Lung-specific MCEMP1 functions as an adaptor for KIT to promote SCF-mediated mast cell proliferation.

Lung mast cells are important in host defense, and excessive proliferation or activation of these cells can cause chronic inflammatory disorders like asthma. Two parallel pathways induced by KIT-stem cell factor (SCF) and FcεRI-immunoglobulin E interactions are critical for the proliferation and activation of mast cells, respectively. Here, we report that mast cell-expressed membrane protein1 (MCEMP1), a lung-specific surface protein, functions as an adaptor for KIT, which promotes SCF-mediated mast cell proliferation. MCEMP1 elicits intracellular signaling through its cytoplasmic immunoreceptor tyrosine-based activation motif and forms a complex with KIT to enhance its autophosphorylation and activation. Consequently, MCEMP1 deficiency impairs SCF-induced peritoneal mast cell proliferation in vitro and lung mast cell expansion in vivo. Mcemp1-deficient mice exhibit reduced airway inflammation and lung impairment in chronic asthma mouse models. This study shows lung-specific MCEMP1 as an adaptor for KIT to facilitate SCF-mediated mast cell proliferation.

Preclinical Four-Dimensional Functional Lung Imaging and Quantification of Regional Airflow: A New Standard in Lung Function Evaluation in Murine Models.

The current standard for lung function evaluation in murine models is based on forced oscillation technology, which provides a measure of the total airway function but cannot provide information on regional heterogeneity in function. Limited detection of regional airflow may contribute to a discontinuity between airway inflammation and airflow obstruction in models of asthma. Here, we describe quantification of regional airway function using novel dynamic quantitative imaging and analysis to quantify and visualize lung motion and regional pulmonary airflow in four dimensions (4D). Furthermore, temporo-spatial specific ventilation (ml/ml) is used to determine ventilation heterogeneity indices for lobar and sublobar regions, which are directly compared to ex vivo biological analyses in the same sublobar regions. In contrast, oscillation-based technology in murine genetic models of asthma have failed to demonstrate lung function change despite altered inflammation, whereas 4D functional lung imaging demonstrated diminished regional lung function in genetic models relative to wild-type mice. Quantitative functional lung imaging assists in localizing the regional effects of airflow. Our approach reveals repeatable and consistent differences in regional airflow between lung lobes in all models of asthma, suggesting that asthma is characterized by regional airway dysfunctions that are often not detectable in composite measures of lung function. 4D functional lung imaging technology has the potential to transform discovery and development in murine models by mapping out regional areas heterogeneously affected by the disease, thus deciphering pathobiology with greater precision.

Barrier Housing and Gender Effects on Allergic Airway Disease in a Murine House Dust Mite Model.

Allergic airway disease models use laboratory mice housed in highly controlled and hygienic environments, which provide a barrier between the mice and a predetermined list of specific pathogens excluded from the facility. In this study, we hypothesized that differences in facility barrier level and, consequently, the hygienic quality of the environment that mice inhabit impact the severity of pulmonary inflammation and lung function. Allergen-naive animals housed in the cleaner, high barrier (HB) specific pathogen-free facility had increased levels of inflammatory cytokines and higher infiltration of immune cells in the lung tissue but not in the bronchoalveolar lavage compared with mice housed in the less hygienic, low barrier specific pathogen-free facility. In both genders, house dust mite-induced airway disease was more severe in the HB than the low barrier facility. Within each barrier facility, female mice developed the most severe inflammation. However, allergen-naive male mice had worse lung function, regardless of the housing environment, and in the HB, the lung function in female mice was higher in the house dust mite model. Severe disease in the HB was associated with reduced lung microbiome diversity. The lung microbiome was altered across housing barriers, gender, and allergen-exposed groups. Thus, the housing barrier level impacts microbial-driven disease and gender phenotypes in allergic asthma. The housing of laboratory mice in more clean HB facilities aggravates lung immunity and causes a more severe allergic lung disease.

Comprehensive phenotyping of endothelial cells using flow cytometry 1: Murine.

The endothelium forms a selective barrier between circulating blood or lymph and surrounding tissue. Endothelial cells play an essential role in vessel homeostasis, and identification of these cells is critical in vascular biology research. However, characteristics of endothelial cells differ depending on the location and type of blood or lymph vessel. Endothelial cell subsets are numerous and often identified using different flow cytometric markers, making immunophenotyping these cells complex. In part 1 of this two part review series, we present a comprehensive overview of markers for the flow cytometric identification and phenotyping of murine endothelial subsets. These subsets can be distinguished using a panel of cell surface and intracellular markers shared by all endothelial cells in combination with additional markers of specialized endothelial cell types. This review can be used to determine the best markers for identifying and phenotyping desired murine endothelial cell subsets.

Comprehensive phenotyping of endothelial cells using flow cytometry 2: Human.

