Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.     

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225–230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I^−/− mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I^−/− × apoE^−/− mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225–230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225–230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.     

Apolipoprotein A-I Modulates Processes Associated with Diet-Induced Nonalcoholic Fatty Liver Disease in Mice

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein (HDL). We investigated the involvement of apoA-I in diet-induced accumulation of triglycerides in hepatocytes and its potential role in the treatment of nonalcoholic fatty liver disease (NAFLD). ApoA-I–deficient (apoA-I^−/−) mice showed increased diet-induced hepatic triglyceride deposition and disturbed hepatic histology while they exhibited reduced glucose tolerance and insulin sensitivity. Quantification of FASN (fatty acid synthase 1), DGAT-1 (diacylglycerol O-acyltransferase 1), and PPARγ (peroxisome proliferator-activated receptor γ) mRNA expression suggested that the increased hepatic triglyceride content of the apoA-I^−/− mice was not due to de novo synthesis of triglycerides. Similarly, metabolic profiling did not reveal differences in the energy expenditure between the two mouse groups. However, apoA-I^−/− mice exhibited enhanced intestinal absorption of dietary triglycerides (3.6 ± 0.5 mg/dL/min for apoA-I^−/− versus 2.0 ± 0.7 mg/dL/min for C57BL/6 mice, P < 0.05), accelerated clearance of postprandial triglycerides and a reduced rate of hepatic very low density lipoprotein (VLDL) triglyceride secretion (9.8 ± 1.1 mg/dL/min for apoA-I^−/− versus 12.5 ± 1.3 mg/dL/min for C57BL/6 mice, P < 0.05). In agreement with these findings, adenovirus-mediated gene transfer of apoA-I_Milano in apoA-I^−/− mice fed a Western-type diet for 12 wks resulted in a significant reduction in hepatic triglyceride content and an improvement of hepatic histology and architecture. Our data extend the current knowledge on the functions of apoA-I, indicating that in addition to its well-established properties in atheroprotection, it is also an important modulator of processes associated with diet-induced hepatic lipid deposition and NAFLD development in mice. Our findings raise the interesting possibility that expression of therapeutic forms of apoA-I by gene therapy approaches may have a beneficial effect on NAFLD.     

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT

The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I^−/− mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-β-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I^−/− × apoE^−/− mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-β-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1^−/− or LCAT^−/− mice. Coexpression of apoA-IV and LCAT in apoA-I^−/− mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.     

Enhanced Hepatic apoA-I Secretion and Peripheral Efflux of Cholesterol and Phospholipid in CD36 Null Mice

CD36 facilitates oxidized low density lipoprotein uptake and is implicated in development of atherosclerotic lesions. CD36 also binds unmodified high and very low density lipoproteins (HDL, VLDL) but its role in the metabolism of these particles is unclear. Several polymorphisms in the CD36 gene were recently shown to associate with serum HDL cholesterol. To gain insight into potential mechanisms for these associations we examined HDL metabolism in CD36 null (CD36^−/−) mice. Feeding CD36^−/− mice a high cholesterol diet significantly increased serum HDL, cholesterol and phospholipids, as compared to wild type mice. HDL apolipoproteins apoA-I and apoA-IV were increased and shifted to higher density HDL fractions suggesting altered particle maturation. Clearance of dual-labeled HDL was unchanged in CD36^−/− mice and cholesterol uptake from HDL or LDL by isolated CD36^−/− hepatocytes was unaltered. However, CD36^−/− hepatocytes had higher cholesterol and phospholipid efflux rates. In addition, expression and secretion of apoA-I and apoA-IV were increased reflecting enhanced PXR. Similar to hepatocytes, cholesterol and phospholipid efflux were enhanced in CD36^−/− macrophages without changes in protein levels of ABCA1, ABCG1 or SR-B1. However, biotinylation assays showed increased surface ABCA1 localization in CD36^−/− cells. In conclusion, CD36 influences reverse cholesterol transport and hepatic ApoA-I production. Both pathways are enhanced in CD36 deficiency, increasing HDL concentrations, which suggests the potential benefit of CD36 inhibition.     

