HMGB1 Promotes Lysosome-Dependent Cell Death Induced Via Dry Eye by Disrupting Lysosomal Homeostasis

Hypertonic stress can induce apoptosis, inflammation, and dry eye disease (DED) through the upregulation of HMGB1 expression. However, its role in mediating and maintaining lysosomal homeostasis and suppressing DED development in living and in vitro models is unclear.

Immunofluorescence, nucleoplasmic separation, and electron microscopic analysis were used to compare the effects of hypertonic stress on lysosomal function, nuclear HGMB1 expression, and lysosomal localization in three different dry eye models. The live model was established by the subcutaneous injection of scopolamine (SCOP) into C57BL/6J female mice, and the in vitro model used human corneal epithelial cell (HCEC) cultures that were maintained at a hyperosmotic level of 450 milliosmolar (mOsm). RNAi technology was used to knockdown HMGB1 expression levels, altering hyperosmotic stress-induced changes in survival and autophagy of corneal epithelial cells in vitro. Lysosomal protease inhibitors were applied to suppress the increase of corneal tissue inflammation in the dry eye mouse model.

This hypertrophic stress upregulated karyoplasmic HMGB1 expression in HCECs. This change disrupted lysosomal structural and functional integrity, which facilitated the release of HMGB1 into the cytoplasm. SiRNAs downregulated HMGB1 expression levels and reversed increases in HMGB1 transfer from the nucleus to the cytoplasm and lysosomal alkalinizing function. These changes promoted increases in cathepsin leakage from lysosomes and increased mouse corneal epithelial apoptosis, whereas autophagy decreased. In a mouse model, administration of a cathepsin inhibitor reduced both ocular inflammation and other aspects of dry eye symptomology.

Hyperosmotic-induced HMGB1 upregulation and increased HMGB1 transfer into the cytoplasm from the nucleus underlie the loss of lysosomal membrane integrity, which increases both cathepsin release and corneal epithelial apoptosis. HMGB1 downregulation instead ameliorated these pathogenic responses.