Uropathogenic Escherichia coli is the causative agent for >80% of uncomplicated urinary tract infections (UTIs). Uropathogenic E. coli strains express a number of virulence and fitness factors that allow successful colonization of the mammalian bladder. To combat this, the host has distinct mechanisms to prevent adherence to the bladder wall and to detect and kill uropathogenic E. coli in the event of colonization. In this study, we investigated the role of IL-17A, an innate-adaptive immunomodulatory cytokine, during UTI using a murine model. Splenocytes isolated from mice infected by the transurethral route robustly expressed IL-17A in response to in vitro stimulation with uropathogenic E. coli Ags. Transcript expression of IL-17A in the bladders of infected mice correlated with a role in the innate immune response to UTI, and γδ cells seem to be a key source of IL-17A production. Although IL-17A seems to be dispensable for the generation of a protective response to uropathogenic E. coli, its importance in innate immunity is demonstrated by a defect in acute clearance of uropathogenic E. coli in IL-17A−/− mice. This clearance defect is likely a result of deficient cytokine and chemokine transcripts and impaired macrophage and neutrophil influx during infection. These results show that IL-17A is a key mediator for the innate immune response to UTIs.
Uncomplicated urinary tract infections (UTIs) occur in otherwise healthy individuals who lack urinary anatomical abnormalities. Bacteria derived from fecal matter access the urinary tract through the urethra and subsequently colonize the bladder, resulting in inflammation and symptoms clinically characterized as cystitis (1). Left untreated, bacteria can ascend the ureters and colonize the kidneys, resulting in a more serious secondary infection termed acute pyelonephritis (2). UTIs accounted for >6.8 million physician office visits and 1.3 million emergency room visits in the year 2000, resulting in an estimated cost of $2.4 billion annually (3). Additionally, 40–50% of women will experience one or more UTIs in their lifetime, and 10–15% of these women will experience recurrent infection (4). Uropathogenic Escherichia coli is the primary causative agent of uncomplicated UTIs (4). This subset of extraintestinal pathogenic E. coli expresses a number of virulence determinants that contributes to successful colonization of the urinary tract. These determinants include factors needed for flagellar motility (5) and an array of adherence organelles and iron-uptake systems (6).
Several mechanical forces are thought to function to minimize UTIs, including urine flow and voiding, mucus shedding, and epithelial cell sloughing (7, 8). When uropathogenic E. coli overcomes these physical barriers by adhering to the epithelium, a robust innate immune response is generated. TLR4, TLR5, and TLR11 have been shown to be important for uropathogenic E. coli recognition and immune mobilization (9–11). Because of a stop codon in the available human gene sequences, TLR11 may only function in mice (12). A number of secreted factors, such as antimicrobial peptides, Tamm-Horsfall protein, cytokines IL-6, TNF-α, IL-1β, G-CSF, and IL-17, and chemokines CXCL1, CXCL2, CXCL3, CXCL8, and CCL4 are detected in the mammalian bladder upon infection (13–18). Among cellular infiltrates, neutrophils are the most abundant early responders to infected bladders (19). Additionally, Ag-presenting macrophages (20) and dendritic cells (21) and innate-like lymphocytes, such as γδ T cells (22), have been implicated in the UTI host defense.
IL-17A or IL-17R has been shown to play a critical role in autoimmune disease (23, 24) and in bacterial (25–31), fungal (32–35), and even viral (36–38) infection. Because of the roles played in both arms of the immune system, IL-17A has emerged as an innate-adaptive immunomodulatory cytokine. With regard to the innate immune response to infection, IL-17A acts by indirectly enhancing neutrophil migration to infected tissue. Specifically, transcripts for cytokines involved in granulopoiesis and chemotaxis in cells treated with IL-17A exhibit enhanced mRNA stability (39–42). In addition to being the signature cytokine secreted by CD4+ Th17 cells, other cell types were reported to secrete IL-17A, including cytotoxic T cells, γδ T cells, invariant NKT cells, neutrophils, eosinophils, and monocytes (23). Although the exact pathogen-associated molecular patterns that trigger the secretion of IL-17A are not defined, components of microbial cell wall, host receptors that recognize such components (i.e., the mannose receptor), and TLR-related signaling pathways are implicated (26, 33).
