A pilot study on the effect of SARS-CoV-2 spike protein on IL-1β-mediated inflammation in peripheral blood immune cells from AIED patients

Abstract

Background: Immune-mediated hearing loss (IMHL) patients (comprised of autoimmune inner ear disease (AIED) and sudden sensorineural hearing loss (SSNHL)) may be at higher risk for hearing loss following Coronavirus disease (COVID-19) infection and/or vaccination.

Methods: We compared inflammatory cytokine expression in response to SARS-CoV2 spike protein between two groups of patients with IMHL: IMHL patients that temporally demonstrated worsening SNHL following COVID vaccination or infection as compared to IMHL patients with worsening SNHL unrelated to COVID exposure: (IMHL-COVID ( +)) (n = 11) (IMHL-COVID (-)) (n = 10). In these two groups, we treated isolated PBMCs with increasing amounts of SARS-CoV-2 spike protein and compared responses to stimulation with positive and negative controls.

Results: Peripheral Blood Mononuclear Cells (PBMC) from IMHL-COVID ( +) patients had increased expression and release of both IL-1β and IL-6 in response to spike protein as compared to IMHL-COVID (-) patients. However, when the IMHL-COVID ( +) group was broken down into AIED patients compared to SSNHL, it became apparent that the greatest responses were from the AIED patients (p < 0.005 for IL-6 mRNA expression and p < 0.003 for IL-6 release when compared between any two similar groups using Wilcoxon Rank-Sum Test). When we broke down the COVID ( +) group to infection versus vaccination, the immune responses in the infection group (N = 3 AIED, 1 SSNHL) were stronger.

Conclusions: COVID-19 exposure with reported changes in hearing sensitivity in IMHL patients resulted in pro-inflammatory responses in response to spike protein. The inflammatory responses were greatest in AIED patients, and greater following infection rather than vaccination. Therefore, based on these studies, we would recommend AIED patients take additional precautions to avoid COVID exposure. Furthermore, we do recommend COVID vaccination during periods of hearing stability, as the immune responses are even more robust in response to infection in this vulnerable group.

Fig. 1

SARS-CoV-2 spike protein treatment resulted in stronger induction in IL-1β and IL-6 mRNA levels in the PBMCs of IMHL-COVID (+) group. PBMCs from IMHL-COVID (-) patients (n = 9) and IMHL-COVID (+) patients (n = 11) were treated with spike proteins at the concentrations of 12 μg/ml, 12 ng/ml, 12 pg/ml, MBP as nonspecific protein, LPS as positive control and compared with no treatment. The expression levels were analyzed by Q-RT–PCR. IL-1β—SARS-CoV-2 spike protein treatment resulted in mild increase in IL-1β mRNA levels in the PBMCs of both groups with slightly higher in IMHL-COVID (+) group. IL-6—SARS-CoV-2 spike protein treatment results in IL-6 mRNA induction in both groups and was more robust in IMHL-COVID (+) patients compared to IMHL-COVID (-) patients. IL-1Ra—SARS-CoV-2 spike protein treatment results in mild induction in IL-1Ra mRNA levels in the PBMCs of both groups

Fig. 2

SARS-CoV-2 spike protein treatment resulted in IL-1β and IL-6 release in the PBMCs of IMHL-COVID (+) group. PBMCs from IMHL-COVID (-) patients (n = 10) and IMHL-COVID (+) patients (n = 11) were treated with spike proteins at the concentrations of 12 μg/ml, 12 ng/ml, 12 pg/ml, MBP as nonspecific protein, LPS as positive control and compared with no treatment. Levels of IL-1β/IL-6/IL-1Ra in the conditioned supernatant were determined by sandwich ELISA. IL-1β—SARS-CoV-2 spike protein treatment resulted in IL-1β release in both groups with slightly higher in IMHL-COVID (+) group. IL-6—SARS-CoV-2 spike protein induces greater levels of IL-6 release in IMHL-COVID (+) patients compared to IMHL-COVID (-) patients and was more robust in IMHL-COVID (+) patients compared to IMHL-COVID (-) patients. IL-1Ra—SARS-CoV-2 spike protein induces IL-1Ra release in the PBMCs of both groups in a dose dependent manner. IMHL-COVID (-) patients showed slightly higher levels compared to IMHL-COVID (+) patients

