Hypothesis

Combining urolithin A (UA) with a microbiome‑derived butyrate precursor produces a synergistic increase in mitophagy in human CD8+ T cells that exceeds the effect of either compound alone, by linking UA‑triggered ER calcium release to butyrate‑mediated inhibition of HDAC3, thereby amplifying PINK1/Parkin‑dependent mitochondrial clearance and reducing senescent phenotypes.

Mechanistic Rationale

UA induces ER calcium release via ITR‑1/ITPR, driving mitochondrial calcium uptake through MCU‑1/MCU and activating DRP‑1‑mediated fission, which primes damaged mitochondria for PINK1/PDR‑1‑dependent mitophagy[1]. Butyrate, a short‑chain fatty acid generated by colonic fermentation of fiber, is a potent class I HDAC inhibitor; it specifically suppresses HDAC3 activity, leading to hyperacetylation of mitochondrial proteins and increased susceptibility to autophagic targeting[5]. We propose that butyrate‑mediated HDAC3 inhibition lowers the threshold for PINK1 stabilization on depolarized mitochondria, while UA‑driven calcium flux provides the activating signal for PINK1 recruitment. The convergence of these two signals—calcium‑dependent priming and acetylation‑dependent sensitization—creates a feed‑forward loop that accelerates mitophagic flux beyond additive expectations.

Testable Predictions

1. In primary human CD8+ T cells, co‑treatment with UA (10 µM) and sodium butyrate (1 mM) will increase mitochondrial clearance (measured by mt‑Keima assay) by ≥150 % relative to UA or butyrate alone after 24 h.
2. The synergistic effect will be abolished by MCU knockdown or by overexpressing a deacetylase‑resistant mutant of PINK1 (K254R), indicating dependence on both calcium uptake and HDAC3 inhibition.
3. Combined treatment will reduce senescence‑associated β‑galactosidase activity and SASP cytokine secretion (IL‑6, IL‑8) to a greater extent than either monotherapy, correlating with increased naïve CD8+ T cell frequency (CD45RA+CCR7+).
4. In a humanized mouse model receiving fecal transplants from high‑UA‑producers versus low‑UA‑producers, supplementation with a butyrate‑producing prebiotic (inulin) will rescue mitophagy and improve viral clearance only in the low‑UA cohort, demonstrating microbiome‑dependent rescue.

Experimental Approach

• Isolate CD8+ T cells from healthy donors (n = 12) and treat with UA, butyrate, or combination for 6 h and 24 h.
• Assess mitophagy using mt‑Keima fluorescence flow cytometry, mitochondrial mass (MitoTracker Green), and membrane potential (TMRM).
• Validate pathway dependence via siRNA knockdown of MCU, CRISPR‑mediated HDAC3 knockout, and overexpression of acetylation‑site mutants of PINK1.
• Measure senescence markers (SA‑β‑gal, p16^INK4a^) and SASP cytokines by ELISA.
• For in vivo validation, germ‑free mice colonized with defined microbiota yielding either high or low UA levels receive inulin (5 % w/w) for 4 weeks; immune phenotypes and mitochondrial health in splenic CD8+ T cells are analyzed.

Potential Outcomes and Falsifiability

If the combination shows no significant increase in mitophagy over the best single agent, or if MCU or HDAC3 manipulation fails to modify the effect, the hypothesis is falsified. Conversely, a robust, mechanism‑dependent synergistic improvement would support the model that integrating ER calcium signaling with HDAC inhibition can be exploited to overcome microbiome variability and enhance mitophagy‑based immune rejuvenation.