Assessing the clinico-immunological profile of patients with obesity and chronic rhinosinusitis.

BACKGROUND: No studies have investigated the systemic and local sinonasal profile of obesity-related chronic rhinosinusitis (CRS), despite its observed association in recent retrospective studies. The objectives of our study were to assess the impact of obesity on the clinical and cytokine profile of patients with CRS and evaluate treatment response with functional endoscopic sinus surgery. METHODS: This was a prospective observational study at the Cleveland Clinic that included patients with CRS (n = 54) between December 2021 and September 2022. Data collection included demographics, body mass index (BMI), comorbidities, baseline sinonasal outcome test scores, baseline radiologic scores (Lund-Mackay), postoperative sinonasal outcome test scores (at 3-4 months), and local and systemic alarmins/T-helper cytokines. RESULTS: Out of the 54 CRS patients, there were 20 CRS patients without nasal polyps (37%) and 34 with nasal polyps (63%). Patients were categorized based on obesity (BMI ≥ 30 kg/m2 ). Obese CRS patients had lower systemic alarmins (interleukin [IL]-33 and Thymic stromal lymphopoietin (TSLP)) compared to non-obese CRS patients (IL-33: 744.2 ± 1164.6 pg/mL vs. 137.5 ± 320.0 pg/mL, p = 0.005; TSLP: 627.7 ± 1806.3 pg/mL vs. 28.1 ± 85.4 pg/mL, p = 0.017). CRS patients with nasal polyps with BMI ≥30 kg/m2 had higher postoperative sinonasal outcome test scores and lower levels of nasal eotaxin-3 and IL-33 compared to BMI <30 kg/m2 counterparts. CONCLUSIONS: In conclusion, patients with obese CRS and nasal polyps displayed diminished levels of intranasal alarmins and reduced intranasal eotaxin-3. These results potentially imply the presence of a unique, obese type 2-low CRS phenotype that warrants further exploration.

Hematopoietic stem cells and extramedullary hematopoiesis in the lungs.

Hematopoietic stem cells are key players in hematopoiesis as the body maintains a physiologic steady state, and the signaling pathways and control mechanisms of these dynamic cells are implicated in processes from inflammation to cancer. Although the bone marrow is commonly regarded as the site of hematopoiesis and hematopoietic stem cell residence, these cells also circulate in the blood and reside in extramedullary tissues, including the lungs. Flow cytometry is an invaluable tool in evaluating hematopoietic stem cells, revealing their phenotypes and relative abundances in both healthy and diseased states. This review outlines current protocols and cell markers used in flow cytometric analysis of hematopoietic stem and progenitor cell populations. Specific niches within the bone marrow are discussed, as are metabolic processes that contribute to stem cell self-renewal and differentiation, as well as the role of hematopoietic stem cells outside of the bone marrow at physiologic steady state. Finally, pulmonary extramedullary hematopoiesis and its associated disease states are outlined. Hematopoiesis in the lungs is a new and emerging concept, and discovering ways in which the study of lung-resident hematopoietic stem cells can be translated from murine models to patients will impact clinical treatment.

Single-cell transcriptomic profiling of microvascular endothelial cell heterogeneity in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a neonatal anomaly that includes pulmonary hypoplasia and hypertension. We hypothesized that microvascular endothelial cell (EC) heterogeneity is different in CDH lungs and related to lung underdevelopment and remodeling. To test this, we evaluated rat fetuses at E21.5 in a nitrofen model of CDH to compare lung transcriptomes among healthy controls (2HC), nitrofen-exposed controls (NC) and nitrofen-exposed subjects with CDH. Single-cell RNA sequencing with unbiased clustering revealed 3 distinct microvascular EC clusters: a general population (mvEC), a proliferative population and a population high in hemoglobin. Only the CDH mvEC cluster had a distinct inflammatory transcriptomic signature as compared to the 2HC and NC endothelial cells, e.g. greater activation and adhesion of inflammatory cells and production of reactive oxygen species. Furthermore, CDH mvECs had downregulated Ca4, Apln and Ednrb gene expression. Those genes are markers for ECs important to lung development, gas exchange and alveolar repair (mvCa4+). mvCa4+ ECs were reduced in CDH (2HC [22.6%], NC [13.1%] and CDH [5.3%], p < 0.0001). Overall, these findings identify transcriptionally distinct microvascular endothelial cell clusters in CDH, including the distinctly inflammatory mvEC cluster and the depleted group of mvCa4+ ECs, which together may contribute to pathogenesis.

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.

Hemoglobin resident in the lung epithelium is protective for smooth muscle soluble guanylate cyclase function.