In an effort to characterize the immune response to known uropathogenic E. coli antigenic outer membrane proteins (43), we discovered that IL-17A was secreted by in vitro-stimulated splenocytes derived from uropathogenic E. coli iron receptor-vaccinated mice (44). Given the importance of IL-17A in controlling mucosal infection (45), the role of IL-17A during the innate and adaptive immune response to UTI was formally investigated. In the murine model, IL-17A is upregulated by in vivo-sensitized secondary lymphoid tissue cells in response to in vitro restimulation, yet IL-17A seems to be dispensable for the generation of protective immunity. IL-17A is also upregulated in the bladder in response to acute infection, and γδ cells are a major source of secreted IL-17A in the bladder tissue. IL-17A seems to play a role in regulating the innate immune response to UTI; mice lacking IL-17A exhibit deficient cytokine transcript upregulation and cellular responses during acute UTI, resulting in suboptimal clearance of uropathogenic E. coli.
Materials and Methods
Mice were maintained in specific pathogen-free conditions, and all experiments were conducted according to protocols approved by University Committee on the Use and Care of Animals at the University of Michigan. C57BL/6 wild type (WT) mice and mice harboring the Tcrdtm1Mom mutation [TCR δ−/−, resulting in deficient γδ TCR expression in all adult lymphoid and epithelial organs (46)] were purchased from The Jackson Laboratory (Bar Harbor, ME). Breeding pairs of IL-17A−/− mice were a gift from Yoichiro Iwakura (The University of Tokyo) (47). All experiments were conducted when animals were 6–15 wk old, and C57BL/6 WT mice with birth dates within 1 wk of the IL-17A−/− or TCR δ−/− knockout mice (both in the C57BL/6 background) were used. For manipulation, mice were anesthetized with 100 mg ketamine, 10 mg xylazine/kg body weight. When necessary, mice were euthanized using a lethal dose of isoflurane, and appropriate organs were harvested for analysis.
For infections and sensitizations, a 50-μl uropathogenic E. coli suspension in PBS was inoculated transurethrally using a sterile 0.28-mm polyethylene catheter connected to a Harvard Apparatus infusion pump. All infections, sensitizations, and challenges consisted of 5 × 107 CFU per mouse administered through the transurethral route. To determine organ CFU, bladders were harvested from euthanized animals, weighed, and homogenized in PBS with a General Laboratory Homogenizer (Omni International, Kennesaw, GA). Homogenates were plated on Luria-Bertani agar containing 0.5 g/l NaCl using the Spiral Biotech Autoplate 4000 spiral plater. Colonies were enumerated using the Spiral Biotech QCount, with a limit of detection of 100 CFU/g tissue.
Bacterial strains and whole-cell lysate preparation
E. coli CFT073 was used for all infections. This prototypic uropathogenic E. coli strain was isolated from the urine and blood of a patient with acute pyelonephritis (48), and it has been fully sequenced and annotated (49). For whole-cell lysate preparation, a single colony of E. coli CFT073 was inoculated into 250 ml sterile human urine (pooled from five healthy donors) and cultured statically at 37°C until the late stationary phase. Bacterial cells were harvested by centrifugation (8,000 × g, 5 min, 4°C), washed, resuspended in 10 ml PBS, and incubated at room temperature with 2 μl benzonase solution (10 U/μl, Sigma-Aldrich, St. Louis, MO) for 30 min. Lysis was executed by two passes through an American Instrument French pressure cell press at a pressure of 20,000 pounds per square inch. Lysate was cleared by centrifugation (8,000 × g, 5 min, 4°C) and sterilized using a 0.22-μm filter (Millipore, Bedford, MA). Protein in the lysate was quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL).
Spleens (1.5 × 106 cells/well) or the inner inguinal (lumbar) lymph nodes (5 × 105 cells/well) responsible for draining the pelvic viscera were harvested and made into single-cell suspensions by forcing organs through 40-μm BD Falcon cell strainers. Lymph nodes from the same groups of animals were pooled and plated in replicate wells. Erythrocytes were lysed using 8.02 mg/ml NH4Cl, 0.84 mg/ml NaHCO3, and 0.37 mg/ml EDTA in distilled water. Final cellular suspensions were made in RPMI 1640 medium (with Gibco l-glutamine) with 1% sodium pyruvate, 1% l-glutamine, 1% penicillin/streptomycin, 1% nonessential amino acids, 10% FBS, and 0.001% 50 mM β-mercaptoethanol. Cells were cultured with medium, 5 μg/ml α-CD3 [clone 145-2C11, a gift from Dr. Cheong-Hee Chang (University of Michigan)], or 25 μg/ml E. coli CFT073 whole-cell lysate and incubated at 37°C, 5% CO2 for 72 h, at which point supernatants were harvested and stored at −20°C for ELISA.
