Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1

Article abstract of DOI:10.1371/journal.pone.0188614

Background: An association of acetaminophen use and asthma was observed in the International Study of Asthma and Allergies in Childhood study. However there are no clear mechanisms to explain an association between acetaminophen use and immunologic pathology. In acidic conditions like those in the stomach and inflamed airway, tyrosine residues are nitrated by nitrous and peroxynitrous acids. The resulting nitrotyrosine is structurally similar to 2,4-dinitrophenol and 2,4-dinitrochlorobenzene, known haptens that enhance immune responses by covalently binding proteins. Nitrated acetaminophen shares similar molecular structure. Objective: We hypothesized the acetaminophen phenol ring undergoes nitration under acidic conditions, producing 3-nitro-acetaminophen which augments allergic responses by acting as a hapten for environmental allergens. Methods: 3-nitro-acetaminophen was formed from acetaminophen in the presence of acidified nitrite, purified by high performance liquid chromatography, and assayed by gas-chromatography mass spectrometry. Purified 3-nitro-acetaminophen was reacted with Dermatophagoides pteronyssinus (Der p1) and analyzed by mass spectrometry to identify the modification site. Human peripheral blood mononuclear cells proliferation response was measured in response to 3-nitro-acetaminophen and to 3-nitro-acetaminophen-modified Der p1. Results: Acetaminophen was modified by nitrous acid forming 3-nitro-acetaminophen over a range of different acidic conditions consistent with airway inflammation and stomach acidity.

Full text of DOI:10.1371/journal.pone.0188614

RESEARCH ARTICLE
Conjugation of nitrated acetaminophen to
Der p1 amplifies peripheral blood monocyte
response to Der p1
Ryan G. Thomas1,2☯, Brenda M. Rivera Reyes1,2☯, Benjamin M. Gaston1,2, Nelki B. Rivera
Acosta1, Ilya R. Bederman2, Laura A. Smith2, Morgan T. Sutton2, Benlian Wang3,
John F. Hunt4, Tracey L. Bonfield2*
1 Department of Pediatrics, Division of Pulmonology, University Hospitals Cleveland Medical Center,
Rainbow Babies and Children’s Hospital, Cleveland, Ohio, United States of America, 2 Department of
Pediatrics, Division of Pulmonology, Case Western Reserve University, Cleveland, United States of America,
3 Center of Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United
States of America, 4 Airbase Therapeutics, Charlottesville, Virginia, United States of America
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☯ These authors contributed equally to this work.
* Tracey.Bonfield@case.edu
Abstract
OPEN ACCESS
Background
Citation: Thomas RG, Rivera Reyes BM, Gaston
BM, Rivera Acosta NB, Bederman IR, Smith LA, et
al. (2017) Conjugation of nitrated acetaminophen
to Der p1 amplifies peripheral blood monocyte
response to Der p1. PLoS ONE 12(12): e0188614.
https://doi.org/10.1371/journal.pone.0188614
An association of acetaminophen use and asthma was observed in the International Study
of Asthma and Allergies in Childhood study. However there are no clear mechanisms to
explain an association between acetaminophen use and immunologic pathology. In acidic
conditions like those in the stomach and inflamed airway, tyrosine residues are nitrated by
nitrous and peroxynitrous acids. The resulting nitrotyrosine is structurally similar to 2,4-dini-
trophenol and 2,4-dinitrochlorobenzene, known haptens that enhance immune responses
by covalently binding proteins. Nitrated acetaminophen shares similar molecular structure.
Editor: Markus M Bachschmid, Boston University,
UNITED STATES
Received: June 9, 2017
Accepted: November 12, 2017
Published: December 11, 2017
Objective
We hypothesized the acetaminophen phenol ring undergoes nitration under acidic condi-
tions, producing 3-nitro-acetaminophen which augments allergic responses by acting as a
hapten for environmental allergens.
Copyright: © 2017 Thomas et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Methods
3-nitro-acetaminophen was formed from acetaminophen in the presence of acidified nitrite,
purified by high performance liquid chromatography, and assayed by gas-chromatography
mass spectrometry. Purified 3-nitro-acetaminophen was reacted with Dermatophagoides
pteronyssinus (Der p1) and analyzed by mass spectrometry to identify the modification site.