Apolipoprotein A-I Modulates Regulatory T Cells in Autoimmune LDLr?/?, ApoA-I?/? Mice

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr^−/−, apoA-I^−/− (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 μg of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.     

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr^−/−, PCPE2^−/− mice, which had elevated HDL levels compared with LDLr^−/− mice with similar LDL concentrations. We found that LDLr^−/−, PCPE2^−/− mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr^−/− mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr^−/−, PCPE2^−/− mice was similar to that reported for LDLr^−/−, apoA-I^−/− mice, which lack any apoA-I/HDL. Furthermore, LDLr^−/−, PCPE2^−/− mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr^−/− mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.     

IL-17A Modulates Oxidant Stress-Induced Airway Hyperresponsiveness but Not Emphysema

IL-17A induces the release of pro-inflammatory cytokines and of reactive oxygen species which could lead to neutrophilic inflammation. We determined the role of IL-17 receptor (IL-17R) signalling in oxidant-induced lung emphysema and airway hyperresponsiveness. IL-17R^−/− and wild-type C57/BL6 mice were exposed to ozone (3 ppm; 3 hours) for 12 times over 6 weeks. Bronchial responsiveness to acetylcholine was measured, and lungs were retrieved. Mean linear intercept (L_m) and isometric contractile responses of intrapulmonary airways to acetylcholine were determined. In wild-type mice but not in IL-17R^−/−, chronic ozone exposure caused airway hyperresponsiveness. The increase in L_m after chronic ozone exposure of wild-type mice was also observed in IL-17R^−/− mice. The increased maximal contractile response to acetylcholine seen in airways of wild-type mice exposed to ozone was abolished in IL-17R^−/− mice. p38-mitogen-activated protein kinase (MAPK) and dexamethasone-dependent increase in contractile response was reduced in airways from IL-17R^−/− ozone-exposed mice. Lung inflammation scores were not altered in IL-17R^−/− mice exposed to ozone compared to wild-type mice. The increased release of IL-17 and IL-1β, and the activation of p38 MAPK in the lungs of ozone-exposed mice was reduced in IL-17R^−/− mice. IL-17R signalling underlies the increase in airway hyperresponsiveness seen after ozone exposure, mediated by the increased contractility of airway smooth muscle. The emphysema and lung inflammation induced by ozone is not dependent on IL-17.     

apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE^−/−) or apoA-I-deficient (apoA-I^−/−)×apoE^−/− mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE^−/− mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE^−/− mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE^−/− mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.     

Dysfunctional HDL containing L159R ApoA-I leads to exacerbation of atherosclerosis in hyperlipidemic mice

The mutation L159R apoA-I or apoA-I_L159R (FIN) is a single amino acid substitution within the sixth helical repeat of apoA-I. It is associated with a dominant negative phenotype, displaying hypoalphaproteinemia and an increased risk for atherosclerosis in humans. Mice lacking both mouse apoA-I and LDL receptor (LDL^−/−, apoA-I^−/−) (double knockout or DKO) were crossed > 9 generations with mice transgenic for human FIN to obtain L159R apoA-I, LDLr^−/−, ApoA-I^−/− (FIN-DKO) mice. A similar cross was also performed with human wild-type (WT) apoA-I (WT-DKO). In addition, FIN-DKO and WT-DKO were crossed to obtain WT/FIN-DKO mice. To determine the effects of the apoA-I mutations on atherosclerosis, groups of each genotype were fed either chow or an atherogenic diet for 12 weeks. Interestingly, the production of dysfunctional HDL-like particles occurred in DKO and FIN-DKO mice. These particles were distinct with respect to size, and their enrichment in apoE and cholesterol esters. Two-dimensional gel electrophoresis indicated that particles found in the plasma of FIN-DKO mice migrated as large α₃-HDL. Atherosclerosis analysis showed that FIN-DKO mice developed the greatest extent of aortic cholesterol accumulation compared to all other genotypes, including DKO mice which lack any apoA-I. Taken together these data suggest that the presence of large apoE enriched HDL particles containing apoA-I L159R lack the normal cholesterol efflux promoting properties of HDL, rendering them dysfunctional and pro-atherogenic. In conclusion, large HDL-like particles containing apoE and apoA-I_L159R contribute rather than protect against atherosclerosis, possibly through defective efflux properties and their potential for aggregation at their site of interaction in the aorta. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Apolipoprotein A-I Deficiency Increases Cerebral Amyloid Angiopathy and Cognitive Deficits in APP/PS1ΔE9 Mice

