Immunol Res

DOI 10.1007/s12026-015-8696-3

AUTOIMMUNITY/INFLAMMATION

N-Acetylcysteine attenuates tumor necrosis factor

alpha levels in autoimmune inner ear disease
patients
Shresh Pathak1,3,5 • Corey Stern1 • Andrea Vambutas1,2,3,4,5

Andrea Vambutas

Ó Springer Science+Business Media New York 2015

Abstract Autoimmune inner ear disease (AIED) is a poorly understood disease marked by bilateral, rapidly progressive hearing
loss triggered by unknown stimuli, which is corticosteroid responsive in 60 % of patients. Although the mechanism of the disease is
not precisely understood, a complex interaction of cytokines is believed to contribute toward the inflammatory disease process and
hearing loss. Previously, we showed the role of TNF-a in steroid-sensitive and IL-1b in steroid-resistant immune-mediated hearing
loss. N-Acetylcysteine (NAC), a broad spectrum antioxidant, has been effective in other autoimmune disorders. Other studies have
shown NAC to have a protective adjunct role in human idiopathic sudden hearing loss, where the addition of NAC resulted in better
hearing recovery than with steroids alone, although the mechanism of this protection was not elucidated. In the present study, we
observed PBMCs from AIED patients exhibited higher baseline TNF-a and MPO levels compared with normal healthy controls.
NAC effectively abrogates LPS-mediated TNF-a release from PBMC of both AIED patients and controls. We demonstrated that in
AIED patients, the TNF-a downstream signaling pathway appears aberrantly regulated, influencing both MPO and IL-8
expression. Given that NAC effectively abrogated LPS-mediated TNF-a release and exerted minimal effects on the downstream
targets of this pathway, we feel NAC may be a rational adjunct therapy for this enigmatic disease, worthy of clinical exploration.

Keywords N-Acetylcysteine (NAC)
Tumor necrosis factor alpha (TNF-a)
Myloperoxidase (MPO)
Autoimmune
inner ear disease (AIED)
Abbreviations
Electronic supplementary material The online version of this AIED Autoimmune inner ear disease
article (doi:10.1007/s12026-015-8696-3) contains supplementary NAC N-Acetyl-L-cysteine
material, which is available to authorized users.
GSH L-c-Glutamyl-L-cysteinyl-glycine
& Andrea Vambutas ROS Reactive oxygen species
vambutas@nshs.edu MPO Myeloperoxidase
1 TPO Thyroperoxidase
The Feinstein Institute for Medical Research, 350
Community Dr, Manhasset, NY 11030, USA EAE Experimental autoimmune encephalomyelitis
2
The Apelian Cochlear Implant Center, Department of
Otolaryngology, North Shore-LIJ Health System,
New Hyde Park, NY, USA
3
Department of Otolaryngology, Hofstra North Shore-LIJ Introduction
School of Medicine at Hofstra University, Hempstead, NY,
USA
4
Autoimmune inner ear disease (AIED) is an enigmatic
Department of Molecular Medicine, Hofstra North Shore-LIJ
disease of periodic episodes of bilateral progressive hear-
School of Medicine at Hofstra University, Hempstead, NY,
USA ing decline activated by unknown stimuli [1]. Recently, we
5 have identified a role of TNF-a in steroid-sensitive and IL-
Department of Otorhinolaryngology-Head and Neck Surgery,
Montefiore Medical Center, Albert Einstein College of 1b in steroid-resistant immune-mediated hearing loss [2,
Medicine, Bronx, NY, USA 3]. Furthermore, in a small cohort of patients, local

