Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun:16:179-188.
doi: 10.1016/j.redox.2018.02.020. Epub 2018 Mar 1.

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells

Affiliations

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells

Larissa A C Carvalho et al. Redox Biol. 2018 Jun.

Abstract

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity.

Keywords: Hypochlorous acid; Microbicidal; Myeloperoxidase; Pseudomonas aeruginosa; Uric acid; dHL-60.

PubMed Disclaimer

Figures

fx1
Graphical abstract
Fig. 1
Fig. 1
Uric acid affects bacterial clearance (A), IL-1β (B) and TNF-α (C) release by dHL-60 cells. dHL-60 cells (2 × 106) were challenged with PA14 (MOI 1:10) for 1, 2 and 3 h at 37 °C. After each time point serial dilutions were spread on agar plates and the colony-forming units (CFU) were determined after overnight incubation at 37 °C (A). The quantification of IL-1β (B) and TNF-α (C) in the supernatants was performed by enzyme-linked immunosorbent assay (ELISA) 3 h after challenging with PA14. Each bar represents mean ± SEM of three independent experiments. Statistical analyses were performed by one-way ANOVA followed by Newman-Keuls post hoc test; *p < 0.05 compared with no dHL-60 group in (A) or with no PA14 group in (B) and (C); #p < 0.05 compared with dHL-60 + PA14 no uric acid group. UA, uric acid.
Fig. 2
Fig. 2
Uric acid does not affect dHL-60 cell viability. (A) dHL-60 cells were incubated with PA14 (MOI 1:10) for one or two hours at 37 °C. Dead cells were stained with PI and the fluorescence (λex = 535 nm, λem = 620 nm) was measured by flow cytometry. Staurosporine (St) was used as a positive control. (B) Cytotoxicity was measured by lactate dehydrogenase (LDH) activity in supernatants of dHL-60 incubated or not with PA14 (MOI 1:10) and uric acid (UA) for 3 h at 37 °C. LDH activity is presented as the percentage relative to the positive control (dHL-60 in presence of lysis buffer). Each bar represents the mean ± SEM of three independent experiments. Statistical analyses were performed by one-way analyses of Variance (ANOVA) followed by Newman-Keuls; *p < 0.05 from control group.
Fig. 3
Fig. 3
Uric acid decreases the production of hypochlorous acid (HOCl) by the myeloperoxidase/Cl-/H2O2system. (A) dHL-60 cells (5 × 106) were incubated with PA14 (MOI 1:10) and uric acid for one hour at 37 °C. (B) Myeloperoxidase (100 nM), NaCl (150 mM), taurine (5 mM) and H2O2 (100 µM) were incubated in minimum medium in the absence or presence of uric acid for 30 min at room temperature. Reaction was stopped by incubating with 50 µg/mL catalase. HOCl was indirectly quantified through the oxidation of TNB (ε412 nm = 14,200 M−1cm−1) to the colorless DTNB by taurine-chloroamine. Each bar represents mean ± SEM of three independent experiments. Statistical analyses were performed by one-way ANOVA followed by Newman-Keuls posthoc test; *p < 0.05 compared to control group (no uric acid, UA).
Fig. 4
Fig. 4
Uric acid inhibits the production of HOCl in dHL-60 challenged with PA14. (A) dHL-60 (5 × 106) were incubated or not with 0.5 mM uric acid or 0.05 mM 4-aminobenzoic acid hydrazide (ABAH) and challenged with PA14 (MOI 1:10) for 1 h at 37 °C in the presence of R19-S (10 µM). Production of HOCl into the phagosome was visualized by confocal microscopy (λex = 515 nm, λem = 545 nm). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar 5 µm. (B) Mean of fluorescence intensity of five different fields in a 16-bit image confocal microscopy. Graph represents the mean ± SEM of three independent experiments. Statistical analyses were performed by one-way ANOVA followed by Bonferroni's test; *p < 0.05; compared to control (PA14) group. UA, uric acid; A.U., arbitrary units.
Fig. 5
Fig. 5
Uric acid increases superoxide production. (A) dHL-60 (1 × 106 cells) were incubated with 5 µM DHR, uric acid (0.2 or 0.5 mM) and PA14 (MOI 1:10) for 1 h. Oxidation of DHR was measured by fluorescence in a 96-well plate reader (λex = 500 nm, λem = 536 nm). (B) Kinetics of superoxide production in dHL-60 cells incubated with PA14 (MOI 1:10), uric acid (0.5 mM), DPI (20 μM) and DHE (10 μM) at 37 °C. Superoxide production was measured by 2-OH-E+ fluorescence (λex = 396 nm, λem = 579 nm). (C) Area under the curve (AUC) of total fluorescence up to 240 min. Each bar represents the mean ± SEM of three experiments. Statistical analyses were performed by one-way analyses of VARIANCE (ANOVA) followed by Newman-Keuls; *p < 0.05 and **p < 0.001 from control group (No PA14 in A or PA14 in C) and #p < 0.05 from PA14 + 0.5 mM UA. DPI: diphenyleneiodonium, UA: uric acid, A.U., arbitrary units.
Fig. 6
Fig. 6
Effect of uric acid on GSH and GSSG levels. dHL-60 cells (5 × 106) were incubated with uric acid (UA, 0.2 or 0.5 mM) and challenged with PA14 (MOI 1:10). Samples were injected onto LC/MS/MS and the mass transitions (m/z 308.0911 → 179.0462) and (m/z 613.1592 → 355.0741) for GSH and GSSG, respectively, were monitored. Each bar represents the mean ± SEM of three independent experiments. Statistical analysis were performed by one-way ANOVA analysis of VARIANCE, followed by Newman-Keuls post-hoc test, *p < 0.05 compared to control without PA14.
Fig. 7
Fig. 7
Uric acid decreases HOCl levels in the phagosome. Uric acid (UA) competes with chloride by the myeloperoxidase-Compound I (+•PorFeIV=O). Uric acid also donates one electron to myeloperoxidase-Compound II (PorFeIV=O), completing the peroxidase cycle of the enzyme (PorFeIII). This competition and the direct reaction of uric acid with hypochlorous acid (HOCl) are likely the main mechanisms responsible by the decrease in HOCl levels. Uric acid has been described by indirectly activate NADPH oxidase (Nox) and increase superoxide (O2•-) production. Superoxide can react with uric acid free radical (UA) to form urate hydroperoxide. Superoxide is also a substrate for myeloperoxidase to generate Compound III (PorFeIVO2) and yet can dismutate to generate the hydrogen peroxide substrate. The rate constants for some reactions are presented.

Similar articles

Cited by

References

    1. Hurst J.K. What really happens in the neutrophil phagosome? Free Radic. Biol. Med. 2012;53:508–520. - PMC - PubMed
    1. Winterbourn C.C., Kettle A.J. Redox reactions and microbial killing in the neutrophil phagosome. Antioxid. Redox Signal. 2013;18:642–660. - PubMed
    1. Winterbourn C.C., Kettle A.J., Hampton M.B. Reactive oxygen species and neutrophil function. Annu. Rev. Biochem. 2016;85:765–792. - PubMed
    1. Ginsburg I., Kohen R. Cell damage in inflammatory and infectious sites might involve a coordinated "cross-talk" among oxidants, microbial haemolysins and ampiphiles, cationic proteins, phospholipases, fatty acids, proteinases and cytokines (an overview) Free Radic. Res. 1995;22:489–517. - PubMed
    1. Babior B.M., Kipnes R.S., Curnutte J.T. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Investig. 1973;52:741–744. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources