Human neutrophil peptides 1-3 protect the murine urinary tract from uropathogenic Escherichia coli challenge

Experimental UTI challenges were performed by transurethrally injecting an inoculum of 50 μL into the bladder of female 6- to 12-wk-old mice. Alternatively, the equivalent volume of sterile phosphate-buffered saline (PBS) was administered transurethrally to control mice. For double inoculum experiments, repeat infections were performed 3 h following the first administration. Mice were killed by CO₂ inhalation and bilateral nephrectomy for organ harvesting and processing. Further information and references on methodologies and materials used in the experiments performed can be found in SI Appendix. Figures were plotted using GraphPad Prism 8 (GraphPad Software). *P value <0.05, **P value <0.01, ***P value <0.001, and ****P value <0.0001 were considered statistically significant. Statistical significances between groups were determined by software’s two-tailed Student’s t test unless specified in the figure legend or methods section. The schematic art images used in figures were obtained from Servier Medical art (https://servier.com/Powerpoint-image-bank). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.

We performed whole-genome sequencing of genomic DNA from both wild-type (WT) and transgenic human DEFA^4/4 gene mice using long-range sequencing technology (10× Genomics). The sequence alignment analysis shows that DEFA1A3-containing reads map to chromosome 13 of the mouse genome. We then aligned the flanking sequences of these reads onto the University of California Santa Cruz genome assembly of the mouse (GRCm38/mm10). Analysis determined a single insertion site of the DEFA1A3 cassette to be located within the second intron of the mouse gene VMn1r195 on chromosome 13 (Fig. 1A). The copy number (CN) of genes was estimated to be eight CNs for the insertion cassette localized in the DEFA^4/4 genome on the C57BL/6J strain mouse background. To confirm our alignment sequencing results, we performed polymerase chain reaction (PCR) genotyping using a primer-probe set designed out of the flanking sequences of the insertion sites for the target repeats. Agarose gel products resulted in 729-bp amplicon bands for nonrepeat carriers. A band of 513 bp was reproduced to determine repeat carriers in the transgenic human DEFA^4/4 gene mouse (Fig. 1 B and C).

We evaluated the effects of transgene insertion in the transgenic human DEFA^4/4 gene mouse compared to its WT counterpart utilizing inflammatory and AMP expression using mRNA markers of the target urinary tract. We further support these findings by performing gastrointestinal (GI) microbiota composition using 16S sequencing alignment by phylum, and immune cell profile using myeloid and nonmyeloid surface markers expressed in kidneys. We found no statistically significant differences in the inflammatory profiling of the kidney and bladder compared to WT or in the GI microbiota amplicons sequenced (SI Appendix, Figs. S1 and S2).

