Surfactant Lipid Homeostasis Research Articles

Alcohol-induced lipid dysregulation impairs glycolytic responses to LPS in alveolar macrophages.

Several conditions are marked by increased susceptibility to, and enhanced severity of, bacterial infections. Alcohol use disorder, one of these conditions, is known to predispose to bacterial pneumonia by suppressing the lung's innate immune system, and more specifically by disrupting critical alveolar macrophage (AM) functions. Recently, we established that chronic ethanol consumption also perturbs surfactant lipid homeostasis in the lung and that elevated concentrations of free fatty acids contribute to blocking essential AM functions, such as agonist-induced cytokine expression. In this study, we extend these observations by showing that elevated free fatty acid levels impair metabolic responses to lipopolysaccharide (LPS) in AMs. In particular, we show that the glycolytic reprogramming characteristic of LPS-stimulated AMs is blunted by the saturated fatty acid palmitate, whereas oleate, an unsaturated fatty acid, or ethanol alone, had no effect on this adaptive metabolic response. Additionally, we found that elevated concentrations of palmitate induced mitochondrial oxidative stress and that glycolytic reprogramming and cytokine production to LPS could be partially restored in AMs by either pharmacologically blocking palmitate entry into mitochondria or administering a mitochondrial-specific antioxidant. Taken together, these findings suggest that alcohol and elevated levels of saturated fatty acids conspire to impair pulmonary innate immunity by altering metabolic responses in AMs. Additionally, our findings suggest that targeting the mechanisms involved in fatty acid metabolism can restore pulmonary immunity and possibly limit bacterial pneumonia in individuals with alcohol use disorder.

ABCG1 regulates pulmonary surfactant metabolism in mice and men

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.

Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene

The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3E292V)) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3E292V gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3E292V knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3E292V) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3E292V–rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3E292V–rNeo+/+) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3E292V–rNeo mice were rendered more vulnerable to exogenous injury. Three weeks following intratracheal bleomycin challenge, mAbca3–rNeo mice demonstrated allele-dependent susceptibility to bleomycin including enhanced weight loss, augmented airspace destruction, and increased fibrosis. Removal of the rNeo cassette from mAbca3 alleles resulted in restoration of BAL PL content to wild-type levels and an absence of changes in lung histology up to 32 weeks of age. These results support the importance of surfactant PL homeostasis as a susceptibility factor for both intrinsic and exogenously induced lung injury/remodeling.

Epithelial SCAP/INSIG/SREBP signaling regulates multiple biological processes during perinatal lung maturation.

Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (ScapΔ/Δ, resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2 Δ/Δ, resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/β-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners.

A systems approach to mapping transcriptional networks controlling surfactant homeostasis

BackgroundPulmonary surfactant is required for lung function at birth and throughout life. Lung lipid and surfactant homeostasis requires regulation among multi-tiered processes, coordinating the synthesis of surfactant proteins and lipids, their assembly, trafficking, and storage in type II cells of the lung. The mechanisms regulating these interrelated processes are largely unknown.ResultsWe integrated mRNA microarray data with array independent knowledge using Gene Ontology (GO) similarity analysis, promoter motif searching, protein interaction and literature mining to elucidate genetic networks regulating lipid related biological processes in lung. A Transcription factor (TF) - target gene (TG) similarity matrix was generated by integrating data from different analytic methods. A scoring function was built to rank the likely TF-TG pairs. Using this strategy, we identified and verified critical components of a transcriptional network directing lipogenesis, lipid trafficking and surfactant homeostasis in the mouse lung.ConclusionsWithin the transcriptional network, SREBP, CEBPA, FOXA2, ETSF, GATA6 and IRF1 were identified as regulatory hubs displaying high connectivity. SREBP, FOXA2 and CEBPA together form a common core regulatory module that controls surfactant lipid homeostasis. The core module cooperates with other factors to regulate lipid metabolism and transport, cell growth and development, cell death and cell mediated immune response. Coordinated interactions of the TFs influence surfactant homeostasis and regulate lung function at birth.

