Effect of betahistine on pro‐inflammatory cytokine expression in autoimmune inner ear disease and Meniere's disease patients

Abstract

Objective

Betahistine is a partial H1 receptor agonist and a potent H3 receptor antagonist commonly used for the treatment of MD and peripheral vertigo. The aim of this study was to investigate the impact of betahistine on the salt induced cytokine expression profiles of AIED and MD patients.

Methods

Peripheral blood mononuclear cells (PBMCs) were obtained from 24 patients with autoimmune inner ear disease (AIED) or Meniere's disease (MD) during an acute exacerbation of hearing loss. These PBMCs were cultured with 80 mM NaCl or a combination of 80 mM NaCl and betahistine and IL‐1β and IL‐6 expression were measured by real time PCR and ELISA.

Results

In most patients, IL‐1β expression in response to NaCl exceeded the unstimulated condition and this expression was abrogated by the addition of betahistine, which was statistically significant at p = .004. mRNA expression of IL‐1β was not reduced when samples were treated with both salt and betahistine compared to samples treated with salt alone, inferring the mechanism of betahistine‐mediated IL‐1β suppression is post‐translational. Similarly, IL‐6 cellular release was augmented with salt exposure and reduced with co‐culture of betahistine. Unlike IL‐1 however, betahistine appeared to reduce IL‐6 mRNA expression.

Conclusion

We observe that betahistine abrogates salt‐induced IL‐1β expression, suggesting an additional treatment option for AIED and MD patients with inflammatory mediated disease.

Level of evidence

Level 4.

The aim of this study was to investigate the impact of betahistine on the salt induced cytokine expression profiles of AIED and MD patients. PBMCs were obtained from 24 patients with AIED or MD during an acute exacerbation of hearing loss and these PBMCs were cultured with 80 mM NaCl or a combination of 80 mM NaCl and betahistine. In most patients, IL‐1β expression by ELISA in response to NaCl exceeded the unstimulated condition and this expression was abrogated by the addition of betahistine, suggesting the possibility of an additional treatment option for AIED and MD patients with inflammatory mediated disease.

1. INTRODUCTION

Both autoimmune inner ear disease (AIED) and Meniere's disease (MD) are characterized by progressive, fluctuating, and asymmetric sensorineural hearing loss (SNHL), tinnitus and vertigo. ¹ Patients with AIED exhibit vertigo in approximately 50% of episodes of hearing fluctuation, whereas MD patients are expected to manifest vertigo during all attacks. ² Nonetheless, treatment for both conditions may include corticosteroids, salt restriction, diuretics, betahistine and biologics. ³ Clinical responsiveness to these interventions is variable. For instance, corticosteroid responsiveness in the AIED population is known to decline to 14% after three years. ¹ The response rate to oral corticosteroids for hearing recovery in the MD population is cited at roughly 45%, ⁴ , ⁵ however, vertigo control through intratympanic steroid injection has been shown to be as high as 91% in one publication. ⁶ Previous works in our laboratory have aimed to characterize the cytokine expression profiles of these patients to guide future treatments.

We have previously published reports that peripheral blood mononuclear cells (PBMCs) from MD and corticosteroid resistant AIED patients demonstrate increased production, processing, and release of interleukin‐1 (IL‐1β). ⁷ MD patients uniquely process IL‐1β to a 28 kDa product which further induces interleukin‐6 (IL‐6) and other inflammatory cytokines in response to sodium salt. ⁸ We have also shown that this IL‐1β processing to the pro‐inflammatory 28 kDa product is inhibited by triamterene‐hydrochlorothiazide (T‐HCTZ). ⁷

The aim of this study was to investigate the impact of betahistine on the salt induced cytokine expression profiles of AIED and MD patients. Betahistine is a partial H1 receptor agonist and a potent H3 receptor antagonist that is widely used in Europe for the treatment of AIED and MD symptoms. ⁹ Benefits include high tolerance and a low side effect profile ¹⁰ ; however, it is not FDA approved in the United States and the mechanism by which betahistine provides symptomatic relief is not yet understood. We hypothesized that the addition of betahistine to PBMC's stimulated by salt would result in decreased expression of the pro‐inflammatory cytokines IL‐1β and IL‐6 in AIED and MD patients.

