Suppression of bladder epithelial cytokine responses by uropathogenic Escherichia coli - PubMed (original) (raw)

Suppression of bladder epithelial cytokine responses by uropathogenic Escherichia coli

David A Hunstad et al. Infect Immun. 2005 Jul.

Abstract

Urinary tract infections are most commonly caused by uropathogenic strains of Escherichia coli (UPEC), which invade superficial bladder epithelial cells via a type 1 pilus-dependent mechanism. Inside these epithelial cells, UPEC organisms multiply to high numbers to form intracellular bacterial communities, allowing them to avoid immune detection. Bladder epithelial cells produce interleukin-6 (IL-6) and IL-8 in response to laboratory strains of E. coli in vitro. We investigated the ability of UPEC to alter epithelial cytokine signaling by examining the in vitro responses of bladder epithelial cell lines to the cystitis strains UTI89 and NU14. The cystitis strains induced significantly less IL-6 than did the laboratory E. coli strain MG1655 from 5637 and T24 bladder epithelial cells. The cystitis strains also suppressed epithelial cytokine responses to exogenous lipopolysaccharide (LPS) and to laboratory E. coli. We found that insertional mutations in the rfa and rfb operons and in the surA gene all abolished the ability of UTI89 to suppress cytokine induction. The rfa and rfb operons encode LPS biosynthetic genes, while surA encodes a periplasmic cis-trans prolyl isomerase important in the biogenesis of outer membrane proteins. We conclude that, in this in vitro model system, cystitis strains of UPEC have genes encoding factors that suppress proinflammatory cytokine production by bladder epithelial cells.

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Figures

FIG. 1.

FIG. 1.

UPEC strains induce less IL-6 from cultured bladder epithelial cells than does E. coli K-12. Confluent bladder cell monolayers were incubated for 2 h with various doses of UPEC strain UTI89 or NU14 or the laboratory E. coli strain MG1655, and IL-6 levels in culture supernatants were measured by sandwich ELISA. (A) IL-6 produced by 5637 cells upon infection with UPEC strain UTI89 or NU14 is equal to unstimulated levels (solid lines), while MG1655 induced significantly more IL-6 at all doses examined (dashed line) (*, P < 0.05; **, P < 0.01). (B) In T24 cells, infection with UPEC induces a modest IL-6 response (solid lines), while stimulation with E. coli K-12 strain MG1655 (dashed line) yields significantly higher IL-6 levels (*, P < 0.03). Experiments were repeated at least three times.

FIG. 2.

FIG. 2.

UPEC strains suppress epithelial cytokine responses to exogenously added gram-negative bacterial LPS or E. coli K-12. (A) Confluent monolayers of 5637 cells were left untreated, incubated with 5 μg/ml E. coli O55:B5 LPS, or incubated with LPS plus 107 CFU of the indicated E. coli strains. The UPEC strains UTI89 and NU14 (solid bars) significantly reduced the IL-6 produced in response to exogenous LPS (*, P < 0.01 versus LPS), while MG1655 (striped bar) did not demonstrate such reduction. (B) Confluent monolayers of T24 cells were treated similarly with 500 ng/ml E. coli O55:B5 LPS or with LPSplus 107 CFU of the indicated E. coli strains. The UPEC strain NU14 (*, P < 0.01) and its isogenic fimH mutant NU14-1 (**, P < 0.05) (solid bars) both suppressed T24 cytokine responses to exogenous LPS. (C) Confluent monolayers of 5637 cells were left untreated, treated with 106-CFU/ml MG1655, or treated with MG1655 plus 107 CFU/ml of the indicated UPEC strains. Live UPEC strain UTI89 or NU14 (solid bars) suppressed IL-6 responses to MG1655 (*, P < 0.01 versus MG1655 alone), while heat-killed UTI89 and NU14 (striped bars) have lost this capability. Experiments were repeated at least three times.

FIG. 3.

FIG. 3.

Disruption of LPS biosynthetic operons in UPEC results in cytokine induction from bladder epithelial cells. Confluent monolayers of 5637 cells were treated with the indicated doses of wild-type UTI89 (squares), the transposon insertion mutant 11F5 (circles) or 16C1 (triangles), or the E. coli K-12 strain MG1655 (diamonds; dashed line). At doses of 106 CFU/ml and above, induction of epithelial IL-6 by the LPS mutants 11F5 and 16C1 was significantly higher than that by UTI89 (*, P < 0.05) and approximated the induction by MG1655. Experiments were repeated at least three times.

FIG. 4.

FIG. 4.

Insertional inactivation of surA results in increased cytokine induction from bladder epithelial cells. Confluent monolayers of 5637 (A) and T24 (B) cells were treated with the indicated doses of wild-type UTI89 (squares), UTI89 surA (open circles), or UTI89 surA/pAER1 (filled circles). The surA mutant displays increased cytokine induction, particularly at doses of 106 CFU/ml and above (*, P < 0.02 versus UTI89), and this phenotype reverts to wild type upon complementation with the plasmid pAER1. Experiments were repeated at least three times.

FIG. 5.

FIG. 5.

Mutations in LPS biosynthetic genes or in surA abrogate UPEC's ability to suppress cytokine responses to MG1655. Confluent monolayers of 5637 cells were left untreated, treated with 106-CFU/ml MG1655, or treated with MG1655 plus 107 CFU/ml of the indicated UPEC strains. Unlike the UPEC strain UTI89 (solid bar), the LPS biosynthetic mutants 11F5 and 16C1 and the surA mutant (striped bars) fail to suppress epithelial cytokine responses to MG1655. Experiments were repeated at least three times.

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