lungs (scale bars, 50 µm). F. The number of neutrophils per field. *P<0.05; ***P<0.001. Results are represented as means ± SEM. In A–F, we used LPS purified from the E. coli strain isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice .
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References

    1. Danese S, Dejana E, Fiocchi C (2007) Immune regulation by microvascular endothelial cells: directing innate and adaptive immunity, coagulation, and inflammation. J Immunol 178: 6017–6022. - PubMed
    1. Andonegui G, Zhou H, Bullard D, Kelly MM, Mullaly SC, et al. (2009) Mice that exclusively express TLR4 on endothelial cells can efficiently clear a lethal systemic Gram-negative bacterial infection. J Clin Invest 119: 1921–1930. - PMC - PubMed
    1. Harding M, Kubes P (2012) Innate immunity in the vasculature: interactions with pathogenic bacteria. Curr Opin Microbiol 15: 85–91. - PubMed
    1. Ley K, Laudanna C, Cybulsky MI, Nourshargh S (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7: 678–689. - PubMed
    1. Rao RM, Yang L, Garcia-Cardena G, Luscinskas FW (2007) Endothelial-dependent mechanisms of leukocyte recruitment to the vascular wall. Circ Res 101: 234–247. - PubMed

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This work was supported by a grant from the Korean Ministry of Education, Science and Technology, FPR08B1-240 of the 21C Frontier Functional Proteomics Program, and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No.20120005634). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.