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Pathogenic Lifestyles of E. coli Pathotypes in a Standardized Epithelial Cell Model Influence Inflammatory Signaling Pathways and Cytokines Secretion - PubMed

  • ️Fri Jan 01 2016

Pathogenic Lifestyles of E. coli Pathotypes in a Standardized Epithelial Cell Model Influence Inflammatory Signaling Pathways and Cytokines Secretion

Javier Sanchez-Villamil et al. Front Cell Infect Microbiol. 2016.

Abstract

Inflammatory response is key for the host defense against diarrheagenic Escherichia coli and contributes to the pathogenesis of the disease but there is not a comparative study among different diarrheagenic pathotypes. We analyzed the inflammatory response induced by five diarrheagenic pathotypes in a HT-29 cell infection model. The model was unified to reproduce the pathogenesis of each pathotype. To compare the inflammatory responses we evaluated: (i) nuclear NF-κB and ERK1/2 translocation by confocal microscopy; (ii) kinetics of activation by each pathway detecting p65 and ERK1/2 phosphorylation by Western blotting; (iii) pathways modulation through bacterial infections with or without co-stimulation with TNF-α or EGF; (iv) cytokine profile induced by each pathotype with and without inhibitors of each pathway. EHEC but mainly EPEC inhibited translocation and activation of p65 and ERK1/2 pathways, as well as cytokines secretion; inhibition of p65 and ERK1/2 phosphorylation prevailed in the presence of TNF-α and EGF, respectively. Intracellular strains, EIEC/Shigella flexneri, caused a strong translocation, activation, and cytokines secretion but they could not inhibit TNF-α and EGF stimulation. ETEC and mainly EAEC caused a moderate translocation, but a differential activation, and high cytokines secretion; interestingly TNF-α and EGF stimulation did no modify p65 and ERK1/2 activation. The use of inhibitors of NF-κB and/or ERK1/2 showed that NF-κB is crucial for cytokine induction by the different pathotypes; only partially depended on ERK1/2 activation. Thus, in spite of their differences, the pathotypes can also be divided in three groups according to their inflammatory response as those (i) that inject effectors to cause A/E lesion, which are able to inhibit NF-κB and ERK1/2 pathways, and cytokine secretion; (ii) with fimbrial adherence and toxin secretion with a moderate inhibition of both pathways but high cytokines secretion through autocrine cytokine regulation; and (iii) the intracellular bacteria that induce the highest pathways activation and cytokines secretion by using different activation mechanisms. This study provides a comprehensive analysis of how the different pathogenesis schemes of E. coli pathotypes manipulate inflammatory signaling pathways, which leads to a specific proinflammatory cytokine secretion in a cell model infection that reproduce the hallmarks of infection of each pathotype.

Keywords: ERK1/2; IL-8; NF-κB; TNF-α; inflammatory response; pathogenic E. coli.

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Figures

Figure 1
Figure 1

Reproduction of the phenotypic features (adhesion, cytotoxicity and actin cytoskeleton rearrangements) induced by different E. coli pathotypes and nuclear translocation of NF-κB in HT-29 cells as intestinal model. HT-29 cells were infected (MOI 10) during 4 h with the different E. coli pathotypes as indicated. As controls, cells were treated with E. coli HB101, a non-pathogenic strain, or with TNF-α, a NF-κB translocation inducer. After treatment, cells were washed, fixed and stained with rhodamine-phalloidin for actin filaments and TO-PRO-3 for DNA detection, while NF-κB translocation was detected by immunofluorescence with anti-phospho-NF-κB (phospho-p65) antibodies followed by FITC-goat anti-rabbit IgG antibodies. The preparations were analyzed and documented with a confocal microscope (63X). Arrowheads indicate the location of bacterial adhesion and arrows point out cytoskeleton rearrangements (pedestal formation induced by EPEC, EHEC), rounding cell (EAEC), and cell invasion (intracellular actin tails by EIEC and S. flexneri). Bar 15 μm.

