Hermansky-Pudlak syndrome is a devastating multisystem disease afflicting roughly 1,000 patients worldwide but especially 1 in 1,800 inhabitants of Puerto Rico, caused by a genetic mutation in the HPS1 gene that has propagated in the population through a founder effect (32
). Pulmonary findings commonly described in adults with HPS from case series and case reports include macrophage-predominant inflammation, ceroid deposits within macrophages, and progressive honeycombing on high-resolution computed tomography indicative of progressive pulmonary fibrosis (3
). Longitudinal studies establishing the natural history of HPS lung disease are limited and have included only adults after presentation with restrictive lung remodeling. With this in mind, we sought to improve our understanding of the pulmonary disease pathogenesis in HPS through the use of an animal model, specifically to identify early events and their molecular basis.
Our first objective was to describe the ontogeny of lung disease in the pale ear/pearl (EPPE) mouse model, a model characterized by others as spontaneously developing fibrotic lung remodeling (8
). The EPPE mouse model, a cross between two HPS mouse strains, was developed previously to facilitate studies of HPS lung disease (4
). The pale ear and pearl parent strains, although demonstrating lamellar body enlargement and an inflammatory cell infiltrate, did not develop fibrosis unless challenged with bleomycin (6
). Single-mutation animals exhibited increased susceptibility to LPS challenge with more exuberant cytokine responses (5
). Although macrophages from adult pale ear or pearl animals exhibited some evidence of activation, specifically increased tumor necrosis factor-α and IL-12p40, it was attributed to an abnormal alveolar milieu because these macrophages rapidly became quiescent when cultured in vitro
, and cell-free BAL from these animals was able to activate otherwise normal alveolar macrophages.
The advantage of the EPPE mouse model is that it recapitulates all the features of adult HPS lung disease with the presence of giant lamellar bodies in alveolar epithelial cells, inflammation, and lung remodeling including fibrosis, albeit also in the context of airspace enlargement not reported in human HPS (4
). The present study uses this animal model to establish the temporal relationships between each facet of the disease. We have demonstrated a reproducible ontogeny for the onset of HPS lung disease in EPPE mice that begins with lamellar body enlargement and tissue phospholipid accumulation followed by alveolar inflammation, with these events being separated by up to 4 weeks and with each increasing in severity into adulthood. Furthermore, alveolar inflammation precedes lung remodeling, consisting of both airspace enlargement and subsequent collagen deposition (8
). Our longitudinal survey demonstrated that alveolitis began much earlier than previously suggested by BAL studies in both HPS mouse models carrying single mutations, and adult patients with HPS1 (14
). These data provide strong support for a more comprehensive evaluation of the pediatric HPS population, and suggest that earlier treatment protocols (before the development of clinically apparent restrictive lung disease) may be important in modifying the disease course.
Our longitudinal studies uncovered an interesting period in the natural history of HPS in the EPPE mouse. We identified a period during which there is evidence of a growing aberrant type 2 cell phenotype signified by lamellar body distention, increasing tissue phospholipid deposition, and type 2 cell hyperplasia, which precedes the initiation of the macrophage-predominant alveolar infiltrate. Thus, the second objective of our study was to explore this period for potential disease mechanisms. There has been increasing evidence that epithelial cell injury is an important factor in the development of HPS lung disease. Both Mahavadi and colleagues (8
) and Young and colleagues (6
) have reported evidence of epithelial cell injury, and prior work by Young and colleagues (5
) suggests that the alveolar inflammation is a reactive and not a primary feature of HPS. Our data strongly support the alveolar type 2 cell as the initiator of lung disease in HPS. Isolated type 2 cells from EPPE mice as early as 2 weeks of age exhibit a proinflammatory phenotype with the production of at least two molecular mediators, specifically MCP-1 and inducible nitric oxide synthase (iNOS). Although it is likely that other cytokines and chemokines are produced by the alveolar epithelium from EPPE animals, our studies indicate that together MCP-1 and SNO-SP-D contribute the majority of the chemotactic activity in the alveolar space early in HPS lung disease, accounting for up to 75% of the chemotaxis induced by EPPE mouse BAL as early as 4 weeks of age. Although MCP-1 production by alveolar macrophages may play a more prominent role in macrophage activation with advanced disease in patients with HPS1 (14
), our data are the first to suggest that the alveolar epithelium may play a prominent role in elaborating inflammatory mediators early in HPS disease.
