Intestinal inflammation and microbiota modulation impact cochlear function: emerging insights in gut-ear axis

Abstract

Background: Although several evidence demonstrates a "gut-microbiota-brain axis", suggesting a bidirectional communication between gut microbiota and the central nervous system, less is known about a possible link between the gut and the peripheral nervous system, including the inner ear.

Methods: Here, we investigated the impact of intestinal inflammation and the modulation of gut microbiota through fecal microbiota transplantation on hearing sensitivity. Female C57BL/6 mice were assigned to four groups: control (Ctrl), DSS-induced colitis (DSS), FMT from patients with active ulcerative colitis (FMT aUC), and FMT from patients with ulcerative colitis in remission (FMT rUC). Auditory function was evaluated by auditory brainstem responses (ABR). Morphological and molecular analyses on cochlear tissues were performed using immunofluorescence, histological staining, and Western blot to assess inflammation, oxidative stress, and blood-labyrinth barrier integrity. Donor microbiota composition was characterized by 16S rRNA sequencing, and systemic inflammation was evaluated by measuring serum lipopolysaccharide (LPS) levels.

Results: We found that intestinal dysbiosis is associated with functional, morphological, and molecular alterations in the cochlea, such as increased oxidative stress, inflammation, and altered blood-labyrinth barrier permeability. This leads to macrophage infiltration and immune response activation through the MyD88/NF-κB pathway. Notably, these effects were exacerbated by FMT from subjects with aUC, while FMT from patients with rUC provided a protective effect on cochlear functions.

Conclusions: Overall, our findings suggest that gut inflammation, microbiota alteration, or its therapeutic modulation can impact inner ear pathology: worsening gut inflammatory status negatively affects hearing sensitivity, while the restoration of gut microbiota positively impacts auditory function.

Keywords: Cochlea; Gut dysbiosis; Gut microbiota; Inflammation.

Fig. 1

Experimental design. C57BL/6 mice of 2 months of age at the beginning of the study were randomly assigned to “Ctrl”, “DSS”, “FMT rUC” and “FMT aUC” groups. Baseline hearing thresholds were evaluated the day before (D0) the beginning of treatments. Functional (ABR), morphological (immunohistochemistry) and molecular evaluations (Western Blot and immunofluorescence) were performed at the end of treatments. ABR: Auditory Brainstem Responses; FMT: Fecal Microbiota Transplantation. Created by using Biorender Academic Lab License

Fig. 2

Characterization of intestinal inflammation. A: Average Disease Activity Index (D.A.I.) on day 15, calculated by assessing weight, fecal consistency, body weight and the presence of occult fecal blood. B: Average LPS serum levels in “DSS”, “FMT rUC” and “FMT aUC” groups. LPS: Lipopolysaccharide. C: Concentration of pro-inflammatory cytokines in intestinal mucosa on day 15. D: Histological analysis was performed according to Rachmilewitz score. E: Stereological analysis of cell density in both DSS tissue vs. control, from 5 different colon mice. Cell density was determined by dividing cell counts by the area of the ROI. Bars show the mean of cell counts ± standard deviation (left panel). H/E staining of colon tissues from control mice (Ctrl) or treated with DSS (DSS). Magnification 40× (right panel) *p = 0.05;**p < 0.01; ***p < 0.001

Fig. 3

Ecological analysis of the gut microbiota of the two donors. A: α-diversity index (Shannnon Weiner index) of the two donors: one with active UC (aUC) and the other with UC in remission (rUC). B: Heatmap of the relative abundance of the major phyla of gut microbiota from the donor aUC and the donor rUC. C: Heatmap of the relative abundance of the major genera of gut microbiota from the donor aUC and the donor rUC

Fig. 4

Functional analysis and morphological evaluations in the organ of Corti: hair cell loss. A, B: Graphs showing ABR threshold values (means ± SEM) across all frequencies analyzed. A: Treatment with ABX alone does not significantly affect auditory thresholds compared to the Ctrl group. B: DSS treatment leads to a slight but significant increase of threshold values of approximately 15 dB in the mid-high frequencies (20–32 kHz) compared to the Ctrl group. FMT aUC further increases the auditory threshold of about 20–25 dB, in almost all frequencies analysed, while the FMT rUC group shows a recovery of hearing function. Symbols indicate significant differences among groups ($#*p < 0.05; $$** p < 0.01; ***p < 0.001). C-F: Representative images of surface preparations of the organ of Corti in the middle cochlear turn, hair cell distribution labeled with F-actin (green fluorescence). G: Cochleograms (means ± SEM) showing the percentage of outer hair cell (OHC) survival in all cochlear turns. Scale bar: 25 μm. Asterisks indicate significant differences among groups (*p < 0.05; ** p < 0.01; ***p < 0.001)