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administration of the TNF-a inhibitor etanercept by autoimmune encephalomyelitis (EAE) [27]. In animal
intratympanic injection was able to maintain hearing when models of autoimmune thyroiditis, adjunct treatment with
systemic corticosteroid therapy was tapered [4], although NAC reduced oxidative stress and the immune infiltration,
use of systemic etanercept met with less promising efficacy thereby leading to a restoration of thyroid morphology
[5, 6]. [28]. NAC therapy also reduced disease severity in a
Reactive oxygen species (ROS) act as central mediators clinical trial of systemic lupus erythematosus (SLE),
in the etiology of many autoimmune diseases. Among although the mechanism was ascribed to blocking the
numerous ROS-generating molecules, inducible nitric mammalian target of rapamycin in T cells [29]. Based on
oxide synthase (iNOS) and myloperoxidase (MPO) are our prior observations that steroid-sensitive AIED patients
hypothesized to play key roles. MPO is a pro-oxidant exhibit higher pre-treatment levels of TNF-a [2], we
enzyme released from granules of activated leukocytes, hypothesized that this elevated TNF-a induces expression
neutrophils, monocytes and macrophages at inflammatory of MPO which results in oxidative damage and further
sites. When released as part of the innate host defense, it hearing loss. We further hypothesized that treatment with
generates free radicals and ROS [7, 8]. MPO plays a vital NAC can partially abrogate TNF-a-mediated induction of
role in the autoimmune disease process by escalating MPO and subsequent oxidative damage making it a good
oxidative stress and oxidizing lipoproteins [9]. There are candidate to be used for adjunct therapy.
several autoimmune diseases, such as Wegener’s granulo-
matosis [10], systemic lupus erythematosus (SLE) [11],
Goodpasture syndrome [12], Churg–Strauss syndrome [13] Materials and methods
and Cogan’s disease, a rare autoimmune vasculitis causing
sensorineural hearing loss [14], associated with the devel- Subjects
opment of anti-neutrophil cytoplasm (ANCA) autoanti-
bodies against MPO. Furthermore, the perilymph of the This study was approved and monitored by the Institutional
inner ear is replete with apolipoproteins and therefore a Review Board (IRB) of the North Shore Long Island
potential target of MPO [15]. Jewish Health system. Patients were identified by several
N-Acetylcysteine (NAC), a broad spectrum antioxidant, neurotologists. All individuals who were included in study
is a precursor in the synthesis of reduced glutathione gave signed informed consent. Inclusion criteria were
(GSH) and acts as a free radical scavenger that protects based on Niparko et al. [30] study group guidelines. A total
cells from oxidative damage [16]. NAC exerts anti-in- of 64 AIED patients and 31 control subjects were recruited
flammatory effects [17]. NAC has been identified to be for these studies. The control cohort consisted of subjects
otoprotective and served as a beneficial adjunctive therapy of similar age, gender and ethnic background, who denied a
in hearing restoration. NAC has been shown to have a history of hearing loss or autoimmune disease.
protective adjunct role in idiopathic sudden hearing loss,
where the addition of NAC to corticosteroid therapy Human PBMC culture and stimulation
resulted in better hearing recovery than with corticosteroids
alone [18]. Furthermore, NAC ameliorated hearing loss in PBMCs from AIED patients and control subjects were col-
animal models of noise-induced hearing loss in several lected in an identical fashion and were separated by Ficoll
studies [19–22]. NAC was found to be effective in abro- density gradient as previously described [3]. Isolated
gating LPS-induced TNF alpha release in cardiomyocytes PBMCs were washed twice with 1 9 RPMI (GIBCO) and
in dose-dependent manner [23] and in neuroblastoma cell incubated in RPMI 1640 supplemented with 10 % (v/v) FBS
lines [24]. (Atlanta Biologicals GA; a single lot of FBS was used for all
Pulmonary studies have shown that NAC can reduce experiments), 100 units/ml penicillin, 100 lg/ml strepto-
MPO levels in the serum/plasma of a small cohort of mycin and 4.1 mM glutamine and plated at 1 9 106 cells/ml
healthy smokers and the concentrations of MPO were in 24-well plate (Costar). Preliminary experiments were
reduced after NAC treatment for 8 weeks [25]. The chronic done to identify the kinetics of TNF-a release after stimu-
use of NAC inhibited MPO activity in septic lung injury rat lation with LPS, with maximal release plateauing between 4
model [26]. and 24 h (Supplemental Fig. 1). PBMCs (1 9 106 per ml)
Adjunct NAC therapy has also been effective in abating isolated from control subjects were subjected to treatment
autoimmune disease in both animal models of autoimmune with and without 1 lg/ml LPS for different time points (1–
disease and human autoimmune disease, although the 24 h). For rest of the experiments, PBMCs were treated with
mechanism of action described has been varied. NAC in either 5 mM N-acetyl-L-cysteine (NAC), 1 lg/ml LPS
combination with aminoguanidine was found to be effec- E. coli (0111:B4) (both from Sigma), 10 ng/ml recombinant
tive in reducing oxidative stress in experimental TNF-a, recombinant myloperoxidase (1.5 lg/ml) (both