A hallmark of Alzheimer disease (AD) is the deposition of amyloid β (Aβ) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aβ₄₀ aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Aβ₄₂ and decreases Aβ₄₂ toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1ΔE9 to apoA-I^KO mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1ΔE9 mice. Further characterization of APP/PS1ΔE9/apoA-I^KO mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble Aβ oligomer levels, Aβ plaque load, or levels of insoluble Aβ in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aβ isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I^KO mice, insoluble Aβ₄₀ is increased more than 10-fold, and Aβ₄₂ is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1ΔE9/apoA-I^KO mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased Aβ toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1ΔE9 mice in parallel to significantly increased cerebral amyloid angiopathy.     

Short and dysfunctional telomeres protect from allergen‐induced airway inflammation

Asthma is a chronic inflammatory disease affecting 300 million people worldwide. As telomere shortening is a well‐established hallmark of aging and that asthma incidence decreases with age, here we aimed to study the role of short telomeres in asthma pathobiology. To this end, wild‐type and telomerase‐deficient mice with short telomeres (third‐generation (G3 Tert ^−/− mice)) were challenged with intranasal house dust mite (HDM) extract. We also challenged with HDM wild‐type mice in which we induced a telomere dysfunction by the administration of 6‐thio‐2´‐deoxyguanosine (6‐thio‐dG). Following HDM exposure, G3 Tert ^−/− and 6‐thio‐dG treated mice exhibited attenuated eosinophil counts and presence of hematopoietic stem cells in the bone marrow, as well as lower levels of IgE and circulating eosinophils. Accordingly, both G3 Tert ^−/− and 6‐thio‐dG treated wild‐type mice displayed reduced airway hyperresponsiveness (AHR), as indicated by decreased airway remodeling and allergic airway inflammation markers in the lung. Furthermore, G3 Tert ^−/− and 6‐thio‐dG treated mice showed lower differentiation of Club cells, attenuating goblet cell hyperplasia. Club cells of G3 Tert ^−/− and 6‐thio‐dG treated mice displayed increased DNA damage and senescence and reduced proliferation. Thus, short/dysfunctional telomeres play a protective role in murine asthma by impeding both AHR and mucus secretion after HDM exposure. Therefore, our findings imply that telomeres play a relevant role in allergen‐induced airway inflammation.     

An Apolipoprotein A-I Mimetic Peptide Designed with a Reductionist Approach Stimulates Reverse Cholesterol Transport and Reduces Atherosclerosis in Mice

Apolipoprotein A-I (apoA-I) mimetic peptides are considered a promising novel therapeutic approach to prevent and/or treat atherosclerosis. An apoA-I mimetic peptide ELK-2A2K2E was designed with a reductionist approach and has shown exceptional activity in supporting cholesterol efflux but modest anti-inflammatory and anti-oxidant properties in vitro. In this study we compared these in vitro properties with the capacity of this peptide to modify rates of reverse cholesterol transport and development of atherosclerosis in mouse models. The peptide enhanced the rate of reverse cholesterol transport in C57BL/6 mice and reduced atherosclerosis in Apoe^−/− mice receiving a high fat diet. The peptide modestly reduced the size of the plaques in aortic arch, but was highly active in reducing vascular inflammation and oxidation. Administration of the peptide to Apoe^−/− mice on a high fat diet reduced the levels of total, high density lipoprotein and non-high density lipoprotein cholesterol and triglycerides. It increased the proportion of smaller HDL particles in plasma at the expense of larger HDL particles, and increased the capacity of the plasma to support cholesterol efflux. Thus, ELK-2A2K2E peptide reduced atherosclerosis in Apoe^−/− mice, however, the functional activity profile after chronic in vivo administration was different from that found in acute in vitro studies.     

