Hermansky-Pudlak syndrome (HPS) comprises of a group of rare hereditary disorders primarily affecting transport within lysosome related organelles. Up to now, 16 (mice) and 8 (human) genes, respectively, have been found to cause this disease. Amongst other clinical symptoms, patients with HPS may develop lung fibrosis (HPS associated Interstitial Pneumonia; HPSIP). As a consequence of such organ involvement, quality of life is then greatly reduced and prognosis is poor. The underlying molecular reasons for the development of HPSIP had yet not been settled. However, on an ultrastructural level, patients with HPSIP impose with an abnormal enlargement and increased number of lamellar bodies, a lysosome related organelle of alveolar type II cells (AECII) that represents the intracellular storage and secretion form of pulmonary surfactant.
Drawn against this background, the genotype-phenotype correlation was analyzed in mice with different single and combined HPS mutations. This work primarily focussed on the extent of pulmonary fibrosis and performed an in depth surfactant analysis in the lungs of HPS1, HPS2, HPS6, HPS1/6, HPS1/2 and respective wild type mice. In addition, altered signalling pathways and cellular consequences were studied.
Histological studies, undertaken after 3 and 9 months of age, revealed that only HPS1/2 mice, but none of the other HPS mice, would develop lung fibrosis, being quite extensive by 9 months of age. In these mice, but in none of the other mice, AECII appeared swollen and seemed to contain more and larger lamellar bodies. On the other hand, especially the HPS1/6 mice revealed spontaneous development of airspace enlargement. Defective intracellular surfactant trafficking and secretion was a prominent finding in HPS 1/2 mice and caused a highly significant intracellular accumulation of the mature hydrophobic surfactant proteins SP-B and SP-C as well as phospholipids. Among the phospholipids, especially the dipalmitoylated phosphatidylcholine (DPPC), the most abundant surfactant compound, was significantly elevated in lung tissues of HPS 1/2 mice. Isolated AECII from these mice revealed similar results. In contrast, although these above mentioned surfactant compounds appeared slightly increased in the mono mutant and the HPS 1/6 mice, there was a significant difference between HPS1/2 on the one, and the other mice on the other hand with regard to the extent of disturbed intracellular transport and accumulation of surfactant compounds.
As a possible consequence of such defective transport and secretion of surfactant, AECII apoptosis was observed extensively in 3 and 9 month old HPS1/2 mice by in-situ apoptosis assay and by immunohistochemistry of cleaved caspase 3. Hypothesizing that apoptosis of AECII in HPS1/2 mice would develop due to severe cellular stress, lysosomal and ER stress markers in these mice were investigated. The lysosomal protease, cathepsin D, was highly elevated in lung homogenates and isolated AECII of HPS1/2 mice, but, again, in none of the other mutant mice. Such increase in cathepsin D was already visible at the age of 3months. These results indicate an early onset lysosomal stress specifically in HPS1/2 mice. Apart from this, HPS 1/2 mice also exhibited signs of severe ER stress at a later age (9 months). In detail, CHOP, a proapoptotic factor specifically induced by unresolved ER-stress, was found to be highly and exclusively elevated HPS1/2 mice. In addition, lipidomic profiling of 9 month old lung tissues from all HPS mice revealed increased levels of glucosylceramides only in HPS1/2 mice, which also hints towards a serious cellular stress in these mice.
Data from the HPS1/2 mouse model was aptly supported by those obtained from HSPIP patients. As compared to lung transplant donor lungs, paraffin sections of lungs from two HPSIP patients (HPS1 mutation) showed a highly increased immunostaining for cathepsin D in AECII, alongside with induction of caspase-3 and CHOP immunostaining in this cell-type, thereby confirming the cellular stress mechanisms as observed in HPS1/2 mice.
Taken together, this is the the first time to decipher HPS1/2 mice as a valuable animal model of human HPSIP. With regard to the clinical similarities and common pathomechanistic principles between HPSIP and patients with Idiopathic Pulmonary Fibrosis (IPF), HPS 1/2 mice also offer as the currently best available model of IPF. This study showed that defective intracellular surfactant trafficking in AECII of these mice leads to the development of lysosomal and ER stress, finally resulting in AECII apoptosis, and at the end - development of lung fibrosis. Analysis of markers of lysosomal and ER-stress in human HPSIP samples fully corroborated such pathomechanistic concept and lends further credit to the general concept that apoptosis of the AECII represents a key step in the development of lung fibrosis in non-inflammatory triggered forms of Interstitial Lung Diseases. On the basis of these results, further elucidation of the molecular mode of action and the binding partners of the concerned HPS gene products (adaptor protein 3 and BLOC 3) appears reasonable and therapeutic strategies aiming to either restore the defective transport mechanism or to block the cellular stress response or AECII apoptosis are warranted.
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