[Show abstract][Hide abstract] ABSTRACT: Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations of the tumor suppressor genes Tuberous Sclerosis Complex (TSC1 or TSC2). LAM affects almost exclusively women and is characterized by neoplastic growth of atypical smooth muscle-like TSC2-null LAM cells in the pulmonary interstitium, cystic destruction of lung parenchyma, and progressive decline in lung function. In this study, we hypothesized that TSC2-null lesions promote a pro-inflammatory environment which contributes to lung parenchyma destruction. Using a TSC2-null female murine LAM model, we demonstrate that TSC2-null lesions promote alveolar macrophage accumulation, recruitment of immature multinucleated cells, an increased induction of pro-inflammatory genes NOS2, IL-6, CCL2/MCP-1, CXCL1/KC, and upregulation of IL-6, KC, MCP-1 and TGF-β1 levels in bronchoalveolar lavage fluid (BAL). BAL also contained an increased level of surfactant protein (SP)-D but not SP-A, significant reduction of SP-B levels, and a resultant increase in alveolar surface tension. Consistent with the growth of TSC2-null lesions, nitric oxide (NO) levels were also increased and, in turn, modified SP-D through S-nitrosylation forming SNO-SP-D, a known consequence of lung inflammation. Progressive growth of TSC2-null lesions was accompanied by elevated levels of MMP-3 and MMP-9. This report demonstrates a link between growth of TSC2-null lesions and inflammation-induced surfactant dysfunction that might contribute to the lung destruction in LAM.
American Journal of Respiratory Cell and Molecular Biology 12/2014; 53(1). DOI:10.1165/rcmb.2014-0224OC · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutation of threonine for isoleucine at codon 73 (I73T) in the human surfactant protein C (hSP-C) gene (SFTPC) accounts for a significant portion of SFTPC mutations associated with interstitial lung disease (ILD). Cell lines stably expressing tagged proSP-C isoforms were generated to test the hypothesis that deposition of hSP-C(I73T) within the endosomal system promotes disruption of a key cellular quality control pathway, macroautophagy. By fluorescence microscopy, hSP-C(WT) co-localized with exogenously expressed hABCA3, an indicator of normal trafficking to lysosomal related organelles. In contrast, hSPCI73T was dissociated from hABCA3 but co-localized to the plasma membrane and the endosomal network. Cells expressing hSP-C(I73T) exhibited increases in size and number of cytosolic GFP/LC3 vesicles, some of which co-labeled with DsRed/hSP-C(I73T). By transmission EM, hSP-C(I73T) cells contained abnormally large autophagic vacuoles containing organellar and proteinaceous debris which phenocopied ultrastructural changes in alveolar type 2 cells in a lung biopsy from a SFTPC I73T patient. Biochemically, hSP-C(I73T) cells exhibited increased expression of Atg8/LC3, SQST/p62, and RAB7 consistent with a distal block in autophagic vacuole maturation confirmed by flux studies using bafilomycin A1 and rapamycin. Functionally, hSP-C(I73T) cells showed an impaired degradative capacity for an aggregation-prone, (HTT) huntingin-1 reporter substrate. The disruption of autophagy-dependent proteostasis was accompanied by increases in mitochondria biomass and PARK2 (parkin) expression coupled with a decrease in mitochondrial membrane potential. We conclude that hSP-C(I73T) induces an acquired block in macroautophagy-dependent proteostasis and mitophagy which could contribute to the increased vulnerability of the lung epithelia to second hit injury as seen in ILD.
[Show abstract][Hide abstract] ABSTRACT: Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis.
In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS).
Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd(-/-)) and iNOS single knockout mice (NOS2(-/-)) as well as wild type (WT) littermates.
DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd(-/-) demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS.
iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.
PLoS ONE 01/2014; 9(1):e85722. DOI:10.1371/journal.pone.0085722 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the ER folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility and reduced the expression level of wild type ABCA3 in a dose dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that while single motif mutants exhibited a vesicular distribution pattern similar to wild type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability and its disruption results in protein destabilization and proteasomal degradation.
[Show abstract][Hide abstract] ABSTRACT: Background:
Surfactant protein D (SP-D) is a collection that plays important roles in modulating host defense functions and maintaining phospholipid homeostasis in the lung. The aim of current study was to characterize comparatively the SP-D response in bronchoalveolar lavage (BAL) and serum in three murine models of lung injury, using a validated ELISA technology for estimation of SP-D levels.
Mice were exposed to lipopolysaccharide, bleomycin, or Pneumocystis carinii (Pc) and sacrificed at different time points.
In lipopolysaccharide-challenged mice, the level of SP-D in BAL increased within 6 h, peaked at 51 h (4,518 ng/ml), and returned to base level at 99 h (612 ng/ml). Serum levels of SP-D increased immediately (8.6 ng/ml), peaked at 51 h (16 ng/ml), and returned to base levels at 99 h (3.8 ng/ml). In a subacute bleomycin inflammation model, SP-D levels were 4,625 and 367 ng/ml in BAL and serum, respectively, 8 days after exposure. In a chronic Pc inflammation model, the highest level of SP-D was observed 6 weeks after inoculation, with BAL and serum levels of 1,868 and 335 ng/ml, respectively.
We conclude that serum levels of SP-D increase during lung injury, with a sustained increment during chronic inflammation compared with acute inflammation. A quick upregulation of SP-D in serum in response to acute airway inflammation supports the notion that SP-D translocates from the airways into the vascular system, in favor of being synthesized systemically. The study also confirms the concept of using increased SP-D serum levels as a biomarker of especially chronic airway inflammation.