C/EBPα is required for pulmonary cytoprotection during hyperoxia

A number of transcriptional pathways regulating fetal lung development are active during repair of the injured lung. We hypothesized that C/EBPalpha, a transcription factor critical for lung maturation, plays a role in protection of the alveolar epithelium following hyperoxic injury of the mature lung. Transgenic Cebpalpha(Delta/Delta) mice, in which Cebpalpha was conditionally deleted from Clara cells and type II cells after birth, were developed. While no pulmonary abnormalities were observed in the Cebpalpha(Delta/Delta) mice (7-8 wk old) under normal conditions, the mice were highly susceptible to hyperoxia. Cebpalpha(Delta/Delta) mice died within 4 days of exposure to 95% oxygen in association with severe lung inflammation, altered maturation of surfactant protein B and C, decreased surfactant lipid secretion, and abnormal lung mechanics at a time when all control mice survived. mRNA microarray analysis of isolated type II cells at 0, 2, and 24 h of hyperoxia demonstrated the reduced expression of number of genes regulating surfactant lipid and protein homeostasis, including Srebf, Scap, Lpcat1, Abca3, Sftpb, and Napsa. Genes influencing cell signaling or immune responses were induced in the lungs of Cebpalpha(Delta/Delta) mice. C/EBPalpha was required for the regulation of genes associated with surfactant lipid homeostasis, surfactant protein biosynthesis, processing and transport, defense response to stress, and cell redox homeostasis during exposure to hyperoxia. While C/EBPalpha did not play a critical role in postnatal pulmonary function under normal conditions, C/EBPalpha mediated protection of the lung during acute lung injury induced by hyperoxia.

Surfactant Protein D Protects against Acute Hyperoxic Lung Injury

Surfactant protein D (SP-D) is a member of the collectin family of soluble, innate, host defense molecules with demonstrated immunomodulatory properties in vitro. Constitutive absence of SP-D in mice is associated with lung inflammation, alteration in surfactant lipid homeostasis, and increased oxidative-nitrative stress. To test the hypothesis that SP-D would protect against acute lung injury from hyperoxia in vivo. Transgenic mice overexpressing rat SP-D constitutively (SP-D OE) or conditionally via regulation with doxycycline (SP-D Dox-on) were subjected to continuous hyperoxic challenge for up to 14 days. Compared with littermate control mice (wild-type [WT]), SP-D OE mice exposed to 80% O(2) demonstrated substantially increased survival accompanied by significant reductions in wet to dry lung ratios and bronchoalveolar lavage (BAL) protein. Although SP-D OE and WT mice exhibited a twofold increase in total BAL cells and neutrophilia in response to hyperoxia, the SP-D OE group had lower levels of BAL proinflammatory cytokines and chemokines, including IL-6, tumor necrosis factor-alpha, and monocyte chemotactic protein-1; increased mRNA levels of the transcription factor NF-E2 related factor-2 (NRF-2) and phase 2 antioxidants hemoxygenase-1 (HO-1), glutathione peroxidase-2 (GPx-2) and NAD(P)H quinone oxidoreductase-1 (Nqo-1); and decreases in lung tissue thiobarbituric acid-reactive substances. As proof of principle, the protective role of SP-D on hyperoxic injury was confirmed as SP-D Dox-on mice exposed to 85% O(2) demonstrated increased mortality upon withdrawal of doxycycline. Local expression of SP-D protects against hyperoxic lung injury through modulation of proinflammatory cytokines and antioxidant enzymatic scavenger systems.

GP130-STAT3 Regulates Epithelial Cell Migration and Is Required for Repair of the Bronchiolar Epithelium

Following injury, bronchiolar cells undergo rapid squamous metaplasia, followed by proliferation and re-establishment of the complex columnar epithelium that is characteristic of the normal airway. Mechanisms that regulate the repair of bronchiolar epithelium are of considerable relevance for understanding the pathogenesis of both acute and chronic lung diseases associated with airway remodeling. This study was designed to identify the role of the GP130-STAT3 signaling pathway during repair of the bronchiolar epithelium. STAT3 (signal transducer and activator of transcription 3) and GP130 (glycoprotein 130) were each selectively deleted from the pulmonary epithelial cells of transgenic mice in vivo, producing Stat3(Delta/Delta) and Gp130(Delta/Delta) mice, respectively. Airway injury was induced in adult mice by administration of naphthalene, a toxicant of nonciliated respiratory epithelial cells (Clara cells). Nuclear STAT3 staining was induced in bronchiolar epithelial cells following naphthalene-mediated injury in control (Stat3(flox/flox)) mice. Whereas nearly complete repair of the bronchiolar epithelium was observed in control mice within 13 days, restoration of cell shape, cell density, and the pattern of ciliated and nonciliated cells did not occur in the peripheral bronchioles of either Stat3(Delta/Delta) or Gp130(Delta/Delta) mice. Expression of dominant-negative STAT3 inhibited airway epithelial cell migration during repair in vitro; wild-type STAT3 expression activated such migration. In the present study, we show that GP130-STAT3 signaling functions in a cell-autonomous manner to restore cell shape and numbers required for repair of the bronchiolar epithelium following injury.

