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. Author manuscript; available in PMC: 2017 Sep 1.
Published in final edited form as: J Immunol. 2016 Jul 27;197(5):1720–1732. doi: 10.4049/jimmunol.1600336

Uricase Inhibits Nitrogen Dioxide-Promoted Allergic Sensitization to Inhaled Ovalbumin Independent of Uric Acid Catabolism

Jennifer L Ather *, Edward J Burgess *,§, Laura R Hoyt *, Matthew J Randall *, Mridul K Mandal †, Dwight E Matthews †,§, Jonathan E Boyson ‡,§, Matthew E Poynter *,§
PMCID: PMC4992621  NIHMSID: NIHMS800150  PMID: 27465529

Abstract

Nitrogen dioxide (NO2) is an environmental air pollutant and endogenously-generated oxidant that contributes to the exacerbation of respiratory disease and can function as an adjuvant to allergically sensitize to an innocuous inhaled antigen. Since uric acid has been implicated as a mediator of adjuvant activity, we sought to determine whether uric acid was elevated and participated in a mouse model of NO2-promoted allergic sensitization. We found that uric acid was increased in the airways of mice exposed to NO2 and that administration of uricase inhibited the development of ovalbumin (OVA)-driven allergic airway disease subsequent to OVA challenge as well as the generation of OVA-specific antibodies. However, uricase was itself immunogenic, inducing a uricase-specific adaptive immune response that occurred even when the enzymatic activity of uricase had been inactivated. Inhibition of the OVA-specific response was not due to the capacity of uricase to inhibit OVA uptake or processing and presentation by dendritic cells, but at a later step that inhibited OVA-specific CD4+ T cell proliferation and cytokine production. Whereas blocking uric acid formation by allopurinol did not affect outcomes, administration of ultra-clean human serum albumin at protein concentrations equivalent to that of uricase inhibited NO2-promoted allergic airway disease. These results implicate that whereas uric acid levels are elevated in the airways of NO2-exposed mice, the powerful inhibitory effect of uricase administration on allergic sensitization is mediated more through antigen-specific immune deviation than on suppression of allergic sensitization, a mechanism to be considered in the interpretation of results from other experimental systems.

Keywords: allergic airway disease, asthma, uric acid, uricase, inflammation

Introduction

Nitrogen dioxide (NO2) is a byproduct of combustion, both indoor and outdoor, a component of air pollution, and has been linked to the development and severity of respiratory disease including allergic asthma (1, 2). Nitrogen dioxide can also be endogenously generated during infection (3, 4), and can be absorbed all along the respiratory tract. The inhalation and generation of reactive nitrogen species in the lung can lead to oxidative damage of the epithelium, airway acidification, and subsequently impact the pulmonary immune response (5). Animal models have demonstrated that NO2 exposure can contribute to airway epithelial damage, lung tissue fibrosis, and allergic sensitization, depending on the duration of exposure and dose of NO2 provided (6–9). We have previously demonstrated that a single 1 hour 15 ppm dose of NO2 activates airway epithelial NF-κB (8), and when administered just prior to aerosolized ovalbumin (OVA) antigen inhalation is sufficient to sensitize mice to OVA (8), resulting in a mixed Th2/Th17 response that requires TLR2 and MyD88 (8), is mediated through CD11c+ antigen presenting cells (10), and requires both caspase-1 and IL-1 signaling for the Th17 response (11, 12).

The necessity for caspase-1 activity and IL-1 signaling in our NO2-promoted allergic sensitization model suggests some contribution of the NLRP3 inflammasome in the pulmonary response to inhaled NO2 (12), a notion supported by the finding that IL-1α neutralization did not impede NO2-promoted allergic sensitization (12). IL-1β is transcribed as a ~30 kDa precursor protein that typically requires caspase-1 cleavage in order to be secreted in its mature, active form (13). Caspase-1 activity is governed by inflammasome oligomerization, and the NLRP3 inflammasome has been linked to Th2 and Th17 polarization in several allergic asthma models (14–16), although not in all (17).

Uric acid is the most abundant circulating antioxidant in humans and other mammals, accounting for half of the serum antioxidant capacity (18). Typically a byproduct of purine catabolism, uric acid is a small, heterocyclic compound that in its precipitated crystal form is the culprit behind gout (19), wherein it functions as a damage-associated molecular pattern (DAMP) to instigate pro-inflammatory responses. In gout, high concentrations of uric acid crystals activate the NLRP3 inflammasome, causing the production of IL-1β and attendant joint inflammation (20, 21). Uric acid may also be generated locally by the enzyme xanthine oxidase, which is expressed by lavaged cells of house-dust mite exposed mice (22), and is transcriptionally regulated by NF-κB (23). The clinically- and experimentally-used adjuvant, Alum, promotes antigen-specific adaptive immune responses, including those that are Th2-dominated and used to model allergic asthma (24–26) through the local production of uric acid (25), and also promote the elaboration of uric acid into the lavageable airspaces that contribute to allergic sensitization and asthma exacerbation (24, 27). Given that NO2 exposure can lead to cell and tissue damage, and also that NO2 can interact directly with uric acid (28), we hypothesized that uric acid may be present and functional subsequent to NO2 inhalation, contributing to NO2-promoted allergic sensitization.

Methods

Mice

Six to eight week old female C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Female OTII transgenic mice (C57BL/6 background; bred at the University of Vermont from stocks originally from Jackson Laboratories) were 6–8 weeks old at the beginning of experimentation. Mice were housed in an American Association for the Accreditation of Laboratory Animal Care (AALAC)-approved facility, maintained on a 12h light/dark cycle, and provided food and water ad libitum. All animal studies were approved by the University of Vermont Institutional Animal Care and Use Committee. All mice were euthanized with sodium pentobarbital (150 mg/kg by i.p. injection; Wilcox Pharmacy, Rutland, VT).