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from R&D systems), 10 ng/ml recombinant IL-8 (Miltenyi Thyroid peroxidase (TPO) ELISA
Biotec, Auburn, CA), alone or in combination with NAC.
The optimal concentrations of these reagents were previ- Plasma TPO levels were measured according to the man-
ously identified by culturing PBMC with increasing con- ufacturer’s instructions (Abnova). Concentrations were
centrations of these reagents to identify the maximal calculated using a 4-parameter logistic curve. All samples
concentration that could be used without affecting cell via- were run in duplicate. The variance between replicate sets
bility. All PBMC samples were cultured overnight (16 h) at was 0.005.
37 °C in 5 % CO2 and compared with similarly cultured,
unstimulated PBMC. Cell viability was measured after 16 h
by trypan blue dye exclusion method using the Cellometer IgG ELISAs for antibodies to MPO and TPO
Auto T4 automated cell counter (Nexcelom Biosciences). At
the end of all incubations, samples were centrifuged and Plasma anti-MPO IgG and anti-TPO IgG levels were
supernatants were collected and stored at -20 °C. measured by ELISA according to the manufacturer’s
instructions (Abnova). Anti-MPO IgG concentrations were
Monocyte and neutrophil isolation calculated using a 4-parameter logistic curve. For the
plasma anti-TPO IgG concentration, a standard curve was
Monocytes and neutrophils were isolated from same constructed using a quadratic fit. All samples were run in
patient from same lot of blood by MACS monocyte iso- duplicate. The variance between replicate sets was 0.001
lation kit IITM and MACSxpressTM Neutrophil Isolation and 0.01, respectively.
Kit both from (Miltenyi Biotec, Auburn,CA). The purity of
negatively selected monocytes and neutrophils was deter-
mined by flow cytometry (FACSCanto II; BD Biosciences) Western blotting
using anti-CD14PE Abs (BD Immunocytometry Systems,
San Jose, CA) and anti-CD 15 FITC along with Pacific Isolated proteins from monocytes, neutrophils and
BlueTM Mouse Anti-Human CD16 Clone 3G8 (both from PBMCs were separated on 12 % Mini-PROTEANÒ
BD Biosciences), respectively. Purity for these experiments TGXTM Precast Gel (Bio-Rad) and then transferred
exceeded 80 and 90 %, respectively. electrophoretically to PVDF membrane (Bio-Rad). The
blot was then blocked with 5 % nonfat dry milk (NFDM)
ELISA methodology in tris-buffered saline (TBS) with Tween 20 (0.5 %, v/v)
for 1 h at room temperature and subsequently incubated
TNF-a and IL-8 ELISA overnight at 4 degree with anti-MPO at dilution of 1:750
(DAKO) in TBS-Tween 20 (0.5 %, v/v) [TBST] with 5 %
Supernatant TNF-a (GE Healthcare) and IL-8 (R&D sys- (w/v) NFDM. Following three washes of 5 min each with
tems) levels were quantified using a sandwich ELISA as TBST, the blots were incubated with HRP-conjugated
per the manufacturer’s instructions. An 8-point standard goat anti-rabbit IgG (R&D systems) (1:5000) in blocking
curve was constructed for each assay using a quadratic fit, buffer for 45 min at room temperature. After three washes
and data were interpolated using BioLinx 2.2 software with TBST, the blot was developed with Clarity ECL
(Synatech Laboratories Inc). Each sample was run in Western Blot Substrate kit (Bio-Rad) and exposed to
duplicate. Greater than 70 % of all samples were run on Kodak Scientific Imaging X-OMAT LS Film from Care-
replicate plates: The variance between replicate sets was stream Health (Rochester, NY). Anti-actin antibody
0.08 and 0.001 for TNF-a and IL-8. (Sigma) was used as loading control.

MPO ELISA
Statistical analysis
Plasma and supernatant MPO levels were quantified using
Human MPO Instant ELISA (eBioscience) as per the man- The data are expressed as mean ± standard error of the
ufacturer’s instructions. The sensitivity of the assay was mean (SEM) and analyzed using GraphPad Prism 5
0.026 ng/ml. An 8-point standard curve was constructed for (GraphPad Software, San Diego, CA). The differences
each assay using a quadratic fit, and data were interpolated between groups were evaluated using the Mann–Whitney
using BioLinx 2.2 software (Synatech Laboratories Inc). U test or by one-way analysis of variance (ANOVA). Post
Each sample was run in duplicate. A least 19 samples from hoc testing was performed using a Bonferroni’s compar-
AIED patients and four control subjects were replicated on ison on selected columns. p values less than or equal to
different dates. The variance between replicate sets was 0.03. 0.05 were considered significant.

