Regulation of Fn14 Receptor and NF-κB Underlies Inflammation in Meniere's Disease

Abstract

Meniere's disease (MD) is a rare disorder characterized by episodic vertigo, sensorineural hearing loss, tinnitus, and aural fullness. It is associated with a fluid imbalance between the secretion of endolymph in the cochlear duct and its reabsorption into the subarachnoid space, leading to an accumulation of endolymph in the inner ear. Epidemiological evidence, including familial aggregation, indicates a genetic contribution and a consistent association with autoimmune diseases (AD). We conducted a case-control study in two phases using an immune genotyping array in a total of 420 patients with bilateral MD and 1,630 controls. We have identified the first locus, at 6p21.33, suggesting an association with bilateral MD [meta-analysis leading signal rs4947296, OR = 2.089 (1.661-2.627); p = 1.39 × 10^-09]. Gene expression profiles of homozygous genotype-selected peripheral blood mononuclear cells (PBMCs) demonstrated that this region is a trans-expression quantitative trait locus (eQTL) in PBMCs. Signaling analysis predicted several tumor necrosis factor-related pathways, the TWEAK/Fn14 pathway being the top candidate (p = 2.42 × 10^-11). This pathway is involved in the modulation of inflammation in several human AD, including multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. In vitro studies with genotype-selected lymphoblastoid cells from patients with MD suggest that this trans-eQTL may regulate cellular proliferation in lymphoid cells through the TWEAK/Fn14 pathway by increasing the translation of NF-κB. Taken together; these findings suggest that the carriers of the risk genotype may develop an NF-κB-mediated inflammatory response in MD.

Keywords: Meniere’s disease; NF-κB signaling; NFKB1; TNFRSF12A; TWEAK/Fn14 pathway; sensorineural hearing loss; vertigo.

Figure 1

Two loci associated with bilateral sensorineural hearing loss (SNHL). (A) Manhattan plot for association study findings from Immunochip genotyped bilateral cases and controls. (B) Association area at the region on chromosome 2 and (C) association area at the region on chromosome 6. Both (B,C) −logP values of single nucleotide variants (SNVs) associated with bilateral SNHL are shown on the left y-axis and the recombination rates expressed in centimorgans (cM) per Mb, are shown on the right y-axis. Positions in Mb are on the x-axis (NCBI Build GRCh38). Linkage disequilibrium for each SNV with the top SNV, displayed as a large purple diamond, is indicated by its color. The plots were drawn using LocusZoom tool (http://locuszoom.sph.umich.edu/locuszoom/).

Figure 2

Gene expression in peripheral blood mononuclear cells. (A) Heatmap of 973 differentially expressed genes (DEG). Samples and genes (columns and rows, respectively) are reordered on the basis of the normalized expression value and give rise to groups of genes and samples with similar expression levels, according to the color key. The samples (column) were clustered into two groups according to rs4947296: three individuals with CC genotype (risk) and seven individuals with TT genotype (protective). (B) The shortest path from Fn14 (TNFRSF12A) to NFKB genes. DEG in mononuclear cells (adjusted p < 0.001), according to the homozygous genotype, were used to predict involved pathways. The network was retrieved from three MetaCore pathways [“apoptosis and survival Apoptotic tumor necrosis factor (TNF)-family pathways,” “signal transduction NF-κB activation pathways,” and “apoptosis and survival Anti-apoptotic TNFs-NF-κB-Bcl-2 pathway”] enriched in our pathway enrichment analysis. Log fold change is color-coded, where red nodes indicate upregulated genes, whereas blue nodes indicate downregulated genes. Activation interactions are indicated by arrow heads, whereas inhibitory interactions are indicated by blunted heads. Black edges indicate physical binding interaction, purple edges indicate phosphorylation, and brown edges indicate ubiquitination. Genes with thick purple margin are DEG. (C) Quantitative RT-PCR validation of genes involved in the TWEAK/Fn14 pathway (NFKB1, Fn14, BIRC3, FADD, NFKBIE, FOS, APAF1, CASP3, CASP6, CASP9, CYCS, and IKBKG) (*p < 0.03, **p < 0.0005).

Figure 3

Genotype-conditioned lymphoblastoid cell lines (LCL) for rs4947296. (A) Proliferation assay of homozygous LCL treated with tumor necrosis factor-like weak inducer of apoptosis (TWEAK; 0, 50, 100, 250, and 500 ng/mL) and measured using PrestoBlue™ (*p < 0.05). Comparisons between groups were achieved using a two-sided Student’s t-test. (B) Relative gene expression according to the genotype, after treatment with 250 ng/mL of TWEAK, p-values: TNFRSF12A = 0.44; NFKB1 = 1.36 × 10⁻⁴; ICAM = 0.91; LFA-1 = 5 × 10⁻⁶; TJP1 = 3.2 × 10⁻⁵ (*p < 1 × 10⁻⁴, **p < 5 × 10⁻⁶) (C) Cluster size within LCLs with and without stimulation (**p = 5 × 10⁻⁷, *p = 0.02). CC, risk genotype; TT, protective genotype.

Figure 4

NF-κB transcription factor protein studies in genotype-conditioned lymphoblastoid cell lines (LCLs). (A) Total NF-κB ELISA performed in homozygous LCL for rs4947296 at basal levels and after stimulation with TWEAK at 250 ng/mL (*p < 0.02, *p < 0.006). (B) Phosphorylation assay for NF-κB p65 on serine 536 in homozygous LCL at basal levels and after stimulation with TWEAK at 250 ng/mL. (C) Representative NF-κB western blot of homozygous LCL before and after treatment with 250 ng/mL of TWEAK. (D) NF-κB western blot showing significant differences between both genotype cells at basal levels as well as differences within the protective genotype cells before and after stimulation (*p < 0.05). CC, risk genotype; TT, protective genotype.

Figure 5

Fn14 and NF-κB expression in homozygous lymphoblastoid cell lines (LCLs). Confocal microscopy images showing representative clusters of LCLs with Fn14 and NF-κB immunolabeling after treatment with 250 ng/mL of TWEAK. CC, risk genotype; TT, protective genotype.

Figure 6

Inflammation model in Meniere’s disease (MD). (A) TWEAK/Fn14 pathway activates non-canonical NF-κB signaling in lymphoid cells in MD. (B) Potential sites of inflammatory damage are the blood–brain barrier (BBB), the endolymphatic sac, the spiral ligament, and the reticular lamina in the neurosensory epithelium of the cochlea.