Activation of PPARγ reverses a defect of surfactant synthesis in mice lacking two types of fatty acid binding protein

Lung surfactant is a lipid–protein-film covering the inner alveolar surface. We have previously shown that double knock-out (d-ko) mice lacking both the epidermal-type (E-) and the heart-type (H-) fatty acid binding protein (FABP) exhibit a defect of surfactant synthesis in alveolar type II cells that can be corrected by feeding pioglitazone, a drug that activates peroxisome proliferator-activated receptor gamma (PPARγ). Here, we demonstrate first that healthy surfactant at collapse pressure produces protrusions composed of bilayers but not folds, second that the d-ko effect profoundly perturbs lipid/hydrophobic protein composition, pressure-area isotherm, and structural organisation of the surfactant at nanoscale, parameters that are critical for the normal breathing cycle. In support of these data in vivo measurements of lung function reveal that maximum compliance in d-ko vs. wild-type mice is significantly reduced. Further, we show that the biophysical phenotype can be corrected substantially with pioglitazone. Finally, we show that d-ko alveolar cells up-regulate liver-type (L-) FABP, a member of the FABP family that we have previously shown to interact with PPARγ. Taken together, these data suggest that PPARγ agonists could be a tool to repair surfactant damage caused by dysfunctional alveolar lipid metabolism, and provide in vivo support for L-FABP aided signaling.     

L-FABP is exclusively expressed in alveolar macrophages within the myeloid lineage: evidence for a PPARalpha-independent expression

Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particular, PPARgamma is involved in monocyte/macrophage differentiation. Members of the fatty acid-binding protein (FABP) family have been reported to function as transactivators for PPARs. Therefore, the expression of PPARs and FABPs in the myeloid lineage was investigated by real-time PCR and immunofluorescence analysis. We found adipocyte-, epidermal-, and heart-type FABP to be ubiquitously expressed within the myeloid lineage. In contrast, liver-type FABP was exclusively detected in murine alveolar macrophages (AM), confirmed on protein level by double fluorescence analysis. The PPAR subtypes also showed a temporally and spatially regulated expression pattern in myeloid cells: the beta-subtype was expressed in bone marrow, peritoneal, and alveolar macrophages, whereas it was not detected in dendritic cells (DCs). The gamma1-isoform was present in all cells, however, at different levels, whereas the gamma2-isoform was expressed in alveolar macrophages and dendritic cells. A low level PPARalpha mRNA could be detected in peritoneal macrophages and immature dendritic cells but not in mature dendritic cells and bone marrow macrophages. Interestingly, PPARalpha mRNA was also absent in the alveolar macrophages although liver-type FABP was expressed, indicating that gene expression of liver-type FABP was independent of PPARalpha. Since liver-type FABP is known as transactivator of PPARgamma the simultaneous expression of both proteins may have general implications for the activation of PPARgamma in alveolar macrophages.     

Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(-/-)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(-/-) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(-/-), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(-/-) mice. SA from SP-D(-/-) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(-/-) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(-/-) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.     

ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar proteinosis

Patients with pulmonary alveolar proteinosis (PAP) display impaired surfactant clearance, foamy, lipid-filled alveolar macrophages, and increased cholesterol metabolites within the lung. Neutralizing autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) are also present, resulting in virtual GM-CSF deficiency. We investigated ABCG1 and ABCA1 expression in alveolar macrophages of PAP patients and GM-CSF knockout (KO) mice, which exhibit PAP-like pulmonary pathology and increased pulmonary cholesterol. Alveolar macrophages from both sources displayed a striking similarity in transporter gene dysregulation, consisting of deficient ABCG1 accompanied by highly increased ABCA1. Peroxisome proliferator-activated receptor gamma (PPARgamma), a known regulator of both transporters, was deficient, as reported previously. In contrast, the liver X receptor alpha, which also upregulates both transporters, was highly increased. GM-CSF treatment increased ABCG1 expression in macrophages in vitro and in PAP patients in vivo. Overexpression of PPARgamma by lentivirus-PPARgamma transduction of primary alveolar macrophages, or activation by rosiglitazone, also increased ABCG1 expression. These results suggest that ABCG1 deficiency in PAP and GM-CSF KO alveolar macrophages is attributable to the absence of a GM-CSF-mediated PPARgamma pathway. These findings document the existence of ABCG1 deficiency in human lung disease and highlight a critical role for ABCG1 in surfactant 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.     

Fatty acid binding protein 3 as a potential mediator for diabetic nephropathy in eNOS deficient mouse

In human diabetic nephropathy, glomerular injury was found to comprise lipid droplets, suggesting that abnormal lipid metabolism might take place in the development of diabetic glomerular injury. However, its precise mechanism remains unclear. Fatty acid binding protein (FABP) is currently considered as a key molecule for lipid metabolism. Since diabetic eNOS knockout (KO) mouse is considered to be a good model for human diabetic nephropathy, we here investigated whether FABP could mediate glomerular injury in this model. We found that glomerular injuries were associated with inflammatory processes, such as macrophage infiltration and MCP-1 induction. Microarray assay with isolated glomeruli revealed that among 10 isoforms in FABP family, FABP3 mRNA was most highly expressed in diabetic eNOSKO mice compared to non-diabetic eNOSKO mice. FABP3 protein was found to be located in the mesangial cells. Overexpression of FABP3 resulted in a greater response to palmitate, a satulated FA, to induce MCP-1 in the rat mesangial cells. In turn, the heart, a major organ for FABP3 protein in normal condition, failed to alter its expression level under diabetic condition in either wild type or eNOSKO mice. In conclusion, FABP3 is induced in the mesangial cells and likely a mediator to induce MCP-1 in the diabetic nephropathy.     