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Results ELISA to compare their MPO plasma levels. MPO levels

were significantly increased in the plasma of AIED patients
NAC effectively abrogates LPS-mediated TNF-a compared with controls [231 ng/ml of AIED patients
release from PBMC of both AIED patients and plasma versus 47.76 ng/ml of plasma from control subjects
controls (p \ 0.0001 by the Mann–Whitney U test)] (Fig. 2). The
data suggest that increase in MPO levels is associated with
PBMCs isolated from AIED (N = 12) patients and healthy AIED disease.
control subjects (N = 5) were either treated with 1 lg/ml
LPS, 5 mM NAC, co-cultured with the combination of 1 lg/ MPO can be marginally induced by TNF-a and
ml LPS plus 5 mM NAC or left untreated for 16 h. NAC, in blocked by NAC
combination with LPS, reduced TNF-a release when com-
pared with LPS alone in AIED patients and control subjects: In To determine whether MPO induction was TNF-a medi-
LPS-stimulated PMBC from AIED subjects, NAC reduced ated, PBMCs isolated from AIED (N = 8) patients and
TNF-a release from 653 to 280 pg/ml (a 57 % reduction), and healthy control subjects (N = 6) were either treated with
in PBMC from control subjects, NAC reduced TNF-a release 10 ng/ml TNF-a or 5 mM NAC, co-cultured with 10 ng/ml
from 445 to 173 pg/ml (a 61 % reduction) (Fig. 1, p = 0.03, TNF-a plus 5 mM NAC or left untreated for 16 h. NAC
Mann–Whitney U test). NAC had a marginal effect on had a marginal effect on unstimulated PBMC. NAC
unstimulated PBMC, largely because minimal expression of reduced MPO in unstimulated PMBC from AIED patients
TNF-a secretion was detected (in PMBC from AIED subjects, and control subjects by 20 and 21 %, respectively
NAC reduced TNF-a release from 21 to 12 pg/ml, and in (Fig. 3a). Similarly, basal MPO release from untreated
PBMC from control subjects, NAC reduced TNF-a release PBMC of AIED patients (N = 12) was 3.3 times greater as
from 13 to 8 pg/ml). Based on our prior observations of ele- compared with release from PBMC of control subjects.
vated TNF-a secreted protein levels in a subset of AIED NAC reduced the TNF-a-mediated marginal increase in
patients, we queried whether myeloperoxidase (MPO) was MPO release in AIED patients p \ 0.05 (one-way ANOVA
elevated in these AIED patients as well. followed by post hoc analysis using a Bonferroni’s com-
parison on selected columns) (Fig. 3a). LPS-stimulated
AIED patients have elevated plasma levels of MPO PBMC cultures from AIED patients were largely refractory
to NAC treatment, as NAC treatment resulted in only a
Plasma samples from 50 AIED patients and from 24 nor- minimal decrease in MPO levels from LPS-stimulated
mal healthy control subjects were tested by sandwich PBMC (Fig. 3a). We suspect this failure of NAC to miti-
gate the LPS effect may be a result of LPS induction of

Fig. 1 NAC abrogates LPS-mediated TNF-a release from PBMC of

both AIED patients and controls. PBMCs isolated from AIED patients
(N = 12) and healthy control subjects (N = 5) are treated with 1 lg/ Fig. 2 AIED patients have higher plasma levels of MPO compared
ml LPS, 5 mM NAC, 5 mM NAC ? 1 lg/ml LPS or left untreated with control subjects. Plasma samples from AIED patients (N = 50)
for 16 h. LPS-mediated TNF-a release is significantly reduced by and normal healthy (N = 24) control subjects are tested by sandwich
NAC in AIED patients (p = 0.03, Mann–Whitney U test) compared ELISA to compare their MPO levels. MPO plasma levels are
with LPS alone. In case of control subjects, there is trend toward significantly increased in AIED patients (p \ 0.0001) compared with
reduction in TNF release; however, significance is not achieved controls by the Mann–Whitney U test with 95 % CI. Error bars
(p = 0.09). Error bars show ± SEM show ± SEM

