Blocking RAGE expression after injury reduces inflammation in mouse model of acute lung injury.

BACKGROUND: Receptor for Advanced Glycated Endproducts (RAGE) plays a major role in the inflammatory response to infectious and toxin induced acute lung injury. We tested the hypothesis that a RAGE blocking antibody when administered after the onset of injury can reduce lung inflammation compared to control antibody. METHODS: Male and female C57BL/6 (WT) mice were used. Forty-six received lipopolysaccharide (LPS) and 26 PBS by nasal instillation on day one, repeated on day three. On day 2, 36 mice receiving LPS were divided into two groups of 18, one treated with 200 µg of non-immune isotype control IgG and the second group treated with 200 µg of anti-RAGE Ab, each dose divided between IV and IP. Ten of the 46 were not treated. On day 4, before euthanasia, mice were injected with fluorescein isothiocyanate (FITC) labelled albumen. BALF and serum samples were collected as well as lung tissue for immunohistochemistry (IHC). BALF was analyzed for cell (leukocyte) counts, for FITC BALF/serum ratios indicating pulmonary vascular leak, and for cytokines/chemokines using bead based multiplex assays. Quantitative IHC was performed for MPO and RAGE. RESULTS: Ten LPS mice showed minimal inflammation by all measures indicating poor delivery of LPS and were excluded from analysis leaving n = 11 in the LPS + IgG group and n = 12 in the LPS + anti-RAGE group. BALF cell counts were low in the PBS administered mice (4.9 ± 2.1 × 105/ml) and high in the LPS injured untreated mice (109 ± 34) and in the LPS + IgG mice (91 ± 54) while in comparison, LPS + anti-RAGE ab mice counts were significantly lower (51.3 ± 18 vs. LPS + IgG, P = 0.03). The BALF/serum FITC ratios were lower for the LPS + anti-RAGE mice than for the LPS + IgG mice indicating less capillary leakiness. Quantitative IHC RAGE staining was lower in the LPS + anti-RAGE ab mice than in the LPS + IgG treated mice (P = 0.02). CONCLUSIONS: These results describe a four-day LPS protocol to sustain lung injury and allow for treatment and suggests that treatment aimed at blocking RAGE when given after onset of injury can reduce lung inflammation.

The Role of Secreted Frizzled-related Protein-1 in Allergic Asthma.

Although allergic asthma is a highly prevalent chronic inflammatory condition, the underlying pathogenesis driving T-helper cell type 2 inflammation is not well understood. Wnt/ß-catenin signaling has been implicated, but the influence of individual members of the pathway is not clear. We hypothesized that SFRP-1 (secreted frizzled-related protein-1), a Wnt signaling modulator, plays an important role in the development of allergic inflammation in asthma. Using an in vivo house dust mite asthma model, SFRP-1-/- mice were sensitized, and their BAL fluid was collected to evaluate airway inflammation. SFRP-1-/- mice exhibited less inflammation with reduced cellular infiltration and concentration of IL-5 in bronchoalveolar lavage fluid compared with wild-type (WT) mice. Similar findings were observed in WT mice treated with SFRP-1 inhibitor, WAY316606. Alveolar macrophages from sensitized SFRP-1-/- mice demonstrated reduced alternative polarization compared with WT, indicating that macrophages could mediate the alteration in inflammation seen in these mice. These findings suggest that SFRP-1 is an important potentiator of asthmatic airway inflammation.

Attenuation of pulmonary injury by an inhaled MMP inhibitor in the endotoxin lung injury model.

Acute respiratory distress syndrome (ARDS) is characterized by pulmonary edema and poor gas exchange resulting from severe inflammatory lung injury. Neutrophilic infiltration and increased pulmonary vascular permeability are hallmarks of early ARDS and precipitate a self-perpetuating cascade of inflammatory signaling. The biochemical processes initiating these events remain unclear. Typically associated with extracellular matrix degradation, recent data suggest matrix metalloproteinases (MMPs) are regulators of pulmonary inflammation. To demonstrate that inhalation of a broad MMP inhibitor attenuates LPS induced pulmonary inflammation. Nebulized CGS27023AM (CGS) was administered to LPS-injured mice. Pulmonary CGS levels were examined by mass spectroscopy. Inflammatory scoring of hematoxylin-eosin sections, examination of vascular integrity via lung wet/dry and bronchoalveolar lvage/serum FITC-albumin ratios were performed. Cleaved caspase-3 levels were also assessed. Differential cell counts and pulse-chase labeling were utilized to determine the effects of CGS on neutrophil migration. The effects of CGS on human neutrophil migration and viability were examined using Boyden chambers and MTT assays. Nebulization successfully delivered CGS to the lungs. Treatment decreased pulmonary inflammatory scores, edema, and apoptosis in LPS treated animals. Neutrophil chemotaxis was reduced by CGS treatment, with inhalation causing significant reductions in both the total number and newly produced bromodeoxyuridine-positive cells infiltrating the lung. Mechanistic studies on cells isolated from humans demonstrate that CGS-treated neutrophils exhibit decreased chemotaxis. The protective effect observed following treatment with a nonspecific MMP inhibitor indicates that one or more MMPs mediate the development of pulmonary edema and neutrophil infiltration in response to LPS injury. In accordance with this, inhaled MMP inhibitors warrant further study as a potential new therapeutic avenue for treatment of acute lung injury.