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.     

Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFG(b1)/EGF in murine induced pluripotent stem cell-derived alveolar Type II-like cells

Induced pluripotent stem (iPS) cells are derived from reprogrammed somatic cells and are similar to embryonic stem (ES) cells in morphology, gene/protein expression, and pluripotency. In this study, we explored the potential of iPS cells to differentiate into alveolar Type II (ATII)-like epithelial cells. Analysis using quantitative real time polymerase chain reaction and immunofluorescence staining showed that pulmonary surfactant proteins commonly expressed by ATII cells such as surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC) were upregulated in the differentiated cells. Microphilopodia characteristics and lamellar bodies were observed by transmission electron microscopy and lipid deposits were verified by Nile Red and Periodic Acid Schiff staining. C3 complement protein, a specific feature of ATII cells, was present at high levels in culture supernatants demonstrating functionality of these cells in culture. These data show that the differentiated cells generated from iPS cells using a culture method developed previously (Rippon et al., 2006) are ATII-like cells. To further characterize these ATII-like cells, we tested whether they could undergo epithelial to mesenchymal transition (EMT) by exposure to drugs that induce lung fibrosis in mice, such as bleomycin, and the combination of transforming growth factor beta1 (TGF(b1)) and epidermal growth factor (EGF). When the ATII-like cells were exposed to either bleomycin or a TGF(b1)-EGF cocktail, they underwent phenotypic changes including acquisition of a mesenchymal/fibroblastic morphology, upregulation of mesenchymal markers (Col1, Vim, a-Sma, and S100A4), and downregulation of surfactant proteins and E-cadherin. We have shown that ATII-like cells can be derived from skin fibroblasts and that they respond to fibrotic stimuli. These cells provide a valuable tool for screening of agents that can potentially ameliorate or prevent diseases involving lung fibrosis.     

Restoration of PPARγ reverses lipid accumulation in alveolar macrophages of GM-CSF knockout mice

Pulmonary alveolar proteinosis (PAP) is a lung disease characterized by a deficiency of functional granulocyte macrophage colony-stimulating factor (GM-CSF) resulting in surfactant accumulation and lipid-engorged alveolar macrophages. GM-CSF is a positive regulator of PPARγ that is constitutively expressed in healthy alveolar macrophages. We previously reported decreased PPARγ and ATP-binding cassette transporter G1 (ABCG1) levels in alveolar macrophages from PAP patients and GM-CSF knockout (KO) mice, suggesting PPARγ and ABCG1 involvement in surfactant catabolism. Because ABCG1 represents a PPARγ target, we hypothesized that PPARγ restoration would increase ABCG1 and reduce macrophage lipid accumulation. Upregulation of PPARγ was achieved using a lentivirus expression system in vivo. GM-CSF KO mice received intratracheal instillation of lentivirus (lenti)-PPARγ or control lenti-eGFP. Ten days postinstillation, 79% of harvested alveolar macrophages expressed eGFP, demonstrating transduction. Alveolar macrophages showed increased PPARγ and ABCG1 expression after lenti-PPARγ instillation, whereas PPARγ and ABCG1 levels remained unchanged in lenti-eGFP controls. Alveolar macrophages from lenti-PPARγ-treated mice also exhibited reduced intracellular phospholipids and increased cholesterol efflux to HDL, an ABCG1-mediated pathway. In vivo instillation of lenti-PPARγ results in: 1) upregulating ABCG1 and PPARγ expression of GM-CSF KO alveolar macrophages, 2) reducing intracellular lipid accumulation, and 3) increasing cholesterol efflux activity.     

Regulation of lung surfactant secretion by microRNA-150

P2X7 receptor (P2X7R) is a purinergic ion-channel receptor. We have previously shown that the activation of P2X7R in alveolar type I cells stimulates surfactant secretion in alveolar type II cells. In this study, we determined whether miR-150 regulates P2X7R-mediated surfactant secretion. The miR-150 expression level in alveolar type II cells was much higher than alveolar type I cells, which was inversely correlated with the P2X7R protein level. An adenovirus expressing miR-150 significantly reduced the P2X7R protein expression in E10 cells, an alveolar type I cell line. Furthermore, pre-treatment of E10 cells with the adenovirus reduced the surfactant secretion induced by E10 cell conditioned medium. Our study demonstrates that miR-150 regulates surfactant secretion through P2X7R.     