2. MATERIALS AND METHODS

These studies were approved by the Institutional Review Board at Northwell Health. All patients signed written consent for inclusion into these studies. To qualify for enrollment, all patients must have a clinical diagnosis of AIED or MD and have demonstrated a recent sensorineural decline in their hearing. PBMCs were obtained from these patients following an acute attack. The timing from acute attack to office visit was variable, but patients were excluded if their hearing returned to baseline at the time of their office visit. All patients had prior MRIs demonstrating an absence of retro‐cochlear pathology as the etiology of their hearing loss. The demographics of included patients are shown in Table 1.

TABLE 1.

Patient demographics.

Age/Gender	Dx	AI diagnosis	Vertigo	Migraine	Meds at recruitment	Steroid response	Audio pattern	BH Use	Positive antibodies	IL‐1 Exp	CRP/ESR	Steroid in last 30d
76M White	AIED	Ankylosing Spondylitis	Y	N	T‐HCTZ Azelastine Colchicine Naproxen Uloric Nexium	Y	AU: moderate SNHL, flat	N	Atypical ANCA indeterminate	N	WNL/WNL	N
31M White	MD	NO	Y	N	BH T‐HCTZ Prednisone Meclizine Benadryl Zyrtec	N	AD: wnl AS: severe mixed HL, upsloping	Y	N/A	N	WNL/DNT	N
45F White	AIED	Hashimoto	N	N	NAC	N	AD: wnl AS: moderate SNHL, upsloping	N	TPO	N	H/DNT	N
44F White	AIED	NO	N	N	Amitriptyline	N	AU: moderate SNHL, upsloping (downsloping after 4k)	N	ANA, TPO	N	WNL/WNL	Y
51F White	AIED/b/l MD	NO	Y	N	BH T‐HCTZ NAC Meclizine Diazepam Synthroid Lipitor Misoprostol Sertraline	N	AD: severe mixed HL, upsloping AS: moderate SNHL, upsloping	Y	TPO, TgAb	N	WNL/WNL	N
41F White	AIED	NO	N	N	BH Anakinra Prednisone Acetazolamide Clonazepam	Y	AU: moderately severe SNHL, upsloping	Y	N/A	N	WNL/WNL	Y
41F White	AIED	NO	N	N	Minastin	Y	AU: SNHL, downsloping	N	N/A	Y	WNL/H	Y
74F White	AIED	NO	Y	N	BH NAC Famotidine Synthyroid Plaquenil Zolpidem Albuterol	Y	AD: severe SNHL, up then downsloping AS: severe SNHL, upsloping	Y	TgAb	Y	WNL/WNL	N
69M White	AIED	SSc	N	N	Naltrexone	N	AD: mild to severe SNHL, downsloping AS: mild to moderate mixed HL, downsloping to severe SNHL	N	SCL70	Y	WNL/WNL	N
62M White	MD	NO	Y	N	HCTZ, Acetazolamide Alprazolam Ambien Lotrel Methocarbamol	N	AD: wnl AS: mild SNHL, upsloping	Y	N/A	Y	DNT/DNT	N
66M White	MD	NO	Y	Y	BH T‐HCTZ Amitriptyline Aspirin 81 Atorvastatin	Y	AD: wnl AS: moderate SNHL, flat	Y	N/A	Y	WNL/WNL	N
59F White	AIED/b/l MD	NO	Y	Y	T‐HCTZ Meclizine	N	AD: DNT AS: severe SNHL, upsloping	Y	N/A	Y	WNL/WNL	N
60F White	AIED/b/l MD	NO	Y	Y	Dyazide Acetazolamide Meclizine Neurontin	Y	AU: moderate SNHL, downsloping	Y	N/A	Y	WNL/DNT	N
69M White	AIED/b/l MD	NO	Y	Y	BH Anakinra Prednisone Meclizine Eliquis Proscar Tacrolimus Tamsulosin	N	AD: mild to severe SNHL, downsloping AS: moderately severe SNHL, downsloping	Y	N/A	Y	WNL/DNT	Y
58F White	MD	NO	Y	N	None	Not given	AU: moderate SNHL upsloping to normal, then downsloping to moderately severe	N	N/A	Y	WNL/H	N
70M White	MD	NO	Y	N	Dyazide	Y	AD: normal to moderately severe SNHL, downsloping AS: moderate SNHL upsloping to normal, then downsloping to severe	N	N/A	Y	DNT/DNT	N
57F White	AIED	Psoriasis	N	N	Humira	Y	AD: moderately severe SNHL, downsloping AS: moderate SNHL, upsloping	N	N/A	Y	WNL/WNL	N
48M White	AIED	NO	Y	N	T‐HCTZ	N	AD: moderate SNHL, upsloping AS: mild SNHL, upsloping to normal, then downsloping to moderately severe	N	N/A	Y	WNL/WNL	N
59M White	AIED	NO	Y	N	T‐HCTZ Anakinra Prednisone Lorazepam Alfuzosin Cialis Alprazolam Eszopiclone Simvastatin Pantoprazole	N	AU: severe SNHL, upsloping	N	N/A	Y	WNL/WNL	Y
61M White	AIED	NO	Y	N	T‐HCTZ Metoprolol Rosuvastatin Xarelto	N	AD: normal AS: moderately severe SNHL, upsloping	N	Gliadin IgA	Y	WNL/WNL	Y
65F White	MD	NO	Y	N	BH Meclizine Alprazolam Cyclobenzaprine Risedronate Zolpidem	Y	AD: moderately severe SNHL, upsloping AS: moderate SNHL, flat	Y	TPO Atypical ANCA indeterminate	Y	WNL/DNT	N
64M White	AIED	NO	Y	N	BH T‐HCTZ Prednisone Claritin	N	AU: moderate SNHL, downsloping	Y	ANA, TPO, Gliadin IgA/IgG	Y	WNL/DNT	Y
22M White	AIED	NO	N	N	None	N	AD: normal to severe CHL, downsloping AS: wnl	N	N/A	Y	WNL/WNL	N
71M White	AIED	NO	N	N	Eliquis Valsartan	Could not tolerate	AU: normal to severe SNHL, downsloping	N	N/A	Y	WNL/H	N

