Altered autophagy in Hermansky-Pudlak syndrome associated interstitial pneumonia

Introduction: Autophagy is a self-destructive, lysosomal catabolic pathway and is known from several decades as an adaptation to starvation. Basal autophagy is extremely important in turn-over of long lived proteins. Defective or severely altered macroautophagy has been reported and has been implicated in the pathogenesis of many lysosome related disorders. Hermansky-Pudlak syndrome (HPS) is one such lysosome realated disorder, affecting several lysosome-related organelles of the body. Several HPS mutations have been identified so far, but patients only with HPS types -1 & -4 develop pulmonary fibrosis called Hermansky-Pudlak syndrome associated Interstitial Pneumonia (HPSIP), the main reason of death in such patients. HPSIP lungs typically show enlarged alveolar typeII cells (AECII) with giant lamellar bodies, the lysosome related organelles of AECII. We recently reported spontaneous development of lung fibrosis in a mouse model of HPSIP (HPS1/2 mice), severe surfactant accumulation and apoptosis of AECII due to severe lysosomal stress and ER stress in these mice as well as in human HPS1.

Aim: We aim to study the autophagy pathway under conditions of HPSIP both in humans and in mice.

Materials and Methods: Left lungs from mouse HPS1, HPS2, HPS1/2 and WT controls and lungs from human HPS1 patients and healthy donors were fixed in formalin and embedded in paraffin. Serial sections were immunohistochemically stained for autophagy related proteins LC3, p62 and TFEB and for AECII marker, pro SP-C. Shock frozen right lungs were homogenized and used for western blotting of the same proteins.

Results: LC3II protein, the macroautophagy marker did not alter in lung homogenates of HPS1, HPS2, HPS1/2 as compared to WT controls by western blot. Immunohistochemistry revealed that the AECII of HPS1/2 mice and human HPS1 specifically did not stain for LC3 protein, while a convincing signal was observed within macrophages of the same sections and within AECII & macrophages of WT mice and healthy donors. In addition, on western blot, a significant increase in p62 in HPS1/2 mice was observed compared to WT mice. Transcription Factor-EB (TFEB) protein levels were also altered in HPS1/2 mice.

Conclusion: Our results point towards a defective autophagy under HPSIP conditions. An in depth analysis of this pathway is still needed to understand the disease pathomechanisms.