We examined the role of the DEFA1A3 gene locus in the transgenic human DEFA^4/4 gene mouse by modeling UTI responses in the human kidney upon UPEC challenge with the CFT073 pyelonephritis strain. We conducted time-point studies to evaluate the regulation of alpha-defensins transcription kinetics in the transgenic human DEFA^4/4 gene mouse kidney and bladder tissues. DEFA1A3 mRNA transcripts significantly increased in the kidney and bladder by 1.98 ± 0.16-fold increase (P < 0.0001) and 10.47 ± 2.83-fold increase (P < 0.0001), respectively, compared to unstimulated controls (Fig. 2 A and B). The kidney peak transcription occurred 6 h postinfection (hpi), while bladder expression peaked at 24 hpi. Interestingly, down-regulation of DEFA1A3 transcription in kidney and bladder occurred at multiple early time points. Transcriptional regulation of kidney and bladder tissues of transgenic human DEFA^4/4 gene mouse were restored to baseline levels by 48 h following UPEC pyelonephritis challenge. We also evaluated other peptides important to innate immunity that are induced in the kidney following UPEC challenge (7). We compared the induction of pentraxin 3 (Ptx3), lipocalin 2 (Lcn2), myeloperoxidase (Mpo), and β-defensins 1 (Defb) mRNA AMPs upon experimental pyelonephritis challenge at the acute time points between the DEFA^4/4 and C57BL/6J mouse kidneys (SI Appendix, Fig. S3). Interestingly, Mpo and Defb mRNA expression was induced as early as 1 and 3 hpi, respectively. Significant induction of Lcn2 and Ptx3 occurred at 6 hpi and returned to baseline levels after 24 h of challenge. Moreover, mean expression levels were not statistically different between WT and DEFA^4/4-infected kidneys for the AMP mRNA markers assessed. These findings indicate that DEFA1A3 does not induce other components of the innate immune system involved in the defense against UTIs. To determine whether our model recapitulates HNP1-3 secretion into urine during infection as seen in UTI patients, we designed a sandwich enzyme-linked immunosorbent assay (ELISA) with urine samples from infected transgenic DEFA^4/4 mice (6). Due to limitations in mouse clean-catch urine collection methods, samples of four mice per group were pooled to quantify urinary HNP1-3 levels. Urine samples from DEFA^4/4 at 24 h post-UPEC challenge showed elevation of HNP1-3 compared to saline-injected controls regardless of the UPEC strains inoculated (P = 0.341). Urine HNP1-3 levels significantly increased for both UTI89 (P = 0.004) and CFT073 (P = 0.001) experimental UTI challenges when compared to saline controls (Fig. 2C). Immunofluorescence experiments visually showed enhanced expression of HNP1-3 mostly colocalized in aquaporin-2 (AQP2) positive collecting ducts in kidney sections from transgenic human DEFA^4/4 gene mouse upon UPEC pyelonephritis challenge at 6 hpi compared to baseline expression in saline-injected mice (Fig. 2D). At this time point, we also observed other nonstructural HNP1-3–expressing cells upon infection, irrespective of myeloid cell migration (Fig. 2E). The expression of HNP1-3 and AQP2 dual-positive cells appears in apical collecting duct cells postpyelonephritis challenge in DEFA^4/4 mice compared to saline controls. Additionally, we evaluated immunohistochemistry staining of transgenic human DEFA^4/4 gene mouse bladders. Although we did not detect baseline HNP1-3 expression in epithelial cells, there was a significant influx of HNP1-3–expressing nonstructural cells visually localized around the bladder lumen (Fig. 2 F and G). To further understand the contribution of different cellular sources of alpha-defensins under the UTI setting, we performed in vivo experimental UTI challenges of our transgenic mice and evaluated mRNA expression levels of UPEC groups of three cellular subsets (CD45⁺, CD45⁻cKIT⁺, and CD45⁻cKIT⁻). Interestingly, the myeloid (CD45⁺) and intercalated cell populations (CD45⁻cKIT⁺) had the highest DEFA1A3 transcription at baseline. The myeloid cell populations (CD45⁺) had no change in transcriptional expression of DEFA1A3 under the pyelonephritis UTI murine challenge at 6 hpi (P = 0.99), whereas the intercalated cell population increased by 14.17-fold ± 14.81 (P = 0.33) when compared to saline transgenic controls (SI Appendix, Fig. S4A). We further confirmed this transcription pattern in enriched human intercalated cells using similar cell population sorting methodologies (8) and identified a significant up-regulation of DEFA1A3 mRNA with a mean increase of 2.93-fold ± 0.62 (P = 0.02) following in vitro stimulation with UPEC; the CFT073 strain compared to cells under saline treatment derived from four separate human kidney tissue samples (SI Appendix, Fig. S4B).

