N-Acetylcysteine attenuates tumor necrosis factor alpha levels in autoimmune inner ear disease patients

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-α in steroid-sensitive and IL-1β 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-α and MPO levels compared with normal healthy controls. NAC effectively abrogates LPS-mediated TNF-α release from PBMC of both AIED patients and controls. We demonstrated that in AIED patients, the TNF-α downstream signaling pathway appears aberrantly regulated, influencing both MPO and IL-8 expression. Given that NAC effectively abrogated LPS-mediated TNF-α 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: Autoimmune inner ear disease (AIED); Myloperoxidase (MPO); N-Acetylcysteine (NAC); Tumor necrosis factor alpha (TNF-α).

Fig. 1. NAC abrogates LPS-mediated TNF-α release from PBMC of both AIED patients and controls

PBMCs isolated from AIED patients (N=12) and healthy control subjects (N=5) were treated with 1 μg/ml LPS, 5 mM NAC, 5 mM NAC + 1 μg/ml LPS or left untreated for 16 h. LPS-mediated TNF-α release was significantly reduced by NAC in AIED patients (p = 0.03, Mann–Whitney U test) compared to LPS alone. In case of control subjects, there was trend towards reduction of TNF release, however significance was not achieved (p=0.09). Error bars show ± SEM.

Fig. 2. AIED patients have higher plasma levels of MPO compared to control subjects

Plasma samples from AIED patients (N=50) and normal healthy (N=24) control subjects were tested by sandwich ELISA to compare their MPO levels. MPO plasma levels were significantly increased in AIED patients (p<0.0001) compared with controls by the Mann–Whitney U test with 95% CI. Error bars show ± SEM

Fig. 3. MPO is generated by both neutrophils and monocytes fractions of PBMC and NAC reduces TNF-α-mediated MPO release from PBMC of both AIED patients and controls

a. A sandwich ELISA was done on stimulated PBMCs isolated from AIED patients and healthy control subjects. PBMC from AIED patients were either treated with 10 ng/ml TNF-α (8 AIED patients) or 1 μg/ml LPS (16 AIED patients) +/− 5 mM NAC, and compared to untreated PBMC, or PBMC from control subjects (N=6) cultured under the same conditions for 16 h. NAC in combination with TNF-α was able to reduce MPO release when compared with TNF-α alone in PBMC from AIED patients and control subjects. Comparisons were made by one-way analysis of variance (ANOVA) p < 0.0001, followed by post hoc testing using a Bonferroni’s comparison test on selected columns. TNF-α induced MPO release compared with TNF-α + NAC achieved statistical significance p < 0.05 in AIED patients. b. Neutrophils (Neut), monocytes (Mono) and total PBMCs were isolated from an AIED patient and treated with LPS 1 μg/ml or (N=4) and healthy control subjects (N=3) were either treated with 10 ng/ml recombinant TNF-α or 5 mM NAC, co-cultured with 10 ng/ml recombinant TNF-α + 5 mM NAC, 1 μg/ml LPS, co-cultured with 1 μg/ml LPS + 5 mM NAC, or left untreated for 16 h. TNF-α and LPS were able to induce IL-8 release from PBMCs of AIED patients. Addition of NAC to either TNF-α or LPS cultures resulted in only a minimal reduction of IL-8 release.

Fig. 4

Fig. 5. IL-8 or MPO do not augment TNF-α release

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

Fig. 6. MPO IgG plasma levels were absent in AIED patients and control subjects

Plasma samples from 29 AIED patients and 10 normal healthy control subjects were tested by ELISA were compared with a positive control (MPO antibodies in serum/buffer) and a negative control (PBS, BSA detergent sodium azide 0.09%) to identify whether AIED patients made anti-MPO IgG antibodies. Anti-MPO IgG plasma levels were in the same range as of negative control for both AIED patients as well as control subjects. Error bars show ± SEM.

Fig. 7. AIED patients have marginally elevated TPO levels; however exhibit enhanced anti-TPO antibodies compared with age matched controls

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

Fig. 8

Schematic diagram illustrating the proposed signaling pathway.