Abbreviations: ANCA, antineutrophil cytoplasmic antibody; ANA, antinuclear antibody; AIED, autoimmune inner ear disease; AI, autoimmune; BH, betahistine; CHL, conductive hearing loss; CRP, C‐reactive protein; DNT, did not test; Dx, diagnosis; ESR, erythrocyte sedimentation rate; T‐HCTZ, triamterene‐hydrochlorothiazide; H, high; IL‐1, interleukin‐1; MD, Meniere's disease; NAC, N‐acetyl‐cystine; N, no; SSc, scleroderma; SNHL, sensorineural hearing loss; TgAb, thyroglobulin antibody; TPO, thyroid peroxidase; WNL, within normal limits; Y, yes.

2.1. Preparation of human PBMCs and treatments

Fresh blood was collected from the patients with AIED and MD in sodium heparin tubes. PBMCs were isolated using Ficoll‐Paque PLUS (Cytiva) as per manufacturer's protocol. PBMCs were washed two times with RPMI 1640 (Thermo Fisher Scientific). Cell number was determined in a Z2 Coulter particle counter (Beckman Coulter, Brea, CA) then plated in 24‐well plate (Costar) at 1 × 10⁶/mL in an RPMI‐1640 medium (GIBCO) supplemented with fetal bovine serum (Atlanta Biologicals) L‐glutamine and penicillin/streptomycin (GIBCO) (complete medium). Cells were either stimulated with (endotoxin‐free cell‐culture grade) 80 mM NaCl (Sigma‐Aldrich) alone or in combination with 20 μM Betahistine (Sigma‐Aldrich). This concentration of betahistine was the optimal concentration at which inhibition was demonstrated without affecting cell viability. Lipopolysaccharide (LPS) (Sigma‐Aldrich) was used at 100 ng/mL as a positive control. For every treatment, cells were incubated for 16 h at 37°C/5% CO₂.