In vivo NO2 exposure and NO2-promoted allergic sensitization and challenge

For NO2 exposure, a single 1-hour dose of 15 ppm NO2 was administered (12) and mice were analyzed at several times thereafter. Comparisons were made between mice exposed to NO2 or subjected to time in a similar exposure chamber through which HEPA-filtered room air was flowed. For NO2-promoted allergic sensitization, a single 1-hour exposure to 15ppm of NO2 on day 1 was followed by 30 minutes of nebulized 1% OVA, Fraction V (Sigma-Aldrich, St. Louis, MO) in saline, on days 1, 2, and 3 (29). All mice were OVA-challenged on days 14, 15, and 16, as described (30). Analyses were performed at 48 hours after the final OVA challenge, on day 18.

Uricase, human serum albumin, and allopurinol doses and delivery methods

Recombinant Candida uricase produced in E. coli was purchased from Sigma-Aldrich and delivered intranasally to isoflurane-anesthetized mice at 10 U per mouse in 40 μl sterile saline. For some studies, uricase was inactivated by exposing 40 μl/tube of 250 U/ml solutions in sterile saline to 254 nm UV light generated by a UV crosslinker (Stratalinker 1800, Stratagene, San Diego, CA) at a distance of 18 cm for 180 minutes. Human serum albumin (RMBIO, Missoula, MT) was delivered intranasally to isoflurane-anesthetized mice at 2 mg per mouse (equivalent to the protein content of 10 U uricase) in 40 μl sterile saline. Allopurinol (Sigma-Aldrich) was freshly dissolved in saline at 2.5 mg/ml and delivered sub-cutaneously at 25 mg/kg (31) 1 hour before and at 6 and 24, as well as in the allergic airway disease study at 48 hours, after NO2 inhalation.

Assessment of airway responsiveness to methacholine

Mice were anaesthetized with i.p. sodium pentobarbital (90 mg/kg), the trachea was cannulated, the mice were connected to a flexiVent™ computer controlled small animal ventilator (SCIREQ, QC, Canada), and the mice were ventilated at 200 breaths/minute with a 0.25 ml tidal volume. Next, the mice were paralyzed with an i.p. injection of pancuronium bromide (0.8 μg/kg). The animals were stabilized over about ten minutes of regular ventilation at a positive end-expiratory pressure (PEEP) of 3 cmH2O. A standard lung volume history was then established by delivering two total lung capacity maneuvers (TLC) to a pressure limit of 25 cmH2O and holding for three seconds. Next, two baseline measurements of respiratory input impedance (Zrs) were obtained, followed by an inhalation of aerosolized PBS (control) for 10 seconds, achieved by an in-line piezo electric nebulizer (Aeroneb, Aerogen, Galway, Ireland). Zrs was then measured every 10 seconds for 3 minutes (18 measurements of Zrs in total). This complete sequence of maneuvers and measurements was then repeated for aerosol exposures to four ascending doses of aerosolized methacholine (12.5, 25, 50, and 100 mg/ml). Data were fit to the single-compartment model (7) to provide values for resistance (R), reflecting constriction in the lungs, and elastance (E), reflecting the elastic rigidity of the lungs. Data were also fit to the constant phase model to provide values reflecting Newtonian airway resistance (RN), tissue damping (G), and tissue resistance (H). R, E, RN, G, and H were calculated for each mouse by averaging three measurements at each methacholine dose: the peak value and those immediately preceding and following it. The average values (± SEM) in each of the mouse groups, at each incremental methacholine dose, are reported.

Bronchoalveolar lavage (BAL) collection and processing

Lungs were lavaged with 1 mL DPBS (Sigma-Aldrich, St. Louis, MO). The BAL fluid was centrifuged at 400 × g, the supernatant was collected and frozen, and the total cells in the pellet were resuspended in PBS and enumerated by counting with an Advia 120 Hematology System (Bayer HealthCare, Leverkusen, Germany). Differential analysis was performed by cytospin and H&E stain from approximately 200 cells per slide.

In vitro antigen restimulation and cytokine quantitation

Following mechanical disruption of the spleen through a 70 μm mesh filter, splenocytes were isolated using Lymphocyte Separation Media (MP Biomedicals, Solon, OH), as previously described (32). Cells were counted with an Advia 120 Hematology System and 4×106 cells/ml were cultured in RPMI-1640 supplemented with 10% FBS (Cell Generation, Fort Collins, CO), penicillin/streptomycin, L-glutamine, folic acid, and 2-ME and treated with 200 μg/ml OVA, UV-inactivated uricase, or human serum albumin. Supernatants were collected after 96 hours of incubation at 37°C in 5% CO2. Analysis of cytokine content from cell supernatants was performed using ELISA kits for IL-5, IL-13, IL-17A, and IFNγ (R&D Systems, Minneapolis, MN).

Quantitative RT-PCR

Total RNA was extracted from snap frozen whole lungs or the single large lung lobe using the PrepEase RNA Isolation kit (USB Corp., Cleveland, OH) and reverse transcribed to cDNA using the iScript kit (Bio-Rad, Hercules, CA). Primers were designed for mouse xanthine dehydrogenase (Xdh) (5′-AGGGGATTCCGGACCTTTG-3′ and 5′-GCAGCAGTTTGGGTTGTTTC-3′), Muc5ac (5′-CCATGCAGAGTCCTCAGAACAA-3′ and 5′-TTACTGGAAAGGCCCAAGCA-3′), and Cxcl1 (5′-AAGCCAACCACTCCCATGAC-3′ and 5′-TGCGAAAGCATCAACAACAC-3′), Csf3 (5′-GAGCAGTTGTGTGCCACCTA-3′ and 5′-GCTCAGGTCTAGGCCAAGTG-3′), Ccl20 (5′-CGTCGTCTCTTCCTTGCTTT-3′ and 5′-TTGACAAGTCCACTGGGACA-3′), and Saa3 (5′-CAGGATGAAGCCTTCCATTG-3′ and 5′-CATGACTGGGAACAACAGGA-3′) using NCBI Primer-BLAST and synthesized by Integrated DNA Technologies (Coralville, IA). Quantitative RT-PCR was performed using SYBR Green Supermix (Bio-Rad) and normalized to Gapdh (5′-ACGACCCCTTCATTGACCTC-3′ and 5′-TTCACACCCATCACAAACAT-3′) or Actb (5′-TCCTTCGTTGCCGGTCCACA-3′ and 5′-CGTCTCCGGAGTCCATCACA-3′) using the ΔΔCT method, as previously described (33).

