Lower Airway Dysbiosis Augments Lung Inflammatory Injury in Mild-to-Moderate Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

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.

Deaccelerated Myogenesis and Autophagy in Genetically Induced Pulmonary Emphysema.

Patients with chronic obstructive pulmonary disease (COPD)-pulmonary emphysema often develop locomotor muscle dysfunction, which entails reduced muscle mass and force-generation capacity and is associated with worse outcomes, including higher mortality. Myogenesis contributes to adult muscle integrity during injury-repair cycles. Injurious events crucially occur in the skeletal muscles of patients with COPD in the setting of exacerbations and infections, which lead to acute decompensations for limited periods of time, after which patients typically fail to recover the baseline status they had before the acute event. Autophagy, which is dysregulated in muscles from patients with COPD, is a key regulator of muscle stem-satellite- cells activation and myogenesis, yet very little research has so far mechanistically investigated the role of autophagy dysregulation in COPD muscles. Using a genetically inducible interleukin-13-driven pulmonary emphysema model leading to muscle dysfunction, and confirmed with a second genetic animal model, we found a significant myogenic dysfunction associated with the reduced proliferative capacity of satellite cells. Transplantation experiments followed by lineage tracing suggest that an intrinsic defect in satellite cells, and not in the COPD environment, plays a dominant role in the observed myogenic dysfunction. RNA sequencing analysis and direct observation of COPD mice satellite cells suggest dysregulated autophagy. Moreover, while autophagy flux experiments with bafilomycin demonstrated deacceleration of autophagosome turnover in COPD mice satellite cells, spermidine-induced autophagy stimulation leads to a higher replication rate and myogenesis in these animals. Our data suggest that pulmonary emphysema causes disrupted myogenesis, which could be improved with stimulation of autophagy and satellite cells activation, leading to an attenuated muscle dysfunction.

High mobility group AT-hook 2 regulates osteoblast differentiation and facial bone development.

The mutation and deletion of high mobility group AT-hook 2 (Hmga2) gene exhibit skeletal malformation, but almost nothing is known about the mechanism. This study examined morphological anomaly of facial bone in Hmga2-/- mice and osteoblast differentiation of pre-osteoblast MC3T3-E1 cells with Hmga2 gene knockout (A2KO). Hmga2-/- mice showed the size reduction of anterior frontal part of facial bones. Hmga2 protein and mRNA were expressed in mesenchymal cells at ossification area of nasal bone. A2KO cells differentiation into osteoblasts after reaching the proliferation plateau was strongly suppressed by alizarin red and alkaline phosphatase staining analyses. Expression of osteoblast-related genes, especially Osterix, was down-regulated in A2KO cells. These results demonstrate a close association of Hmga2 with osteoblast differentiation of mesenchymal cells and bone growth. Although future studies are needed, the present study suggests an involvement of Hmga2 in osteoblast-genesis and bone growth.

Wingless/integrase-1 signaling in allergic asthma and pediatric lung diseases.

PURPOSE OF REVIEW: To provide an update on the current understanding of the role of wingless/integrase-1 (Wnt) signaling in pediatric allergic asthma and other pediatric lung diseases. RECENT FINDINGS: The Wnt signaling pathway is critical for normal lung development. Genetic and epigenetic human studies indicate a link between Wnt signaling and the development and severity of asthma in children. Mechanistic studies using animal models of allergic asthma demonstrate a key role for Wnt signaling in allergic airway inflammation and remodeling. More recently, data on bronchopulmonary dysplasia (BPD) pathogenesis points to the Wnt signaling pathway as an important regulator. SUMMARY: Current data indicates that the Wnt signaling pathway is an important mediator in allergic asthma and BPD pathogenesis. Further studies are needed to characterize the roles of individual Wnt signals in childhood disease, and to identify potential novel therapeutic targets to slow or prevent disease processes.

Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary T-Helper Cell Type 17 Response that Mitigates Susceptibility to Streptococcus pneumoniae.

Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen.Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.

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.

Biased Generation and In Situ Activation of Lung Tissue-Resident Memory CD4 T Cells in the Pathogenesis of Allergic Asthma.

Asthma is a chronic inflammatory disease mediated by allergen-specific CD4 T cells that promote lung inflammation through recruitment of cellular effectors into the lung. A subset of lung T cells can persist as tissue-resident memory T cells (TRMs) following infection and allergen induction, although the generation and role of TRM in asthma persistence and pathogenesis remain unclear. In this study, we used a mouse model of chronic exposure to intranasal house dust mite (HDM) extract to dissect how lung TRMs are generated and function in the persistence and pathogenesis of allergic airway disease. We demonstrate that both CD4+ and CD8+ T cells infiltrate into the lung tissue during acute HDM exposure; however, only CD4+ TRMs, and not CD8+ TRMs, persist long term following cessation of HDM administration. Lung CD4+ TRMs are localized around airways and are rapidly reactivated upon allergen re-exposure accompanied by the rapid induction of airway hyperresponsiveness independent of circulating T cells. Lung CD4+ TRM activation to HDM challenge is also accompanied by increased recruitment and activation of dendritic cells in the lungs. Our results indicate that lung CD4+ TRMs can perpetuate allergen-specific sensitization and direct early inflammatory signals that promote rapid lung pathology, suggesting that targeting lung CD4+ TRMs could have therapeutic benefit in alleviating recurrent asthma episodes.