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Fig. 3 MPO is generated by both neutrophil and monocyte fractions from AIED patients and control subjects. Comparisons are made by
of PBMC, and NAC reduces TNF-a-mediated MPO release from one-way analysis of variance (ANOVA) p \ 0.0001, followed by post
PBMC of both AIED patients and controls. a A sandwich ELISA is hoc testing using a Bonferroni’s comparison test on selected columns.
done on stimulated PBMCs isolated from AIED patients and healthy TNF-a induced MPO release compared with TNF-a ? NAC
control subjects. PBMC from AIED patients are either treated with achieved statistical significance p \ 0.05 in AIED patients. b
10 ng/ml TNF-a (8 AIED patients) or 1 lg/ml LPS (16 AIED Neutrophils (Neut), monocytes (Mono) and total PBMCs are isolated
patients) ± 5 mM NAC and compared with untreated PBMC, or from an AIED patient and treated with LPS 1 lg/ml or kept untreated
PBMC from control subjects (N = 6) cultured under the same as indicated. Lysates were analyzed by Western blot for the relative
conditions for 16 h. NAC in combination with TNF-a is able to expression of MPO to b-actin
reduce MPO release when compared with TNF-a alone in PBMC

other pro-inflammatory cytokines other than TNF-a and

that the NAC effect may be limited to TNF-a (Fig. 3a).

Neutrophils and monocytes produce MPO

To see whether increased MPO levels could be ascribed to

a specific cell type, we isolated monocytes and neutrophils
from an AIED patient and by Western blot compared their
intracellular MPO contents with total PBMC isolated from
same patient from same lot of blood. Western blotting
indicated that MPO is expressed by both neutrophils and
monocytes (Fig. 3b). However, the majority of MPO is
produced by the neutrophil fraction (Fig. 3b).

TNF-a induces IL-8 release, which is marginally

reduced by NAC co-culture
Fig. 4 TNF-a induces IL-8 release, which is not reduced by NAC.
PBMCs isolated from AIED patients (N = 4) and healthy PBMCs isolated from AIED patients (N = 4) and healthy control
subjects (N = 3) were either treated with 10 ng/ml recombinant TNF-
control subjects (N = 3) were either treated with 10 ng/ml
a or 5 mM NAC, co-cultured with 10 ng/ml recombinant TNF-a ? 5
recombinant TNF-a, LPS, 5 mM NAC, co-cultured with mM NAC, 1 lg/ml LPS, co-cultured with 1 lg/ml LPS ? 5 mM
10 ng/ml recombinant TNF-a plus 5 mM NAC or 1 lg/ml NAC, or left untreated for 16 h. TNF-a and LPS were able to induce
LPS or co-cultured with 1 lg/ml LPS plus 5 mM NAC and IL-8 release from PBMCs of AIED patients. Addition of NAC to
either TNF-a or LPS cultures resulted in only a minimal reduction of
compared with untreated PBMC. NAC’s effect on
IL-8 release. Figure 4 TNF-a induces IL-8 release, which is not
unstimulated PBMC in AIED subjects and control subjects reduced by NAC. PBMCs isolated from AIED patients (N = 4) and
could not be judged as practically there was no basal healthy control subjects (N = 3) were either treated with 10 ng/ml
secretion in both cases. Both TNF-a and LPS were able to recombinant TNF-a or 5 mM NAC, co-cultured with 10 ng/ml
recombinant TNF-a ? 5 mM NAC, 1 lg/ml LPS, co-cultured with
induce IL-8 release from PBMCs of AIED patients,
1 lg/ml LPS ? 5 mM NAC, or left untreated for 16 h. TNF-a and
although, again, the LPS effect was substantially greater. LPS were able to induce IL-8 release from PBMCs of AIED patients.
The addition of NAC to either TNF-a or LPS-stimulated Addition of NAC to either TNF-a or LPS cultures resulted in only a
cultures failed to substantially reduce IL-8 levels suggest- minimal reduction of IL-8 release

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Fig. 5 IL-8 or MPO do not augment TNF-a release. a PBMCs isolated from healthy control subjects (N = 5) are either treated with
isolated from AIED patients (N = 4) and healthy control subjects 10 ng/ml recombinant IL-8, 5 mM NAC, co-cultured with 10 ng/ml
(N = 5) are either treated with 1.5 lg/ml recombinant MPO, 5 mM IL-8 ? 5 mM NAC or left untreated for 16 h. IL-8 is unable to
NAC, co-cultured with 1.5 lg/ml MPO ? 5 mM NAC or left induce TNF-a release from PBMCs of control subjects
untreated for 16 h. MPO treatment does not induce TNF-a. b PBMCs

ing NAC exerts a greater effect on TNF-a expression rather

than IL-8 (Fig. 4).