Uricase enzyme activity and uric acid quantitation by Amplex Red assay

Uricase activity and uric acid content were measured using an assay kit from Molecular Probes (Eugene, OR), according to manufacturer’s instructions.

Uric acid quantitation by liquid chromatography-mass spectrometry (LCMS)

Uric acid (UA, purity >99%) was purchased from Sigma Aldrich (St. Louis, MO, USA). [1,3-15N2]-uric acid (purity >98%) used as the internal standard was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Acetonitrile (ACN), formic acid (FA), potassium hydroxide (KOH) and methanol were obtained from Fisher Scientific (Pittsburgh, PA, USA). A stock solution of UA was prepared at a concentration of 1.8 mM in 0.3M KOH. The solutions of various dilutions (20, 10, 5, 1, 0.5 and 0 μM) were prepared in ACN/H2O (1:1). The internal standard 15N2-UA was prepared at a concentration of 2.0 mM in 0.3M KOH and diluted in ACN/H2O (1:1) to a concentration of 15 μM. All solutions were kept at −20°C prior to the analysis. Equal volumes (250 μL) of uric acid and 15N2-UA standards solutions were mixed for preparing the calibration curves for concentration calculation of UA in the unknown samples. A 250-μL aliquot of biological fluid was mixed with 250 μL of 15 μM 15N2-UA. 500 μL of ACN was added, vortexed for 1 min, and centrifuged for 15 min at 4500xg for protein precipitation. The supernatant was removed and dried in a new vial using a SpeedVac concentrator (SavantTM, Thermo Scientific, Waltham, MA). The dried samples were dissolved in 100 μL ACN/H2O (1:1) for the LCMS analysis. The LCMS analyses were carried out with a Shimadzu HPLC (Shimadzu, Kyoto, Japan) connected to an ABI 4000 QTRAP triple quadrupole mass spectrometer (ABSciex, Redwood City, CA) equipped with an ESI interface. The operation of the LCMS and data analysis were performed using ABSciex Analyst Software. The second quadrupole was operated in a collision induced dissociation MS/MS mode for performing data collection by selected reaction monitoring (SRM). LC analyses were performed using a Hypersil Gold HILIC column (100 mm x1 mm, 1.9 μm particles, Thermo Scientific). The mobile phase A was MeOH-H2O (3:1) containing 0.1 % FA; mobile phase B was ACN containing 0.1% FA (B). The mobile phase flow was 80 μL/min, and the injection volume was 1 μL. The HPLC gradient began at 25% A and was linearly ramped to 50% A over the next 15 min, held for 1 min, then reversed to the original composition of 25% A over 5 min, after which it was kept constant for 10 min to re-equilibrate the column. LCMS measurements were performed via SRM using negative ESI mode. The m/z ion selection for UA was 167/124 and 169/125 for 15N2-UA. The declustering potential and collision energies were −65 V, and −22 eV respectively. Peak areas were integrated for the UA and 15N2-UA SRM peaks, and a standard curve produced for the SRM area ratio of (UA 167/124)/(15N2-UA 169/125). Using this calibration curve the UA content was measured from each sample’s measured (UA 167/124)/(15N2-UA 169/125) SRM area ratio.

Serum collection and immunoglobulin analysis

Following euthanasia, approximately 300 μl of blood was collected via cardiac puncture of the right ventricle using a 25g needle attached to a 1 ml syringe, transferred into serum separator tubes (Becton Dickinson, Franklin Lanes, NJ), centrifuged, and serum was kept frozen at −80°C. For IgG1 and IgG2c, indirect ELISAs were performed. 96-well plates were coated overnight at 4°C with 2 μg/ml ovalbumin, UV-inactivated uricase, or human serum albumin in PBS (pH 7.2–7.4), washed with 0.05% Tween-20 in PBS, and blocked for 2 hours at 4°C with 1% BSA in PBS. Plates were washed and serum serially diluted in blocking solution was applied to the wells in triplicate and incubated overnight at 4°C. Plates were washed and 2 μg/ml biotinylated anti-mouse IgG1 or IgG2c secondary antibodies (Pharmingen) in 1% BSA/PBS were incubated in the plates at room temperature for 1 hour. Plates were washed and 0.05 U/ml streptavidin/peroxidase (Roche) was incubated in the plates at room temperature for 1 hour. Plates were washed, developed using reagents from R&D Systems (Minneapolis, MN), stopped with 1N H2SO4, and optical densities (ODs) were read using a BioTek Instruments PowerWaveX (Winooski, VT) at 450 nm with background subtraction at 570 nm.

Dendritic cell cultures

The mouse DC2.4 cell line (34) was kindly provided by Dr. Brent Berwin, Dartmouth College. For the analysis of OVA uptake and processing, DC2.4 cells plated at 1×106 cells/ml were incubated with 10 μg/ml AlexaFluor488-conjugated OVA or DQ-OVA (Molecular Probes), respectively, along with 0, 10, 50, or 250 μg/ml UV-inactivated uricase, and analyzed on an LSR II FACS flow cytometer (BD Biosciences, San Jose, CA) at the specified times and data were analyzed using FlowJo software (Tree Star, Ashland, OR). For analysis of surface displaceability of antigen in MHC class II, DC2.4 cells were cultured with 10 μg/ml ovalbumin and 0, 10, 50, or 250 μg/ml UV-inactivated uricase for 24 hours, washed 3 times with cold PBS, treated with 33 μM FITC-conjugated OVA323-339 peptide (AnaSpec, Fremont, CA) in PBS for 6 hours at 4°C, and FITC fluorescence was measured using a BioTek Instruments Synergy HTX plate reader (Winooski, VT).