In vascular research, clinical samples and samples from animal models are often used together to foster translation of preclinical findings to humans. General concepts of endothelia and murine-specific endothelial phenotypes were discussed in part 1 of this two part series. Here, in part 2, we present a comprehensive overview of human-specific endothelial phenotypes. Pan-endothelial cell markers, organ specific endothelial antigens, and flow cytometric immunophenotyping of blood-borne endothelial cells are reviewed.

Characterization and origins of cell-free mitochondria in healthy murine and human blood.

Intact cell-free mitochondria have been reported in microparticles (MPs) in murine and human bodily fluids under disease conditions. However, cellular origins of circulating extracellular mitochondria have not been characterized. We hypothesize that intact, cell-free mitochondria from heterogeneous cellular sources are present in the circulation under physiological conditions. To test this, circulating MPs were analyzed using flow cytometry and proteomics. Murine and human platelet-depleted plasma showed a cluster of MPs positive for the mitochondrial probe MitoTracker. Transgenic mice expressing mitochondrial-GFP showed GFP positivity in plasma MPs. Murine and human mitochondria-containing MPs were positive for the platelet marker CD41 and the endothelial cell marker CD144, while hematopoietic CD45 labeling was low. Both murine and human circulating cell-free mitochondria maintained a transmembrane potential. Circulating mitochondria were able to enter rho-zero cells, and were visualized using immunoelectron microscopic imaging. Proteomics analysis identified mitochondria specific and extracellular vesicle associated proteins in sorted circulating cell-free human mitochondria. Together the data provide multiple lines of evidence that intact and functional mitochondria originating from several cell types are present in the blood circulation.

Arginine metabolic control of airway inflammation.

Inducible nitric oxide synthase (iNOS) and arginase-2 (ARG2) share a common substrate, arginine. Higher expression of iNOS and exhaled NO are linked to airway inflammation in patients. iNOS deletion in animal models suggests that eosinophilic inflammation is regulated by arginine metabolism. Moreover, ARG2 is a regulator of Th2 response, as shown by the development of severe eosinophilic inflammation in ARG2-/- mice. However, potential synergistic roles of iNOS and ARG2 in asthma have not been explored. Here, we hypothesized that arginine metabolic fate via iNOS and ARG2 may govern airway inflammation. In an asthma cohort, ARG2 variant genotypes were associated with arginase activity. ARG2 variants with lower arginase activity, combined with levels of exhaled NO, identified a severe asthma phenotype. Airway inflammation was present in WT, ARG2-/-, iNOS-/-, and ARG2-/-/iNOS-/- mice but was greatest in ARG2-/-. Eosinophilic and neutrophilic infiltration in the ARG2-/- mice was abrogated in ARG2-/-/iNOS-/- animals. Similarly, angiogenic airway remodeling was greatest in ARG2-/- mice. Cytokines driving inflammation and remodeling were highest in lungs of asthmatic ARG2-/- mice and lowest in the iNOS-/-. ARG2 metabolism of arginine suppresses inflammation, while iNOS metabolism promotes airway inflammation, supporting a central role for arginine metabolic control of inflammation.

Interdependence of hypoxia and ß-adrenergic receptor signaling in pulmonary arterial hypertension.

The ß-adrenergic receptor (ßAR) exists in an equilibrium of inactive and active conformational states, which shifts in response to different ligands and results in downstream signaling. In addition to cAMP, ßAR signals to hypoxia-inducible factor 1 (HIF-1). We hypothesized that a ßAR-active conformation (R**) that leads to HIF-1 is separable from the cAMP-activating conformation (R*) and that pulmonary arterial hypertension (PAH) patients with HIF-biased conformations would not respond to a cAMP agonist. We compared two cAMP agonists, isoproterenol and salbutamol, in vitro. Isoproterenol increased cAMP and HIF-1 activity, while salbutamol increased cAMP and reduced HIF-1. Hypoxia blunted agonist-stimulated cAMP, consistent with receptor equilibrium shifting toward HIF-activating conformations. Similarly, isoproterenol increased HIF-1 and erythropoiesis in mice, while salbutamol decreased erythropoiesis. ßAR overexpression in cells increased glycolysis, which was blunted by HIF-1 inhibitors, suggesting increased ßAR leads to increased hypoxia-metabolic effects. Because PAH is also characterized by HIF-related glycolytic shift, we dichotomized PAH patients in the Pulmonary Arterial Hypertension Treatment with Carvedilol for Heart Failure trial (NCT01586156) based on right ventricular (RV) glucose uptake to evaluate ßAR ligands. Patients with high glucose uptake had more severe disease than those with low uptake. cAMP increased in response to isoproterenol in mononuclear cells from low-uptake patients but not in high-uptake patients' cells. When patients were treated with carvedilol for 1 wk, the low-uptake group decreased RV systolic pressures and pulmonary vascular resistance, but high-uptake patients had no physiologic responses. The findings expand the paradigm of ßAR activation and uncover a novel PAH subtype that might benefit from ß-blockers.