Whereas MCP-1 has clearly been linked to the pathogenesis of HPS lung disease in studies of patients with HPS1 (14
), our data establish the importance of SP-D in the pathogenesis of HPS lung disease in both mice and humans. SP-D has emerged as a key modulator of inflammation in the alveolar space (36
). In its multimeric form, the lectin-binding domains interact with surface receptors such as SIRPα on effector cells, attenuating NF-κB–mediated proinflammatory gene expression (37
). In a variety of models of pulmonary inflammation using exogenous inflammatory stimuli (bleomycin, Pneumocystis
), we have shown previously that alveolar inflammation leads to local production of NO metabolites via increased iNOS expression by activated macrophages, promoting S-nitrosylation of SP-D and disrupting SP-D tertiary structure (9
). Studies of patients with asthma similarly show that SNO-SP-D was associated with pulmonary inflammation and was predictive of response to segmental allergen challenges (24
), expanding the usefulness of SNO-SP-D as a biomarker of inflammation. The mechanism by which SNO-SP-D promotes a proinflammatory state stems from trimeric SNO-SP-D having accessibility to surface receptors, such as CD91, that trigger proinflammatory responses such as macrophage migration and induction of iNOS (9
). This signaling pathway, which is highly sensitive to small amounts of SNO-SP-D, is not otherwise accessible to multimeric SP-D (9
). By contrast, oxidized SP-D, which results in cross-linking of SP-D, results in inactivation of LPS agglutination by SP-D (38
) and does not stimulate inflammatory responses (24
Our initial studies of the EPPE mouse model (7
), in which we characterized surfactant abnormalities in adult mice but did not demonstrate differences in SP-D, highlight an important issue. It is becoming clear from animal models and human studies that the complex oligomeric structure of SP-D and its liability for disruption in disease models call for a comprehensive analysis of SP-D (36
). The availability of new antisera (39
) with better specificity for murine and human SP-D than we used previously, and the advent of new techniques including the biotin-switch assay to detect S-nitrosyl protein modifications and native gel electrophoresis to examine the quaternary structure of SP-D, enabled us to revisit the role of SP-D in HPS. In the current study, we found both increases in total SP-D and SNO-SP-D in HPS BAL from both EPPE mice and patients with HPS1. In our prior study we normalized total SP-D immunoblotting data to BAL total protein and total phospholipid. This normalization presented a problem in retrospect because of elevated total protein and reduced total phospholipid in BAL from EPPE mice, as we and others have reported (4
), leading us to conclude that total SP-D was not altered in the EPPE mice. In the present study we instead reported total SP-D in equivalent volumes of BAL from EPPE mice () and patients with HPS1 (), and found dramatic differences that progressed with age in EPPE mice, and with disease severity in patients with HPS1. In examining SNO-SP-D and changes in quaternary structure, we instead started with samples containing equal amounts of total SP-D, so that the analysis was not confounded by the already increased SP-D in BAL from EPPE mice and patients with HPS1. Together, this more thorough examination in both the EPPE mouse and patients with HPS1 has uncovered an important role for SNO-SP-D as an amplifier of inflammation in HPS lung disease.
The effects of SNO-SP-D in EPPE mice and patients with HPS1 are reminiscent of our prior studies in bleomycin-treated mice, in which enhanced chemotaxis in vitro
due to BAL SNO-SP-D was mitigated by removal of S
-nitrosothiols, using ascorbate or by immunodepletion SP-D (9
). It is interesting that whereas SNO-SP-D has been shown to mediate both migration and activation of macrophages in response to bleomycin injury, in our current study the dominant effect of SNO-SP-D is on macrophage migration early in the pathogenesis of HPS lung disease in EPPE mice. Another interesting difference between our prior work using the bleomycin model and our current studies in EPPE animals relates to the source of NO. S-nitrosylation of proteins largely occurs in the context of increased NO production (22
), which in the lung occurs typically from activated macrophages expressing iNOS (23
). In bleomycin-treated animals, S-nitrosylation resulted from enhanced NO production via iNOS induced in alveolar macrophages (9
). In young EPPE animals, the source of NO production uncharacteristically arose from the alveolar epithelium. NO production from the induction of type 2 cell iNOS has been described previously in response to external inflammatory stimuli, in cell lines (29
) and with injury from bleomycin in vivo
). In HPS lung disease, the stimulus is endogenous, possibly from pathways within alveolar type 2 cells activated in response to lamellar body enlargement.
The role of inflammation in the pathogenesis of pulmonary fibrosis has been controversial, with more recent evidence validating the central role of repetitive epithelial injury in fibroproliferation (40
). Studies of type 2 cell–targeted expression of diphtheria toxin receptor suggest that not only epithelial injury but injury to important progenitor cells in the alveolus initiate fibroproliferation (41
). Pulmonary fibrosis from overexpression of transforming growth factor (TGF)-β1
, using adenovirus, emphasizes the role of the epithelial–mesenchymal transition (EMT) to the process of fibroproliferation (42
), and conditional overexpression of TGF-α now makes a strong case for non-EMT pathways in the process of pulmonary fibrosis (43
). However, two of these models of fibrosis have similarly been associated with alveolar inflammation (44
). Thus, inflammation may simply be a proxy for alveolar epithelial injury, or may function as an amplifier of injury that contributes to the process of remodeling, as seen in acute exacerbations of idiopathic pulmonary fibrosis (46
The data presented in this study point to a chronic, unremitting injury of alveolar epithelial cells in HPS, leading to the elaboration of proinflammatory mediators. Like others (6
), we are unable to determine from our present studies whether the observed inflammatory signals in EPPE animals directly contribute to the development of fibrosis in animal models of HPS, or are simply an earlier marker of a failing epithelial cell. Although inflammation may be a secondary manifestation of alveolar type 2 cell dysfunction, it represents a reliable early end point for future studies as well as a potential therapeutic target. Moreover, SNO-SP-D as a mediator of inflammation in HPS is an attractive candidate for a new biomarker in HPS lung disease that tracked well with disease progression in both the mouse model as well as in patients with HPS1. Taken together, these studies clarify the ontogeny of HPS lung disease in a reliable mouse model for future studies of disease pathogenesis and preclinical testing of therapeutic agents, and establish key mediators of the disease as well as a potential biomarker for expanded investigation in patients with HPS.