In this study, we examined the actions of the prototypic UPEC isolate NU14 and a panel of diverse E. coli strains on urothelial cytokine secretion. We found that, in contrast to a laboratory strain, NU14 elicited a reduced CXCL-8 response and suppressed urothelial secretion of CXCL-8 in response to stimulation with TNF-α. We also tested a panel of diverse E. coli strains representing clonal groups commonly associated with UTI and including cystitis, pyelonephritis, and fecal isolates and found that the majority of strains in the panel failed to induce urothelial CXCL-8 and were also capable of inhibiting TNF-α-induced CXCL-8 and CXCL-6 secretion. The ability of UPEC and other clinical E. coli strains to inhibit inflammatory chemokine secretion in vitro suggests that the capacity to modulate immune responses is common among pathogenic E. coli strains.
Recently, transposon mutagenesis of the UPEC strain UTI89 revealed that suppression of chemokine/cytokine secretion is mediated by the LPS biosynthetic operons rfa
along with the periplasmic membrane chaperone SurA in vitro (20
). These results suggest that modification to the bacterial LPS core O antigen may play a role in suppression of TLR4-mediated cytokine secretion. Salmonella
species are known to modify the O antigen, thereby escaping TLR4/CD14 activation of the NF-κB pathways, suggesting the possibility of a conserved mechanism between these closely related pathogens (17
Similarly, Selvaraj and Prasadarao found that RS218, a strain homologous to and of the same serotype as UPEC isolate NU14 (22
), was able to suppress chemokine/cytokine secretion from human monocytes (41
). These investigators also found that suppression of chemokine secretion was heat labile and dependent upon expression of OmpA and IbeA. The cellular mechanisms underlying suppression by RS218 in monocytes and by NU14 in urothelial cells both involve decreased IκB phosphorylation (27
). These similarities suggest that NU14 and RS218 target similar cellular pathways in monocytes and urothelial cells to inhibit NF-κB activation and that this suppression requires several components of the bacterial outer membrane.
The stimulation of urothelial chemokine secretion, whether by E. coli or by TNF-α, LPS, or IL-1β, occurs in an NF-κB-dependent manner through signaling pathways that result in IκB phosphorylation. UPEC suppression of these NF-κB stimuli suggests that the cellular target of suppression occurs either at a common signaling pathway component upstream of IκB phosphorylation or through activation of an inhibitory pathway which is capable of inhibiting IκB phosphorylation by these stimuli. Future studies to identify the precise host cell target(s) of the NF-κB-suppressive activity of UPEC and other clinical E. coli isolates may also shed light upon the identities of the bacterial effectors and their mechanisms of action.
We also report that UPEC can suppress inflammatory responses in a murine model of early stages in UTI. Our experimental observations suggest that UPEC inhibits the transcription of inflammatory chemokines in the bladder relative to the nonpathogenic K-12 strain MG1655. Inhibition of the NF-κB-regulated KC, MIP-2, and CXCL-6 genes was determined using whole-bladder samples. Thus, it is possible that along with urothelial cells, other cell types, such as neutrophils, may be targets of UPEC suppression of chemokine secretion. Another clinical UPEC isolate, 1177, induces MIP-2 secretion from urothelial cells in a mouse model of UTI (12
). Peak MIP-2 secretion, however, originated from recruited neutrophils, which required MIP-2 to cross the urothelial cell layer into the bladder lumen (12
). Our data are consistent with a requirement for MIP-2 secretion in neutrophil recruitment into the urine, as indicated by the correlation between reduced MIP-2 levels in the bladders and urine of mice infected with NU14 and low myeloperoxidase levels relative to levels in mice infected with MG1655.
Ultimately, UPEC does stimulate inflammatory responses in the urinary tract. Infection with UPEC results in net secretion of CXCL-8 from urothelial cells, but the kinetics and magnitude of this induction are diminished relative to levels following infection with the same dose of nonsuppressor E. coli
strains. Our models of early events in the pathogenesis of UTI by UPEC indicate that modulation of urothelial innate immune responses may be important in delaying or diminishing neutrophil influx to the bladder lumen. Modulation of chemokine secretion may allow additional time for invasion of urothelial cells by UPEC, where UPEC has been shown to form intracellular bacterial communities (IBC) in a mouse model of UTI (23
). IBC may provide an early refuge from phagocytosis by neutrophils and later serve as latent reservoirs which are a potential source of recurrent UTI (3
). This ability may underlie the increased colonization of the mouse bladder by strain NU14 compared to colonization by MG1655, which is unable to form IBC.
In summary, we demonstrated that clinical E. coli isolates possess the capacity to modulate urothelial cytokine expression in vitro. This capacity is shared among strains from diverse phylogenetic groups and different clinical origins. In vivo infection with NU14 results in reduced induction of inflammatory chemokines and cytokines, which correlates with decreased recruitment of neutrophils into the bladder and urine and increased bacterial colonization in a mouse model of UTI. Modulation of cytokine secretion is independent of the presence of type 1 pili and 21 other known virulence factors, suggesting that novel virulence factors mediate pathogenesis in the urinary tract by ExPEC.