Fig. 5

Cochlear oxidative stress and redox imbalance associated with gut dysbiosis. A-D: Representative images of cochlear cryosections stained with DHE (red fluorescence) to detect ROS amount. Scale bar: 100 μm. E-G: Representative dot blot images showing 3-NT (E), 4-HNE (F) and GSH (G) levels in all experimental groups. I-K: Histograms (means ± SEM) showing the results of the densitometric analyses normalized to Ponceau S. H, L: Representative images of iNOS western blot bands (H) with the results (means ± SEM) of the densitometric analysis (L). M-T: Representative images of cochlear cryosections showing iNOS (M-P, green fluorescence) and GSH (Q-T, red fluorescence) expression in the principal cochlear structures, in all experimental groups. DAPI staining indicates cell nuclei. Scale bar: 100 μm. Asterisks indicate significant differences among groups (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 6

Cochlear damage associated with gut microbiota alteration involves inflammation through MyD88/NF-kB pathway. A, B: Representative western blot images showing the expression of NF-κB, MyD88 (A) and IL-1β (B) in all experimental groups. C-E: Bar graphs showing the results of densitometric analyses for NF-κB (C), MyD88 (D) and IL-1β (E), normalized to total protein level (GAPDH). F: Schematic representation of MyD88/NF-κB/ IL-1 pathway. Created by using Biorender Academic Lab License. G-N: Representative images of cochlear cryosections showing NF-B (G-J, red fluorescence) and IL-1β (K-N, green fluorescence) expression in the principal cochlear structures, in all experimental groups. DAPI staining indicates cell nuclei. Scale bar: 100 μm. Data are expressed as mean ± SEM. Asterisks indicate significant differences among groups (*p < 0.05; ** p < 0.01; *** p < 0.001)

Fig. 7

Cochlear damage associated with gut microbiota alteration involves macrophage/lymphocyte infiltration. A-D: Representative images of freshly explanted stria vascularis marked with IBA-1 (green fluorescence), desmin (red fluorescence) and stained with DAPI (blue fluorescence) to identify cell nuclei. Arrows indicate macrophages surrounding blood vessels E: Scheme illustrating perivascular macrophages (PVM) and pericytes around blood vessels. F: Representative confocal image (60×) of a PVM (green fluorescence) in the FMT aUC group. Scale bar: 25 μm. G: Histograms showing the number of IBA-1 positive cells in stria vascularis whole mount. H: Representative western blot images showing the expression of CD45 in all experimental groups. I: Bar graphs showing the results of densitometric analyses for CD45, normalized to total protein level (GAPDH). Data are expressed as mean ± SEM. Asterisks indicate significant differences among groups (*p < 0.05; ** p < 0.01; *** p < 0.001)

Fig. 8

Gut microbiota state affects tight junctions and vascular integrity of stria vascularis. A-D: Representative western blot images showing ZO-1 (A) and occludin (B) levels in all experimental groups. C-D: Bar graphs representing results of densitometric analyses normalized to GAPDH. Data are expressed as mean ± SEM. E-H: Representative images of stria vascularis whole mount marked with occludin (green fluorescence) and stained with DAPI (blue fluorescence) to identify cell nuclei. Scale bar: 10 μm. I-L: Representative images of cochlear cryosections showing stria vascularis labeled with Na^+/K⁺-ATPase (green fluorescence) and DAPI (blue fluorescence). Scale bar: 25 μm. M: Representative western blot images of Na⁺/K⁺-ATPase level in all experimental groups. N: Bar graphs (mean ± SEM) representing the densitometric analyses normalized to total protein level (GAPDH). Asterisks indicate significant differences among groups (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 9

Cochlear vascular dysfunction and blood-labyrinth barrier increased permeability. A-D: Representative images of freshly explanted stria vascularis marked with desmin (green fluorescence) to visualize pericytes and stained with DAPI (blue fluorescence) to identify cell nuclei. E-H: Representative images of stria vascularis whole mount stained with Alexa Fluor 546 mouse IgG to visualize the capillary network. Arrows in F and H indicate mouse immunoglobulin fluorescence out of the vessels. Scale bar: 25 μm. I, J: Representative western blot images showing desmin expression level (I) and densitometric analysis (J) with respect to total protein level (GAPDH). Asterisks indicate significant differences among groups (*p < 0.05; **p < 0.01; ***p < 0.001)