Flow cytometry analysis of CD4+ T cell proliferation

For the generation of bone marrow-derived dendritic cells (BMDCs), bone marrow was flushed from the femurs and tibiae of C57BL/6J mice and cultured on 6-well plates at 1×106 cells/well (3 ml/well) in RPMI-1640 containing 10% serum and 5% conditioned media from X63-GMCSF myeloma cells transfected with murine GM-CSF cDNA (kindly provided by Dr. Brent Berwin, Dartmouth College). Media was replaced on days 2 and 4 and the adherent and lightly-adherent BMDCs, predominantly CD11b+CD11c+, were collected on day 6. CD4+ T cells were negatively selected from the spleens and peripheral lymph nodes of OTII TCR transgenic mice, according to manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). Transgenic CD4+ T cells were labeled with CFSE according to manufacturer’s protocol (Molecular Probes), washed, and incubated at 1×106 cells/ml along with BMDCs plated at 1×105 cells/ml, 10 μg/ml OVA, and 0, 10, 50, or 250 μg/ml UV-inactivated uricase for 72 hours. The loosely-adherent T cells were harvested and were analyzed by flow cytometry.

J774 cell cultures

J774 macrophages purchased from American Type Culture Collection (ATCC, Manassas, VA) were maintained in DMEM (Gibco, Grand Island, NY) supplemented with 10% FBS (Gibco), 1% L-Glutamine (Gibco), and 1x Primocin (Invivogen, San Diego, CA). For experiments, cells were plated at 2.5×105 cells/well in 250μl of media in a 48-well plate and allowed to grow overnight. The following day, the media was removed, fresh media was added and cells were treated for 18 hours with the indicated agonists. Cell supernatants were harvested, spun down at 3,300xg for 10 minutes to pellet cellular debris, transferred to new tubes, and analyzed for TNF content by ELISA (BD Biosciences, San Jose, CA).

Statistical analyses

Data were analyzed by two-tailed unpaired t test, one-way ANOVA or two-way ANOVA and Bonferroni post hoc test using GraphPad Prism 6 for Windows (GraphPad). Data are presented as mean ± SEM. A p value <0.05 was considered statistically significant. * or # = p≥0.05; ** = p≥0.01; *** = p≥0.001; **** = p≥0.0001.

Results

NO2 exposure induces uric acid

We exposed female C57BL/6 mice to 1 hour of 15 ppm NO2 and measured uric acid 2, 24, or 48 hours later using an Amplex Red-based enzymatic assay system. By 24 hours, uric acid was elevated in the lavageable airspace (BAL fluid), and this secretion persisted at 48 hours (Fig. 1A). An additional study was performed to measure BAL levels of uric acid by the more direct and specific LCMS method. Mice were again exposed to 1 hour of 15 ppm NO2 and analyzed 6, 20, 48, or 68 hours later. Again, uric acid levels in the lavageable airspaces continued to increase over time following NO2 exposure (Fig. 1B). Uric acid can be generated by the enzyme xanthine oxidase (Xdh), and other groups have published that Xdh levels correspond to uric acid release (22). However, our analysis of gene expression from the lungs of mice exposed to NO2 and analyzed 2, 24, or 48 hours after exposure indicated no significant changes in Xdh expression (Fig 1C), suggesting that lung damage induced as a consequence of NO2 exposure (35) causes leakage of uric acid-containing plasma into the lavageable airspaces rather than the increases in uric acid coming about as a consequence of local production.

Figure 1. NO2 exposure induces uric acid accumulation in the lung.

Figure 1

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Mice were exposed to room air or 15ppm NO2 for 1 hour and were analyzed for uric acid content in BAL fluid by Amplex Red (A) or liquid chromatography–mass spectrometry (B) assays, or were analyzed for xanthine oxidoreductase (Xdh) gene expression in the lungs by quantitative RT-PCR (C), at several times thereafter. N=3–4 mice per group and data are representative of experiments performed twice. Comparisons are versus air.

Uricase treatment inhibits NO2-induced allergic sensitization

Having established that NO2 induced the robust release of uric acid into the BAL, we sought to determine whether this DAMP played a mechanistic role in our model of NO2-promoted allergic sensitization. Mice were sensitized and challenged according to the scheme shown in Figure 2A. Briefly, after a single exposure to 15 ppm NO2 for 1 hour, mice were administered 1% w/v OVA for 30 minutes for 3 days. Some groups received 10U of intranasal uricase, as have been used by others (22), on all three days of the sensitization phase. Two weeks later, all mice were challenged with nebulized OVA for three days, and mice were analyzed 48 hours later. Indicative of having developed asthma-like disease, mice sensitized via NO2 and OVA (NO2/OVA) displayed increased lung resistance and elastance in response to inhaled methacholine challenge (methacholine responses from naïve control mice are depicted by the horizontal bars), whereas mice subjected to the NO2/OVA protocol and administered inhaled uricase during sensitization did not (Fig. 2B). Similar results were seen for RN, G, and H (Supplemental Fig. 1A). Furthermore, NO2/OVA mice developed a mixed Th2/Th17 BAL inflammatory profile as indicated by the presence of both eosinophils and neutrophils (Fig. 2C). In contrast, mice that received uricase during their NO2/OVA sensitization displayed significantly reduced levels of BAL eosinophils and neutrophils. When gene expression levels from the lungs of these mice were compared, uricase treatment diminished the expression of the mucin gene Muc5ac, and the chemokines Cxcl1 and Ccl20, whereas levels of the neutrophil-maturation cytokine Csf3 were unchanged (Fig. 2D). When restimulated in vitro with OVA, splenocytes from NO2/OVA sensitized mice released IL-13 and IL-17A into the cell media, levels of which were significantly decreased from cells of mice that received uricase, whereas the low levels of IFNγ were unaffected by uricase treatment (Fig. 2E). Finally, mice treated with uricase during sensitization did not develop substantial titers of OVA-specific IgG1 in the serum, compared to the robust levels from the NO2/OVA mice (Fig. 2F). Overall, it appeared that uricase treatment during sensitization was sufficient to abrogate the parameters of Th2 and Th17 disease associated with our NO2/OVA model. However, contrary to our expectations, we measured the presence of a substantial anti-uricase IgG1 response in those mice subjected to uricase treatment (Fig. 2G), suggesting that either the uricase itself was immunogenic or that the in vivo NO2 exposure was creating an environment conducive to generating an anti-uricase immune response.

Figure 2. Uricase treatment inhibits NO2-promoted allergic airway disease.