Human peripheral blood mononuclear cells proliferation response was measured in
response to 3-nitro-acetaminophen and to 3-nitro-acetaminophen-modified Der p1.
Data Availability Statement: All relevant data are
within the paper.
Funding: BG received funding from the National
Heart Lung and Blood Institute grant numbers
1P01HL101871 and 1U10HL109250 (https://www.
nhlbi.nih.gov/) as well as the Children’s Lung
Foundation. RT received funding from the
Fellowship Research Award Program Grant,
Rainbow Babies & Children’s Foundation (http://
www.uhhospitals.org/rainbow/about/rainbow-
foundation). The funders had no role in study
Results
Acetaminophen was modified by nitrous acid forming 3-nitro-acetaminophen over a range
of different acidic conditions consistent with airway inflammation and stomach acidity. The
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
design, data collection and analysis, decision to
publish, or preparation of the manuscript
Der p1 protein-hapten adduct creation was confirmed by liquid chromatography-mass spec-
trometry proteomics modifying cysteine 132. Peripheral blood mononuclear cells exposed
to 3-nitro-acetaminophen-modified Der p1 had increased proliferation and cytokine produc-
tion compared to acetaminophen and Der p1 alone (n = 7; p < 0.05).
Competing interests: The authors have declared
that no competing interests exist.
Conclusion
These data suggests 3-nitro-acetaminophen formation and reaction with Der p1 provides a
mechanism by which stomach acid or infection-induced low airway pH in patients could
enhance the allergic response to proteins such as Der p1.
Introduction
Multivariate analysis of the International Study of Asthma and Allergies in Childhood
(ISAAC) shows a dose dependent association between parental reported use of acetaminophen
(AP) for fever in the first year of life and an increased risk of asthma, rhinoconjunctivitis, and
eczema at 6–7 [1]. Similar results were found in the follow-up ISAAC analysis of 13–14 year
old patients [2], a birth cohort in Ethiopia [3], and in adults [4].
Two important objections have been raised to AP being causally associated with asthma.
First, the infections causing fever may also be casually associated with asthma [5]. Sordillo and
coworkers showed the association of AP use for fever and asthma was not significantly greater
than ibuprofen when controlling for infections [6]. An Asthmanet study [7] also failed to find
an increased risk of asthma in children randomized to AP versus ibuprofen for fever. These
studies emphasize infection as an important contributor to asthma risk.
The second objection has been the absence of a mechanism for this effect. One theory is AP
depletes intracellular glutathione stores. Studies have shown up to a 53% decrease in glutathi-
one stores in the lungs following a dose of AP [8]. However lower respiratory tract glutathione
levels are 140 fold higher than serum [9] and glutathione resynthesis time is short [10]. Fur-
thermore, airway glutathione in asthmatics is elevated [11,12] compared to controls. Impor-
tantly, the animal studies showing pulmonary depletion of gluathione after AP administration
used toxic doses [13]. Glutathione depletion by AP seems unlikely to impact antioxidant
defenses in the lung sufficiently to induce asthma/atopy.
We hypothesized that the combination of AP and inflammation amplifies the immune
response to antigens. The inflamed airway is acidic [14–19]. When protonated, nitrite and per-
oxynitrite form the potent nitrating agents nitrous acid and peroxynitrous acid which readily
nitrate tyrosine residues in endogenous and exogenous proteins. 3-nitro-tyrosine has been
identified in inflamed tissue in asthma [20], cystic fibrosis [21, 22], allergic rhinitis [23] and
chronic obstructive pulmonary disease [24] and is an in vivo marker of nitrative stress [25].
The chemical structure of 3-nitro-tyrosine is similar to those of 2,4-dinitrophenol and
2,4-dinitrochlorobenzene, both highly potent haptens [26–28] known for enhancing immune
responses by binding to proteins (Fig 1). Immune response to environmental allergens can be
amplified by tyrosine nitration. Nitration of the birch tree pollen allergen Bet v 1 enhanced the
presentation of Bet v 1-derived peptides by human dendritic cells [29].