3BO3, and 0.64 mg/ml NaOH) overnight at 4°C. Nonspecific binding sites were blocked with 2% BSA in PBS at 37°C for 1 h. Dilutions of purified IL-17A or test samples were made in dilution buffer (0.05% Tween 20, 2% FBS in PBS), and 50 μl was applied to wells at 37°C for 1 h. Biotinylated detection Abs were diluted to 0.25 μg/ml in dilution buffer and applied to wells at 37°C for 45 min. Streptavidin-HRP (Southern Biotechnology Associates, Birmingham, AL) was diluted 1:5000 in dilution buffer, and 100 μl was applied to wells at 37°C for 30 min. o-Phenylenediamine easy-tablets from Acros Organics (2 mg/tablet) were dissolved (4 tablets, 5 μl H2O2 in 12 ml dH2O), and 100 μl was applied to wells at room temperature until color developed. The reaction was stopped by the addition of 100 μl 6 N H2SO4, and absorbance was measured with a Bio-Tek Instruments μ Quant plate reader at 490 nm. Plates were washed by flooding each well four times with wash buffer between each step.
RNA isolation, cDNA synthesis, quantitative RT-PCR, and RT-PCR
−ΔCt, where ΔCt equals the cycle threshold of test gene − the cycle threshold of GAPDH).
50), Cδ (5152); Vγ1.2 with Cγ (53); Vγ2-4 (54) and Vγ5 (5′-CCT ACT TCT AGC TTT CTT GC-3′) with Jγ1 (5′-CTT ACC AGA GGG AAT TAC TAT GAG-3′); and Vδ1-3 and 5-8 (52) and Vδ4 (53) with Jδ1 (51). Nomenclature used in the manuscript is according to Garman et al. (55). As with qPCR, RT-PCR reactions were conducted with appropriate no template and no reverse transcriptase controls for cDNA and qPCR reactions (data not shown).
Bladder tissue was harvested at necropsy and fixed in 10% neutral buffered formalin for 24 h. Tissues were trimmed and processed by standard histological methods and were stained with H&E. Light microscopic histopathological assessment was performed by a board-certified veterinary pathologist blinded to the group assignment of the samples. The presence or absence of inflammation and the predominating type and tissue distribution of inflammatory cells were assessed qualitatively. Additional points assessed were the presence/absence of bacteria, subjective assessment of bacterial quantity and distribution, and mucosal changes.
Urine was collected by massaging the mouse abdomen while holding the urethra over a sterile Eppendorf tube. Urine was mixed 10:1 with Turk’s stain (0.05 mg/ml crystal violet, 3% glacial acetic acid in distilled water), and neutrophils were enumerated using a hemacytometer.
Cellular staining and flow cytometry
Bladders isolated from euthanized mice were cut into small pieces with a scalpel. Tissue was digested for 50 min at 37°C with agitation in 0.5% heat-inactivated FBS, 20 mM HEPES, pH 7, 0.057 Kunitz U/μl DNase I (Sigma-Aldrich), and 1 mg/ml collagenase A (Roche) in RPMI 1640 medium, with repeated passage through an 18.5-gauge needle 25 min into the incubation. Erythrocytes were lysed as described above. E-lysed homogenates were filtered through 40-μm cell strainers and washed once with flow cytometry buffer (1% FBS, 0.01% NaN3
Graphing and statistical analyses were done using GraphPad Prism 5. Data were represented as mean or median values based on the D’Agostino and Pearson omnibus normality test. Where applicable, the Mann-Whitney U test, paired t test, or Fisher exact test was used to determine statistical significance with two-way ANOVA and 95% confidence intervals.