Figure 2
Figure 2

Reproduction of the phenotypic features induced by different E. coli pathotypes and nuclear translocation of ERK1/2 in HT-29 cells. HT-29 cells were infected (MOI 10) during 4 h with the different E. coli pathotypes as indicated. As controls, cells were treated with E. coli HB101, a non-pathogenic strain, or with EGF, an ERK1/2 translocation inducer. After treatment, cells were processed for confocal microscopy as indicated in Figure 1, but ERK1/2 translocation was detected by immunofluorescence using anti-phospho-ERK1/2 antibodies followed by FITC-goat anti-mouse IgG antibodies. Arrowheads indicate the location of bacterial adhesion and arrows point out cytoskeleton rearrangements (pedestal formation induced by EPEC, EHEC), rounding cell (EAEC), and cell invasion (intracellular actin tails by EIEC and S. flexneri). Bar 15 μm.

Figure 3
Figure 3

TNF-α and EGF induce a strong activation of NF-κB and ERK1/2 signaling pathways respectively, but not a non-pathogenic strain, in HT-29 cells. (A,B) Effects of TNF-α and EGF on epithelial cells. HT-29 cells were stimulated with TNF-α or EGF (10 ng/ml) during 10, 15, 30, 60, 120, and 240 min. (C,D) Effects of E. coli HB101 on epithelial cells and its co-stimulation with either TNF-α or EGF. HT-29 cells were inoculated with the non-pathogenic strain E. coli HB101 at a MOI of 10 for 0.5, 1, 2, and 4 h and divided into two groups. The first group was directly processed after E. coli HB101 inoculation. In the second group, after incubation, cells were treated with Gentamicin (100 μg/ml) to kill bacteria for 1 h prior to adding TNF-α (C) or EGF (D) (10 ng/ml) for 10 min. Cellular extracts were analyzed by Western Blot using primary specific antibodies for NF-κB (phospho-p65, phospho-IκB-α and IκB-α) or ERK1/2 (phospho-ERK1/2 and total ERK1/2) and actin, following by HRP-goat anti-rabbit IgG or HRP-rabbit anti-mouse IgG2a. Uninfected cells stimulated with TNF-α or EGF represent 100% of activation for NF-κB (C) or ERK1/2 (D) (as positive controls). All proteins evaluated were normalized with actin. Densitometry analysis shows the means ± SEM for at least three independent experiments. ***p < 0.001 comparing stimulated versus co-stimulated at each time point, using two-way ANOVA test and post-hoc Tukey test.

Figure 4
Figure 4

NF-κB stimulation by the different E. coli pathotypes on HT-29 cells and its co-stimulation with TNF-α. HT-29 cells were infected with the different E. coli pathotypes, EPEC (A), EHEC (B), ETEC (C), EAEC (D), EIEC (E), and S. flexneri (F) at a MOI of 10 for 0.5, 1, 2, and 4 h and divided into two groups. The first group was directly processed after the infection. In the second group, after incubation, cells were treated with Gentamicin (100 μg/ml) to kill bacteria for 1 h prior to adding TNF-α (10 ng/ml) during 10 min. Cellular extracts were analyzed by Western Blot using primary specific antibodies for NF-κB (phospho-p65) and actin, following by HRP-goat anti-rabbit IgG antibody. Uninfected cells stimulated with TNF-α (as a positive control). All proteins evaluated were normalized with actin. Densitometry analysis shows the means ± SEM for at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 comparing stimulated versus co-stimulated at each time point, using two-way ANOVA test and post-hoc Tukey test. For easy comparison, gray bars indicate infected cells and dark gray those co-stimulated cells.