Fig. 3

IMHL-COVID (+) patients (n = 11) patients were segregated into two groups AIED-COVID (+) (n = 6) and SSNHL-COVID (+) (n = 5) based on previous hearing decline unrelated to COVID-19. IL-1β—SARS-CoV-2 spike protein at 12 μg/ml concentration showed mild induction IL-1β mRNA expression in both groups of IMHL-COVID (+) patients with slightly higher levels in AIED-COVID (+).IL-6—SARS-CoV-2 spike protein at 12 μg/ml concentration showed robust induction of IL-6 mRNA expression in AIED-COVID (+) group (n = 6) when compared with SSNHL-COVID (+) patient group (n = 5). Wilcoxon rank sum test with significant P values (< 0.005) shown. IL-1Ra—SARS-CoV-2 spike protein at 12 μg/ml concentration showed mild induction IL-1Ra mRNA expression in both groups of COVID (+) IMHL patients. AIED-COVID (+) group (n = 6) showed almost twofold higher induction with spike protein at 12 μg/ml when compared with SSNHL-COVID (+) group (n = 5)

Fig. 4

IMHL-COVID (+) patients (n = 11) patients were segregated into two groups AIED-COVID (+) and SSNHL-COVID (+) based on previous hearing decline unrelated to COVID-19. IL-1β—SARS-CoV-2 spike protein treatment resulted in moderate increase in IL-1β release in AIED-COVID (+) group whereas almost no induction in SSNHL-COVID (+) group. IL-6—SARS CoV2 spike protein showed robust induction of IL-6 release in AIED-COVID (+) group compared to the SSNHL-COVID (+) group. Wilcoxon's Rank Sum was used to compare between the two groups indicated a significant effect (p < 0.003) for the comparison between untreated, LPS, 12 μg/ml, 12 ng/ml and 12 pg/ml treatments in both groups. IL-1Ra- SARS-CoV-2 spike protein at 12 μg/ml concentration showed mild induction IL-1Ra mRNA expression in both groups of COVID (+) IMHL patients. AIED-COVID (+) group (n = 6) showed almost sevenfold higher induction with spike protein at 12 μg/ml when compared with SSNHL-COVID (+) group (n = 5)

Fig. 5

IMHL-COVID (+) patients (n = 11) patients were segregated into two groups (vaccination (n = 7) and infection (n = 4)) based on their hearing loss status due to COVID-19 vaccine administration or COVID-19 infection. IL-1β—SARS-CoV-2 spike protein at 12 μg/ml concentration showed mild induction of IL-1β mRNA expression in both groups of IMHL-COVID (+) patients with slightly higher levels in the infection group compared to vaccination group. IL-6—SARS-CoV-2 spike protein at 12 μg/ml concentration showed robust induction of IL-6 mRNA expression in infection group (n = 4), when compared with the vaccination group (n = 7). IL-1Ra-SARS-CoV-2 spike protein at 12 μg/ml concentration showed mild induction in both groups

Fig. 6

IMHL-COVID (+) patients (n = 11) patients were segregated into two groups (vaccination (n = 7) and infection (n = 4)) based on their hearing loss status due to COVID-19 vaccine administration or COVID-19 infection. IL-1β—SARS-CoV-2 spike protein at 12 μg/ml concentration showed strong induction of IL-1β release in infection group (n = 4) group, more than fourfold when compared to vaccination group (n = 7). IL-6—SARS-CoV-2 spike protein at 12 μg/ml concentration showed robust release of IL-6 in infection group (n = 4), more than sixfold when compared to vaccination group (n = 7). IL-1Ra- SARS-CoV-2 spike protein showed dose dependent release of IL-1Ra in both groups. Spike protein at 12 μg/ml concentration showed robust release of IL-1Ra in infection group (n = 4) when compared to vaccination group (n = 7)