A Synthetic Peptide 2Abz23S29 Reduces Bacterial Titer and Induces Pro-Inflammatory Cytokines in a Murine Model of Urinary Tract Infection

Abstract

Introduction: A urinary tract infection (UTI), which is often caused by uropathogenic E. coli (UPEC) strains, affects many people worldwide annually. UPEC causes the production of pro-inflammatory cytokines by the bladder epithelial cells; however, it has been proven that the UPEC can inhibit the early activation of the innate immune system.

Methods: This study aimed to examine the antibacterial and immunomodulatory effects of different doses of truncated alpha-defensins (human neutrophil peptide (HNP)-1) analog 2Abz²³S²⁹ on the mouse UTI model. Experimentally uropathogenic E. coli CFT073-infected mice were treated with low-dose 2Abz²³S²⁹ (250µg/mL), high-dose 2Abz²³S²⁹ (750µg/mL), ciprofloxacin (cip) (800µg/mL), or high-dose 2Abz²³S²⁹plus cip once a day 24 h post-infection. The 2Abz²³S²⁹ and cip treatment were given for two consecutive days.

Results: The in vivo results showed that fewer UPEC were recovered from the bladders of mice treated transurethrally with 2Abz²³S²⁹. Moreover, low-dose 2Abz²³S²⁹ significantly decreased the level of the interleukin-6 (IL-6), whereas high-dose 2Abz²³S²⁹ increased pro-inflammatory cytokines including IL-6, macrophage inflammatory protein/2 (MIP/2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in infected bladders of mice. Besides, the levels of cytokines IL-6 and MIP/2 in infected mice treated with a combination of high-dose 2Abz²³S²⁹ and cip were significantly higher than the untreated mice. In contrast, CFT073-infected mice treated with a combination of high-dose 2Abz²³S²⁹ and cip showed no changes in cytokines TNF-α and IL-1β levels, indicating that ciprofloxacin may play an anti-inflammatory role.

Conclusion: Collectively, apart from the direct antibacterial role of 2Abz²³S²⁹, our data illustrated that 2Abz²³S²⁹ modulates pro-inflammatory cytokine production of bladder in a dose-dependent manner, which has implications for the development of new anti-infective agents.

Keywords: human neutrophil peptide (HNP)-1; pro-inflammatory cytokines; urinary tract infection.

Figure 1

Schematic overview of the experimental set up used for in vivo UTI experiment. At the beginning of the experiment (A), mice were subdivided into four groups. Two infected groups and two uninfected groups that received H₂O or 2Abz²³S²⁹ treatment (n=6 per group, except uninfected group that received H₂O n=3). A transurethral injection of 50 µL of H₂O or 2Abz²³S²⁹ at a low dosage (250µg/mL) was performed after 24 h post-infection, only once. All mice were euthanized on the third day, and bladders were harvested for bacterial count and evaluation of pro-inflammatory cytokines. In the second part of the study (B), mice were randomly subdivided into six groups (n=6 per group), infected untreated group: no treatment, the ciprofloxacin (cip) groups: treated with oral or transurethral cip, the 2Abz²³S²⁹group: treated with transurethral 2Abz²³S²⁹, and the cip plus 2Abz²³S²⁹ groups: treated with oral or transurethral cip and transurethral 2Abz²³S²⁹. A single transurethral high-dose of 2Abz²³S²⁹and ciprofloxacin was 750 µg/mL, and 40 µg in 50 µL PBS, respectively. For a better comparison between groups, ciprofloxacin was also injected transurethrally. Treatments were performed once a day for two consecutive days. All mice were euthanized on the fourth day, and bladders were harvested for bacterial count and evaluation of pro-inflammatory cytokines.

Figure 2

Killing kinetic curve of E. coli CFT073 at 1×MIC for ciprofloxacin and 2Abz²³S²⁹. Bacteria (5×10⁵ CFU/mL) were cultured in the presence of 2Abz²³S²⁹ or ciprofloxacin for various times at 37°C. The untreated bacteria represented as a control. 2Abz²³S²⁹ has shown faster bactericidal effect than ciprofloxacin.

Figure 3

Toxicity of 2Abz²³S²⁹ in human bladder carcinoma cell line 5637. Cytotoxicity assay representing the mean percentage absorbance at 570 nm after incubation human bladder carcinoma cell line 5637 with different concentrations of 2Abz²³S²⁹. Cell viability was measured by MTT assay. All data are displayed as means ± SD from three independent experiments performed in duplicate.

Figure 4

Efficacy of low-dose 2Abz²³S²⁹ peptide in bacterial load (A) and level of pro-inflammatory cytokines (B and C) in the E. coli-infected mouse model. Female BALB/c mice were infected with 1 × 10⁷ CFU E. coli strain CFT073 by transurethral injection. After 24 h, infected and uninfected mice were treated with single low dose of 2Abz²³S²⁹ (250 µg/mL) or H₂O administered. The mice were sacrificed, and the bladders were homogenized 48 h post-infection. Bladder homogenate supernatant was examined for bacterial load and ELISA detection of pro-inflammatory cytokine MIP/2 and IL-6. Data are representative of at least two independent experiments (n=6 per group, except H₂O treated without challenge group n=3). Bars represent the median values. ns indicates not significant, *p<0.05, **p<0.01, as determined by Mann–Whitney U-test.

Figure 5

Continued.

Figure 5

Efficacy of high-dose 2Abz²³S²⁹ peptide, ciprofloxacin, or a combination of both in bacterial load (A) and level of pro-inflammatory cytokines (B–E) in the E. coli-infected mouse model. Female BALB/c mice were infected with 1 × 10⁷ CFU E. coli strain CFT073 by transurethral injection, and double dose of 2Abz²³S²⁹ peptide and/or ciprofloxacin administered after 24 h and 48 h, once a day. The mice were sacrificed, and the bladders were homogenized 72 h post-infection. Bladder homogenate supernatant was examined for bacterial load and ELISA detection of pro-inflammatory cytokine MIP/2, IL-6, TNF-α, and IL-1β. Data are representative of at least two independent experiments (n=6 per group). Bars represent the median values. ns indicates not significant, *p<0.05, **p<0.01, as determined by Mann–Whitney U-test.