Figure 5
Figure 5

ERK1/2 stimulation by the different E. coli pathotypes on HT-29 cells and its co-stimulation with EGF. HT-29 cells were infected with the different E. coli pathotypes, EPEC (A), EHEC (B), ETEC (C), EAEC (D), EIEC (E), and S. flexneri (F) at a MOI of 10 for 0.5, 1, 2, and 4 h and divided into two groups. The first group was directly processed after the infection. In the second group, after incubation, cells were treated with Gentamicin (100 μg/ml) to kill bacteria for 1 h prior to adding EGF (10 ng/ml) during 10 min. Cellular extracts were analyzed by Western Blot using primary specific antibodies for ERK1/2 and phosphorylated-ERK1/2, following by HRP-rabbit anti-mouse IgG2a antibody. Uninfected cells stimulated with EGF (as a positive control). ERK1/2 activation values were normalized with total ERK1/2. Densitometry analysis shows the means ± SEM for at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 comparing stimulated versus co-stimulated at each time point, using two-way ANOVA test and post-hoc Tukey test. For easy comparison, gray bars indicate infected cells and dark gray those co-stimulated cells.

Figure 6
Figure 6

IL-8 and TNF-α secretion induced by the different E. coli pathotypes in HT-29 cell model. HT-29 cells were infected with the different E. coli pathotypes (MOI 10) during 0.5, 1, 2, or 4 h. After the infection, cell supernatants were removed and analyzed using Human inflammatory cytokines CBA kit (BD Biosciences). Data were acquired on a BD FACS Fortessa flow cytometer. All IL-8 (A) and TNF-α (B) data are means ± SEM of the three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 comparing each pathotype by group, using Student's t-test.

Figure 7
Figure 7

Infection of human macrophages THP-1 cell line with the different E. coli pathotypes induce a differential cytokine secretion of IL-8, IL-1β, TNF-α, IL-6, and IL-10. THP-1 cells were differentiated into macrophages-like cells using PMA at a concentration of 10 ng/mL during 48 h. After differentiation, adherent cells were infected with the different E. coli pathotypes (MOI 10) during 2 or 4 h. After infection, cell supernatants were removed and analyzed using Human inflammatory cytokines CBA kit. Data were acquired on a BD™ FACS Fortessa flow cytometer. Graphs represent cytokine secretion of IL-8 (A) and TNF-α (B), as IL-1β (C), IL-6 (D), and IL-10 (E). All data are means ± SEM of the three independent experiments. For statistical comparison, the pathotypes were grouped according to their way of interacting with the host cell as in Figure 6: *p < 0.05, **p < 0.01, ***p < 0.001 comparing each pathotype group or ∧p < 0.05, ∧∧p < 0.01, ∧∧∧p < 0.001 comparing each pathotype versus non-pathogenic E. coli HB101, using Student's t-test.

Figure 8
Figure 8

Effect of the inhibitors of NF-κB and ERK1/2 in the cytokine secretion induced by the different E. coli pathotypes. HT-29 cells were pretreated with inhibitors of NF-κB (BAY 11-7082) or ERK1/2 (PD98059) pathways or both, during 1 h at a concentration of 100 μM. After treatment, cells were carefully washed and infected with the different E. coli pathotypes (MOI 10) during 2 and 4 h. After the infection, the supernatants were removed and analyzed using Human inflammatory cytokines CBA kit. Data were acquired on a BD LSR Fortessa™ FACS flow cytometer. Graphs represent cytokine secretion of IL-8 (A) and TNF-α (B). All data are means ± SEM of the three independent experiments. ***p < 0.001 comparing infected versus pretreated with inhibitors, using two-way ANOVA test and post-hoc Tukey test.

Figure 9
Figure 9

Summary of the inflammatory responses induced by the different pathotype on epithelial cells. HT-29 cells differentially responded to the infection by the different pathotypes. In spite of their differences, the pathotype can also be divided in three groups according to their inflammatory response: (i) EPEC and EHEC that inject effectors to cause A/E lesion, which are able to inhibit NF-κB and ERK1/2 pathways, and cytokine secretion; (ii) EAEC and ETEC with fimbrial adherence and toxin secretion with a moderate inhibition of both pathways but high cytokines secretion through autocrine cytokine regulation; and (iii) S. flexneri and EIEC, the intracellular bacteria, that induce the highest pathways activation and cytokines secretion by using different activation mechanisms. The secretion of IL-8 and TNF-α induced by the different E. coli pathotypes was partially inhibited by ERK1/2 inhibitor (PD98059) and completely inhibited by NF-κB inhibitor (BAY 11-7082).

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