Single-photon emission computed tomography/computed tomography imaging of RAGE in smoking-induced lung injury.

BACKGROUND: Expression of the Receptor for Advanced Glycation Endproducts (RAGE) initiates pro-inflammatory pathways resulting in lung destruction. We hypothesized that RAGE directed imaging demonstrates increased lung uptake in smoke-exposure. METHODS: After exposure to room air or to cigarette smoke for 4-weeks or 16-weeks, rabbits were injected with 99mTc-anti-RAGE F(ab')2 and underwent Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) imaging. Lung radiotracer uptake was calculated as percent injected dose (%ID). Lungs were dissected for gamma well counting and histological analysis. RESULTS: 99mTc-anti-RAGE F(ab')2 SPECT/CT imaging demonstrated increased lung expression of RAGE with smoke exposure compared to room air control at 4-weeks: Room air right (R) 0.75 ± 0.38%ID, left (L) 0.62 ± 0.32%ID vs. Smoke exposed R 0.17 ± 0.03, L 0.17 ± 0.02%ID (p = 0.02 and 0.028, respectively). By 16-weeks of smoke exposure, the uptake decreased to 0.19 ± 0.05%ID R and 0.17 ± 0.05%ID L, significantly lower than 4-week imaging (p = 0.0076 and 0.0129 respectively). Staining for RAGE confirmed SPECT results, with the RAGE ligand HMGB1 upregulated in the macrophages of 4-week smoke-exposed rabbits. CONCLUSIONS: RAGE-directed imaging identified pulmonary RAGE expression acutely in vivo in an animal model of emphysema early after smoke exposure, with diminution over time. These studies document the extent and time course of RAGE expression under smoke exposure conditions and could be utilized for disease monitoring and examining response to future RAGE-targeted therapies.

A critical role for ABC transporters in persistent lung inflammation in the development of emphysema after smoke exposure.

Macrophage infiltration is common to both emphysema and atherosclerosis, and cigarette smoke down-regulates the macrophage cholesterol efflux transporter ATP binding cassette (ABC)A1. This decreased cholesterol efflux results in lipid-laden macrophages. We hypothesize that cigarette smoke adversely affects cholesterol transport via an ABCA1-dependent mechanism in macrophages, enhancing TLR4/myeloid differentiation primary response gene 88 (Myd88) signaling and resulting in matrix metalloproteinase (MMP) up-regulation and exacerbation of pulmonary inflammation. ABCA1 is significantly down-regulated in the lung upon smoke exposure conditions. Macrophages exposed to cigarette smoke in vivo and in vitro exhibit impaired cholesterol efflux correlating with significantly decreased ABCA1 expression, up-regulation of the TLR4/Myd88 pathway, and downstream MMP-9 and MMP-13 expression. Treatment with liver X receptor (LXR) agonist restores ABCA1 expression after short-term smoke exposure and attenuates the inflammatory response; after long-term smoke exposure, there is also attenuated physiologic and morphologic changes of emphysema. In vitro, treatment with LXR agonist decreases macrophage inflammatory activation in wild-type but not ABCA1 knockout mice, suggesting an ABCA1-dependent mechanism of action. These studies demonstrate an important association between cigarette smoke exposure and cholesterol-mediated pathways in the macrophage inflammatory response. Modulation of these pathways through manipulation of ABCA1 activity effectively blocks cigarette smoke-induced inflammation and provides a potential novel therapeutic approach for the treatment of chronic obstructive pulmonary disease.-Sonett, J., Goldklang, M., Sklepkiewicz, P., Gerber, A., Trischler, J., Zelonina, T., Westerterp, M., Lemaître, V., Okada, V., D'Armiento, J. A critical role for ABC transporters in persistent lung inflammation in the development of emphysema after smoke exposure.

Single-Photon Emission Computed Tomography/Computed Tomography Imaging in a Rabbit Model of Emphysema Reveals Ongoing Apoptosis In Vivo.

Evaluation of lung disease is limited by the inability to visualize ongoing pathological processes. Molecular imaging that targets cellular processes related to disease pathogenesis has the potential to assess disease activity over time to allow intervention before lung destruction. Because apoptosis is a critical component of lung damage in emphysema, a functional imaging approach was taken to determine if targeting apoptosis in a smoke exposure model would allow the quantification of early lung damage in vivo. Rabbits were exposed to cigarette smoke for 4 or 16 weeks and underwent single-photon emission computed tomography/computed tomography scanning using technetium-99m-rhAnnexin V-128. Imaging results were correlated with ex vivo tissue analysis to validate the presence of lung destruction and apoptosis. Lung computed tomography scans of long-term smoke-exposed rabbits exhibit anatomical similarities to human emphysema, with increased lung volumes compared with controls. Morphometry on lung tissue confirmed increased mean linear intercept and destructive index at 16 weeks of smoke exposure and compliance measurements documented physiological changes of emphysema. Tissue and lavage analysis displayed the hallmarks of smoke exposure, including increased tissue cellularity and protease activity. Technetium-99m-rhAnnexin V-128 single-photon emission computed tomography signal was increased after smoke exposure at 4 and 16 weeks, with confirmation of increased apoptosis through terminal deoxynucleotidyl transferase dUTP nick end labeling staining and increased tissue neutral sphingomyelinase activity in the tissue. These studies not only describe a novel emphysema model for use with future therapeutic applications, but, most importantly, also characterize a promising imaging modality that identifies ongoing destructive cellular processes within the lung.