We quantified and compared the bacterial burden of kidneys and bladders for the infected transgenic human DEFA^4/4 gene and C57BL/6J mice (WT). Experiments were conducted on transurethrally infected mice with two UPEC strains for selective infectivity toward the kidney (CFT073) or bladder (UTI89). To evaluate early invasion and persistence of UPEC in urinary tract tissues, we evaluated colony forming unit (CFU) per gram of bladders and left and right kidneys at 6 and 24 hpi between mouse genotypes. First, transgenic human DEFA^4/4 gene mice transurethrally challenged with cystitis strain UTI89 demonstrated decreased bladder and kidney burdens at 6 h. The kidney bacterial clearance was higher in DEFA^4/4 mice compared to WT (Fig. 3 A and B). In the challenge with the more virulent pyelonephritis strain CFT073, the protection of DEFA^4/4 mice kidney and bladder tissues was more evident when compared to the WT mouse. At early time points, both the kidney and bladder demonstrate lower burdens, but only the mean bladder CFU burdens were significantly lower in the DEFA^4/4 mouse. At 24 h, the bladders had significantly lower bacterial burdens compared to WT controls (Fig. 3 C and D). Similar to the UTI89 UPEC cystitis strain challenge, the CFT073 UPEC pyelonephritis strain challenge showed no significant differences at later time points evaluated 1 wk postinfection (SI Appendix, Fig. S5 A and B). After 1 wk postinfection, we did not observe differences in signs of renal scarring between infected transgenic and WT mice in kidney Masson’s trichrome stained slides (SI Appendix, Fig. S5 C–E). Due to limited renal infection burden in the human gene transgenic mouse model of UTIs compared to WT, we further induced experimental pyelonephritis twice by repeating the UTI challenge in the transgenic mice 3 h following the first challenge and were able to find an inverse correlation between peak kidney DEFA1A3 mRNA expression at 6 hpi with CFU counts for the bladder (r² = 0.545) and kidney (r² = 0.608) tissue lysates, respectively (Fig. 3 E and F). Additionally, we evaluated the gene-dosage effect of the DEFA1A3 genome copy number (GCN) insertions under the pyelonephritis UTI murine challenge at 6 hpi using littermate homozygous DEFA^4/4, heterozygote DEFA^4/0, and wild-type genotypes (8, 4, and 0 GCNs per diploid genome, respectively). We found significantly lower bacterial burden and bacterial clearance in the hemizygous DEFA^4/0 mouse group with four GCNs when compared to littermate zero human gene transgenic mouse urinary tract tissues lacking DEFA1A3 expression, respectively, for bladder (P = 0.0004) and kidneys (P = 0.022) at 6 hpi. No significant differences were found between eight and four GCN groups in mean bacterial burden of bladder (P = 0.20) or kidneys (P = 0.81), although more kidneys were protected from infection in the eight GCN (DEFA^4/4) when compared to four GCN (DEFA^4/0) group (Fig. 3 G and H). In order to determine whether renal sources of HNP1-3 alone are enough to protect the urinary tract, we performed kidney transplant isographs into WT recipient mice with DEFA^4/4 transgenic kidneys and used WT transplants to WT recipients as controls for infection studies (Fig. 3I). Following experimental pyelonephritis challenge, the DEFA^4/4 transgenic kidney transplanted WT recipient mice group exerted significant reduction in mean bladder total CFU burden (P = 0.002), and kidney bacterial protection in the transgenic kidney transplanted mice when compared to control transplants at 6 hpi (Fig. 3 J and K). The resistance of a WT kidney into a WT mouse to ascending UPEC infection using our methodology is consistent with our prior studies using a transplant infection model (9).

From the single and double inoculation experimental pyelonephritis challenges in DEFA^4/4 and WT mice, we examined kidney histology in order to measure the extent of pyelonephritis in infected kidneys. Kidney sections stained with hematoxylin and eosin dyes (H&E) were evaluated by implementing a previously reported inflammatory scoring system (10, 11). We blindly scored tissue sections assessing the medullary and cortex kidney regions from each kidney from respective genotypes (SI Appendix, Fig. S6 A–H). We did not find inflammatory differences between infected DEFA^4/4 and WT mice at 6 or 24 hpi (SI Appendix, Fig. S6I). At these acute time points, inflammation was most prominent in the kidneys from mice with double inoculations regardless of the mouse genotypes at 6 and 24 hpi. Then, we correlated inflammatory scores, kidney CFU burdens, and relative mRNA expression of interleukin 6 (Il6), C-C motif ligand 2 (Ccl2), interleukin 1-beta (Il1β), and tumor necrosis factor alpha (Tnfα) mRNA inflammatory markers in C57BL6/J kidneys. We are able to demonstrate significant correlations between murine inflammatory kidney scores, CFU bacterial titers, Il6, and Il1β mRNA inflammatory markers following single and double CFT073, UPEC challenges as early as 6 hpi (SI Appendix, Table 1). Correlated in C57BL6/J mice, inflammatory scores correlated the most with Il6 and Il1β mRNA fold expression (R² = 0.728, P = 0.003 and R² = 0.828, P = 0.0007, respectively). Additionally, Il6 and Il1β strongly correlated to kidney CFU bacterial burdens (R² = 0.881, P = 0.0002 and R² = 0.602, P = 0.014). Using these inflammatory assessment methodologies, enabled us to correlate infection and inflammation with the relative expression of Il6 and Il1β cytokines.