2.2. RNA isolation and quantitative real‐time PCR

Total RNA was extracted from PBMCs using RNA Mini easy kit (Qiagen). Purity and yield were determined by using NanoDrop ND‐1000 (Thermo Fisher Scientific). One‐step RT‐qPCR was performed using the iTaq One‐Step Kit as directed by the manufacturer instructions on ABI 7900HT Fast Real‐time PCR System (Applied Biosystems). C _t values were calculated by the RQ Manager software 1.2. GAPDH gene expression was used as the housekeeping gene, and data was presented as ΔΔCT relative to baseline. For each experiment there were at least two biological replicates. For GAPDH (Assay ID: Hs99999905_m1), and IL‐6 (Assay ID: Hs00985639_m1) gene expression, pre‐validated human TaqMan primer and probe sets were used. IL‐1β primer pair was designed by proprietary primer design algorithm (Universal Probe Library Assay Design Center). UPL probe number 78 was used with the primer set. Primers used to measure the RNA expression of IL‐1β were (forward primer) ctgtcctgcgtgttgaaaga (reverse primer) ttgggtaatttttgggatctaca.

2.3. ELISA

The amount of IL‐1β and IL‐6 in the conditioned media was determined by using a human IL‐1β and IL‐6 ELISA (Enzyme linked immunosorbent assay) kit (Quantikine; R&D Systems, Minneapolis, MN) as per manufacture's protocol. Data was analyzed using a logistic four‐parameter fit. For each condition at least two replicates were used.

2.4. Western blot

Protein contents were measured using a BCA protein assay kit (Pierce, Rockford, IL, USA) as per manufacturer's protocol. Twenty micrograms of lysate were loaded onto the gel. Samples were resolved on a 12% gel (Bio‐Rad), proteins were then transferred to a poly vinylidene fluoride (PVDF) membrane (Bio‐Rad). Blot was incubated overnight at 4°C using anti‐IL‐1β (MAB 201). Next day the blots were washed and incubated with the mouse horseradish peroxidase (HRP) conjugated secondary antibody (1:4000) for 1 h at room temperature. Immunoreactive proteins were detected using the Super Signal West Femto Substrate (Thermo Fisher Scientific). Blots were stripped and reprobed with β‐actin as a loading control. The relative band densities were determined using ImageJ software (NIH, Bethesda) then normalized to the density of each control.

2.5. Statistics

Descriptive statistics, including the mean, standard deviation, median, interquartile range, minimum, and maximum, were calculated for continuous variables in the overall cohort and stratified by treatment. Similarly, frequencies and percentages were tabulated for categorical variables in the overall cohort and stratified by treatment. A linear mixed model was utilized to determine whether there was a difference in the protein and mRNA levels of IL‐1β and IL‐6 among the three treatment groups. Log transformation of data was performed to meet the assumption of the analysis method. The results were back transformed into their original units for interpretation. All analyses were carried out using SAS V9.4 (SAS Institute, Cary, NC). A p‐value <.05 was considered statistically significant in the group effect of the linear mixed model. Bonferroni correction was performed to adjust for multiple comparisons in the pairwise comparison and a p‐value <.025 was considered statistically significant.

3. RESULTS

Between January 4, 2021 and January 18, 2023, 24 patients were enrolled. Demographic information for all patients is shown in Table 1. The median age among the entire group was 60.5 years old (range 22–76). 54.1% of patients were male and 45.8% were female. 29.2% of patients were taking betahistine 16 mg TID at the time of recruitment whereas 16.6% of patients had a history of prior betahistine use but were no longer using it at the time of recruitment. Vertigo was present in 66.7% of patients. 29% of patients had been treated with prednisone within 30 days of enrollment. PBMCs were obtained from patients following an acute exacerbation of hearing loss. These PBMC were cultured with 80 mM NaCl or in combination with 80 mM NaCl and betahistine.