Treatment of experimental asthma using a single small molecule with anti-inflammatory and BK channel-activating properties.

Large conductance voltage- and calcium-activated potassium (BK) channels are highly expressed in airway smooth muscle (ASM). Utilizing the ovalbumin (OVA) and house dust mite (HDM) models of asthma in C57BL/6 mice, we demonstrate that systemic administration of the BK channel agonist rottlerin (5 µg/g) during the challenge period reduced methacholine-induced airway hyperreactivity (AHR) in OVA- and HDM-sensitized mice (47% decrease in peak airway resistance in OVA-asthma animals, P<0.01; 54% decrease in HDM-asthma animals, P<0.01) with a 35-40% reduction in inflammatory cells and 20-35% reduction in Th2 cytokines in bronchoalveolar lavage fluid. Intravenous rottlerin (5 µg/g) reduced AHR within 5 min in the OVA-asthma mice by 45% (P<0.01). With the use of an ex vivo lung slice technique, rottlerin relaxed acetylcholine-stimulated murine airway lumen area to 87 ± 4% of the precontracted area (P<0.01 vs. DMSO control). Rottlerin increased BK channel activity in human ASM cells (V50 shifted by 73.5±13.5 and 71.8±14.6 mV in control and asthmatic cells, respectively, both P<0.05 as compared with pretreatment) and reduced the frequency of acetylcholine-induced Ca(2+) oscillations in murine ex vivo lung slices. These findings suggest that rottlerin, with both anti-inflammatory and ASM relaxation properties, may have benefit in treating asthma.

Activation of the TLR4 signaling pathway and abnormal cholesterol efflux lead to emphysema in ApoE-deficient mice.

Smokers with airflow obstruction have an increased risk of atherosclerosis, but the relationship between the pathogenesis of these diseases is not well understood. To determine whether hypercholesterolemia alters lung inflammation and emphysema formation, we examined the lung phenotype of two hypercholesterolemic murine models of atherosclerosis at baseline and on a high-fat diet. Airspace enlargement developed in the lungs of apolipoprotein E-deficient (Apoe(-/-)) mice exposed to a Western-type diet for 10 wk. An elevated number of macrophages and lymphocytes accompanied by an increase in matrix metalloproteinase-9 (MMP-9) activity and MMP-12 expression was observed in the lungs of Apoe(-/-) mice on a Western-type diet. In contrast, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice did not exhibit lung destruction or inflammatory changes. Most importantly, we revealed augmented expression of the downstream targets of the Toll-like receptor (TLR) pathway, interleukin-1 receptor-associated kinase 1, and granulocyte colony-stimulating factor, in the lungs of Apoe(-/-) mice fed with a Western-type diet. In addition, we demonstrated overexpression of MMP-9 in Apoe(-/-) macrophages treated with TLR4 ligand, augmented with the addition of oxidized LDL, suggesting that emphysema in these mice results from the activation of the TLR pathway secondary to known abnormal cholesterol efflux. Our findings indicate that, in Apoe(-/-) mice fed with an atherogenic diet, abnormal cholesterol efflux leads to increased systemic inflammation with subsequent lung damage and emphysema formation.

Knockdown of Alpha-1 Antitrypsin with antisense oligonucleotide does not exacerbate smoke induced lung injury.

Alpha-1 Antitrypsin (AAT) is a serum protease inhibitor that regulates increased lung protease production induced by cigarette smoking. Mutations in the Serpina1 gene cause AAT to form hepatoxic polymers, which can lead to reduced availability for the protein's primary function and severe liver disease. An AAT antisense oligonucleotide (ASO) was previously identified to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knockdown of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups each for the smoking and smoke-flu injury models. The ASO and control (No-ASO) were injected subcutaneously starting with smoking or four days prior to influenza infection and then injected weekly at 50 mg/kg body weight. ASO treatment during a 3-month smoke exposure significantly decreased the serum and lung AAT expression, resulting in increased Cela1 expression and elastase activity. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly worsened by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure, but did not observe an ASO treatment effect. Similarly, with the smoke-flu model, differences were only observed between smoke-flu and room air controls, but not as a result of ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment, while sufficient to protect from liver injury, may not be robust enough to lead to lung injury. The results also suggest that previously described AAT ASO treatment for AAT mutation related liver disease may attenuate hepatic injury without being detrimental to the lungs. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.