Interleukin-6/Stat3 signaling has an essential role in the host antimicrobial response to urinary tract infection

Abstract

The signaling networks regulating antimicrobial activity during urinary tract infection (UTI) are incompletely understood. Interleukin-6 (IL-6) levels increase with UTI severity, but the specific contributions of IL-6 to host immunity against bacterial uropathogens are unknown. To clarify this we tested whether IL-6 activates the Stat3 transcription factor, to drive a program of antimicrobial peptide gene expression in infected urothelium during UTI. Transurethral inoculation of uropathogenic Escherichia coli led to IL-6 secretion, urothelial Stat3 phosphorylation, and activation of antimicrobial peptide transcription, in a Toll-like receptor 4-dependent manner in a murine model of cystitis. Recombinant IL-6 elicited Stat3 phosphorylation in primary urothelial cells in vitro, and systemic IL-6 administration promoted urothelial Stat3 phosphorylation and antimicrobial peptide expression in vivo. IL-6 deficiency led to decreased urothelial Stat3 phosphorylation and antimicrobial peptide mRNA expression following UTI, a finding mirrored by conditional Stat3 deletion. Deficiency in IL-6 or Stat3 was associated with increased formation of intracellular bacterial communities, and exogenous IL-6 reversed this phenotype in IL-6 knockout mice. Moreover, chronic IL-6 depletion led to increased renal bacterial burden and severe pyelonephritis in C3H/HeOuJ mice. Thus, IL-6/Stat3 signaling drives a transcriptional program of antimicrobial gene expression in infected urothelium, with key roles in limiting epithelial invasion and ascending infection.

Keywords: IL-6; Stat3; antimicrobial peptide; intracellular bacterial community; urinary tract infection; urothelium.

Figure 1. Experimental UTI triggers IL-6 secretion, urothelial p-Stat3, and AMP mRNA expression

A) Urine IL-6/Cr levels peak 2 hpi following transurethral UPEC inoculation. Urine was pooled from 6 mice/experimental group at each time point. Bars indicate mean ± standard deviation of triplicate IL-6/Cr values. #: p = 0.0073, Kruskal-Wallis test. B) Western blotting demonstrates a transient increase in p-Stat3 levels in UPEC infected bladders. C) Immunofluorescence microscopy identifies p-Stat3 reactive nuclei (pink staining) localized mainly within the basal and intermediate urothelial layers. Green staining demonstrates E-cadherin, and DAPI-positive nuclei are indicated in blue. The white dotted line indicates the basement membrane. Scale bars represent 16.25 microns, 60x original magnification. D) QRT-PCR identifies bladder AMP mRNA induction during experimental UTI. * p < 0.05, compared to uninfected, Mann-Whitney U test, n=4 bladders/time point. E) RegIIIβ and RegIIIγ proteins (green) localize to the urothelium during urinary tract infection. Scale bars represent 16.25 microns (RegIIIβ) and 25 microns (RegIIIγ), 60x and 40x original magnification, respectively.

Figure 2. IL-6 is necessary for urothelial Stat3 phosphorylation and AMP mRNA production following UTI

A) Western blot detects attenuated p-Stat3 induction 6hpi in IL-6 KO bladders compared to C57BL/6J controls. Total Stat3 is unchanged in both strains. B) Immunofluorescence demonstrates the absence of p-Stat3 reactivity in IL-6 KO urothelium 6 hpi, compared to C57BL/6J controls. Positive p-Stat3 reactivity is marked by pink nuclear staining and highlighted by arrows. Green: E-cadherin; Blue: nuclei; scale bars represent 25 microns, 40x original magnification. C) Impaired expression of RegIIIβ, RegIIIγ, and Hamp transcripts in IL-6 KO bladders 6 hpi compared to WT C57BL/6J mice; * p = 0.0379 and # p = 0.0002 Mann-Whitney U test, n=8 mice/group. D) Reduced RegIIIγ/Cr levels in IL-6 KO versus WT C57BL/6J urine 24 hpi. #p = 0.0286, Mann-Whitney U test, n = 6 mice/group.

Figure 3. IL-6 drives urothelial Stat3 phosphorylation and AMP production in the absence of UTI

A) Primary human urothelial cells exhibit p-Stat3 induction in response to recombinant human IL-6 treatment. B) Intraperitoneal injection of 100 ng recombinant murine IL-6 induces p-Stat3 in WT bladders and kidneys within 1 hour as compared to animals injected with PBS carrier alone. C) IL-6 treatment induces Lcn2, RegIIIβ, and RegIIIγ mRNA in the absence of UTI. Animals underwent intraperitoneal injection of IL-6 or carrier, and were euthanized 4 hours later. *p = 0.0023 and #p = 0.007, Mann-Whitney U test. Data shown represent 4 mice/condition from one of two independent experiments.