Figure 2

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Mice were subjected to NO2-promoted allergic sensitization and administered saline or 10 U of uricase by intranasal instillation. Mice were challenged with three daily doses of 1% inhaled ovalbumin and analyzed 48 hours later (A) for methacholine responsiveness (B; responsiveness of naïve mice is indicated by horizontal lines), inflammatory cells in BAL fluid (C), gene expression in the lungs (D), cytokine production from OVA-restimulated splenocytes (E), and OVA-specific (F) or uricase-specific (G) IgG1 in serum. N=8 mice per group and data are representative of experiments performed twice. Comparisons are versus NO2/OVA.

Uricase instillation induces acute pulmonary inflammation

It was unclear from our data whether uricase was inhibiting allergic sensitization by removing uric acid that was generated in response to NO2 or by initiating a specific immune reaction that would overshadow the response to OVA. Therefore, we tested whether a single dose of uricase administered to mice provoked an innate immune response in the lung. Uricase treated mice, analyzed 24 hours after administration, displayed neutrophilia in the BAL (Fig. 3A), as well as robust increases in the lung gene expression of Cxcl1, Csf3, and Saa3 (Fig. 3B), indicative of an acute-phase and pro-inflammatory response. We next sought to determine whether enzymatic activity of uricase was responsible for its pro-inflammatory activity. Whereas boiling promoted the aggregation of the uricase into clumps unsuitable for delivery to the lung, exposure to UV light significantly and substantially decreased uricase activity (Fig. 3C) without causing clumping. When active uricase or UV-inactivated uricase were subsequently administered via intranasal instillation into mice and inflammation was measured 24 hours later, there was an absence of neutrophils in those animals that received UV-inactivated uricase (Fig. 3D). Furthermore, lung expression of Cxcl1, Csf3, and Saa3 were increased only in response to the active enzyme (Fig. 3E).

Figure 3. Uricase enzyme activity exerts pro-inflammatory effects in the lung.

Figure 3

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Mice were administered saline or 10 U of uricase by intranasal instillation and were evaluated for inflammatory cells in BAL fluid (A) or gene expression in the lungs (B) 24 hours later. Triplicate samples of uricase were untreated or exposed to UV light after which enzyme activity was measured by Amplex Red assay (C). Female C57BL/6J mice were administered 10 U of uricase or UV-inactivated uricase by intranasal instillation and were evaluated for inflammatory cells in BAL fluid (D) or gene expression in the lungs (E) 24 hours later. Gene expression is compared to mice exposed to intranasal saline. N=7–8 mice per group and data are representative of experiments performed twice. Comparisons are versus saline (A–B) or active uricase (C–E).

UV-inactivated uricase inhibits NO2-promoted allergic sensitization

Having determined that intranasal instillation of UV-inactivated uricase did not induce an innate pro-inflammatory response in the lung in comparison to the active enzyme, we sought to determine whether only active uricase could inhibit NO2-promoted allergic sensitization. Accordingly, mice were subjected to NO2 and OVA inhalation, either alone or accompanied by the administration of uricase or UV-inactivated uricase (Fig. 4A). Naïve mice were used as negative controls. NO2/OVA-promoted allergic sensitization resulted in robust eosinophilia and neutrophila in the lavageable airspaces that was abrogated by treatment with either uricase or UV-inactivated uricase (Fig. 4B). Similarly, the methacholine hyperresponsiveness displayed by mice subjected to the NO2/OVA protocol was abrogated by the administration of either uricase or UV-inactivated uricase (Fig. 4C and Supplemental Fig. 1B). In addition, uricase activity was not required for the elimination of IL-5, IL-13, and IL-17A secretion from splenocytes restimulated with OVA (Fig. 4D). However, both uricase and UV-inactivated uricase treatment led to the release of IL-5, IL-13, and IL-17A from splenocytes restimulated with UV-inactivated uricase (Fig. 4E). Likewise, both uricase and UV-inactivated uricase capably decreased OVA-specific IgG1 and IgG2c levels in serum (Fig. 4F), while driving the production of uricase-specific immunoglobulins (Fig. 4G).

Figure 4. Enzymatically inactive uricase treatment inhibits NO2-promoted allergic airway disease.

Figure 4

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Mice were untreated (naïve) or were subjected to NO2-promoted allergic sensitization and were untreated or were administered 10 U of uricase or UV-inactivated uricase by intranasal instillation. Mice were challenged with three daily doses of 1% inhaled ovalbumin and analyzed 48 hours later (A) for inflammatory cells in BAL fluid (B), methacholine responsiveness (C), cytokine production from OVA-restimulated (D) and UV-inactivated uricase-stimulated splenocytes (E), and OVA-specific (F) or uricase-specific (G) IgG1 and IgG2c in serum. N=8 mice per group and data are representative of experiments performed twice. Comparisons are versus naive.

Uricase itself elicits an antigen-specific adaptive immune response

Having established that uricase skews the cytokine and immunoglobulin responses in our model of NO2-promoted allergic asthma, and that this effect is independent of enzyme activity, we explored the possibility that uricase itself can serve as an antigen against which mice will develop allergic responses. One group of mice was sensitized with the NO2/OVA regimen and received UV-inactivated uricase during the sensitization period. Another group of mice was administered uricase on days 0, 1, and 2 without being subjected to NO2 or OVA inhalations during the sensitization period. These two groups of mice were challenged two weeks later with UV-inactivated uricase, instead of OVA (Fig. 5A) and were compared to naïve mice never exposed to any the inhaled agents. All sensitized mice developed asthma-like disease after challenge with UV-inactivated uricase, with eosinophilia and especially robust neutrophilia in the bronchoalveolar lavage compared to naïve controls (Fig. 5B). Additionally, splenocytes from sensitized mice restimulated with UV-inactivated uricase secreted IL-5, IL-13, IL-17A, and IFNγ (Fig. 5C). Analysis of serum immunoglobulins revealed only very low levels of IgG1 specific for OVA (Fig. 5D) but pronounced uricase-specific IgG1 and IgG2c production in mice sensitized and challenged with either uricase exposure regimen (Fig. 5E). Interestingly, most of the measured responses were indistinguishable in the mice exposed to the NO2/OVA + UV-inactivated uricase or uricase sensitization regimens and challenged with UV-inactivated uricase, suggesting that uricase itself functions as a potent immunogen in the setting of inhalational exposure, a situation that may override its uric acid-catabolizing activity to inhibit NO2-promoted allergic sensitization to inhaled OVA.