IL-17A is secreted by spleen and lymph node cells from C57BL/6 mice in response to transurethral infection with uropathogenic E. coli
To determine whether IL-17A is secreted adaptively in response to uropathogenic E. coli Ags, WT C57BL/6 mice were inoculated via the transurethral route with uropathogenic E. coli strain CFT073 or PBS, according to the outlined schedules (Fig. 1). Cells harvested from the spleen (Fig. 1A) and the inner inguinal lymph nodes (Fig. 1B) were stimulated in vitro with medium alone, α-CD3 mAb, or uropathogenic E. coli strain CFT073 whole-cell lysate and incubated for 72 h before harvesting supernatants for ELISA. As expected, splenocytes treated with α-CD3 mAb had a high expression of IL-17A, regardless of whether they originated from PBS- or CFT073-treated animals; unstimulated cells from both treatment groups did not secrete IL-17A (Fig. 1). However, in response to in vitro stimulation with uropathogenic E. coli whole-cell lysate, only the splenocytes from uropathogenic E. coli-infected mice showed significant secretion of IL-17A compared with unstimulated controls (p = 0.0147; Fig. 1A). Additionally, inner inguinal lymph node cells from uropathogenic E. coli-infected mice secreted significantly higher amounts of IL-17A in response to in vitro lysate stimulation than lymphoid cells from PBS-treated animals (p = 0.0105; Fig. 1B). These results indicate that treatment with uropathogenic E. coli Ags stimulates adaptive secretion of IL-17A from in vivo-sensitized cells of systemic and local lymphoid origins.
IL-17A is not necessary for the adaptively acquired protective immune response to UTI
Because IL-17A was secreted in response to uropathogenic E. coli Ags (Fig. 1) and the role that IL-17A plays in bacterial vaccination models (56, 57), we wanted to determine whether IL-17A is required for the generation of a protective immune response to UTI. To do this, we used a reinfection model based on one presented by Thumbikat et al. (58). WT and IL-17A−/− mice were transurethrally sensitized once (1×), twice (2×), or not at all (N) prior to a 48-h challenge with uropathogenic E. coli (Fig. 2). Both WT sensitization groups had significantly fewer bacteria in their bladders compared with WT naive mice (p = 0.0193 for 1× and p = 0.0016 for 2× compared with N; Fig. 2), consistent with a previously published study (58). Sensitized IL-17A−/− mice also exhibited a similar pattern of accelerated clearance (p = 0.0071 for 1× and p = 0.0062 for 2× compared with N; Fig. 2). These results suggest that IL-17A is not required for the generation of a protective immune response to UTI.
IL-17A transcript is upregulated in response to acute bladder infection by uropathogenic E. coli
Because there was no overt defect in the generation of protective immunity in IL-17A−/− mice, we sought to determine whether IL-17A played a role in the innate response to UTI. To do this, we first examined IL-17A transcript dynamics during acute infection. Mice were inoculated transurethrally with uropathogenic E. coli, and their bladders were collected for qPCR analysis at several time points during a 28-d period. uropathogenic E. coli-infected mice demonstrated a dramatic increase in IL-17A, with median values peaking at 48 h postinfection (hpi) (Fig. 3A), suggesting a role for IL-17A in the innate immune response to UTI.
γδ T cells are a significant source of IL-17A during acute UTI in the mouse model
Because TCR δ−/− mice are more susceptible to UTI (22), and γδ TCR+ cell populations are known to express IL-17A in the context of bacterial infection (25, 28–30, 59), we wanted to determine whether TCR δ−/− mice had a deficiency in IL-17A transcript expression upon bladder infection. WT C57BL/6 and TCR δ−/− mice were inoculated transurethrally with PBS or uropathogenic E. coli, and their bladders were analyzed by qPCR at 48 hpi. WT and TCR δ−/− mice exhibited significant upregulation of IL-17A compared with PBS-treated controls (data not shown). However, the median value of IL-17A expression was 3.7-fold higher in the WT mice (1.16 × 10−3 relative to GAPDH for WT compared with 3.1 × 10−4 for TCR δ−/− mice; Fig. 3B). These results demonstrate that mice deficient in the γδ TCR tend to have lower expression of IL-17A at 48 hpi, suggesting that γδ T cells are a source of the IL-17A secreted in response to uropathogenic E. coli bladder colonization.
