Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Oct 11:9:2268.
doi: 10.3389/fmicb.2018.02268. eCollection 2018.

Outer Membrane Vesicles Derived From Escherichia coli Regulate Neutrophil Migration by Induction of Endothelial IL-8

Jaewook Lee  1 , Yae Jin Yoon  1 , Ji Hyun Kim  1 , Nhung Thi Hong Dinh  1 , Gyeongyun Go  1 , Sookil Tae  2 , Kyong-Su Park  1 , Hyun Taek Park  1 , Changjin Lee  1 , Tae-Young Roh  1   2 , Dolores Di Vizio  3 , Yong Song Gho  1
Affiliations

Outer Membrane Vesicles Derived From Escherichia coli Regulate Neutrophil Migration by Induction of Endothelial IL-8

Jaewook Lee et al. Front Microbiol. .

Abstract

Outer membrane vesicles (OMVs) are spherical, proteolipid nanostructures that are constitutively released by Gram-negative bacteria including Escherichia coli. Although it has been shown that administration of E. coli OMVs stimulates a strong pulmonary inflammatory response with infiltration of neutrophils into the lungs in vivo, the mechanism of E. coli OMV-mediated neutrophil recruitment is poorly characterized. In this study, we observed significant infiltration of neutrophils into the mouse lung tissues in vivo, with increased expression of the neutrophil chemoattractant CXCL1, a murine functional homolog of human IL-8, on intraperitoneal administration of E. coli OMVs. In addition, OMVs and CD31-positive endothelial cells colocalized in the mouse lungs. Moreover, in vitro results showed that E. coli OMVs significantly increased IL-8 release from human microvascular endothelial cells and toll-like receptor (TLR)4 was found to be the main component for recognizing E. coli OMVs among human endothelial cell-associated TLRs. Furthermore, the transmigration of neutrophils was suppressed in the lung tissues obtained from TLR4 knockout mice treated with E. coli OMVs. Taken together, our data demonstrated that E. coli OMVs potently recruit neutrophils into the lung via the release of IL-8/CXCL1 from endothelial cells in TLR4- and NF-κB-dependent manners.