Like tyrosine and the pre-haptens discussed above, AP is a phenol readily nitrated under
mildly acidic conditions to form stable 3-nitro-acetaminophen (NAP) [30]. Indeed, this reac-
tion is the basis of the assay kit for serum AP levels [31]. NAP’s chemical structure is similar to
the above haptens (Fig 1). Here, we show NAP is formed in vitro under conditions similar to
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
Fig 1. A. Structural simularity of 3-nitro-tyrosine and 3-nitro-acetaminophen (NAP) with 2,4-dinitrophenol, a
well-known hapten. Acetaminophen shown for reference. B. Depiction of acetaminophen nitration reaction. C.
High Performance Liquid Chromatography (HPLC) elution profile of NAP. Synthesized NAP was purified by
HPLC, arrow denotes the NAP peak that is seen at elution time of 8 minutes. Figure is representative of
results of multiple HPLC elutions and representative of similar profiles obtained at different pH.
https://doi.org/10.1371/journal.pone.0188614.g001
those of the stomach or acidic airway in vivo and modifies Der p1, amplifying in vitro immune
responses to it.
Materials and methods
Reagents
Unless noted, all reagents were purchased from Sigma Aldrich (St. Louis, MO).
Synthesis of NAP
We synthesized NAP from AP (Sigma, St. Louis, MO) by mixing 6.25mL of 26.5nM AP with 5
mL of 6N HCL and 5 mL of 1.4M NaNO2 and 5 mL of 1.3M ammonium sulfamate [32]. HCl
was used to acidify, NaNO2 was used as the nitrating agent, and the ammonium sulfamate was
used to neutralize the excess nitrous acid. Nitroparacetamol Reaction: 200mg of acetaminophen
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
was dissolved in 2 mL of methanol, and evaporated with the final residue dissolved in 50 mL
H20. To the dissolved AP, we added 0.25 mL of 1N, 0.1N, 0.05N, 0.01N, or 0.005N HCl which
was followed by the addition of 0.25 mL of 1.4 mol/L NaN02. Each reaction mixture was neu-
tralized with 0.25 mL 1.3 mol/L ammonium sulfamate. The reaction product was compared to
nitroparacetamol stock standard.
High performance liquid chromatography (HPLC) protocol for NAP
Isolation
NAP was purified using the Flexar PSA PLUSS HPLC system, (PerkinElmer, Waltham, MA)
using a Pecosphere C18 3mcM, 33x4.8mm column (PerkinElmer, Waltham, MA) and Chro-
mera 4.1.0 software (PerkinElmer, Waltham, MA). The mobile phase with 20mM ammonium
acetate buffer and acetonitrile. The detector was set at 245nm wavelength.
Derivation and analysis
NAP identification by derivatization and gas chromotography-mass spectrometry (GC/MS;
Agilent 6890; Santa Clara, CA). Lyophilized HPLC elutes were reacted with 80 μl of bis(tri-
methylsilyl) trifluoroacetamide + 10% trimethylchlorosilane (Regis, Morton Grove, IL) for
30 min at 75˚C. NAP was identified using an Agilent 5973N-MSD equipped with an Agilent
6890 GC system (GC/MS). A DB17-MS capillary column (30 m × 0.25 mm × 0.25 μm) was
used in all assays with a helium flow of 1 ml/min. Samples were analyzed in Scan mode using
electron impact ionization (EI). Ion dwell time was set to 10 msec [33]. Samples were analyzed
with electron impact ionization. The National Institute of Standards and Technology (NIST,
Gaithersburg, MD) Mass spectral library with search program was used with AP as the stan-
dard for verification. The sensitivity of the nitration process to various acidic conditions was
verified using Thermo Fisher TSQ triple-quad LC/MS instrument with direct injection in scan
mode.
Conjugation of NAP with Derp1 and purification and liquid
chromatography mass spectrophotometry proteomics
Purified NAP (10mcg/mL) was reacted with Derp1 (Indoor Biotechnologies, Charlottesville,
VA) (5mcg/mL) at in a 2:1 ratio at pH values of 7, 6 and 4. Samples were incubated at 37˚C for
1 hour. The post-reaction Der p1 and NAP mixture was visualized on SDS-PAGE gel with
Coomassie staining. SDS PAGE was washed in distilled water to remove excess background
stain; the gel band corresponding to Der p1 was excised and cut into 1 mm2 pieces. The gel
pieces were de-stained with 500 mcL of 1:1 acetonitrile and 100 mM ammonium bicarbonate
solution for 2–8 hours. Afterwards, 10 mM reductive tris (2-carboxyethyl) phosphine was
added and free cysteines alkylated with 55 mM iodoacetamide. Acetonitrile and 100 mM
ammonium bicarbonate were used to dehydrate and rehydrate the gel pieces alternatively for
three times. Gel pieces were swelled in 50 mM ammonium bicarbonate containing 10 ng/μL
trypsin (Promega, Madison, WI), and Glu-C and digested overnight. Peptides were extracted
with 60% acetonitrile/5% formic acid and dried in SpeedVac.