Because TCR δ−/− mice express less IL-17A in response to experimental UTI, we sought to quantify the level of IL-17A expression by γδ T cells in infected WT animals by flow cytometry. Bladders were isolated from PBS- or uropathogenic E. coli-inoculated WT C57BL/6 mice at 48 hpi and made into single-cell suspensions for staining and flow cytometric analysis. For comparison, the expression of IL-17A by CD4+ cells was also analyzed. Although the number of infiltrating CD4+ cells was an order of magnitude higher than that of γδ TCR+ cells, the increases in both populations were statistically significant (p = 0.0021 and p = 0.0229, respectively; Fig. 4A). Each population was then interrogated for IL-17A positivity, as depicted by representative plots (Fig. 4B). Only γδ cells exhibited statistically significant increases in IL-17A positivity after uropathogenic E. coli infection (p = 0.0225; Fig. 4C). In addition, the median frequency of γδ cells also staining positive for IL-17A was ∼5%; in some animals, up to 12% of the γδ cell population expressed IL-17A compared with the PBS group (p = 0.0317; Fig. 4D). These results indicate that at 48 hpi, γδ+ T cells are responsible for the upregulated IL-17A transcripts seen in uropathogenic E. coli-infected mouse bladders.
Recently, it was shown that responsiveness to IL-23 is important for the expression of IL-17A by γδ T cells in a non–TCR-dependent fashion (60). To determine whether IL-23R was expressed in the bladder and, thus, could be mediating a role in IL-17A expression by γδ T cells in response to UTI, we quantified IL-23R expression in bladder tissue by qPCR. IL-23R transcript was detected in the bladder of C57BL/6 WT mice, regardless of the state of their infection (Fig. 5A), and levels of IL-23R transcript were similar in WT and IL-17A−/− mice (data not shown). These results indicate that IL-23R is present in the bladder tissue of mice and may play a role in the rapid expression of IL-17A in response to UTI.
In addition to IL-23R expression, we wanted to probe the expression of γ and δ variable chains to determine whether a particular subset of γδ T cells is responsible for the secretion of IL-17A in response to UTI. Bladders from C57BL/6 mice that were transurethrally infected with CFT073 for 48 h were harvested and prepared for RT-PCR. cDNA was first synthesized using gene-specific primers for the common γ or common δ chain, and PCR for six Vγ and eight Vδ chains was performed on the corresponding products. We detected the expression of all of the Vγ [with the exception of the Vγ1.3 pseudogene (61)] and Vδ chains tested in the bladder tissue of infected mice (Fig. 5B). A similar pattern of expression was observed in uninfected mice (data not shown), indicating that variable chain expression did not seem to change in response to infection.
IL-17A plays a role in defending the urinary tract from acute uropathogenic E. coli colonization
To examine the role of IL-17A in the innate control of UTI, bladder and kidney homogenates from WT and IL-17A−/− mice were cultured at the reported peak (24 hpi) of bacterial colonization in C57BL/6 mice (14). At this time point, IL-17A−/− mice had a 3-fold higher median CFU/g bladder tissue (Fig. 6). By 48 hpi, the peak of IL-17A transcript expression in the bladder (Fig. 3A), this trend increased 10-fold to a 35-fold higher median CFU/g tissue in the bladders of IL-17A−/− mice (Fig. 6). Although these trends were reproducible, we sought to investigate later time points, presuming that the colonization phenotypes resulting from the lack of IL-17A may be exacerbated. Indeed, at 72 and 96 hpi, IL-17A−/− mice had significantly more bacteria colonizing their bladders (p < 0.05; Fig. 6). These results indicate that IL-17A−/− mice are more susceptible to cystitis than are isogenic WT mice.
IL-17A is necessary for proinflammatory transcript upregulation in response to UTI
Given that IL-17A mediates inflammatory responses largely by influencing mRNA levels of key cytokines and chemokines posttranscriptionally (39–42), we wanted to determine whether such effects were present in the context of UTI. WT and IL-17A−/− mice were inoculated transurethrally with uropathogenic E. coli, and their bladders were collected at 48 hpi for transcript analysis by qPCR. We measured mRNA levels of a panel of chemokines, one antimicrobial effector protein (inducible NO synthase [iNOS]), (Fig. 7A) and cytokines (Fig. 7B) previously shown to be affected by IL-17A expression (63). Strikingly, transcripts for all of the genes investigated were expressed at a significantly lower level in IL-17A−/− mice compared with their WT counterparts (Fig. 7), indicating that animals lacking IL-17A−/− signaling are not able to efficiently upregulate the appropriate mRNA transcripts in infected bladder.