Figure 5. Uricase inhalation promotes a uricase-specific immune response.

Figure 5

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Mice were subjected to NO2-promoted allergic sensitization and administered 10 U of UV-inactivated uricase by intranasal instillation, or were administered 10 U of uricase by intranasal instillation without exposure to NO2 or OVA. Mice were challenged with three daily doses of 10U of UV-inactivated uricase and analyzed 48 hours later (A) for inflammatory cells in BAL fluid (B), cytokine production from UV-inactivated uricase-stimulated splenocytes (C), and OVA-specific (D) or uricase-specific (E) IgG1 and IgG2c in serum. N=4–8 mice per group and data are representative of experiments performed twice. Comparisons are versus naïve.

UV-inactivated uricase does not inhibit the uptake, processing, or display of OVA antigen by dendritic cells, but does impede OVA-specific CD4+ T cell proliferation and cytokine production

DC2.4 mouse dendritic cells were treated with AlexaFluor488-labeled OVA or DQ-OVA and increasing concentrations of UV-inactivated uricase. At 2, 4, 7, and 24 hours, cells were collected, washed, and analyzed by flow cytometry for Alexa488 fluorescence, indicating uptake of labeled antigen, or BODIPY fluorescence, indicating proteolysis during antigen processing that separated the quencher from the fluorescent dye. Treatment with UV-inactivated uricase, even at concentrations 25 times that of OVA, which is analogous to the relative levels in the airways when uricase is administered as an inhibitor of NO2-promoted allergic sensitization, had no significant effects on OVA antigen uptake (Fig. 6A) or processing (Fig. 6B). In addition, when DC2.4 cells were cultured with OVA and increasing concentrations of UV-inactivated uricase for 24 hours, incubated at 4°C with FITC-conjugated OVA323-339 peptide for 6 hours to displace peptide presented on the cell surface by MHC class II molecules (36), uricase had no significant effect on surface fluorescence (Fig. 6C), supporting the notion that uricase does not affect the relative affinity of MHC class II for peptide. However, co-culture experiments with bone marrow-derived dendritic cells (BMDCs) incubated with OVA and increasing concentrations of UV-inactivated uricase, along with CFSE-labeled OVA peptide-specific OTII CD4+ T cells, indicated a profound effect of UV-inactivated uricase on T cell proliferation (Fig. 6D). Furthermore, secretion of the TH2 cytokines IL-13 and IL-5, as well as secretion of IL-17A, from these CD4+ T cells was also repressed with increasing concentrations of UV-inactivated uricase relative to OVA, indicating a failure of the OVA-specific CD4+ T cell response in the presence of uricase lacking enzymatic activity (Fig. 6E).

Figure 6. Enzymatically-inactive uricase inhibits CD4+ T cell proliferation and cytokine production.

Figure 6

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DC2.4 cells were left untreated or were administered 10 μg/ml Alexa488-labeled OVA or DQ-OVA in the absence or presence of increasing concentrations of UV-inactivated uricase based on weight (10, 50, or 250 μg/ml). At indicated timepoints, BMDCs were washed, fixed, and analyzed by flow cytometry for Alexa488 (A) or unquenched DQ-OVA (B) fluorescence. DC2.4 cells were cultured for 24 hours with OVA in the absence or presence of increasing concentrations of UV-inactivated usicase, incubated for 6 hours with FITC-conjugated OVA323-339 peptide, and assessed for FITC fluorescence (C). Bone marrow-derived dendritic cells co-cultured with CFSE-labeled OVA-specific OTII CD4+ T cells were untreated or treated with 10 μg/ml OVA in the absence or presence of increasing concentrations of UV-inactivated uricase based on weight (10, 50, or 250 μg/ml). After 72 hours, T cell proliferation (C) and cytokine abundance in culture supernatants (D) were measured. N=4 in each culture condition. Comparisons are versus 0 uricase (at each timepoint in A and B).

Allopurinol does not inhibit NO2-promoted allergic sensitization

We observed that NO2 exposure causes the accumulation of uric acid in the lavageable airspaces and that uricase treatment prevents antigen challenge-induced asthma-like disease, although perhaps through a mechanism distinct from its enzymatic activity. We therefore examined whether inhibiting the formation of uric acid through the administration of allopurinol would diminish circulating uric acid levels and its accumulation in BAL fluid following NO2 exposure. Allopurinol administration prior and subsequent to 1 hour of exposure to 15 ppm NO2 (Fig. 7A) did decrease serum uric acid levels (Fig. 7B) and diminished by one-half the levels of uric acid in the lavageable airspaces 48 hours following NO2 exposure (Fig. 7C). Having established the capacity of allopurinol to affect circulating and NO2-induced uric acid accumulation, we next administered allopurinol to test whether uric acid is indeed a mediator of NO2-promoted allergic sensitization. Mice underwent NO2/OVA sensitization, during which they were treated subcutaneously with either vehicle (saline) or 25 mg/kg allopurinol (Fig. 7D). Following OVA challenge, all mice developed asthma-like disease. Allopurinol treatment had no impact on inflammatory cell numbers or types recruited to the lavageable airspaces (Fig. 7E), the levels of IL-5, IL-13, IL-17A, or IFNγ produced by splenocytes restimulated with OVA (Fig. 7F), or the quantities of OVA-specific IgG1 or IgG2c in the serum (Fig. 7G). These results implicate that the capacity of uricase to diminish OVA-specific immune responses in our NO2-promoted allergic disease model is not a consequence of decreasing uric acid levels.

Figure 7. Allopurinol treatment does not inhibit NO2-promoted allergic airway disease.