Protein digestion
Gel pieces of PEPT1 protein bands cut from SDS-PAGE were destained with 50% acetonitrile
in 100 mM ammonium bicarbonate, and then in 100% acetonitrile. The proteins in the gels
were reduced by 20 mM 1,4-Dithiothreitol at room temperature for 30 min followed by alkyl-
ation by 50 mM idoacetamide in 100 mM ammonium bicarbonate for 30 min in the dark.
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
After the treatment, the reagents were removed and the gel pieces were washed with 100 mM
ammonium bicarbonate and then dehydrated in acetonitrile. The dried gel pieces were then
reswelled in sequencing grade modified trypsin in 50 mM ammonium bicarbonate for over-
night digestion. Tryptic peptides were extracted from the gel with 50% acetonitrile in 5% for-
mic acid.
Liquid chromatography -Tandem mass spectrometry (LC-MS/MS)
proteomic analysis
Proteolytic peptide analysis was performed using Orbitrap Elite Hybrid Mass Spectrometer
(Thermo Electron, San Jose, CA) equipped with a Waters nanoAcquity UPLC system (Waters,
Taunton, MA). A full scan at 120,000 resolution was obtained in the Oribtrap for eluted pep-
tides in the range of 300–1800 Da followed by MS/MS scans on the twenty most abundant pre-
cursor ions by collision-induced dissociation at normalized collision energy of 35%. Raw
LC-MS/MS data were searched through Mascot search engine (version 2.2.0, Matrix Science)
against PEPT1 protein primary sequence with Met oxidation, Cys carbamidomethylation and
NAP modification as variable modifications. The mass tolerance was set as 10 ppm for precur-
sor ion and 0.8 Da for product ion. The significance threshold was P < 0.05.
Peripheral blood mononuclear cell (PBMC) stimulation
Whole blood was collected from healthy adult volunteers not currently taking AP, ibuprofen,
or any antihistamine and placed in ethylenediaminetetraacetic acid (EDTA). PBMC’s were iso-
lated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Uppsala,
Sweden), per the manufacturer’s instructions. The cells were washed, counted and plated at a
density of 1x106 cells/well in 1mL RPMI plus 10% heat inactivated fetal bovine serum. Treat-
ment groups included no treatment (negative control), phytohemagluttin, to induce non-
specific activation of T cells [34], (PHA, 50 ng/mL, positive control), Der p1 (50 ng/mL), AP
(50ng/ml), NAP (50 ng/mL), Der p1 with NAP (50 ng/mL each), Der p1 mixed with AP
(50 ng/mL each), Der p1 conjugated with NAP (50 ng/mL), or Der p1 conjugated with AP
(50 ng/mL). These treatments were incubated at 37˚C for 18 hours in 5% CO2, pH 7.4. Super-
natant and cells were harvested after culture. ATP was measured in mol/mL with BacTiterGlo
chemiluminescence assay (Promega, Madison, WI) as a surrogate for cellular activity [35] and
therefore growth kinetics [36]. To account for intrasubject variability the data is presented as a
ratio with negative control as the denominator. Cytokine (TNFa, IL-1β, IL-4, IL-5, IL-6, IL-10,
IL-13, and IL-17) levels were measured in pg/mL with Multiplex Luminex assays (Thermo-
Fischer, Waltham, MA).
Statistics
Data are expressed as mean standard deviation. Comparisons of mean data were made
through analysis of variance, and subsequent multiple comparisons were made using Bonfer-
oni correction. A value of p< 0.05 was considered significant.
Approvals
All studies involving human use were approved by the Institutional Review Board of Univer-
sity Hospitals Cleveland Medical Center (IRB#: 01-16-08) Informed consent was obtained for
the blood draws.