Infected WT and IL-17A−/− mice exhibit qualitatively similar responses to UTI when examined histologically
Because IL-17A−/− mice had decreased cytokine and chemokine expression, we wanted to examine the bladders of WT and IL-17A−/− mice histologically to determine whether there were any gross pathological or qualitative differences in inflammation. Longitudinal sections of bladders from WT and IL-17A−/− mice that were uninfected or infected for 48 h (the peak of IL-17A expression) were stained with H&E and visualized microscopically. The sections revealed similar histopathological effects in response to UTI in both backgrounds (Fig. 8). More specifically, bladders from the uninfected mice were histologically within normal limits, without inflammation or other alteration (Fig. 8A, 8B, 8E, 8F). However, bladders from the infected mice had expansion of the lamina propria by edema fluid, accompanied by perivascular and interstitial inflammation (Fig. 8C, 8D, 8G, 8H, black arrowheads). These occurrences ranged from mild to severe in WT and IL-17A−/− mice. Additionally, umbrella cell sloughing was apparent in the infected animals (compare apical surface of the transitional epithelium in Fig. 8E and 8F to that in Fig. 8G and 8H). Inflammatory infiltrates consisted primarily of neutrophils, although sometimes a mixed monocytic and neutrophilic infiltrate was observed (high-power images not shown). Occasional intraepithelial inflammation was also noted in both backgrounds (Fig. 8H, open arrowhead). Small numbers of adherent bacteria were observed in slides from infected animals, whereas larger numbers of adherent and intraluminal bacteria were present in sections from IL-17A−/− mice (Fig. 8H, “B” with arrow), indicative of a decreased ability to eliminate bacteria and consistent with higher bacterial loads in the knockout animals.
IL-17A is required for optimal macrophage and neutrophil infiltration in response to UTI
Neutrophils are the first cell type to migrate to the bladder in the event of UTI, and they are crucial for controlling infection at early time points (10, 19, 64). Because no differences in neutrophil numbers were seen histologically, a more quantitative approach to determine the neutrophil response in WT and IL-17A−/− mice was taken. We counted the number of neutrophils in the urine postinfection using a hemacytometer; the peak of this measurement is 6 hpi in mice and humans (19, 65). At 6 hpi, IL-17A−/− mice had significantly fewer neutrophils in their urine (p = 0.0480; Fig. 9). Additionally, IL-17A−/− mice lacked a population of high-responder mice that was present in the WT cohort; these animals had >1.5 × 106 neutrophils/ml in their urine at 6 hpi (compare 45% of WT animals to 17% of IL-17A−/− animals, p = 0.0262; Fig. 9). Because IL-17A transcript and protein are detectable in the bladder at this early time point (Fig. 3A) (14), these results indicate that IL-17A may be important for very early neutrophil migration to the bladder in response to uropathogenic E. coli infection.
To investigate the innate cellular response to UTI in the absence of IL-17A at a later time point (48 hpi), we used flow cytometry to quantify the number of macrophages and neutrophils localized to the bladder tissue in WT and IL-17A−/− mice. Macrophages were determined by interrogating the bladder for F4/80+MHC class II+ cells, whereas neutrophils were defined as Ly-6G+CD11b+MHC class II− cells. Representative plots showing the gating for macrophages and neutrophils in WT and IL-17A−/− bladder cells are shown (Fig. 10A, 10C). After quantification, the macrophage (p = 0.0145; Fig. 10B) and the neutrophil (p = 0.0031; Fig. 10D) cell populations were significantly lower in IL-17A−/− mice compared with WT mice. These data reveal that IL-17A plays an important role in the recruitment of macrophages and neutrophils to the bladder in response to uropathogenic E. coli infection.
With the exception of asymptomatic bacteriuria (66), the bladder mucosa has been widely accepted as a sterile environment. Mammalian hosts use a number of mechanisms to keep this niche microbe-free, because infection by bacterial and fungal pathogens can lead to serious clinical consequences. In this study, we characterized the role of the cytokine IL-17A during UTI using a murine model. IL-17A was upregulated specifically in response to uropathogenic E. coli Ags by secondary lymphoid tissue cells from uropathogenic E. coli-infected C57BL/6 mice. IL-17A transcripts were also highly upregulated in the bladders of acutely infected mice; γδ T cells were a major source. Although no role for IL-17A in vaccination-induced UTI protection was observed, we noted a deficiency in bladder neutrophil influx during the very early stages of acute cystitis and higher bacterial burdens in IL-17A−/− mice. Knockout mice also had impaired proinflammatory transcript expression and fewer macrophages and neutrophils infiltrating the bladder tissue. Taken together, these results define IL-17A as an important factor in the innate immune response to uropathogenic E. coli-mediated UTI.