Figure 7

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Mice were administered saline vehicle or 25 mg/ml allopurinol and exposed to 1 hour or 15 ppm NO2 (or air control) at the indicated times (A). Forty-eight hours after the NO2 or air exposure, serum (B) and BAL fluid (C) were collected and analyzed for uric acid content by Amplex Red assay. Mice were subjected to NO2-promoted allergic sensitization and subcutaneously administered vehicle (saline) or 25 mg/kg allopurinol at the indicated times relative to NO2 exposure (D). Mice were challenged with three daily doses of 1% inhaled ovalbumin and analyzed 48 hours later for inflammatory cells in BAL fluid (E), cytokine production from OVA-stimulated splenocytes (F), and OVA-specific IgG1 and IgG2c in serum (G). N=5 (A–C) or 4 (D–G) mice per group. Comparisons are between the indicated group and naïve (*) or NO2+vehicle (#).

An excess of clean antigen inhibits NO2-promoted allergic sensitization

Since enzymatic activity was not responsible for the inhibitory effect of administering uricase to the lung during NO2-promoted allergic sensitization, and despite the demonstration that UV exposure renders uricase non-inflammatory in vivo, the fact that uricase is a Candida-derived protein generated in E. coli meant that bacterial contaminants may participate in skewing the adaptive immune response upon administration. Therefore, we sought to use a very clean protein to determine whether administering similar concentrations of an innocuous protein would mitigate the OVA-specific immune response in a manner similar to that of uricase or UV-inactivated uricase. If so, the effect would be due to the large amount of “inhibitory” protein administered during NO2-promoted allergic sensitization rather than enzymatic activity or contaminants. First, we used J774 macrophage cells to determine their response to uricase, UV-inactivated uricase, and a preparation of human serum albumin (HSA; intended for use in pharmaceutical manufacturing). In comparison to 100 ng/ml LPS, 50 μg/ml uricase or 50 μg/ml UV-inactivated uricase elicited small but significant production of TNF from J774 cells, whereas 50 μg/ml human serum albumin (HSA) elicited no detectable levels of TNF, similar to unstimulated cells (Fig. 8A). We next sought to determine whether HSA could inhibit NO2-promoted allergic sensitization. Accordingly, mice were subjected to NO2 and OVA inhalation, accompanied either by the administration of saline or an amount of HSA protein equivalent to the protein content of uricase or UV-inactivated uricase used in previous experiments (Fig. 8B). Mice administered HSA displayed reduced methacholine responsiveness, similar to the levels from naïve mice (horizontal bars), compared to the mice exposed to saline vehicle along with NO2 and OVA (Fig. 8C and Supplemental Fig. 1C). Furthermore, whereas NO2/OVA-promoted allergic sensitization resulted in robust eosinophilia and neutrophila in the lavageable airspaces, these responses were significantly decreased by HSA treatment (Fig. 8D). Additionally, HSA administration inhibited IL-5, IL-13, and IL-17A secretion from splenocytes restimulated with OVA (Fig. 8E), but did not induce HSA-specific cytokine production (Fig. 8F). However, while HSA significantly decreased OVA-specific serum IgG1 levels (Fig. 8G), it also promoted the production of HSA-specific IgG1 (Fig. 8H), indicating that an anti-HSA response was generated in vivo. These results support the conclusion that the administration of large amounts of clean antigen during NO2-promoted allergic sensitization is sufficient to skew the response away from the OVA antigen to which the mice were initially sensitized, manifesting in substantially blunted OVA-specific recall responses upon challenge and diminished OVA-driven allergic airway disease.

Figure 8. Treatment with a clean protein inhibits NO2-promoted allergic airway disease.

Figure 8

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J774 macrophages were untreated or exposed to indicated agonists for 18 hours, after which TNF was measured from culture supernatants (A). Mice were subjected to NO2-promoted allergic sensitization and were untreated or were administered 2 mg human serum albumin (HSA; an amount of protein equivalent to that in 10 U of uricase or UV-inactivated uricase) in 40 μl by intranasal instillation. Mice were challenged with three daily doses of 1% inhaled ovalbumin and analyzed 48 hours later (B) for methacholine responsiveness (C; responsiveness of naïve mice is indicated by horizontal lines), inflammatory cells in BAL fluid (D), cytokine production from OVA-restimulated (E) and HSA-restimulated splenocytes (F), and OVA-specific (G) or HSA-specific (H) IgG1 in serum. nd = not detected. N=8 mice per group. Comparisons are versus untreated (A) or between groups (C–H).

Discussion

As an air pollutant and an endogenously-generated oxidant, the detrimental effects of NO2 on respiratory disease include induction of acute lung injury, impairment of innate immune defenses, silo-fillers disease, induction of allergic responses, and exacerbation of asthma (37). As avoiding the persistent levels of NO2 present in ambient air is not possible for many affected populations (38), there remains an unmet need to understand the mechanisms of NO2 pathogenesis in the induction of respiratory and systemic disease. Part of the lung injury induced by NO2 is oxidative modification of airway surface liquid, damage to epithelium, and leakage of plasma components into the airspaces (3, 39). Plasma is an abundant source of uric acid, an antioxidant that is superbly capable of interacting with and detoxifying NO2 (40, 41). However, uric acid may possess other qualities that makes it a possible pathogenic mediator of tissue injury. We had previously reported that heat shock protein 70 (Hsp70), a damage-associated molecular pattern (DAMP) protein capable of activating Toll-Like Receptor (TLR)2 and TLR4, was elevated in the lavageable airspaces of NO2-exposed mice and that TLR2 and its intracellular adaptor MyD88, but not TLR4, were required for the generation of an antigen-specific adaptive immune response to NO2-promoted allergic sensitization (8). A number of damage-associated molecules function to stimulate innate immune responses to clear areas of injury and avoid infection, and the presence of uric acid in the airways has been reported to be sufficient and necessary to promote adaptive immune responses that lead to asthma-like disease as well as to asthma exacerbation (22). A part of the mechanism through which uric acid functions is via stimulation of dendritic cells, activation of the NLRP3 inflammasome, and secretion of IL-1β (20, 24). We have reported that NLRP3 inflammasome activation participates in the induction of the adaptive immune response subsequent to NO2 and antigen inhalation, and that IL-1 receptor is critical for generation of the ensuing Th17 response (12). Consequently, we sought to determine whether uric acid was present in the airways of NO2-exposed mice and whether it is a participant in the generation of the antigen-specific immune response in NO2-promoted allergic airway disease.