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
Results
Synthesis, purification and identification of NAP
NAP was produced by nitration of AP in nitrite at pH 4, 2, and 0.3 and purified by HPLC with
15 minutes of elution time in a mobile phase of 20 mM ammonium acetate and 5% acetoni-
trile. The NAP peak eluted at 8 minutes (n = 10 replicates, Fig 1). NAP was identified by
GC/MS (Fig 2A and 2B, representing 3 replicates). The MS profile was similar to one previ-
ously published by Lakshmi, et al32. As a standard analytical quality AP was converted to its tri-
methylsilyl (TMS) derivative and analyzed by GC/MS. Fig 2C and 2D shows GC/MS trace
with prominent molecular ion 296 and typical loss of CH3+ ion 280. Fig 3 shows validation of
AP TMS derivative by NIST database matching (98%; n = 3 replicates). Fig 3 shows the NAP
TMS derivative. Panels A and B show GC/MS spectra matching data published by Lakshmi
et al [32]. Molecular ion 340 corresponds to NAP TMS molecular weight and ion 325 repre-
senting loss of CH3+ group. Panels C and D show the same samples analyzed after 3 months of
storage of lyophilized NAP. The relative signal was 10% of the original but usable for identifi-
cation and quantification. To determine the effect of HCL concentration on NAP synthesis AP
reactions were done using HCL covering. The AP nitration reactions were done using HCL
concentrations of 1N, 0.1N, 0.05N, 0.01N or0.005N (panel E1-5). AP nitration and generation
of NAP was evident at 0.05N HCL and above demonstrating the concentration dependent
impact of HCL concentration on the synthesis of NAP.
Conjugation of Der p1 with NAP
Haptens are electrophilic and can directly react with protein nucleophiles [37]. Amino acids
with the most-reactive nucleophilic side chains are lysine, cysteine and histidine [38]. We
expected covalent binding of NAP to Der p1 because of the reactivity of the electrophilic moi-
ety on NAP with the nucleophilic amino acids on Der p1. Conjugation was accomplished by
incubating 25μg of Der p1 with 50μg of NAP at 37˚C for one hour at pH 7.4, 6.0 and 4.0. No
significant difference in reaction results resulted from changes in pH. LC-MS analysis showed
a molecular mass shift of 194.03Da in the NAP + Der p1 groups as compared to untreated Der
p1 of (Table 1) (n = 3; p<0.05). The modification was mapped to Der p1 Cysteine (Cys) 129 or
132 (there were insufficient ion fragments to distinguish between these sites), however, the
Cys 129 in native Der p1 forms a disulfide bond with Cys 169 and is unlikely the binding site
absent denaturation [39] making Cys132 more likely.
Immunoreactivity of the NAP and Der p1 compounds
To determine if NAP conjugated Der p1 enhances immune response to Der p1, we tested Der
p1 with and without conjugation with NAP using human PBMCs in vitro monitoring cytokine
response and cellular activity (Fig 4). The PBMC response to NAP versus NAP conjugated to
Der p1 demonstrated very different responses. NAP conjugated to Der p1 induced a signifi-
cant IL-6 and IL-1β response (pg/mL, n = 7; p<0.05) (Fig 4A, 4B & 4C), which did not occur
when unconjugated NAP was added concurrently with the Der p1. Further, the conjugation of
Der p1 with NAP did not appear to have a significant impact on the other cytokines measures.
PBMC’s were responsive to the nonspecific stimulant PHA (positive control). NAP conjugated
Der p1 induced a proliferative response in the monocytes compared to the unstimulated sam-
ple (Fig 4D n = 3; p<0.05) with a focus on PBMC ATP production. The NAP combined with
the Der p1 had the same impact on PBMC proliferation and ATP production as NAP conju-
gated to the Der p1. No other sample generated a significant response.