TNF-a-mediated MPO and IL-8 induction is

unidirectional

Given that TNF-a is capable of inducing MPO, we queried

whether MPO could reciprocally induce TNF-a. PBMCs
isolated from AIED patients (N = 4) and healthy control
subjects (N = 5) were either treated with 1.5 lg/ml recom-
binant MPO, 5 mM NAC, co-cultured with 1.5 lg/ml MPO
plus 5 mM NAC or left untreated for 16 h. MPO treatment
does not induce TNF-a (Fig. 5a). Interestingly, co-culture Fig. 6 MPO IgG plasma levels are absent in AIED patients and
control subjects. Plasma samples from 29 AIED patients and ten
with MPO negated the ability of NAC to inhibit TNF-a normal healthy control subjects are tested by ELISA and are
release in AIED PBMC, suggesting polarization of AIED compared with a positive control (MPO antibodies in serum/buffer)
PBMC to a refractory phenotype. Similarly, we also inves- and a negative control (PBS, BSA detergent sodium azide 0.09 %) to
tigated whether IL-8 can induce TNF-a. PBMCs isolated identify whether AIED patients made anti-MPO IgG antibodies. Anti-
MPO IgG plasma levels are in the same range as of negative control
from healthy control subjects (N = 5) were either treated for both AIED patients and control subjects. Error bars show ± SEM
with 10 ng/ml recombinant IL-8, 5 mM NAC, co-cultured
with 10 ng/ml IL-8 plus 5 mM NAC or left untreated for determine whether AIED patients exhibited elevated anti-
16 h. IL-8 was similarly unable to induce TNF-a release MPO plasma IgG levels compared with controls. No sig-
from PBMCs of control subjects (Fig. 5b). A similar pattern nificant anti-MPO IgG antibodies were detected in either
was observed in a small cohort of AIED patients (data not AIED patients or control subjects (Fig. 6). These results
shown). These observations led us to conclude that TNF-a suggest that although MPO is elevated, it is not the target of
induction of MPO and IL-8 is unidirectional, and a feedback an aberrant antibody-mediated immune response.
loop does not appear to be present.
AIED patients have mildly elevated TPO levels and
Anti-MPO IgG antibodies were not detected in autoantibodies to TPO
AIED patients
Since MPO shares great extent of homology with thy-
Antibodies to MPO have been detected in many autoimmune roperoxidase (TPO) and anti-TPO antibodies are readily
diseases. Since plasma MPO levels are elevated in our detected in patients with various autoimmune diseases, we
patient cohort, we hypothesized that our patients may have measured TPO protein levels in plasma of AIED patients
anti-MPO antibodies. Plasma from 29 AIED patients and ten (N = 30) and normal healthy controls (N = 10) by a
normal healthy control subjects was tested by ELISA to sandwich ELISA. TPO plasma levels were slightly greater

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Fig. 7 AIED patients have marginally elevated TPO levels however levels. TPO IgG plasma levels are very high (17-fold) in AIED
exhibit enhanced anti-TPO antibodies compared with age-matched patients’ plasma when compared with control subjects. Error bars
controls. a Plasma samples from 30 AIED patients and ten normal show ± SEM. c Analysis of TPO IgG threshold relative to patient
healthy control subjects are tested by sandwich ELISA to compare age. We segregated our AIED patient (N = 26) TPO IgG ELISA data
their TPO levels. TPO plasma levels are almost same in AIED into two groups on the basis of a threshold of 10 U/ml of TPO IgG.
patients compared with controls (49.02 pg/ml for AIED patients vs The lower threshold group of\10 U/ml (N = 19 patients) had a mean
40.37 pg/ml for control subjects). Error bars show ± SEM. b Plasma value of TPO IgG of 0.67 with a mean age of 54 years old versus the
samples from AIED patients (N = 26) and normal healthy (N = 10) higher threshold group of [10 U/ml (N = 7 patients) with a mean
control subjects are tested by ELISA to compare their TPO IgG value of TPO IgG of 277.94 with a mean age of 41 years old

