Hypothesis

Inflammatory SASP signaling triggers lysosomal membrane permeabilization in aged immune cells, releasing cathepsin proteases that trim protective protein aggregates into fibrillar, immunogenic seeds. These seeds act as damage‑associated molecular patterns that amplify NLRP3 inflammasome activation, converting a transient containment strategy into chronic pathology.

Mechanistic Rationale

• Proteostasis collapse in senescence leads to amorphous aggregates that sequester misfolded proteins (adaptive).
• SASP cytokines (IL‑6, TNFα, IL‑1β) activate NF‑κB and also induce ROS‑dependent lysosomal destabilization.[3][4]
• Cathepsin B/L released into the cytosol cleave aggregated substrates, generating exposed β‑sheet rich fragments.[5]
• Such fragments nucleate further amyloid formation and are readily phagocytosed, triggering TLR/NLRP3 pathways.[6]
• This creates a feed‑forward loop: more SASP → more lysosomal leak → more fibrillar seeds → more inflammation.

Testable Predictions

1. In aged macrophages exposed to SASP‑inducing conditions (e.g., ionizing radiation or etoposide), pharmacological inhibition of cathepsin B (Ca‑074Me) or genetic knockout will reduce cytosolic cathepsin activity without affecting overall lysosomal mass.
2. Under cathepsin inhibition, protein aggregates will remain predominantly amorphous (Thioflavin T low, EM showing lack of ordered fibrils) compared with control conditions where aggregates acquire Thioflavin T positivity and fibrillar ultrastructure.
3. Consequently, inflammasome readouts (ASC speck formation, caspase‑1 cleavage, IL‑1β secretion) will be markedly lower in cathepsin‑deficient cells despite equivalent SASP cytokine levels.
4. Functionally, phagocytic clearance of apoptotic cells and antigen presentation will be preserved, and markers of immunosenescence (p16^INK4a, SA‑β‑gal) will decline.

Experimental Design (outline)

• Model: Bone‑marrow derived macrophages from young (3 mo) and aged (24 mo) mice; also human monocyte‑derived macrophages from donors >65 y.
• Treatment: 2 Gy γ‑irradiation to induce SASP; +/- cathepsin B inhibitor (Ca‑074Me, 10 µM) or vehicle.
• Readouts (24‑48 h):
  • Lysosomal integrity (Galectin‑3 puncta, LysoTracker loss).
  • Cytosolic cathepsin activity (Magic Red assay).
  • Aggregate conformation (Thioflavin T, filter‑trap assay, EM).
  • Inflammasome activation (ASC specks via imaging, caspase‑1 p20 Western, ELISA for IL‑1β/IL‑18).
  • Senescence markers (p16, SA‑β‑gal).
  • Phagocytosis assay (pHrodo‑labeled apoptotic neutrophils).
• Expected outcome: Cathepsin blockade preserves amorphous aggregates, lowers inflammasome output, improves phagocytosis, and reduces senescence markers.

Falsifiability

If cathepsin inhibition fails to shift aggregate morphology from fibrillar to amorphous, or does not diminish inflammasome activation despite verified lysosomal protection, the hypothesis is refuted. Likewise, if aggregate morphology changes but inflammasome activity remains unchanged, the proposed link between fibrillar seeds and immune activation is invalid.

Broader Implications

Confirming this mechanism would reposition protein aggregates not as inert waste but as dynamic substrates whose pathogenicity is shaped by lysosomal protease activity. It would suggest that combining senolytics or SASP blockers with lysosomal stabilizers (e.g., cathepsin inhibitors, Hsp70 inducers) could preserve the proteome’s adaptive aggregation state and break the inflammation‑aggregation vicious cycle in aging immunity.