Expression of IL‐1β in response to NaCl was variable, but in most patients, exceeded the unstimulated condition (p < .05). Figure 1 shows IL‐1β release in all samples. When treated with 80 mM NaCl, there was considerable induction of IL‐1β expression and this expression was abrogated by the addition of betahistine, which was statistically significant at p = .004 by a linear mixed model. A small subset of patients (n = 6) did not show any expression of IL‐1β when treated with salt. As a positive control, all samples showed strong induction of IL‐1β with LPS which was several orders of magnitude higher than induction with salt, discounting a failed reaction to account for the lack of IL‐1β expression. Among the six patients who did not express of IL‐1β in response to salt, four had a diagnosis of AIED, one bilateral MD/AIED, and one classical unilateral MD, and 50% were on betahistine at the time of recruitment.

FIGURE 1.

Release of IL‐1β from PBMCs as measured by ELISA. PBMCs (1 × 10⁶ cells/mL) from 20 patients were either treated with NaCl at the concentration of 80 mM, or in combination with 20 μM betahistine (NaCl + BH), LPS as positive control (not shown) and left untreated for 16 h. IL‐1β release was determined by ELISA from the culture supernatants. Data represents the mean ± SEM in all panels.

Figure 2 shows that mRNA expression of IL‐1β was not reduced when samples were treated with both salt and betahistine compared to samples treated with salt alone, inferring the mechanism of betahistine‐mediated IL‐1β suppression is post‐translational. Western blotting for IL‐1β was performed on a subset of these samples (7 IL‐1β expressers and 4 non‐expressers). Densitometry analysis of western blot bands plotting the ratio of IL‐1β to actin is shown in Figures 3 and 4. Notably, the addition of betahistine exerted an inhibitory effect not only on the 28 kDa IL‐1 band but on total IL‐1, as measured by the reduction of the 31 kDa band in Figure 3. Consistent with ELISA data, the IL‐1β expressers made the 28 kDa IL‐1 band in response to NaCl, which was reduced with co‐culture with betahistine in all but one patient. Interestingly, in this one patient, betahistine still reduced IL‐1 release by ELISA. Also consistent with ELISA data, those patients that did not release IL‐1β from their PBMC similarly had no detectable IL‐1β by western blotting.

FIGURE 2.

MRNA expression of IL‐1β in PBMCs. mRNA expression of IL‐1β in PBMCs from 18 of 20 subjects evaluated in Figure 1A was measured in NaCl, NaCl + BH and compared to the unstimulated condition by Q‐RT‐PCR. Fold change is shown relative to the unstimulated condition. Error bars show ±SEM.

FIGURE 3.

The 31 kDa band of IL‐1β/actin ratio from the densitometry analysis of the Western blot bands from IL‐1β expressers patients (N = 7). As anticipated, the IL‐1β expressers made the 31 kDa IL‐1β band when stimulated with NaCl, which was reduced by more than half with the addition of BH. The data is shown as mean ± SEM.

FIGURE 4.

The 28 kDa band of IL‐1β/actin ratio from the densitometry analysis of the Western blot bands from IL‐1β expressers patients (N = 7). Similarly, as anticipated, the IL‐1β expressers made the 28 kDa IL‐1β band when stimulated with NaCl, which was reduced by more than half with the addition of BH. The data is shown as mean ± SEM.

Similarly to IL‐1, NaCl also induced IL‐6 release from PBMCs of patients. Release was variable between patients. Figure 5 shows that IL‐6 release was augmented with salt exposure and reduced to co‐culture of betahistine. Unlike IL‐1 however, betahistine appeared to reduce IL‐6 mRNA expression (Figure 6). Finally, those who did not express IL‐1β similarly did not express IL‐6 (Figure 7).

FIGURE 5.