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition,Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6^−/− mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6^−/− mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6^−/− mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.     

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1^−/−) mice were used. Apoa1^−/− mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1^−/− mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1^−/− mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.     

A novel approach to oral apoA-I mimetic therapy

Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR^−/−) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.     

Non-Essential Role for TLR2 and Its Signaling Adaptor Mal/TIRAP in Preserving Normal Lung Architecture in Mice

Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an in vivo genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in Tlr4^−/− mice by 6 months of age, the lungs of Tlr2^−/− mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in Tlr4^−/− mice, along with up-regulation of NADPH oxidase 3 (Nox3) mRNA expression. With respect to Mal, the architecture of the lungs of Mal^−/− mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free Mal^−/− mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous Tlr4^−/− mice. In conclusion, our data provide in vivo evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema.     

B Cells Play Key Roles in Th2-Type Airway Immune Responses in Mice Exposed to Natural Airborne Allergens

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH^−/− mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH^−/− mice. The allergen-induced expansion of CD4⁺ T cells was impaired in the lungs and draining lymph nodes of JH^−/− mice. Furthermore, lymphocytes from JH^−/− mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.     

Matrix Metalloproteinase 9 Exerts Antiviral Activity against Respiratory Syncytial Virus

Increased lung levels of matrix metalloproteinase 9 (MMP9) are frequently observed during respiratory syncytial virus (RSV) infection and elevated MMP9 concentrations are associated with severe disease. However little is known of the functional role of MMP9 during lung infection with RSV. To determine whether MMP9 exerted direct antiviral potential, active MMP9 was incubated with RSV, which showed that MMP9 directly prevented RSV infectivity to airway epithelial cells. Using knockout mice the effect of the loss of Mmp9 expression was examined during RSV infection to demonstrate MMP9’s role in viral clearance and disease progression. Seven days following RSV infection, Mmp9 ^-/- mice displayed substantial weight loss, increased RSV-induced airway hyperresponsiveness (AHR) and reduced clearance of RSV from the lungs compared to wild type mice. Although total bronchoalveolar lavage fluid (BALF) cell counts were similar in both groups, neutrophil recruitment to the lungs during RSV infection was significantly reduced in Mmp9 ^-/- mice. Reduced neutrophil recruitment coincided with diminished RANTES, IL-1β, SCF, G-CSF expression and p38 phosphorylation. Induction of p38 signaling was required for RANTES and G-CSF expression during RSV infection in airway epithelial cells. Therefore, MMP9 in RSV lung infection significantly enhances neutrophil recruitment, cytokine production and viral clearance while reducing AHR.     

“Ciliophagy”: The consumption of cilia components by autophagy

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) associated with respiratory epithelial cell cilia shortening and impaired mucociliary clearance (MCC). The underlying cellular and molecular mechanisms for CS-associated cilia shortening have remained incompletely understood. We have previously demonstrated increased autophagy in the lungs of COPD patients; however, whether or not this process is selective for specific autophagic targets in the lung was not elucidated. Based on observations that increased morphological and biochemical indicators of autophagy correlate with cilia shortening in our models, we posited that autophagy might regulate cilia length in response to CS in the lung. We demonstrate that CS-induced cilia shortening occurs through an autophagy-dependent mechanism mediated by the deacetylase HDAC6 (histone deacetylase 6). Autophagy-impaired (Becn1^+/−, map1lc3b^−/−, or Hdac6^-/Y) mice resist CS-induced cilia shortening. Furthermore, cilia components are identified as autophagic substrates during CS exposure. Assessment of airway cilia function using a 3D MCC assay demonstrates that Becn1^+/−, map1lc3b^−/−, and Hdac6^-/Y mice or mice injected with the HDAC6 inhibitor tubastatin A are protected from CS-associated mucociliary dysfunction. We concluded that an autophagy-dependent pathway regulates cilia length during CS exposure, which identifies new pathways and targets in COPD.