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
Fig 2. GC/MS spectra of paracetamol standard. Panel A: Gas chromatography trace. Panel B: Mass spectra trace. Data were acquired in the SCAN
mode (50–800 amu). C: Matching of standard sample to National Institute of Standards and Technology (NIST) standard database. Panel A: Mass
spectra and formula of paracetamol. Panel D: Mass spectra matching to NIST (top NIST, bottom standard). Data were acquired in the SCAN mode
(50–800 amu).
https://doi.org/10.1371/journal.pone.0188614.g002
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
Fig 3. Determination and stability of 3-nitroacetaminophen (NAP)-TMS derivative product. Panels A and B: Gas chromatography
and mass spectra traces, respectively, at time 0. Panels C and D: Gas chromatography and mass spectra traces, respectively, at
3-month time point. Data were acquired in the SCAN mode (50–800 amu). TMS(trimethylsilyl). Panel E demonstrates the LCMS trace
peaks of NAP (arrow) post when the AP nitration was done in the presence of 1N, 0.1N, 0.5N, 0.01N, 0.05N and 0.005N HCL.
https://doi.org/10.1371/journal.pone.0188614.g003
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
Table 1. Liquid chromatography-mass spectrometry analysis of Der p1/3-nitro-acetaminophen (NAP) conjugate. LC-MS analysis of NAP + Der p1
conjugation reaction at pH7 showing mass shift in the NAP treated group. CAM (carbamidomethyl Cys) OxMet (oxidized methionine), Mw (Molecular weight),
Obs (observed), Cal (calculated).
Der p1 Peptide
No
Site
Mw(Obs.)
2643.1622
2659.131
2715.163
2780.1564
2796.1545
2837.1546
Mw(Cal.)
Mass error
(ppm)
MQGGCGSCWAFSGVAATESAYLAYR
125–149
C129 or
C132
Un-modified
2643.1425
2659.1374
2715.1516
2780.1538
2796.1487
2837.1752
7
CAM
Der p1 peptides
-2
4
CAM
OxMet
2CAM
OxMet
NAP
NAP-modified Der p1
peptides
1
2
NAP
OxMet
NAP
-7
CAM
https://doi.org/10.1371/journal.pone.0188614.t001
Fig 4. Peripheral blood monocyte (PBMC) response to Der p1 with and without 3-nitro-acetaminophen (NAP). PBMCs were
obtained from 7 different individuals and cultured without treatment (Blank), after stimulation with acetaminophen (AP; 100ng/ml),
3-nitroacetaminophen (NAP; 50ng/ml), Der p1 (Drp; 50ng/ml), Der p1 conjugated with NAP (N+D; 50ng/ml), Der p1 mixed with NAP (N/
D; 50ng/ml), Der p1 conjugated with AP (A+D; 50ng/ml), and Der p1 mixed with AP (A/D; 50ng/ml). After 24 hours PBMC cytokine (pg/
mL) and adenosine triphosphate (ATP, mol/mL) levels were quantified as a measure of cell proliferation. ATP levels were standardized
to subject baseline to account for intrasubject variability. Der p1 conjugated to NAP induced a significant response in IL-6 (Fig 4A), IL-1β
(Fig 4B) and ATP (Fig 4D). There was no significant difference in any other cytokine with TNFa shown as reference (Fig 4C).
https://doi.org/10.1371/journal.pone.0188614.g004
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
Discussion
The original hypothesis that increased prevalence of asthma was related to AP use was pro-
posed in 1998 [40]. Reports subsequently suggested an association between AP use and rhino-
conjunctivitis, eczema and asthma severity [2]. More recently, the NHLBI AsthmaNet AVICA
Trial found no difference in asthma incidence in children treated with either AP or ibuprofen
for fever from the age of 3–5 [8]. However, the original ISAAC study involved AP exposure in
children less than 1, and Sordillio et al, among others, showed prenatal AP exposure was asso-
ciated with wheeze. The effects of early exposure, and the association of AP use with atopic dis-
ease, remain unclear.
Nitration is the addition of NO2+ to a target. Multiple nitration pathways exist in inflamed
-
airways. The reaction of nitric oxide (NO) with superoxide (O2 ) forms peroxynitrite
(OONO-) which, when protonated forms peroxynitrous acid a potent nitrating agent [41,42].
-
Additionally, abundant airway nitrite (NO2 ), when protonated forms nitrous acid, another
nitrating agent [43]. Eosinophil peroxidase and neutrophil myeloperoxidase enzymatically
nitrate proteins [44]. 3-nitro-tyrosine is detected in inflamed aiways [20,45], exhaled breath
condensate analysis [46–48] induced sputum, and airway lavage samples [21,22,24,49]. AP is
uniformly distributed in most tissues [50] and detected in rat lungs [51]. Under acidic condi-
tions found in the stomach and inflamed airways, we demonstrate AP, like tyrosine, can be
nitrated to form NAP.