Release of IL‐6 from PBMC as measured by ELISA. PBMC from 18 patients were harvested and stimulated for 16 h with either NaCl, NaCl + BH and compared to unstimulated PBMC. IL‐6 release was determined by ELISA from the culture supernatants. Data represents the mean ± SEM in all panels.

FIGURE 6.

MRNA expression of IL‐6 in PBMCs. mRNA expression of IL‐6 in PBMC from 17 of 20 subjects evaluated in Figure 1A was measured in NaCl, NaCl + BH and compared to the unstimulated condition by Q‐RT‐PCR. Fold change is shown relative to the unstimulated condition. Error bars show ±SEM.

FIGURE 7.

Low IL‐1β expressers were also low IL‐6 expressers. Patients were segregated into two groups based on the detectable levels of IL‐1β. Groups with undetectable IL‐1β levels were low IL‐6 expressers.

Although we did not thoroughly interrogate histamine receptor expression as the mechanism for betahistine‐mediated reduction of IL‐1, we did assay expression of H1 receptors by Q‐RT‐PCR. Expression was present in PBMC from all samples and did not appear to vary in response to treatment with NaCl or NaCl + betahistine (not shown).

4. DISCUSSION

We have previously demonstrated that exposure of PBMC to NaCl from MD patients results in IL‐1β expression. ⁷ Similar to our previously reported findings, NaCl exposure results in variable induction of IL‐1. Furthermore, co‐culture with betahistine reduced that expression by both western blotting and ELISA, but not quantitative real time PCR, suggesting the mechanism of inhibition is post‐translational. Interestingly, a cohort of patients had no IL‐1 expression (Figure 7). Notably, the lack of expression could not be correlated with diagnosis, presence of vertigo, presence of migraine, medications taken or clinical serologies obtained. Furthermore, oral steroids used within 30 days prior to recruitment similarly did not correlate with lack of IL‐1 expression. It is likely that two different mechanisms resulting in similar clinical phenotypes may be present. In support of this, other investigators have identified that a subset of Meniere's patients have higher basal levels of inflammatory cytokines than others with 89 having low basal levels and 24 demonstrating high basal levels. ¹¹ This may explain why a subset of patients may not respond to immunosuppressive drugs including corticosteroids where others derive significant benefit from these interventions.

It is not surprising that betahistine exhibited anti‐inflammatory properties. Betahistine has been shown to attenuate joint inflammation, including IL‐6 and TNF‐a expression in a collagen‐induced arthritis mouse model. ¹² The authors did not examine IL‐1 expression in this model. Interestingly, although the H1 receptor is responsible for most of the pro‐inflammatory effect of histamine, ¹³ we did not observe any variation of H1 expression in response to salt in PBMC from these patients.

Expression of IL‐6 was variably observed at both the mRNA level and the protein level in response to NaCl. This was expected, as IL‐1 expression usually results in increased expression of IL‐6, as we have previously observed. ⁷ We have previously demonstrated elevated IL‐6 levels in patients with immune mediated SNHL, a positive correlation with CRP levels. ¹⁴ In hypertensive cerebral vasospasm, elevated CRP and IL‐6 levels have been observed. Notably, treatment with a combination of betahistine and nimodipine was shown to reduce these inflammatory responses. ¹⁵

Given the rarity of these disease processes, this study is limited by a small sample size. Furthermore, the study is additionally limited by the variability in timing between a patient's acute attack and office visit.

5. CONCLUSION

We have observed that betahistine abrogates salt‐induced IL‐1 expression in AIED and MD patients. Our data suggests this mechanism of betahistine‐mediated IL‐1β suppression is post‐translational as mRNA expression of IL‐1β was not reduced when samples were treated with both salt and betahistine compared to samples treated with salt alone. These observations may offer an additional treatment option for AIED and MD patients with inflammatory mediated disease.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Yellin I, Pathak S, Vambutas A. Effect of betahistine on pro‐inflammatory cytokine expression in autoimmune inner ear disease and Meniere's disease patients. Laryngoscope Investigative Otolaryngology. 2024;9(6):e70032. doi: 10.1002/lio2.70032