Although the presence of pathogen-specific Abs in the urine and serum of infected humans and experimental animals has been documented for decades (67), the cascade of immunological events that occurs during the generation of adaptive immunity during UTI has not been established. Although cells from uropathogenic E. coli-sensitized mice highly upregulate IL-17A in vitro, IL-17A was not necessary for the generation of vaccine-induced protective immunity (Fig. 2). This result is in contrast to vaccination models for Streptococcus pneumoniae, Bordetella pertussis, and Pseudomonas aeruginosa, in which IL-17A was required for a protective immunity (56, 57, 68). Because the immune system features redundant pathways, it is unclear whether there is a compensatory factor acting in bladder or lymphoid tissue or whether the downstream effects of IL-17A are not necessary for a protective response to UTI.
In addition to secretion in a recall response setting, IL-17A is upregulated in an innate fashion (Fig. 3A). Similarly, airway IL-17A peaked innately in response to intranasal infection with Chlamydia muridarum, another mucosal pathogen, and this was dependent on bacterial replication (26). In experimental UTI, IL-17A upregulation was also dependent on the ability of uropathogenic E. coli to successfully colonize the urinary tract, because a fecal strain that is unable to colonize the bladder efficiently (EFC4) (48) does not induce IL-17A transcript (data not shown). A recent study by Ingersoll et al. (14) surveyed cytokine and chemokine protein in the bladder of mice during a 2-wk experimental UTI. Although most of the cytokines examined returned to near baseline levels 1 wk postinfection, IL-17 remained elevated (higher than in control mice) throughout the experiment (14). Our transcript data agreed with this and the fact that IL-17A is highly upregulated in response to UTI, with peak levels attained only days postinfection.
A number of cell types has been shown to secrete IL-17A (23). Unlike classical αβ T cells, which recognize Ag that is processed and presented in the context of self-MHC molecules, γδ T cells harbor the ability to directly recognize cognate Ag, allowing for the rapid production of effector molecules (61, 69). Therefore, because of the early upregulation of IL-17A in the bladder (Fig. 3A) (14), we reasoned that CD4+ Th17 cells are not the principal source of IL-17A. Despite being in the T cell minority (Fig. 4A), intracellular staining and flow cytometric analyses demonstrated that γδ T cells were a major source of IL-17A during UTI (Fig. 4B–D). Of note, TCR δ−/− mice were still able to generate some IL-17A transcript over background (Fig. 3B), demonstrating that additional cell types make IL-17A in the bladder of uropathogenic E. coli-infected mice. These results suggest that γδ+ cells recognize uropathogenic E. coli by a currently unidentified ligand and secrete IL-17A in response to uropathogenic E. coli infection.
In the context of UTI, IL-17A seems to play a role in the optimal restriction of bacterial burden (Fig. 6). Infection models for Listeria monocytogenes, disseminated E. coli, Klebsiella pneumoniae, oral and systemic Candida albicans, oral Toxoplasma gondii, C. muridarum, Bacillus subtilis, and others also show that IL-17A signaling is required for acute clearance of the invading organism (25, 26, 29, 31, 32, 34, 70, 71). This collection of data demonstrates the breadth and versatility of IL-17A–mediated pathways in handling various classes of microbes. In contrast, IL-17A was shown to be dispensable for clearance in infection models for systemic Salmonella enterica serovar Enteritidis and pulmonary Mycobacterium bovis bacille Calmette-Guérin (28, 30, 72). The factors determining whether IL-17A signaling is important for clearance in a particular infection model are not clear. They may depend on anatomical location, inherent qualities of the infectious agent, or innate immune signaling in response to pathogen recognition.