Beiträge zur Klinik der Tuberkulose 02/2013; 191(3). DOI:10.1007/s00408-013-9452-x · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The integrity of the alveolar epithelium is a key factor in the outcome of acute lung injury. Here, we investigate alveolar epithelial injury and the expression of epithelial-selective markers in Streptococcus pneumoniae-induced acute lung injury. S. pneumoniae was instilled into rat lungs and alveolar type I (RTI(40)/podoplanin, MMC6 antigen) and alveolar type II (MMC4 antigen, surfactant protein D, pro-surfactant protein C, RTII(70)) cell markers were quantified in lavage fluid and lung tissue at 24 and 72 hours. The alveolar epithelium was also examined using electron, confocal, and light microscopy. S. pneumoniae induced an acute inflammatory response as assessed by increased total protein, SP-D, and neutrophils in lavage fluid. Biochemical and morphological studies demonstrated morphologic injury to type II cells but not type I cells. In particular, the expression of RTI(40)/podoplanin was dramatically reduced, on the surface of type I cells, in the absence of morphologic injury. These data demonstrate that type II cell damage can occur in the absence of type I cell injury without affecting the ability of the lung to return to a normal morphology. These data also demonstrate that RTI(40)/podoplanin is not a type I cell phenotypic marker in experimental acute lung injury caused by S. pneumoniae. Given that RTI(40)/podoplanin is an endogenous ligand for the C-type lectin receptor and this receptor plays a role in platelet aggregation and neutrophil activation, we hypothesize that the reduction of RTI(40)/podoplanin on type I cells might be important for the regulation of platelet and/or neutrophil function in experimental acute lung injury.
Experimental Lung Research 06/2012; 38(5):266-76. DOI:10.3109/01902148.2012.683321 · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Concurrent with the global escalation of the AIDS pandemic, cryptococcal infections are increasing and are of significant
medical importance. Furthermore, Cryptococcus neoformans has become a primary human pathogen, causing infection in seemingly healthy individuals. Although numerous studies have elucidated
the virulence properties of C. neoformans, less is understood regarding lung host immune factors during early stages of fungal infection. Based on our previous studies
documenting that pulmonary surfactant protein D (SP-D) protects C. neoformans cells against macrophage-mediated defense mechanisms in vitro (S. Geunes-Boyer et al., Infect. Immun. 77:2783–2794, 2009), we postulated that SP-D would facilitate fungal infection in vivo. To test this hypothesis, we examined the role of SP-D in response to C. neoformans using SP-D−/− mice. Here, we demonstrate that mice lacking SP-D were partially protected during C. neoformans infection; they displayed a longer mean time to death and decreased fungal burden at several time points postinfection than
wild-type mice. This effect was reversed by the administration of exogenous SP-D. Furthermore, we show that SP-D bound to
the surface of the yeast cells and protected the pathogenic microbes against macrophage-mediated defense mechanisms and hydrogen
peroxide (H2O2)-induced oxidative stress in vitro and in vivo. These findings indicate that C. neoformans is capable of coopting host SP-D to increase host susceptibility to the yeast. This study establishes a new paradigm for
the role played by SP-D during host responses to C. neoformans and consequently imparts insight into potential future preventive and/or treatment strategies for cryptococcosis.
Infection and immunity 04/2012; 80(7):2444-53. DOI:10.1128/IAI.05613-11 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial cell dysfunction is now recognized as an important mechanism in the pathogenesis of interstitial lung diseases. Surfactant Protein C (SP-C), an alveolar type II cell specific protein, has contributed to this concept with the observation that heterozygous expression of SFTPC gene mutations are associated with chronic interstitial lung disease. We have shown that transient expression of aggregation prone mutant SP-C isoforms (SP-C BRICHOS) destabilize ER quality control mechanisms resulting in the intracellular accumulation of aggregating propeptide, inhibition of the ubiquitin/proteasome system, and activation of apoptosis. The goal of the present study was to define signaling pathways linking the unfolded protein response (UPR) and subsequent ER stress with intrinsic apoptosis events observed following mutant SP-C expression. In vitro expression of the SP-C BRICHOS mutant, SP-C(Δexon4), was used as a model system. Here we show stimulation of a broad ER stress response in both transfected A549 and HEK293 cells with activation of all 3 canonical sensing pathways, IRE1/XBP-1, ATF6, and PERK/eIF2α. SP-C(Δexon4) expression also resulted in activation of caspase 3, but failed to stimulate expression of the apoptosis mediating transcription factors ATF4/CHOP. However, inhibition of either caspase 4 or c-jun kinase (JNK) each blocked caspase 3 mediated cell death. Taken together, these results suggest that expression of SP-C BRICHOS mutants induce apoptosis through multiple UPR signaling pathways, and provide new therapeutic targets for the amelioration of ER stress induced cytotoxicity observed in fibrotic lung remodeling.
The international journal of biochemistry & cell biology 01/2012; 44(1):101-12. DOI:10.1016/j.biocel.2011.10.003 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known.
The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM).
Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps.
BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-β1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-β1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-β1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice.
These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.
American Journal of Respiratory and Critical Care Medicine 12/2011; 185(5):525-36. DOI:10.1164/rccm.201103-0561OC · 13.00 Impact Factor