Hypothesis

Chronic sleep disruption prevents the nocturnal glymphatic‑autophagic clearance of extracellular and intracellular danger‑associated molecular patterns (DAMPs). This failure leaves DAMPs to accumulate in the periphery where they are taken up by dendritic cells, priming the NLRP3 inflammasome via a two‑signal model. The primed state lowers the threshold for inflammasome activation, resulting in heightened IL‑1β/IL‑18 release that drives CD8⁺ T‑cell dysfunction and exhaustion. Restoring sleep‑dependent clearance or inhibiting NLRP3 priming should break this loop and preserve T‑cell fitness.

Mechanistic Rationale

During slow‑wave sleep the glymphatic system expands interstitial space, flushing soluble proteins and metabolites from the brain parenchyma【3](https://pmc.ncbi.nlm.nih.gov/articles/PMC7698404/)​. Simultaneously, autophagy degrades intracellular NLRP3 components, limiting inflammasome priming【4](https://doi.org/10.4161/auto.29647)​. Sleep loss diminishes both processes, causing DAMPs such as ATP, mitochondrial DNA, and oxidized lipids to spill into the cerebrospinal fluid and eventually reach systemic circulation. Peripheral dendritic cells internalize these DAMPs. Signal 1—NF‑κB activation via TLRs—upregulates NLRP3 and pro‑IL‑1β transcription【1](https://pmc.ncbi.nlm.nih.gov/articles/PMC6651423/)​. Because autophagy is impaired, newly synthesized NLRP3 is not cleared, amplifying the priming step. Signal 2—caspase‑1‑dependent inflammasome assembly—then proceeds more readily, yielding mature IL‑1β/IL‑18【2](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119179)​. Elevated IL‑1β/IL‑18 act on T cells, promoting PD‑1 upregulation, metabolic dysregulation, and the exhausted phenotype observed in chronic inflammation and aging【6](https://doi.org/10.1016/j.cmet.2013.09.010)​.

Testable Predictions

1. Mice subjected to fragmented sleep will show increased extracellular DAMP levels in CSF and plasma compared with rested controls.
2. Bone‑marrow‑derived dendritic cells from sleep‑fragmented mice will exhibit higher basal NLRP3 protein, elevated pro‑IL‑1β mRNA, and enhanced IL‑1β release after a low‑dose ATP trigger (Signal 2).
3. Adoptive transfer of these primed dendritic cells into naïve recipients will accelerate CD8⁺ T‑cell exhaustion markers (PD‑1, TIM‑3, reduced IFN‑γ) upon antigen challenge.
4. Pharmacological enhancement of glymphatic flow (e.g., intrathecal acetazolamide) or genetic upregulation of autophagy in dendritic cells (Atg5 overexpression) will normalize NLRP3 priming and rescue T‑cell function despite sleep disruption.
5. NLRP3 inhibition (MCC950) administered during the sleep‑rest period will abolish the sleep‑loss‑induced increase in IL‑1β/IL‑18 and prevent T‑cell exhaustion, even when clearance pathways remain compromised.

Experimental Design

• Sleep manipulation: Use a motorized treadmill or gentle handling to induce 6 h of sleep fragmentation per night for 2 weeks in C57BL/6 mice; controls receive undisturbed sleep.
• DAMP quantification: Collect CSF and plasma at zeitgeber time 6; measure ATP, mtDNA, and lipid peroxidation products via luciferase assays, qPCR, and MDA‑ELISA.
• DC phenotyping: Isolate CD11c⁺ DCs from spleen and lymph nodes; assess NLRP3, pro‑IL‑1β (Western blot, qPCR), and caspase‑1 activity (FLICA assay). Stimulate with 1 mM ATP for 30 min and quantify secreted IL‑1β/IL‑18 (ELISA).
• T‑cell assay: Co‑culture DCs with OT‑I CD8⁺ T cells plus OVA₍₂₅₇₋₂₆₄₎ peptide; after 48 h evaluate exhaustion markers (flow cytometry) and cytokine production (IFN‑γ ELISA).
• Intervention groups: (a) acetazolamide (50 mg/kg i.p.) to boost glymphatic influx, (b) dendritic‑cell‑specific Atg5 transgene, (c) MCC950 (10 mg/kg i.p.) administered at ZT18.
• Statistical analysis: Two‑way ANOVA with factors sleep condition and treatment; post‑hoc Tukey test. Power analysis targets n = 8 per group to detect 30 % change with α = 0.05, power = 0.8.

Falsifiability

If sleep‑fragmented mice fail to show elevated CSF/plasma DAMPs, or if dendritic cells from these mice do not display heightened NLRP3 priming and IL‑1β release, the core premise—that sleep loss drives inflammasome priming via clearance failure—is refuted. Likewise, if enhancing glymphatic flow or autophagy does not reduce NLRP3 activity or improve T‑cell outcomes, the mechanistic link between nocturnal clearance and inflammasome regulation would be unsupported. Conversely, observation of the predicted changes would substantiate the hypothesis and highlight a actionable window where sleep hygiene and NLRP3 antagonism could jointly mitigate immunosenescence.