Figure 4. AMP expression is significantly reduced in Stat3^Δ mice

A) RT-PCR demonstrates unique detection of a truncated Stat3 mRNA in Stat3^Δ bladders after tamoxifen injection. B) Reduced baseline total Stat3 protein levels as well as total Stat3 protein and pStat3 at 6 hpi in Stat3^Δ and Stat3^flox bladders; C) Significantly decreased Hamp, RegIIIβ, and RegIIIγ mRNA levels in Stat3^Δ bladders 6 hpi versus Stat3^flox controls (*p = 0.0022, Mann-Whitney U test). n=6 mice/condition from two independent experiments. D) RegIIIγ protein secretion is undetectable in Stat3^Δ urine 6 hpi, compared to Stat3^flox controls; n.d.= not detectable; #p = 0.03, student’s t test, n = 6 mice/group from two independent experiments.

Figure 5. IL-6 deficiency leads to increased bacteriuria and IBC formation

A) Higher magnitude of UPEC recovered from IL-6 KO urine compared to WT C57BL/6J controls. *p=0.0021; **p=0.0440; #p=0.0045, Mann-Whitney U test. B) Transient increase in bladder UPEC burden 6 hpi in IL-6 KO (^#p = 0.038, Mann-Whitney U test), that was not subsequently sustained. C) Increased IBC in IL-6 KO bladders 6 and 16 hpi compared to WT C57BL/6J controls, that normalizes by 24 hpi. *p = 0.0043; **p=0.0286, Mann-Whitney U test. D) This increased IBC phenotype in IL-6 KO bladders is diminished in the setting of recombinant IL-6 protein given intravesically 1 hour before infection and again 2 hpi prior to mice being sacrificed at 6hpi. *p=0.0317, Mann-Whitney U test. E) There is a similar increase in IBC inStat3^Δ bladders compared to control Stat3^flox bladders. *p=0.0216, Mann-Whitney U test.

Figure 6. IL-6 neutralization in C3H/HeOuJ mice leads to severe pyelonephritis

A) Reduction in serum IL-6 levels induced by UPEC in C3H/HeOuJ mice pretreated with α-IL-6 neutralizing antibody 24 hours before infection versus isotype control antibody (α-HRPN). *p<0.05, Kruskal-Wallis test. n=8 mice/group. B) Reduced bladder and kidney p-Stat3 induction by UPEC in C3H/HeOuJ mice treated with α-IL-6 neutralizing antibody versus isotype control (α-HRPN), at specified times after inoculation. C) Increased renal bacterial burden in mice treated with α-IL-6 neutralizing antibody versus α-HRPN 7 days post UPEC infection. ^#p=0.036, Mann-Whitney U test. D) H&E staining of a representative kidney following α-IL-6 neutralizing antibody treatment (left) demonstrating extensive cortical inflammation 7 dpi. The middle micrograph shows higher power magnification of the area depicted in the inset with areas of neutrophilic infiltrate, neutrophil casts in tubules (white arrows), hemorrhage (white asterisk), and abscess (a) formation. An unaffected glomerulus (g) is visible. An example of a representative kidney following α-HRPN treatment is shown for comparison (right) which demonstrates normal renal architecture. Scale bars represent 200 microns, 4x and 20x original magnification.

Figure 7. IL-6 production, Stat3 phosphorylation, and AMP production are Tlr4-dependent

A) UPEC inoculation in LPS-responsive C3H/HeOuJ mice results in urine IL-6 secretion and accumulation of serum IL-6 24 hpi compared to uninfected (Un) controls. Urine and serum IL-6 was not detected (n.d.) in LPS resistant C3H/HeJ mice at the same time point (*p = 0.0046; **p=0.0015, #p=0.034, ##p=0.001, one-way ANOVA, n ≥ 6 mice/group [urine] or 4 mice/group [serum]). B) Western blotting indicates UPEC induced p-Stat3 expression in C3H/HeOuJ bladders and kidneys to a greater extent than that of C3H/HeJ animals. C) Reduced bladder mRNA levels of AMPs in C3H/HeJ bladders compared to C3H/HeOuJ mice 24 hpi (*p<0.01; n=5 mice/group).