Modulation of host innate immune response in the bladder by uropathogenic Escherichia coli

Abstract

Uropathogenic Escherichia coli (UPEC), the most frequent cause of urinary tract infection (UTI), is associated with an inflammatory response which includes the induction of cytokine/chemokine secretion by urothelial cells and neutrophil recruitment to the bladder. Recent studies indicate, however, that UPEC can evade the early activation of urothelial innate immune response in vitro. In this study, we report that infection with the prototypic UPEC strain NU14 suppresses tumor necrosis factor alpha (TNF-alpha)-mediated interleukin-8 (CXCL-8) and interleukin-6 (CXCL-6) secretion from urothelial cell cultures compared to infection with a type 1 piliated E. coli K-12 strain. Furthermore, examination of a panel of clinical E. coli isolates revealed that 15 of 17 strains also possessed the ability to suppress cytokine secretion. In a murine model of UTI, NU14 infection resulted in diminished levels of mRNAs encoding keratinocyte-derived chemokine, macrophage inflammatory peptide 2, and CXCL-6 in the bladder relative to infection with an E. coli K-12 strain. Furthermore, reduced stimulation of inflammatory chemokine production during NU14 infection correlated with decreased levels of bladder and urine myeloperoxidase and increased bacterial colonization. These data indicate that a broad phylogenetic range of clinical E. coli isolates, including UPEC, may evade the activation of innate immune response in the urinary tract, thereby providing a pathogenic advantage.

FIG. 1.

UPEC isolate NU14 induces less urothelial CXCL-8 secretion than a piliated lab strain. (A) TEU-1 cultures were incubated with media only (Control), 1.5 ng/ml recombinant human TNF-α, or increasing amounts of either NU14 or the type 1 piliated lab strain HB101/p1-17. Cells were incubated with either bacterial strain at an MOI of 50:1, 100:1, 250:1, or 500:1. Supernatants were collected after 4 h, and CXCL-8 secretion was determined by ELISA. NU14 induced significantly less CXCL-8 secretion than HB101/p1-17 at all MOI. (B) TEU-1 cells were incubated with media alone (Control), 1.5 ng/ml TNF-α, NU14, or HB101/p1-17 for various times (MOI of 250:1), and CXCL-8 secretion was determined by ELISA. TNF-α and HB101/p1-17 elicited increased CXCL-8 secretion relative to control or NU14 at 3 or more hours of incubation. Data represent the means ± standard deviations of three separate infections, and each experiment was performed in duplicate. Asterisks indicate statistically significant differences between NU14 and HB101/p1-17.

FIG. 2.

NU14 and clinical E. coli isolates suppress urothelial CXCL-8 and CXCL-6 secretion. (A) TEU-1 cells were treated with E. coli strain NU14 or HB101/p1-17 in the presence (+) or absence (−) of 1.5 ng/ml TNF-α. Following culture for 4 h, culture supernatants were harvested, and CXCL-8 secretion was measured by ELISA. (B) TEU-1 cells were treated for 4 h with TNF-α, NU14, HB101/p1-17, or clinical isolates 8 to 64 from the ECOR panel (MOI of 250:1), culture supernatants were harvested, and CXCL-8 secretion was measured by ELISA. (C and D) TEU-1 cells were treated for 4 h with E. coli strain NU14, HB101/p1-17, or various clinical isolates (8 to 64) in the presence (+) or absence (−) of 1.5 ng/ml TNF-α, and CXCL-8 (C) or CXCL-6 (D) secretion was measured by ELISA. Error bars reflect the standard deviations of triplicate samples. Asterisks indicate statistically significant differences between (A) TNF-α-treated and NU14-infected and (B) NU14- and ECOR strain 14- and 51-infected TEU-1 cells.

FIG. 3.

NU14 suppresses chemokine expression in murine UTI. (A) TEU-1 cells were treated with NU14 or MG1655 (MOI of 250:1). Following 4 h of culture, culture supernatants were harvested and CXCL-8 secretion was determined by ELISA. The asterisk indicates statistically significant differences between NU14 and MG1655. Error bars reflect standard deviations of triplicate samples. (B, C, and D) C57BL/6 mice (controls, n = 2; infected, n = 5) were catheterized, and 1 × 10⁶ CFU of either NU14 or MG1655 was instilled into the bladder. After 4 h, the mice were sacrificed and the bladders were harvested for mRNA isolation. Quantitative real-time PCR was used to determine the changes in gene transcript levels by comparing the relative changes in KC (B), CXCL-6 (C), and MIP-2 (D) in infected mouse samples normalized to levels in uninfected controls. Asterisks indicate statistically significant differences between samples infected with NU14 and samples infected with MG1655.

FIG. 4.

Quantitation of myeloperoxidase (MPO) in mouse urine and bladder homogenates during experimental UTI by ELISA. Infection with NU14 results in less MPO in mouse bladder homogenates and urine than infection with MG1655. Mice were infected with 1 × 10⁶ CFU NU14 or MG1655 via catheter (for bladder experiments, four control mice and nine infected mice; for urine experiments, 10 control mice, eight NU14-infected mice, and 10 MG1655-infected mice). Urine samples were collected, and the bladders were removed and homogenized 6 h postinfection. Bladder (A) and urine (B) MPO levels were determined by ELISA. Asterisks indicate statistically significant differences between uninfected controls and samples infected with strain MG1655.

FIG. 5.

Infection with NU14 results in greater bladder colonization. Female C57BL/6 mice (n = 21) were catheterized and instilled with 1 × 10⁸ CFU of NU14 or MG1655 via transuretheral catheter. After 24 h, the mice were sacrificed, and the bladders were homogenized and plated to determine colonization. The asterisk indicates a statistically significant difference between samples infected with NU14 and samples infected with MG1655 (P = 0.0001).