Difference in the regulation of IL-8 expression induced by uropathogenic E. coli between two kinds of urinary tract epithelial cells

Abstract

Bacterial adherence to epithelial cells is a key virulence trait of pathogenic bacteria. The type 1 fimbriae and the P-fimbriae of uropathogenic Escherichia coli (UPEC) have both been described to be important for the establishment of urinary tract infections (UTI). To explore the interactions between the host and bacterium responsible for the different environments of UPEC invasion, we examined the effect of pH and osmolarity on UPEC strain J96 fimbrial expression, and subsequent J96-induced interleukin-8 (IL-8) expression in different uroepithelial cells. The J96 strain grown in high pH with low osmolarity condition was favorable for the expression of type 1 fimbriae; whereas J96 grown in low pH with high osmolarity condition was beneficial for P fimbriae expression. Type 1 fimbriated J96 specifically invaded bladder 5637 epithelial cells and induced IL-8 expression. On the contrary, P fimbriated J96 invaded renal 786-O epithelial cells and induced IL-8 expression effectively. Type 1 fimbriated J96-induced IL-8 induction involved the p38, as well as ERK, JNK pathways, which leads to AP-1-mediated gene expression. P fimbriated J96-induced augmentation of IL-8 expression mainly involved p38-mediated AP-1 and NF-kappaB transcriptional activation. These results indicate that different expression of fimbriae in J96 trigger differential IL-8 gene regulation pathways in different uroepithelial cells.

Figure 1

Measurement of adhesin mRNA expression and invertible element orientation in UPEC J96 grown under different conditions. (A) Analysis of adhesins FimH and PapG mRNA expression in J96 grown in either pH 7.0 with no NaCl medium or pH 5.5 with 400 mM NaCl medium. RNA samples from J96 were isolated and subjected to RT-PCR analysis. (B) The PCR was performed with chromosomal DNA isolated from J96 grown in pH 7.0 with no NaCl condition or in pH 5.5 with 400 mM NaCl condition, using the INV and FIMA primers to amplify phase-on-oriented DNA, and the FIME and INV primers to amplify phase-off-oriented DNA. The dilutions were used for PCR as follows: undiluted (lanes 1), 1/2 (lanes 2), 1/4 (lanes 3), 1/8 (lanes 4), 1/16 (lanes 5), 1/32 (lanes 6). All PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide.

Figure 2

Invasion of the uroepithelial cells by UPEC. (A) Invasion percentage of bladder 5637 epithelial cells by J96 grown in either pH 7.0 with no NaCl medium (J96-1) or pH 5.5 with 400 mM NaCl medium (J96-P). * p < 0.05 vs. J96-P. (B) Invasion percentage of renal 786-O epithelial cells by either J96-1 or J96-P. * p < 0.05 vs. J96-1. (C) Invasion percentage of 5637 and 786-O epithelial cells by non-fimbriated E. coli 83972 and P-fimbriated 83972 (83972-P). * p < 0.05 vs. 5637 cells infected by 83972-P.

Figure 3

Induction of IL-8 expression in uroepithelail cells by different fimbriated J96 invasion. RNA samples were isolated at the indicated time periods with the infection with indicated fimbrial types, followed by subjecting to real-time PCR analysis. Data are normalized against 18S rRNA level and presented as fold changes in fluorescent density in comparison to that of control ECs (CL) (A-D). The IL-8 protein secretion in conditioned media was determined by ELISA analyses (E,F). 5637 or 786-O cells were kept as controls (CL) or invasion with either type 1 or P fimbriated J96 for the times indicated (A,B), or the cells were invaded with different fimbrial types of J96 for 4 h (C,E) or 12 h (D,F). Data are shown as mean ± standard error of the mean (SEM). * P < 0.05 versus control epithelial cells (CL).

Figure 4

Induction of IL-8 expression in uroepithelail cells by non-fimbriated E. coli 83972 and P-fimbriated 83972 (83972-P) invasion. (A) 5637 or 786-O cells were kept as controls (CL) or invasion with non-fimbriated 83972 or 83972-P for 4 h, RNA samples were isolated and subjected to real-time PCR analysis. Data are normalized against 18S rRNA level and presented as fold changes in fluorescent density in comparison to that of control ECs (CL). (B) The IL-8 protein secretion in conditioned media was determined by ELISA analyses. 5637 or 786-O cells were kept as controls (CL) or invasion with non-fimbriated 83972 or 83972-P for 12 h. Data are shown as mean ± standard error of the mean (SEM). * P < 0.05 versus control epithelial cells (CL).