Phenotype of palmitic acid transport and of signalling in alveolar type II cells from E/H-FABP double-knockout mice: contribution of caveolin-1 and PPARgamma

Based on the assumption that fatty-acid-binding proteins (FABPs) of the epidermal-type (E-FABP) and heart-type (H-FABP) in murine alveolar type II (TII) cells mediate the synthesis of dipalmitoyl phosphatidylcholine (DPPC), the main surfactant phospholipid, we analysed TII cells isolated from wild-type (wt) and E/H-FABP double-knockout (double-ko) mice. Application of labelled palmitic acid to these cells revealed a drop in uptake, beta-oxidation, and incorporation into neutral lipids and total phosphatidylcholine (PC) of TII cells from double-ko mice. Whereas incorporation of labelled palmitic acid into DPPC remained unchanged, degradation studies demonstrated a substantial shift in DPPC synthesis from de novo to reacylation. In addition, increased expression of mRNAs and proteins of caveolin-1 and PPARgamma, and an increase of the mRNA encoding fatty acid translocase (FAT) was observed in the double-ko phenotype. As caveolin-1 interacted with PPARgamma, we assumed that FAT, caveolin-1, and PPARgamma form a signalling chain for fatty acid or drug. Consequently, PPARgamma-selective pioglitazone was added to the diet of double-ko mice. We found that further activation of PPARgamma could 'heal' the E/H-FABP double-ko effect in these TII cells as transport and utilisation of labelled palmitic acid restored a wt phenocopy. This indicated that E-FABP and/or H-FABP are involved in the mediation of DPPC synthesis in wt TII cells.     

Identification of a potential biomarker for FABP4 inhibition: the power of lipidomics in preclinical drug testing

The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E-deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficiency. However, despite the therapeutically attractive long-term effects of FABP4 inhibition, an acute biomarker for drug action is lacking. The authors applied mass spectrometry lipidomics analysis to in vitro and in vivo (plasma and adipose tissue) samples upon inhibitor treatment. They report the identification of a potential biomarker for acute in vivo FABP4 inhibition that is applicable for further investigations and can be implemented in simple and fast-flow injection mass spectrometry assays. In addition, this approach can be considered a proof-of-principle study that can be applied to other lipid-pathway targeting mechanisms.     

Spectral monitoring of surfactant clearance during alveolar epithelial type II cell differentiation

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.     

GM-CSF mediates alveolar epithelial type II cell changes, but not emphysema-like pathology, in SP-D-deficient mice

Surfactant protein D (SP-D) is a member of the collectin subfamily of C-type lectins, pattern recognition proteins participating in the innate immune response. Gene-targeted mice deficient in SP-D develop abnormalities in surfactant homeostasis, hyperplasia of alveolar epithelial type II cells, and emphysema-like pathology. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is required for terminal differentiation and subsequent activation of alveolar macrophages, including the expression of matrix metalloproteinases and reactive oxygen species, factors thought to contribute to lung remodeling. Type II cells also express the GM-CSF receptor. Thus we hypothesized GM-CSF might mediate some or all of the cellular and structural abnormalities in the lungs of SP-D-deficient mice. To test this, SP-D (D-G+) and GM-CSF (D+G-) single knockout mice as well as double knockout mice deficient for both SP-D and GM-CSF (D-G-) were analyzed by design-based stereology. Compared with wild type, D-G+ as well as D+G- mice showed decreased alveolar numbers, increased alveolar sizes, and decreased alveolar epithelial surface areas. These emphysema-like changes were present to a greater extent in D-G- mice. D-G+ mice developed type II cell hyperplasia and hypertrophy with increased intracellular surfactant pools, whereas D+G- mice had smaller type II cells with decreased intracellular surfactant pools. In contrast to the emphysematous changes, the type II cell alterations were mostly corrected in D-G- mice. These results indicate that GM-CSF-dependent macrophage activity is not necessary for emphysema development in SP-D-deficient mice, but that type II cell metabolism and proliferation are, either directly or indirectly, regulated by GM-CSF in this model.     

Surface tension influences cell shape and phagocytosis in alveolar macrophages

The effect of surface tension on alveolar macrophage shape and phagocytosis was assessed in vivo and in vitro. Surface tension was regulated in vivo by conditionally expressing surfactant protein (SP)-B in Sftpb-/- mice. Increased surface tension and respiratory distress were produced by depletion of SP-B and were readily reversed by repletion of SP-B in vivo. Electron microscopy was used to demonstrate that alveolar macrophages were usually located beneath the surfactant film on the alveolar surfaces. Reduction of SP-B increased surface tension and resulted in flattening of alveolar macrophages on epithelial surfaces in vivo. Phagocytosis of intratracheally injected fluorescent microbeads by alveolar macrophages was decreased during SP-B deficiency and was restored by repletion of SP-B in vivo. Incubation of MH-S cells, a mouse macrophage cell line, with inactive surfactant caused cell flattening and decreased phagocytosis in vitro, findings that were reversed by the addition of sheep surfactant or phospholipid containing SP-B. SP-B controls surface tension by forming a surfactant phospholipid film that regulates shape and nonspecific phagocytic activity of alveolar macrophages on the alveolar surface.     

Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro

The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.