Up to 85% of asthmatics are sensitized to house dust mites [52,53]. Dermatophagoides ptero-
nyssinus is a predominant HDM species [54] with Der p1 being its principal allergen. We dem-
onstrate AP covalently binds to Der p1 at Cys 132. When PBMC are exposed to NAP
conjugated Der p1, the PBMC are stimulated to proliferate and produce IL-1, and IL-6. Inter-
estingly, the cytokine response was not type 2 nor was there a strong response to Derp1 alone.
It has been shown that mutation of Cys 132 leads to loss of the enzymatic activity [41] impli-
cated in the type 2 responses to Der p1 [55]. The NAP binds to this same enzymatic active site
of Der p1, potentially similarly affecting immune bias.
Immunologic response to haptenized proteins may be directed at the native protein, the
protein-hapten complex, or the hapten. The cytokine response was only apparent in the con-
text of conjugation between the NAP and Der p1. The PBMC proliferative response, as moni-
tored by the production of ATP, was similar whether the NAP was conjugated to the Der p1 or
not. The combined presence of both NAP and Der p1 enhanced PBMC ATP production com-
pared to when the PBMCs treated with NAP or Der p1 independent of each other. The differ-
ence between the cytokine levels and the ATP response suggests that the ATP response to
NAP and Der p1 is exquisitely sensitive and may reflect the overall sensitivity of the PBMCs to
both NAP and Der p1 in which ATP is a product of immediate activation of the cells. The sub-
sequent cytokine production involves a variety of down-stream events to cellular activation
which are reflected in the specificity of the response to the NAP conjugated to Der p1. We
speculate that immune responses to a given protein may differ based on whether the protein of
interest is nitrated. A patient’s allergic immune response could be influenced by the nitration
status at time of initial presentation.
In summary, AP is nitrated under acidic conditions and reacts with Cys 132 on Derp1
forming a hapten-antigen complex capable of amplifying human monocytic proliferative
responses to antigen stimulation. Acidic nitrating conditions—perhaps particularly in the
presence of AP—may modify antigenicity and human immune responses.
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Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1
Acknowledgments
Thank you to Yuejin Li, David Fletcher and Nicholas Beno for the technical expertise and
contributions.
Author Contributions
Conceptualization: Ryan G. Thomas, Brenda M. Rivera Reyes, Benjamin M. Gaston, John F.
Hunt, Tracey L. Bonfield.
Data curation: Ryan G. Thomas, Brenda M. Rivera Reyes, Benjamin M. Gaston, Ilya R. Beder-
man, Morgan T. Sutton, Benlian Wang, Tracey L. Bonfield.
Formal analysis: Ryan G. Thomas, Benjamin M. Gaston, Ilya R. Bederman, Laura A. Smith,
Benlian Wang, Tracey L. Bonfield.
Funding acquisition: Ryan G. Thomas, Benjamin M. Gaston, Tracey L. Bonfield.
Investigation: Ryan G. Thomas, Brenda M. Rivera Reyes, Benjamin M. Gaston, Nelki B.
Rivera Acosta, Ilya R. Bederman, Morgan T. Sutton, Benlian Wang, Tracey L. Bonfield.
Methodology: Ryan G. Thomas, Brenda M. Rivera Reyes, Benjamin M. Gaston, Ilya R. Beder-
man, Laura A. Smith, Benlian Wang, Tracey L. Bonfield.
Project administration: Ryan G. Thomas.
Resources: Ryan G. Thomas, Tracey L. Bonfield.
Software: Ryan G. Thomas.
Supervision: Benjamin M. Gaston, Tracey L. Bonfield.
Validation: Ryan G. Thomas, Benlian Wang.
Writing – original draft: Ryan G. Thomas, Brenda M. Rivera Reyes.
Writing – review & editing: Ryan G. Thomas, Brenda M. Rivera Reyes, Benjamin M. Gaston,
Ilya R. Bederman, Benlian Wang, John F. Hunt, Tracey L. Bonfield.
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