IL-17A has a well-documented role in affecting the level of cytokine, chemokine, and antimicrobial expression (62). Thus, it was not surprising that proinflammatory bladder transcripts probed in infected IL-17A−/− mice were not expressed as well as in WT counterparts (Fig. 7). The lack of chemokines important for the infiltration of neutrophils (CXCL2 and weakly CCL20), T cells (CXCL10, CCL5), and dendritic cells (CXCL10, CCL20) may contribute to the defect in bacterial clearance seen in the IL-17A−/− animals. Additionally, cytokines crucial for the activation and mobilization of innate immune responses (IL-6 and IFN-γ) were lacking in this background. Although deficiency in these transcripts may be the result of inadequate stimulation of epithelial cells and resident macrophages, lower iNOS levels may be a reflection of impaired neutrophil infiltrate in the IL-17A−/− mice (Fig. 10D). Unexpectedly, IL-4 was also significantly lower in the bladders of IL-17A−/− mice. IL-4 is a canonical Th2 cytokine (73), and given that IL-4 knockout mice do not have an acute uropathogenic E. coli clearance defect (22), a function in UTI immunity has not been defined. Nonetheless, the reported role of IL-4 in B cell activation (74) suggests that IL-4 may be upregulated to stimulate B cell Ab generation in an adaptive response to UTI. In total, it seems that there is a general inflammatory defect in IL-17A−/− mice and that genes of appreciated and unknown importance are affected by the absence of IL-17A during UTI.
Because IL-17A orchestrates neutrophil recruitment to infected tissue, there is precedence for an innate immune mechanism involving rapid secretion of large amounts of IL-17A to bolster neutrophil killing of uropathogenic E. coli (19, 75). A lack of early neutrophil infiltrate in IL-17A−/− mice (Fig. 9) may be the result of impaired neutrophil exit from the bone marrow and migration to the bladder tissue, because growth factor G-CSF and neutrophil-specific chemokine transcripts were deficient in infected IL-17A−/− animals. Interestingly, Ab-mediated knockdown of G-CSF rendered mice more resistant to UTI, and the investigators suggested that macrophage activation status is responsible for this surprising phenotype (14). Although lacking some G-CSF expression, IL-17A−/− mice do not exhibit an enhanced clearance phenotype (Fig. 6), possibly due to the pleiotropic effects of IL-17A deficiency or signaling by the existing G-CSF. We also examined neutrophil levels in WT and IL-17A−/− mice at later time points during infection. Macrophages were included in the analysis to determine whether their total numbers varied with neutrophils. Upon uropathogenic E. coli infection, both populations increased several-fold over PBS mock-infected mice; however, in the IL-17A−/− mice, neutrophil and macrophage counts were lower compared with WT mice (Fig. 10B, 10D). These data reveal that the defect IL-17A−/− mice exhibit in uropathogenic E. coli clearance may be due to fewer macrophages and neutrophils present in the bladder to execute bacterial clearance.
Collectively, these data demonstrate that IL-17A plays a role in the innate immune response to experimental UTI in a mouse model. Because many of the genes influenced by IL-17A have similar function during UTI in mice and humans (76), we expect that IL-17A also plays a role in controlling the bacterial burdens early during UTI in humans. IL-17A accomplishes such control by enhancing the presence of mRNA transcripts important for the infiltration of neutrophils and other inflammatory mediators. The presence of such cell types is crucial to the defense of the urinary tract from epithelial cell adherence and subsequent invasion by uropathogenic E. coli.
Histology was performed by the Pathology Cores for Animal Research, Unit for Laboratory Animal Medicine, University of Michigan. Histopathological assessment was performed by Pathology Cores for Animal Research pathologist Dr. Ingrid Bergin. The authors also thank Drs. Cheong-Hee Chang, Phillip D. King, Nick Lukacs, and Mary O’Riordan for reagents; Dr. Timothy Bauler, Pamela Lincoln, and Dr. M. Hanief Sofi for technical support; Dr. Yoichiro Iwakura for the gift of IL-17A−/− mice; and members of the Mobley Lab for critical review of the manuscript.
Disclosures The authors have no financial conflicts of interest.
This work was supported in part by Public Health Service Grant AI043363 from the National Institutes of Health.
Abbreviations used in this paper:
- blood vessel
- hours postinfection
- inducible NO synthase
- lamina propria
- not detectable
- quantitative RT-PCR
- TCR δ−/−
- Tcrdtm1Mom targeted mutation knockout mice
- transitional epithelium
- transurethral infection
- umbrella cell
- urinary tract infection
- wild type.
- Received July 23, 2009.
- Accepted December 6, 2009.
- Copyright © 2010 by The American Association of Immunologists, Inc.