NaCl exposure results in increased expression and processing of IL-1β in Meniere's disease patients

Abstract

Meniere's disease (MD) is a chronic disease that causes episodic vertigo, fluctuating hearing loss, and aural fullness, initially managed by dietary salt reduction, and use of diuretics. Our prior research in autoimmune inner ear disease (AIED) demonstrated that in peripheral blood mononuclear cell (PBMC) from corticosteroid-resistant AIED patients, increased production, processing and release of interleukin-1β (IL-1β) is observed and hearing could be improved with use of anakinra, an interleukin-1 receptor antagonist. We have further identified that in these AIED patients, IL-1β is uniquely processed to a 28 kDa pro-inflammatory product by caspase-7. In the present study, we characterize the production, processing and release of the pro-inflammatory cytokines IL-1β and IL-6 from PBMC of MD (n = 14) patients in response to sodium chloride (NaCl), and determined the effect of the diuretic triamterene-hydrocholothiazide (T-HCTZ), or anakinra in these patients. We observed that PBMC cultured with NaCl from MD patients show processing of IL-1β to the 28 kDa product, and that this product is abrogated with T-HCTZ. Our observations are consistent with other autoimmune diseases where high concentrations of NaCl caused release of pro-inflammatory cytokines and may provide further insight as to the mechanism of disease progression in MD patients.

Figure 1

(a) NaCl treatment results in dose-dependent increase in IL-1β mRNA levels in the PMBCs of MD patients. PBMCs from MD patients (n = 10) and control subjects (n = 10) were treated with NaCl at the concentrations of 20, 30, 40 and 80 mM, LPS as positive control and compared with no treatment. The IL-1β expression levels were measured by Q-RT–PCR. Statistical analysis by one way ANOVA resulted in p = 0.0005. Applying a Bonferroni’s multiple comparison test still indicated a significant effect (p < 0.05) for comparison between 80 mM NaCl treatment in controls vs. 80 mM NaCl treatment in MD patients. (b) NaCl treatment results in dose-dependent increase in IL-6 mRNA levels in the PMBCs of MD patients. PBMCs from MD patients (n = 10) and control subjects (n = 10) were treated with NaCl at the concentrations of 20, 30, 40 and 80 mM, LPS as positive control along with no treatment. The IL-6 expression levels were measured by Q-RT–PCR. Statistical analysis by one way ANOVA resulted in p = 0.0035. Applying a Bonferroni’s multiple comparison test still indicated a significant effect (p < 0.05) for comparison between 80 mM NaCl treatment in controls vs. 80 mM NaCl treatment in MD patients.

Figure 2

(a) NaCl treatment results in dose-dependent increase in IL-1β release in the PMBCs of MD patients. PBMCs from MD patients (n = 12) and control subjects (n = 12) were treated with either LPS (positive control), NaCl at the concentrations of 20, 30, 40 and 80 mM or left untreated. Supernatants were collected from the conditioned medium of MD patients and control subjects. IL-1β release was determined by ELISA. The bars show the mean protein release in pg/ml with ± SEM. Statistical analysis by one way ANOVA resulted in p = 0.0001. Applying a Bonferroni’s multiple comparison test still indicated a significant effect (p < 0.05) for comparison between 80 mM NaCl treatment in controls vs. 80 mM NaCl treatment in MD patients. (b) NaCl treatment results in dose-dependent increase in IL-6 release in the PMBCs of MD patients. PBMCs isolated from MD patients (n = 12) and control subjects (n = 12) were cultured over a 16 h period in the presence of increasing doses of sodium chloride, LPS (as positive control) or left untreated. Supernatants were collected from the conditioned medium. IL-6 release was determined by ELISA. The bars show the mean protein release in pg/ml with ± SEM. Statistical analysis by one-way ANOVA resulted in p = 0.0001. Applying a Bonferroni’s multiple comparison test still indicated a significant effect (p < 0.05) for comparison between 80 mM NaCl treatment in controls vs. 80 mM NaCl treatment in MD patients.

Figure 3

(a) NaCl treatment results increase in generation of 28 kDa IL-1β fragment in MD patients when compared to control subjects. PBMCs of control subject and MD patient were treated with NaCl at concentrations of 40 and 80 mM, LPS as positive control along with no treatment. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as internal control for immunoblotting. The representative blot for one of five MD patients and one of five control subjects are shown in the figure. (b) Potassium chloride failed to induce 28 kDa band of IL-1β in MD patients. PBMCs from MD patient were treated with Potassium chloride (KCl) (80 mM), NaCl (80 mM) along with LPS as positive control and kept untreated. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as a control for total protein.

Figure 4

(a) T-HCTZ inhibited NaCl induced 28 kDa band of IL-1β in a dose-dependent manner in MD patients. PBMCs from MD patient were treated with increasing concentration of T-HCTZ (10^–6 M to 10^–8 M) in combination with NaCl at concentration 80 mM, along with LPS as positive control and kept untreated. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as a control for total protein. The representative blots for two out of nine MD patients are shown in the figure. (b) NaCl failed to induce IL-1β in control subjects. PBMCs from healthy controls were treated with increasing concentration of T-HCTZ in combination with NaCl at concentration 80 mM, along with LPS as positive control and kept untreated. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as a control for total protein. The representative blots for two out of ten control subjects are shown in the figure. (c) NaCl failed to induce IL-1β in control subjects. The histogram shows the quantitative densitometry of 28 kDa protein of IL-1β (fold over control) normalized over actin expression from ten control subjects and nine different MD patients. The data is shown as mean ± SEM.). Statistical analysis by one-way ANOVA resulted in p = 0.0011. Applying a Bonferroni’s multiple comparison test still indicated a significant effect (p < 0.05) for comparison of T-HCTZ (10⁻⁶ M) and NaCl with NaCl alone.

Figure 5

(a) Anakinra inhibited NaCl induced 28 kDa band of IL-1β in MD patients. PBMCs from MD patients (n = 8) were treated with anakinra 100 ng/ml with or without 80 mM NaCl, along with LPS as positive control and kept untreated. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as a control for total protein. (b) NaCl failed to induce 28 kDa band of IL-1β in control subjects and therefore effect of anakinra on NaCl induction could not accessed. PBMCs from control subjects (n = 10) were treated with anakinra 100 ng/ml alone or in combination with NaCl at concentration 80 mM, 80 mM salt alone, LPS as positive control and kept untreated. The samples were subjected to Western blotting using IL-1β antibody. β-actin was used as a control for total protein. The figure represents one blot out of 10 blots. (c) The 28 kDa band of IL-1β/actin ratio from the densitometry analysis of the Western blot bands from ten control subjects and eight different MD patients. The data is shown as mean ± SEM.