Correction of Pulmonary Abnormalities in Sftpd-/- Mice Requires the Collagenous Domain of Surfactant Protein D

Surfactant protein D (SP-D) is a member of the collectin family of innate defense proteins. Members of this family share four distinct structural domains: an N-terminal cross-linking domain, a collagenous domain, a neck region, and a carbohydrate recognition domain. In this study, the function of the collagenous domain was evaluated by expressing a SP-D collagen deletion mutant protein (rSftpdCDM) in wild type and SP-D null mice (Sftpd(-/-)). rSftpdCDM formed disulfide-linked trimers that further oligomerized into higher order structures. The mutant protein effectively bound carbohydrate and aggregated bacteria in vitro. Whereas rSftpdCDM did not disrupt pulmonary morphology or surfactant phospholipid levels in wild type mice, the mutant protein failed to rescue the emphysema or enlarged foamy macrophages that are characteristic of Sftpd(-/-) mice. Moreover, rSftpdCDM partitioned with small aggregate surfactant in a manner similar to SP-D, but rSftpdCDM did not correct the abnormal surfactant ultrastructure or phospholipid levels observed in Sftpd(-/-) mice. In contrast, rSftpdCDM completely corrected viral clearance and the abnormal inflammatory response that occurs following pulmonary influenza A challenge in Sftpd(-/-) mice. Our findings indicate that the collagen domain of SP-D is not required for assembly of disulfide-stabilized oligomers or the innate immune response to viral pathogens. The collagen domain of SP-D is required for the regulation of pulmonary macrophage activation, airspace remodeling, and surfactant lipid homeostasis.

Reversibility of Pulmonary Abnormalities by Conditional Replacement of Surfactant Protein D (SP-D) in Vivo

Surfactant protein D (SP-D) gene-targeted mice develop severe pulmonary disease associated with emphysema, pulmonary lipidosis, and foamy macrophage infiltrations. To determine the potential reversibility of these abnormalities, transgenic mice were developed in which SP-D was conditionally replaced in the respiratory epithelium of SP-D(-/-) mice. SP-D was not detected in the absence of doxycycline. Treatment with doxycycline after birth restored pulmonary SP-D concentrations and corrected pulmonary pathology at adulthood. When SP-D was replaced in adult SP-D(-/-) mice, alveolar SP-D was restored within 3 days, pulmonary lipid abnormalities were corrected, but emphysema persisted. In corrected adult SP-D(-/-) mice, loss of SP-D caused focal emphysema and pulmonary inflammation but did not cause phospholipid abnormalities characteristic of SP-D(-/-) mice. Thus, abnormalities in surfactant phospholipid homeostasis and alveolar macrophage abnormalities were readily corrected by restoration of SP-D. However, once established, emphysema was not reversed by SP-D. SP-D-dependent processes regulating surfactant lipid homeostasis were disassociated from those mediating emphysema.

The roles of surfactant proteins A and D in innate immunity.

Research over the last decade on the surfactant proteins SP-A and SP-D suggests roles beyond surfactant lipid homeostasis, involving their participation in innate immune defence. SP-A and SP-D bind and agglutinate an impressive array of non-self structures, ranging from bacteria and fungi to allergens and environmental inorganic substrates. Complementing binding. SP-A and SP-D initiate and enhance immune cell ingestion and killing of targets. Recently, some exciting developments have extended and clarified their contributions to innate immunity. Knockout mice for SP-A and SP-D have been developed. The SP-A knockout confirms that SP-A plays a key role in defence against lung pathogens and reveals the underlying defense mechanisms that require SP-A. These surfactant proteins have also been shown to have important roles in modulating the immune response, instructing, yet quenching, the immune reactions in the lung. The crystal structure of SP-D plus functional studies with recombinantly altered forms of SP-A and SP-D has begun to characterise the structural motifs responsible for mediating their immune functions. Linkage and polymorphism analysis is explaining the role these genes may play in lung diseases and infection.