High Mobility Group AT-Hook 2 (HMGA2) Oncogenicity in Mesenchymal and Epithelial Neoplasia.

High mobility group AT-hook 2 (HMGA2) has been associated with increased cell proliferation and cell cycle dysregulation, leading to the ontogeny of varied tumor types and their metastatic potentials, a frequently used index of disease prognosis. In this review, we deepen our understanding of HMGA2 pathogenicity by exploring the mechanisms by which HMGA2 misexpression and ectopic expression induces mesenchymal and epithelial tumorigenesis respectively and distinguish the pathogenesis of benign from malignant mesenchymal tumors. Importantly, we highlight the regulatory role of let-7 microRNA family of tumor suppressors in determining HMGA2 misexpression events leading to tumor pathogenesis and focused on possible mechanisms by which HMGA2 could propagate lymphangioleiomyomatosis (LAM), benign mesenchymal tumors of the lungs. Lastly, we discuss potential therapeutic strategies for epithelial and mesenchymal tumorigenesis based on targeting the HMGA2 signaling pathway.

Hmga2 regulation of tooth formation and association with Sox2 and Nanog expression.

Tooth formation is accomplished under strict genetic programs. Although patients with chromosome 12q14 aberration shows tooth phenotype including the size and eruption timing with bone growth anomaly, its etiology is uncertain. Here, we examined expression of Hmga2, which is encoded at chromosome 12q14, in mouse tooth germs and analyzed the involvement in lower first molar (M1) and mandibular bone development. Hmga2 expression was immunohistochemically detected at enamel organ and the surrounding mesenchyme of the M1 germs. The expression was dynamically changed with gestation and rapidly decreased in postnatal mice. In Hmga2-/- mice, the M1 germs and crowns were diminished in size, and formation and eruption of molars were delayed with mandibular bone growth retardation. Hmga2 cDNA or siRNA transfection showed that Hmga2 transcriptionally up-regulates expression of stem cell factors, Sox2 and Nanog. They were co-localized with Hmga2 in the germs, but differentially distributed at enamel organ and mesenchyme in Hmga2-/- mice. These results demonstrate that Hmga2 expressed in tooth germs regulates the growth, sizing and eruption and stem cell factor expression in different compartment of the germ and associates with mandibular bone growth. Although future studies are needed, the present study demonstrates HMGA2 regulation of tooth genesis with skeletal development.

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.

Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs.

The present study tested the hypothesis that maternal smoke exposure results in fetal lung growth retardation due to dysregulation in various signaling pathways, including the Wnt (wingless-related integration site)/ß-catenin pathway. Pregnant female C57BL/6J mice were exposed to cigarette smoke (100-150 mg/m3) or room air, and offspring were humanely killed on 12.5, 14.5, 16.5, and 18.5 d post coitum (dpc). We assessed lung stereology with Cavalieri estimation; apoptosis with proliferating cell nuclear antigen, TUNEL, and caspase assays; and gene expression with quantitative PCR (qPCR) and RNA sequencing on lung epithelium and mesenchyme retrieved by laser capture microdissection. Results demonstrated a significant decrease in body weight and lung volume of smoke-exposed embryos. At 16.5 dpc, the reduction in lung volume was due to loss of lung mesenchymal tissue correlating with a decrease in cell proliferation (n = 10; air: 61.65% vs. smoke: 44.21%, P < 0.05). RNA sequence analysis demonstrated an alteration in the Wnt pathway, and qPCR confirmed an increased expression of secreted frizzled-related protein 1 (sFRP-1) [n = 12; relative quantification (RQ) 1 vs. 2.33, P < 0.05] and down-regulation of Cyclin D1 (n = 7; RQ 1 vs. 0.61, P < 0.05) in mesenchymal tissue. Furthermore, genome expression studies revealed a smoke-induced up-regulation of Rho-GTPase-dependent actin cytoskeletal signaling that can lead to loss of tissue integrity.-Unachukwu, U., Trischler, J., Goldklang, M., Xiao, R., D'Armiento, J. Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs.

Transgenic overexpression of macrophage matrix metalloproteinase-9 exacerbates age-related cardiac hypertrophy, vessel rarefaction, inflammation, and fibrosis.