We then compared the correlations between inoculated C57BL6/J and DEFA^4/4 kidneys infected at 6 hpi with the pyelonephritis CFT073 strain. Following 6 hpi, kidneys from infected DEFA^4/4 mice wild-type mice had statisitically significant correlation between kidney Il6 and Il1β cytokine mRNA expression and CFU bacterial burdens (SI Appendix, Table 2). Additionally, we were able to demonstrate that following 6 hpi with CFT073 infection, the DEFA^4/4 kidneys exerted significantly lower expression of Il6 and Il1β mRNA inflammatory markers upon infection mice compared to wild-type kidneys (P = 0.0437 and P = 0.0092 for each cytokine) (SI Appendix, Fig. S7 A and B). Cumulatively, these results suggest that the HNP1-3 AMP has direct protection from UPEC challenge and not from a secondary effect of inducing inflammatory pathways.

We utilized natural HNP1-3, a commercially available peptide, to determine the minimum inhibitory concentration breakpoints against enteropathogenic ATCC10789/K-12 (EC), and UPEC UTI89 and CFT073 E. coli strains under Clinical and Laboratory Standards Institute (CLSI) guidelines (SI Appendix, Fig. S8 A and B). We identified strain-selective resistance against HNP1-3 reflective of other antimicrobial peptides (Fig. 4A). To better recapitulate the urinary microenvironment, we adjusted the sodium concentration, level of acidity, and alkalinity of the growth media. As a result, raising the pH of media to 9.0 resulted in an average of 30 to 40% antimicrobial activity regardless of the isolate tested at the respective minimal inhibitory concentration (MIC), which represents the smallest concentration of a substance to stop bacterial growth (SI Appendix, Fig. S8D). We report no change in antimicrobial activity when pH was lowered to 4.0. Additionally, addition of sodium chloride at 150 mM in the growth media reduced antimicrobial activity by an average of 50 to 60% (SI Appendix, Fig. S8E). To assess whether HNP1-3 protects the urothelium from bacterial attachment, confluent human bladder epithelial cells (HBLAK) were challenged with UPEC in the presence and absence of HNP1-3. Compared with the vehicle control, the percent of UPEC adhered to HBLAK cells incubated with HNP-1 was significantly reduced at 2× MIC but not at 1× MIC (P = 0.650) (Fig. 4B). To confirm whether HNP1-3 kills UPEC in vitro, we collected cell culture media inoculated with UPEC in the presence and absence of HNP1-3. After a 2-h incubation with HNP1-3, UPEC survival was significantly decreased with both 1× and 2× MIC (Fig. 4C).

Following determination of MIC breakpoints with broth macrodilution and microdilution assays for LL-37, RNAse7, beta-defensins 1 and beta-defensins 2 against E. coli isolates, combinations of antimicrobial peptides were tested at twofold increases to determine synergistic (<0.5), additive (0.5 to 1.0), or no interaction between AMPs and HNP1-3 (SI Appendix, Fig. S8C). Regardless of the E. coli isolate, the calculated fractional inhibitory concentration indexes (FICIs) indicated synergistic effects of LL-37 following the addition of sub-MIC of HNP1-3 (FICI = 0.37) with the exception of MDR58 (FICI = 0.5), which was additive. Combinations of sub-MICs with RNAse7 demonstrated additive effects among all strains (FICI = 0.5). Interestingly, there was no interaction when adding human beta-defensin 1 or human beta-defensin 2 (FICI = 1.0) (Fig. 4 D–G). Evidenced from the drug resistance phenotype from our in vitro MIC breakpoint studies using the MDR58 strain, we tested in vivo antimicrobial resistance in our pyelonephritis model by inoculating MDR58 into DEFA^4/4 and C57BL6/J mice (Fig. 4 H and I). At 6 hpi, quantified bladder and individual kidney bacterial burdens showed a nonsignificant decrease of mean bacterial colonization in the DEFA^4/4 compared to the wild-type counterpart in both kidneys and bladders (mean = 5.26 ± 0.843 and 5.582 ± 0.582 for bladders and mean = 1.44 ± 2.070 and 2.31 ± 2.561 for kidneys). Additionally, we correlated kidney CFU and previously mentioned inflammatory mRNA markers (SI Appendix, Table S3). Our results indicate that inflammatory mRNA expression in wild-type mice is higher than in transgenic DEFA^4/4 infected with MDR58-infected kidneys, in which induction of Il1β and Il6 gene expression was consistent with mice infected with the CFT073 strain (SI Appendix, Fig. S7 E–H). Taken together, our results support that DEFA1A3 protects the murine urinary tract by combining antimicrobial and potential downstream immunomodulatory effects.