Pulmonary-specific expression of SP-D corrects pulmonary lipid accumulation in SP-D gene-targeted mice.

Targeted disruption of the surfactant protein (SP) D (SP-D) gene caused a marked pulmonary lipoidosis characterized by increased alveolar lung phospholipids, demonstrating a previously unexpected role for SP-D in surfactant homeostasis. In the present study, we tested whether the local production of SP-D in the lung influenced surfactant content in SP-D-deficient [SP-D(-/-)] and SP-D wild-type [SP-D(+/+)] mice. Rat SP-D (rSP-D) was expressed under control of the human SP-C promoter, producing rSP-D, SP-D(+/+) transgenic mice. SP-D content in bronchoalveolar lavage fluid was increased 30- to 50-fold in the rSP-D, SP-D(+/+) mice compared with the SP-D(+/+) parental strain. Lung morphology, phospholipid content, and surfactant protein mRNAs were unaltered by the increased concentration of SP-D. Likewise, the production of endogenous mouse SP-D mRNA was not perturbed by the SP-D transgene. rSP-D, SP-D(+/+) mice were bred to SP-D(-/-) mice to assess whether lung-selective expression of SP-D might correct lipid homeostasis abnormalities in the SP-D(-/-) mice. Selective expression of SP-D in the respiratory epithelium had no adverse effects on lung function, correcting surfactant phospholipid content and decreasing phosphatidylcholine incorporation significantly. SP-D regulates surfactant lipid homeostasis, functioning locally to inhibit surfactant phospholipid incorporation in the lung parenchyma and maintaining alveolar phospholipid content in the alveolus. Marked increases in biologically active tissue and alveolar SP-D do not alter lung morphology, macrophage abundance or structure, or surfactant accumulation.

CCSP deficiency does not alter surfactant homeostasis during adenoviral infection.

Clara cell secretory protein (CCSP) deficiency in mice is associated with increased susceptibility to pulmonary inflammation after hyperoxia or viral infection. Because adenoviral exposure perturbs pulmonary surfactant homeostasis in vivo, we hypothesized that CCSP deficiency would influence surfactant metabolism after pulmonary infection. Alveolar and total lung saturated phosphatidylcholine pool sizes were similar in CCSP-deficient [CCSP(-/-)] and wild-type [CCSP(+/+)] mice before and 7 days after intratracheal administration of adenovirus. Radiolabeled choline and palmitate incorporation into saturated phosphatidylcholine was similar, and there was no alteration by previous infection 7 days before the incorporation measurements. Furthermore, CCSP deficiency did not influence clearance of [(14)C]dipalmitoylphosphatidylcholine and (125)I-labeled recombinant surfactant protein C. Increased persistence of alveolar capillary leak was observed in CCSP(-/-) mice after adenoviral infection. Surfactant lipid homeostasis was not influenced by CCSP before or after administration of adenovirus to the lung. Persistence of alveolar capillary leak in CCSP(-/-) mice after adenovirus provides further evidence for the role of CCSP in the regulation of pulmonary inflammation.

Surfactant Protein-D Regulates Surfactant Phospholipid Homeostasis in Vivo

Surfactant protein D (SP-D) is a 43-kDa member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. The SP-D gene was targeted by homologous recombination in embryonic stem cells that were used to produce SP-D (+/-) and SP-D (-/-) mice. Both SP-D (-/-) and SP-D (+/-) mice survived normally in the perinatal and postnatal periods. Whereas no abnormalities were observed in SP-D (+/-) mice, alveolar and tissue phosphatidylcholine pool sizes were markedly increased in SP-D (-/-) mice. Increased numbers of large foamy alveolar macrophages and enlarged alveoli were also observed in SP-D (-/-) mice. Phospholipid composition was unaltered in SP-D (-/-) mice, but surfactant morphology was abnormal, consisting of dense phospholipid membranous arrays with decreased tubular myelin. The pulmonary lipoidosis in the SP-D (-/-) mice was not associated with accumulation of surfactant proteins B or C, or their mRNAs, distinguishing the disorder from alveolar proteinosis syndromes. Surfactant protein A mRNA was reduced and, SP-A protein appeared to be reduced in SP-D (-/-) compared with wild type mice. Targeting of the mouse SP-D gene caused accumulation of surfactant lipid and altered phospholipid structures, demonstrating a previously unsuspected role for SP-D in surfactant lipid homeostasis in vivo.