Figure 5

Invasion with specific fimbriated J96 induces uroepithelial cells to increase the phosphorylation of ERK, JNK, and p38. (A) 5637 cells were kept as controls (CL) or invaded with type 1 fimbriated J96 (J96-1), or (B) 786-O cells were kept as controls (CL) or invaded with P fimbriated J96 (J96-P) for the times indicated, and the phosphorylations of ERK, JNK, and p38 were determined by using Western blot analyses. Phosphorylated ERK, JNK, and p38 levels are presented as band densities (normalized to total protein levels) relative to CL. The results are mean ± SEM from at least 3 independent experiments. * P < 0.05 versus control EC (CL).

Figure 6

Effect of MAPK inhibitors on the regulation of IL-8 expression in uroepithelail cells. (A) 5637 cells were kept as controls (CL) or invaded with type 1 fimbriated J96 (J96-1) for 4 h. (B) 786-O cells were kept as controls (CL) or invaded with P fimbriated J96 (J96-P) for 4 h. Before being kept as controls or invaded with J96, cells were pretreated with PD98059 (PD), SP600125 (SP), or SB203580 (SB) separately for 1 h. Data are normalized against 18S rRNA level and presented as fold changes in comparison to control cells (CL) and. The results are shown as mean ± SEM. * P < 0.05 versus CL. ^#P < 0.05 versus DMSO-treated cells with J96 invasion. ^&P < 0.01 versus DMSO-treated cells with J96 invasion. (C) and (D) The phosphorylation of p38 in 5637 cells (C) and 786-O cells (D) after 30 min of either J96-1 or J96-P invasion was determined by using Western blot.

Figure 7

J96 invasion induced p65 NF-κB and AP-1 binding activities. (A) NF-κB and AP-1 activation by type 1 fimbriated J96 (J96-1) invasion in 5637 cells, and (B) NF-κB and AP-1 activation by P fimbriated J96 (J96-P) invasion in 786-O cells were determined by TF ELISA assay. All bar graphs represent folds of control cells (CL), mean ± SEM. * P < 0.05 versus p65 NF-κB activation in CL. ^#P < 0.05 versus AP-1 activation in CL. (C) 5637 cells were kept as controls (CL) or invaded with J96-1 for 4 h. (D) 786-O cells were kept as controls (CL) or invaded with J96-P for 4 h. Before being kept as controls or invaded with J96, cells were pretreated with NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC), or AP-1 inhibitor Tanshinone IIA (TIIA) individually for 1 h. Data are normalized to 18S rRNA level and presented as fold changes in comparison to control cells (CL) and. The results are shown as mean ± SEM. * P < 0.05 versus CL. ^#P < 0.05 versus DMSO-treated cells with J96 invasion. ^&P < 0.01 versus DMSO-treated cells with J96 invasion.

Figure 8

MAPK signaling pathways were involved in J96-induced AP-1 activation of uroepithelial cells. Type 1 fimbriated J96 (J96-1) induced AP-1 (A) and NF-κB (C) activation in 5637 cells, and P fimbriated J96 (J96-P) induced AP-1 (B) and NF-κB (D) activation in 786-O cells were determined by TF ELISA assays in cells pretreated with vesicle (DMSO), PD98059 (PD; 30 μM), SP600125 (SP; 20 μM), or SB203580 (SB; 10 μM) individually for 1 h, or transfected with si-CL, si-p38, and then invaded with J96 for 2 h. The results are shown as mean ± SEM. * P < 0.05 versus CL. ^#P < 0.05 versus DMSO-treated cells with J96 invasion. ^&P < 0.01 versus DMSO-treated or si-CL transfected cells with J96 invasion.

Figure 9

Schematic representation of the signaling pathways regulating the expressions of IL-8 in uroepithelial cells in response to different fimbrial types of J96 invasion.