Microbial Metabolite Depletion Drives Vocal Fold ECM Degradation via TLR4‑MMP Axis Independent of Central Nervous System

Hypothesis

Age‑related gut dysbiosis accelerates presbyphonia by reducing circulating short‑chain fatty acids (SCFAs) and increasing lipopolysaccharide (LPS) translocation, which directly activates TLR4 on vocal fold lamina propria fibroblasts, upregulating matrix metalloproteinases (MMPs) and degrading hyaluronan‑rich extracellular matrix. This peripheral mechanism operates independently of central vagal signaling, implying that microbiome‑targeted interventions can rescue vocal fold biomechanics even when neural pathways are compromised.

Mechanistic Rationale

• SCFA loss diminishes fibroblast homeostasis: Butyrate and propionate inhibit HDAC activity in fibroblasts, promoting expression of tissue‑inhibitor of metalloproteinases (TIMPs) and suppressing NF‑κB‑driven MMP transcription. Age‑associated dysbiosis lowers fecal butyrate by ~40 % (see [PMC10798364]), removing this brake on MMP synthesis.
• LPS‑TLR4 signaling amplifies proteolytic tone: Translocated LPS binds TLR4 on lamina propria fibroblasts, triggering MyD88‑dependent NF‑κB activation and upregulating MMP‑1, MMP‑3, and MMP‑9. Concurrently, LPS reduces fibroblast production of hyaluronan synthase 2, thinning the extracellular matrix ground substance.
• Vagal anti‑inflammatory pathway is secondary: While the vagus nerve can modulate inflammation via acetylcholine‑α7nAChR signaling, denervation studies show that SCFA supplementation still restores fibroblast TIMP/MMP balance, indicating a direct metabolite effect that does not require intact vagal tone.
• Feed‑forward loop: MMP‑generated matrix fragments act as damage‑associated molecular patterns (DAMPs) that further stimulate TLR4, creating a self‑reinforcing cycle of ECM breakdown and inflammation.

Testable Predictions

1. Prediction 1: In aged mice, fecal butyrate levels will negatively correlate with vocal fold MMP‑9 activity and positively correlate with hyaluronan content, irrespective of vagal integrity.
2. Prediction 2: Chronic low‑dose LPS infusion into young mice will recapitulate the aged vocal fold phenotype (increased MMPs, reduced hyaluronan, heightened phonation threshold pressure) even when the vagus nerve is intact.
3. Prediction 3: Oral butyrate supplementation in aged, dysbiotic mice will reduce vocal fold MMP activity and improve vibratory efficiency, an effect that persists after subdiaphragmatic vagotomy.
4. Prediction 4: Germ‑free mice colonized with microbiota from aged donors will develop vocal fold ECM degradation, whereas colonization with youth‑derived microbiota will protect against it.

Experimental Approach

• Animal groups: Young (3 mo) and aged (24 mo) mice; subsets receive vagotomy, butyrate‑enriched water (200 mM), LPS osmotic pumps (0.5 mg/kg/day), or fecal microbiota transplantation (FMT) from young or aged donors.
• Outcome measures: (a) Quantitative PCR and zymography for MMP‑1, −3, −9 and TIMP‑1 in dissected vocal folds; (b) ELISA for hyaluronan and LPS in lavage fluid; (c) Biophotonic imaging of vocal fold vibration ex vivo to derive fundamental frequency and phonation threshold pressure; (d) 16S rRNA sequencing to confirm dysbiosis and butyrate‑producing taxa abundance.
• Statistical plan: Two‑way ANOVA (age × treatment) with post‑hoc Tukey; correlation analysis between fecal butyrate and vocal fold MMP activity.

If butyrate rescue works despite vagal interruption, the hypothesis gains strong support that microbiome‑driven metabolic signaling, not central neural feedback, is a primary driver of age‑related vocal fold ECM decline.