Keywords: IL-8; NF-κB; exosomes; extracellular vesicles; neutrophil; outer membrane vesicles; pulmonary inflammation; toll-like receptor 4.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Neutrophil transmigration in murine lungs induced by E. coli OMVs. Wild-type mice were intraperitoneally administered with either PBS or E. coli OMVs (15 μg in total protein amounts per mouse). Five animals were used in each group. Different groups of mice administered with either PBS or OMVs were killed at 6 h after OMV administration, and the lung tissues as well as BAL fluids were obtained from the five mice. (A–C) The lung sections of five mice were immunostained with anti-NIMP-R14 (green; neutrophils) and anti-CD31 (red; endothelial cells) antibodies (A), or anti-NIMP-R14 (green; neutrophils) and anti-SP-C (red; lung epithelial cells) antibodies (B). The sections were then counterstained with Hoechst 33258 (blue; nuclei). Representative fluorescence images are shown here. Scale bars = 50 μm. The number of neutrophils per field was counted from five confocal microscopy images obtained from the lung sections of five mice (C). (D) The concentration of CXCL1 was measured in the BAL fluid by ELISA (n = 5). Data were represented as mean ± SEM. ∗∗∗P < 0.001, calculated by unpaired Student’s t-test.
FIGURE 2
FIGURE 2
Endothelial cells as main functional targets of OMVs. (A) Wild-type mice were intraperitoneally administered with either PBS or E. coli OMVs (15 μg in total protein amount per mouse). Five animals were used in each group. At 3 h after OMV administration, the lung tissues were retrieved. The lung sections of five mice were immunostained with anti-OMVs (green; E. coli OMVs) and anti-CD31 (red; endothelial cells) antibodies. Representative fluorescence images are shown here. Scale bars = 30 μm. White arrows indicate E. coli OMV-positive endothelial cells. (B) After treating E. coli OMVs (0.5 ng/mL in total protein concentration) to various human (A549, BEAS2B, HMEC-1, HMVEC, THP-1, U937, Jurkat, and MOLT-4) and mouse cells (RAW264.7 and NIH-3T3), the concentrations of human IL-8 or mouse CXCL1 were measured in the culture supernatants by ELISA (n = 3). (C) After treating various OMVs (E. coli, P. aeruginosa, and A. baumannii OMVs; 0.5 ng/mL in total protein concentration) to human HMEC-1 endothelial cells, the concentration of human IL-8 was measured in the culture supernatants by ELISA (n = 3). (D) After treating E. coli OMVs (0.5 ng/mL in total protein concentration) to human HMEC-1 endothelial cells, the concentrations of human CXCL10 were measured in the culture supernatants by ELISA (n = 3). Data were represented as mean ± SEM. ∗∗P < 0.01 and ∗∗∗P < 0.001, respectively, calculated by two-way (B,D) or one-way ANOVA (C) with Bonferroni correction for multiple comparisons.
FIGURE 3
FIGURE 3
Internalization of OMVs into endothelial cells. Uptake and internalization of free DiI control or DiI-labeled E. coli OMVs (red fluorescent signal) by HMEC-1 labeled with 5-chloromethylfluorescein (CMFDA, green fluorescent signal) was examined using a confocal microscopy. Representative three-dimensional fluorescence images are shown here. Scale bars = 5 μm. Note that free DiI control or DiI-labeled E. coli OMVs were prepared by a size-exclusion spun column to remove residual DiI: free DiI control itself does not contain any fluorescent signal. Low magnificent two-dimensional fluorescence images and high magnificent two-dimensional fluorescence images with representative three-dimensional fluorescence images are shown in Supplementary Figure 3.
FIGURE 4
FIGURE 4
The roles of TLR4 and NF-κB in IL-8 release from OMV-stimulated endothelial cells. (A) Total RNA was isolated from HMEC-1 treated with PBS or E. coli OMVs (0.5 ng/mL) for 12 h, and mRNA expression of TLRs (TLR1–TLR9), MD2, CD14, NOD1, and NOD2 were analyzed by real-time RT-PCR (n = 3). Fold changes were calculated by dividing the expression of each gene by that of GAPDH. (B) HMEC-1 was treated with E. coli OMVs (0.5 ng/mL in total protein concentration) or TLR agonists for 12 h, and the concentrations of IL-8 were quantified in the culture supernatants by ELISA (n = 3). Human TLR agonists were used as follows: TLR1/2 agonist, Pam3CSK4, 100 ng/mL; TLR2/6 agonist, FSL1, 100 ng/mL; TLR2 agonist, HKLM, 107 cells/mL; TLR3 agonist, poly (I:C), 1 μg/mL; TLR4 agonist, LPS-EK, 10 ng/mL; TLR5 agonist, FLA-ST, 100 ng/mL; TLR7 agonist, imiquimod, 100 ng/mL; TLR8 agonist, ssRNA40, 10 ng/mL; and TLR9 agonist, ODN2006, 500 nM. (C) PBS, E. coli OMVs (10 ng/mL in total protein concentration), or LPS-EK (10 ng/mL) were treated to HMEC-1, with or without TLR4 antagonist (1 μg/mL) for 12 h, and the concentrations of IL-8 were measured in the culture supernatants by ELISA (n = 3). (D) Various concentrations (0, 0.01, 0.1, 1, and 10 ng/mL in total protein concentrations) of E. coli W3110 wild-type OMVs or E. coli W3110 ΔmsbB mutant OMVs were treated to HMEC-1 for 12 h, and the concentrations of IL-8 were measured in the culture supernatants by ELISA. (E) PBS or E. coli OMVs (0.5 ng/mL in total protein concentration) were treated to HMEC-1 for 12 h with 0.05% dimethyl sulfoxide (–) or the following signaling inhibitors (final concentration = 10 μM in 0.05% dimethyl sulfoxide): PD98059 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), LY294002 (PI3K inhibitor), and BAY11-7082 (NK-κB inhibitor), and the concentrations of IL-8 were measured in the culture supernatants by ELISA (n = 3). (F) PBS or E. coli OMVs (0.5 ng/mL in total protein concentration) were treated to HMEC-1 for 12 h with various concentrations of BAY11-7082 (0, 0.1, 1, 5, and 10 μM), and the concentrations of IL-8 were measured in the culture supernatants by ELISA (n = 3). (G,H) HMEC-1 were treated with PBS or E. coli OMVs (0.5 ng/mL in total protein concentration) for 0, 10, 30, 60, 120, or 360 min. Whole cell lysates (20 μg in total protein amount) were subjected to analyzing the expression of phosphorylated-IκB (P-IκB) and β-actin by Western blot. The representative blot of two independent experiments (G) and the average values of the relative ratios calculated by dividing the densitometry quantification values for P-IκB by those of β-actin (H). (I) Various concentrations (0, 0.01, 0.1, 1, 10, and 100 ng/mL) of E. coli LPS, OMVs-UC, or OMVs-BDG were treated to HMEC-1 for 12 h, and the concentrations of IL-8 were measured in the culture supernatants by ELISA. LPS, LPS isolated from E. coli; OMVs-UC, OMVs isolated by the combination of ultrafiltration and ultracentrifugation (UC); OMVs-BDG, OMVs isolated by the combination of ultrafiltration, ultracentrifugation, buoyant density gradient ultracentrifugation (BDG), and ultracentrifugation. (J) Wild-type mice were intraperitoneally administered with PBS, E. coli LPS (11.25 μg per mouse), or E. coli OMVs-UC (15 μg in total protein amounts per mouse) or E. coli OMVs-BDG (15 μg in total protein amounts per mouse). Five mice were used for each group. At 6 h after administration, the mice were killed. The BAL fluids were retrieved, and the concentration of CXCL1 was measured in the BAL fluid by ELISA (n = 5). Data were represented as mean ± SEM. ns, non-significant; ∗∗P < 0.01; ∗∗∗P < 0.001, calculated by one-way (B,H,J) or two-way ANOVA (A,C–F) with Bonferroni correction for multiple comparisons.
FIGURE 5
FIGURE 5
Suppression of OMV-induced neutrophil transmigration in murine lungs of TLR4 knockout mice. Wild-type and TLR4 knockout mice were intraperitoneally administered with PBS or E. coli OMVs (15 μg in total protein amount) for 6 h. Five animals were used in each group. (A,B) The lung sections of five mice at 6 h after E. coli OMV introduction were immunostained with anti-NIMP-R14 (green; neutrophils) and anti-CD31 (red; endothelial cells) or anti-SP-C (red; lung epithelial cells) antibodies, and counterstained with Hoechst 33258 (blue; nuclei). Representative fluorescence images are shown here. Scale bars = 50 μm. The number of neutrophils per field was counted from five confocal microscopy images obtained from the lung sections of five mice (B). (C) The concentration of CXCL1 was measured in the BAL fluid by ELISA (n = 5). Data were represented as mean ± SEM. ∗∗∗P < 0.001, calculated by two-way ANOVA with Bonferroni correction for multiple comparisons.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agace W. W., Patarroyo M., Svensson M., Carlemalm E., Svanborg C. (1995). Escherichia coli induces transuroepithelial neutrophil migration by an intercellular adhesion molecule-1-dependent mechanism. Infect. Immun. 63 4054–4062. - PMC - PubMed
    1. Aird W. C. (2003). The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 101 3765–3777. 10.1182/blood-2002-06-1887 - DOI - PubMed
    1. Akira S., Takeda K. (2004). Toll-like receptor signalling. Nat. Rev. Immunol. 4 499–511. 10.1038/nri1391 - DOI - PubMed
    1. Alaniz R. C., Deatherage B. L., Lara J. C., Cookson B. T. (2007). Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo. J. Immunol. 179 7692–7701. 10.4049/jimmunol.179.11.7692 - DOI - PubMed
    1. Andonegui G., Bonder C. S., Green F., Mullaly S. C., Zbytnuik L., Raharjo E., et al. (2003). Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs. J. Clin. Invest. 111 1011–1020. 10.1172/JCI16510 - DOI - PMC - PubMed

LinkOut - more resources