Advancing age is an independent risk factor for cardiovascular disease. Matrix metalloproteinase-9 (MMP-9) is secreted by macrophages and robustly increases in the left ventricle (LV) with age. The present study investigated the effect of MMP-9 overexpression in macrophages on cardiac aging. We compared 16- to 21-mo-old C57BL/6J wild-type (WT) and transgenic (TG) male and female mice (n = 15-20/group). MMP-9 overexpression amplified the hypertrophic response to aging, as evidenced by increased LV wall thickness and myocyte cross-sectional areas (P < 0.05 for both). MMP-9 overexpression reduced LV expression of the angiogenesis-related factors ICAM-1, integrins α3 and ß3, platelet/endothelial cell adhesion molecule-1, thrombospondin-1, tenascin-c, and versican (all P < 0.05). Concomitantly, the number of vessels in the TG was lower than WT LV (P < 0.05). This led to a mismatch in the muscle-to-vessel ratio and resulted in increased cardiac inflammation. Out of 84 inflammatory genes analyzed, 16 genes increased in the TG compared with WT (all P < 0.05). Of the elevated genes, 14 were proinflammatory genes. The increase in cardiac inflammation resulted in greater accumulation of interstitial collagen in TG (P < 0.05). Fractional shortening was similar between groups, indicating that global cardiac function was still preserved at this age. In conclusion, overexpression of MMP-9 in macrophages resulted in exacerbated cardiac hypertrophy in the setting of vessel rarefaction, which resulted in enhanced inflammation and fibrosis to augment the cardiac-aging phenotype. Our results provide evidence that macrophage-derived MMP-9 may be a therapeutic target in elderly subjects.NEW & NOTEWORTHY The present study was the first to use mice with transgenic overexpression of matrix metalloproteinase-9 (MMP-9) in macrophages to examine the effects of macrophage-derived MMP-9 on cardiac aging. We found that an elevation in macrophage-derived MMP-9 induced a greater age-dependent cardiac hypertrophy and vessel rarefaction phenotype, which enhanced cardiac inflammation and fibrosis.

Official American Thoracic Society/Japanese Respiratory Society Clinical Practice Guidelines: Lymphangioleiomyomatosis Diagnosis and Management.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease that primarily affects women. The purpose of these guidelines is to provide recommendations for the diagnosis and treatment of LAM. METHODS: Systematic reviews were performed to summarize evidence pertinent to our questions. The evidence was summarized and discussed by a multidisciplinary panel. Evidence-based recommendations were then formulated, written, and graded using the Grading of Recommendations, Assessment, Development, and Evaluation approach. RESULTS: After considering the panel's confidence in the estimated effects, the balance of desirable (i.e., benefits) and undesirable (i.e., harms and burdens) consequences of treatment, patient values and preferences, cost, and feasibility, recommendations were formulated for or against specific interventions. These included recommendations for sirolimus treatment and vascular endothelial growth factor D testing and recommendations against doxycycline and hormonal therapy. CONCLUSIONS: Evidence-based recommendations for the diagnosis and treatment of patients with LAM are provided. Frequent reassessment and updating will be needed.

Parenchymal Airspace Profiling: Sensitive Quantification and Characterization of Lung Structure Evaluating Parenchymal Destruction.

Lung morphometry was introduced over 50 years ago to provide quantitative evaluation of the lung structure. The existing parameters, such as mean linear intercept and destructive index, suffer from simplistic data interpretation and a subjective data acquisition process. To overcome these existing shortcomings, parenchymal airspace profiling (PAP) was developed to provide a more detailed and unbiased quantitative method. Following the standard protocols of fixation, embedding, and sectioning, lung micrographs were: (1) marked with nonparenchymal area, preprocessed, and binarized under the researcher's supervision; (2) analyzed with a statistical learning method, Gaussian mixture model, to provide an unbiased categorization of parenchymal airspace compartments, corresponding to a single alveolus, alveolar sac, and ductal/destructive airspace; and (3) further quantified into morphometric parameters, including reference volume, alveolar count, and ductal/destructive fraction (DF) based on stereological principles. PAP was performed on hematoxylin and eosin-stained lung sections from mice and rabbits. Unbiased categorization revealed differences in alveolar size among several mouse strains (NZW/LacJ

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.

Immune Modulation of the T Cell Response in Asthma through Wnt10b.

Asthma is a chronic inflammatory disease, which is characterized by activation of CD4(+) T helper 2 cells orchestrating an allergic airway response. Whereas the role of Wnt family members in regulating T cell maintenance and maturation is established, their contribution to T cell activation in allergic asthma is not known. We hypothesized that Wnt10b plays a role in the modulation of the allergic airway response and affects T cell activation and polarization. Using an in vivo house dust mite asthma model, Wnt10b-deficient (Wnt10b(-/-)) mice were allergen-sensitized and inflammation, as well as T cell activation, was studied in vivo and in vitro. Wnt10b(-/-) mice exhibited an augmented inflammatory phenotype with an increase in eosinophils in the bronchoalveolar lavage and IL-4 and IL-13 in the lungs when compared with wild-type mice. In vitro studies confirmed an increased T helper type 2 polarization and increased T cell activation of Wnt10b(-/-) cells. Accordingly, the percentage of naive T cells was elevated by the addition of recombinant Wnt10b protein. Finally, Wnt10b(-/-) mice exhibited an increase in the percentage of effector T cells in the lungs after house dust mite sensitization, which indicated a heightened activation state, measured by an increased percentage of CD69(hi)CD11a(hi) cells. These findings suggest that Wnt10b plays an important role in regulating asthmatic airway inflammation through modification of the T cell response and is a prospective target in the disease process.