In addition to using an amplex red-based assay, we implemented the direct and highly specific LCMS approach, accompanied by an internal stable isotope standard, to unambiguously quantify the uric acid content in BAL fluid. We found increased levels of uric acid in the BAL fluid as long as 68 hours after NO2 exposure, the levels of which were approximately 1% of the levels (1.89±1.80 mg/dl = 112.43 μM) reported in the plasma of female C57BL/6J mice (42). A modest amount of plasma leak subsequent to NO2 exposure could easily account for these levels of uric acid, as levels as high as 10% of those in the plasma have been reported subsequent to ventilator-induced lung injury, with similar concentrations seen in the BAL fluid of patients with acute lung injury (43). Interestingly, the levels of uric acid increase over times that correspond to the optimal time for delivery of antigen into the airways to promote allergic sensitization (8), as well as the increased abundance of inflammatory dendritic cells in the lungs and the delivery of antigen to draining lymph nodes subsequent to NO2 exposure (10). It initially appeared that airway uric acid was functioning as a mediator of allergic sensitization, as its breakdown through the administration of uricase during the NO2-promoted allergic sensitization regimen nearly completely prevented the OVA-induced recall response that causes methacholine hyperresponsiveness, eosinophilic inflammation, Th2/Th17 cytokine production, and antigen-specific immunoglobulin production (8, 10–12), similar to as has been reported in settings of Alum- (27) and house dust mite-promoted (22) antigen sensitization. However, these studies did not investigate the presence of a uricase-specific immune response, which we observed following uricase inhalation. We found that the uricase-specific immune response was generated even when uricase was enzymatically inactive and incapable of generating an innate immune responses.

Independent of enzymatic activity and its consequences, uricase displays a potent capacity to inhibit the proliferation of and cytokine production from OVA-specific CD4+ T cells in a manner distinct from interfering with antigen uptake or processing and presentation by dendritic cells. Based on our studies, it is unlikely that uricase interferes with the presentation of the OVA323-339 peptide in the context of MHC-II that is required for the activation of OTII CD4+ T cells, a condition that would inhibit proliferation and cytokine production. Nevertheless, the effect of uricase or HSA in vivo to inhibit OVA-specific CD4+ T cell activities could be different than in the in vitro experimental systems we employed. In experimental settings, uricase may be functioning, in part (or in whole in some instances), as an abundant antigen that skews the adaptive immune response away from that induced by other concomitantly-present antigens. Our results showing that the administration of an ultra-clean and non-pro-inflammatory preparation of the protein antigen human serum albumin, at a concentration identical to that of uricase or UV-inactivated uricase, was able to inhibit the generation of NO2-promoted allergic airway disease as effectively as uricase, clearly support this mechanism. This mechanism is analogous to the concept of original antigenic sin (44), wherein subsequent immune responses to a similar set of antigens are predicated by those experienced previously. In the setting described herein, the immune response generated during exposure to both OVA and uricase is dominated by that to uricase such that subsequent exposure to OVA gives the impression that the OVA response was inhibited, whereas the truth is that a uricase response was promoted. While this does effectively prevent OVA-driven pathology, the development of asthma-like disease in our model of NO2-promoted allergic sensitization, it assures that the response to subsequent uricase exposures could be deleterious. Interestingly, uricase is used for the treatment of tumor lysis syndrome in the setting of chemotherapy (an immunosuppressive situation), and also indicated for the treatment of severe gout, although it is not widely used by clinicians because of adverse effects, including immunogenicity. Strategies to attenuate the immunogenicity of uricase include the addition of polyethylene glycol (PEG), as is employed in an approved clinical formulation, although substantial immunogenicity remains (45). In fact, antibodies to PEG-conjugated porcine uricase, pegloticase, were present in the vast majority of patients receiving the drug, titers of which were associated with increased pegloticase clearance and a loss of the uric acid-lowering response, as well as an increased risk of infusion reactions and anaphylaxis (46). Therefore, it is not surprising that similar immune-stimulating effects of uricase were observed in our studies using a non-PEGylated form.

Whereas whether uric acid is a marker or a mediator of the events involved in NO2-promoted allergic airway disease is unclear from the uricase studies, the allopurinol experiments indicate that uric acid is likely only a marker. Our results demonstrate that allopurinol significantly decreased serum uric acid levels and decreased by half the amount of uric acid in the lavageable airspaces following NO2 exposure. We agree that the ability of allopurinol to decrease serum uric acid levels is modest and that it remains possible that a quantity of uric acid sufficient to participate in NO2-promoted allergic sensitization is still present in the airways following NO2 exposure, even in the setting of allopurinol administration. Despite this caveat, competitive inhibition of xanthine oxidoreductase enzymatic activity with allopurinol was unable to prevent NO2-promoted allergic sensitization, making it unlikely that induction of this enzyme was responsible for the elevated uric acid levels present in the airway following NO2 exposure.

Nonetheless, xanthine oxidoreductase and the uric acid transporter, Multi-Drug Resistance protein 4 (MRP4), are expressed by human airway epithelial cells exposed to house dust mite extract or particulate matter, factors that can promote allergic sensitization in a manner inhibitable by uricase treatment (22, 47). Why in some circumstances uric acid is a potent inducer of adaptive immunity and in other cases is not may be due to subtleties of experimental design and interpretation, or due to situational differences in abundances or activities. Our results clearly issue a warning of caution in interpreting results of studies involving uricase as a tool for evaluating the importance of uric acid in the generation of adaptive immune responses, as well as for those studies in which the administration of large amounts of proteins are able to seemingly mitigate adaptive immune responses to other antigens.

Supplementary Material

1

Acknowledgments

This work was funded by National Institute of Health grants R01 HL089177, R01 HL107291, P30 GM103532, and P20 GM103496.

The authors thank Dr. Karen Fortner of the University of Vermont, Department of Medicine, Immunobiology Division, and Dr. Roxana Del Rio Guerra of the University of Vermont Flow Cytometry & Cell Sorting Facility, for assistance with cell staining experiments.

Footnotes

Conflict of Interest

The authors declare no conflicts of interest.

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