in AIED patients and controls (49.02 pg/ml of serum for

AIED patients versus 40.37 pg/ml of serum for control
subjects), although this difference is marginal and would
require a substantially larger cohort to draw any conclu-
sions (Fig. 7a). Similar to the rationale for investigating the
presence of anti-MPO antibodies, we also questioned
whether our AIED cohort had anti-TPO antibodies. Plasma
IgG levels of anti-TPO autoantibodies in the same 26
AIED patients were 17-fold higher than in the ten normal
controls (Fig. 7b). Anti-TPO antibodies are prevalent in
older adults [31], to rule out that possibility we segregated
our TPO IgG ELISA data (Fig. 7c) on the basis of a
threshold of 10 U/ml. We observed that the group with
TPO IgG \ 10 U/ml (mean = 0.67) had an age mean of
54 years old versus the group of [10 U/ml (mean
value = 277.94) with an age mean of 41 years of age. This
discounts the possibility that increased TPO IgG levels in
AIED patients are due to more aged AIED population.

Fig. 8 Schematic diagram illustrating the proposed signaling

Discussion pathway

Previously, we observed that PBMCs from AIED patients AIED patients, the TNF-a downstream signaling pathway
exhibited greater TNF-a release compared with normal appears aberrantly regulated (Fig. 8). This TNF-a-medi-
healthy controls [2]. Here we show that PBMC treatment ated IL-8 and MPO induction appears to be unidirectional
with NAC abrogates approximately 60 % of LPS-mediated because MPO and IL-8 treatment does not induce TNF-a,
TNF-a release from PBMCs of both AIED patients and but interestingly, the addition of MPO may cause refrac-
controls. Other studies have also noticed similar inhibition toriness in NAC-mediated TNF-a inhibition in AIED
with NAC when induced by LPS [23]. We noticed that this patients (Fig. 5). Clinically, NAC treatment has been
effect is dose dependent (data not shown), with the 5 mM observed to enhance the effect of corticosteroids in hearing
NAC concentration being optimal. Given that TNF-a is recovery [18]. We have previously demonstrated that,
able to influence MPO [32] and IL-8 [33] expression in specifically, corticosteroid-sensitive AIED correlated with
other systems, we investigated and ascertained that in high TNF-a levels [2]. NAC has been effective in

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inhibiting TNF-a [23] and IL-8 [34, 35] in other studies. segregated our TPO IgG ELISA data on the basis age, we
Notably, TNF-a appears to be a potential target of NAC, as observed that the younger patients had substantially higher
NAC does not exert a strong effect on the further down- anti-TPO titers. The group with low anti-TPO IgG \ 10 U/
stream targets MPO and IL-8. Furthermore, NAC was ml (mean = 0.67 U/ml) had a mean age of 54 years old
ineffective in reducing IL-1b levels (data not shown). versus the group of [10 U/ml (mean = 277.94 U/ml) with
Others have shown that NAC’s effect may be via sup- a mean age of 41 years old. These results suggest that the
pression of TNF-mediated NFkB activation [36–38]. high anti-TPO IgG values are not due to a more aged AIED
Whether this effect is specific to a particular cell type is population, but rather may be associated with development
unknown, however, neutrophils have been observed to of an autoimmune disease (Fig. 8), as many AIED patients
have increased NFkB activation and IL-8 expression [39], have a wide variety of autoantibodies detected in their sera
although PPARs act to inhibit inflammation through NFkB [47]. It is unlikely, however, that these autoantibodies have
in monocytes, B and T cells [40], suggesting it may not be a pathogenic role in the disease process. Presence of these
limited to one cellular subset. Plasma levels of MPO in and other autoantibodies in AIED is variable, and neither
AIED patients were almost fivefold higher when compared assists in clarifying the diagnosis of AIED nor do they
with normal healthy subjects (Fig. 2), indicating that high dictate treatment [47].
MPO levels in AIED could be a result of increased TNF-a Our studies demonstrated that TNF-a is capable of
expression, although elevation of MPO is likely the result inducing IL-8 release (Fig. 4). IL-8 instilled into the round
of a number of factors. Future experiments will determine window membrane of the inner ear of rats resulted in an
the precise effect of anti-TNF-neutralizing antibodies on inflammatory infiltrate; however, interestingly, at the time
MPO expression in these patients, although TNF inhibition of resultant sensorineural hearing loss, the neutrophil
has not been clinically effective in AIED patients [5, 6], infiltrate had resolved, leaving only resident macrophages,
despite possible benefit in patients with Cogan’s disease suggesting their potential role in the development of
[14]. As anticipated, MPO expression is predominantly immune-mediated hearing loss [48]. IL-8 expression is
observed in the neutrophil fraction and to a lesser degree in enhanced in Th17 polarizing conditions, consistent with the
the monocytes fraction which is consistent with prior development of an autoimmune phenotype [49], especially
observations [41]. Future studies will characterize the cell in the background of TNF-a expression [50]. Interestingly,
frequency in AIED patients, as neither we nor other reduced IL-8 levels have been observed in patients with
investigators have determined whether any irregularities thyroid disease [51]. Given that AIED patients that appear
exist. Others have shown that MPO is important in LDL to have anti-TPO antibodies, and elevated IL8 in response
oxidation and resultant monocyte differentiation into to TNF, future studies will further investigate the role of
classical or alteratively activated macrophages based on the IL-8 in these patients.
amount of oxidized LDL [42], suggesting that MPO Taken together, our data demonstrate the ability of NAC
activity may dictate monocyte differentiation and disease to inhibit TNF-a expression in these AIED patients and
progression or remission in other systems based on the may elucidate why adjuvant therapy with NAC has shown
local microenvironment. It is certainly possible that pro- to be clinically beneficial in hearing recovery. Given that
longed treatment with NAC may improve MPO repression. we have observed that TNF-a influences the expression of
Given that we detected elevated MPO in our patient cohort, MPO and IL-8 in these patients and may potentiate
and MPO autoantibodies have been widely observed in autoimmune disease, the equilibrium between the release
different autoimmune diseases, we decided to investigate of oxidants and their inactivation by antioxidants could
MPO antibody levels in our patient cohort. We were unable therefore be decisive in clinical disease progression or
to detect MPO autoantibodies in these patients as measured remission. The above data emphasize that use of antioxi-
by plasma anti-MPO IgG ELISA (Fig. 6), which highlights dants like NAC may be an important adjunct therapy for
the difference between this patient cohort and Cogan’s the treatment of this poorly understood disease, and further
patients as they have anti-MPO antibodies [14]. MPO and understanding of the molecular mechanism of action may
TPO share up to 42 % sequence homology [43–45]. The elucidate further biologic targets for intervention.
relationship between TPO and MPO is also manifested by In conclusion, we demonstrate the ability of NAC to
potentially shared epitopes in autoimmune thyroid disease inhibit TNF-a expression in AIED patients, which may
patients between these two antigens [46]. Although TPO explain the clinical benefit of adjuvant NAC antioxidant
levels in the plasma were marginally higher in AIED therapy hearing recovery.
patients compared with controls, AIED patients had sig-
nificantly higher anti-TPO IgG levels when compared with Acknowledgments This study was supported by a National Insti-
tutes of Health grant R21/R33DC011827 (AV) and Merrill & Phoebe
normal healthy controls (Fig. 7). Although anti-TPO anti- Goodman Otology Research Center.
bodies are prevalent in older adults [31], when we

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Author Contributions The experiments were conceived and autoimmune inner ear disease. Autoimmun Rev. 2013;12(3):396–
designed by SP and AV. The experiments were performed by SP and 400. doi:10.1016/j.autrev.2012.07.012.
CS. The data were analyzed by SP and AV. AV contributed 15. Thalmann I, Kohut RI, Ryu J, Comegys TH, Senarita M, Thal-
reagents/materials/analysis tools; SP and AV wrote the paper. mann R. Protein profile of human perilymph: in search of markers
for the diagnosis of perilymph fistula and other inner ear disease.
Otolaryngol Head Neck Surg Off J Am Acad Otolaryngol Head
Compliance with ethical standards Neck Surg. 1994;111(3 Pt 1):273–80.
16. Cotgreave IA. N-acetylcysteine: pharmacological considerations
Conflict of interest Dr. Vambutas, the Feinstein Institute for and experimental and clinical applications. Adv Pharmacol (San
Medical Research, holds a patent application for the use of IL-1b Diego, Calif). 1997;38:205–27.
receptor antagonists for the treatment of AIED and related diseases. 17. Blesa S, Cortijo J, Mata M, Serrano A, Closa D, Santangelo F,
et al. Oral N-acetylcysteine attenuates the rat pulmonary
inflammatory response to antigen. Euro Respir J.
2003;21(3):394–400.
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