Hemoglobin (Hb) present in the lung epithelium is of unknown significance. However Hb being an nitric oxide (NO) scavenger can bind to NO and reduce its deleterious effects. Hence we postulated an NO scavenging role for this lung Hb. Doing transwell co-culture with bronchial epithelial cells, A549/16-HBE (apical) and human airway smooth muscle cells (HASMCs as basal), we found that Hb can protect the smooth muscle soluble guanylyl cyclase (sGC) from excess NO. Inducing the apical A549/16-HBE cells with cytokines to trigger iNOS expression and NO generation caused a time dependent increase in SNO-sGC and this was accompanied with a concomitant drop in sGC-α1ß1 heterodimerization. Silencing Hbαß in the apical cells further increased the SNO on sGC with a faster drop in the sGC heterodimer and these effects were additive along with further silencing of thioredoxin 1 (Trx1). Since heme of Hb is critical for NO scavenging we determined the Hb heme in a mouse model of allergic asthma (OVA) and found that Hb in the inflammed OVA lungs was low in heme or heme-free relative to those of naïve lungs. Further we established a direct correlation between the status of the sGC heterodimer and the Hb heme from lung samples of human asthma, iPAH, COPD and cystic fibrosis. These findings present a new mechanism of protection of lung sGC by the epithelial Hb, and suggests that this protection maybe lost in asthma or COPD where lung Hb is unable to scavenge the NO due to it being heme-deprived.

Hypoxia Sensing of ß-Adrenergic Receptor Is Regulated by Endosomal PI3Kγ.

BACKGROUND: Impaired beta-adrenergic receptor (ß1 and ß2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3Kγ feedback mechanism in regulating hypoxia-mediated ß1 and ß2AR dysfunction and cardiac remodeling. METHODS: Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for ß1 and ß2AR density and function, PI3Kγ activity and ß1 and ß2AR-associated PP2A activity. Metabolic labeling was performed to assess ß1 and ß2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. RESULTS: Hypoxia increased ß1 and ß2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated ß2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased ß1 and ß2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal ß1- and ß2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased ß1 and ß2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with ß1AR-selective agonist dobutamine post-hypoxia. CONCLUSIONS: Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in ß1- and ß2AR phosphorylation and amelioration of cardiac dysfunction.

Pathological Roles for Endothelial Colony-Forming Cells in Neonatal and Adult Lung Disease.

Endothelial colony-forming cells (ECFCs) are vascular resident and circulating endothelial cell subtypes with potent angiogenic capacity, a hierarchy of single-cell clonogenic potentials, and the ability to participate in de novo blood vessel formation and endothelial repair. Existing literature regarding ECFCs in neonatal and adult pulmonary diseases is confounded by the study of ambiguously defined "endothelial progenitor cells," which are often not true ECFCs. This review contrasts adult and fetal ECFCs, discusses the effect of prematurity on ECFCs, and examines their different pathological roles in neonatal and adult pulmonary diseases, such as bronchopulmonary dysplasia, congenital diaphragmatic hernia, pulmonary artery hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease. Therapeutic potential is also discussed in light of available preclinical data.

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.

Using the Autofluorescence Finder on the Sony ID7000TM Spectral Cell Analyzer to Identify and Unmix Multiple Highly Autofluorescent Murine Lung Populations.

Autofluorescence (AF) is a feature of all cell types, though some have more than others. In tissues with complex heterogeneous cellularity, AF is frequently a source of high background, masking faint fluorescent signals and reducing the available dynamic range of detectors for detecting fluorescence signals from markers of interest in a flow cytometry panel. Pulmonary flow cytometry presents unique challenges because lung cells are heterogeneous and contain varying amounts of high AF. The goal of this study was to demonstrate how a novel AF Finder tool on the Sony ID7000™ Spectral Cell Analyzer can be used to identify and screen multiple AF subsets in complex highly AF tissues like murine lungs. In lung single cell suspensions, the AF Finder tool identified four distinct AF spectra from six highly AF subsets. The subtraction of these distinct AF spectra resulted in a resolution increase by several log decades in several fluorescent channels. The major immune and lung tissue resident cells in a murine model of asthma were easily identified in a multi-color panel using AF subtraction. The findings demonstrate the practicality of the AF Finder tool, particularly when analyzing samples with multiple AF populations of varying intensities, in order to reduce fluorescence background and increase signal resolution in spectral flow cytometry.

Considerations for user consultation in a flow cytometry shared resource laboratory.

User consultation is an essential first step in assuring high-quality flow cytometric data. A central challenge to shared resource laboratory (SRL) staff is how to best guide new and current users to meet each projects' needs. One solution to this challenge is to follow a standard user consultation platform addressing all critical steps between the conception of the experiment and the actual acquisition of samples. Here we describe considerations to help an SRL understand the researcher's goals and how best the SRL staff can provide expert advice in a structured manner. User consultation has an educational nature, informing users about current best practices in cytometry that apply to their specific utilization. A consultation report also improves communication between the SRL, principal investigator, and lab members of the collaborating researcher. Development of best SRL practices is spearheaded by the ISAC SRL committee and this communication sets the foundation to initiate such report for user consultation. Implementation of best practices during user consultation will improve rigor and reproducibility in cytometry.

Menstrual cycle impacts lung structure measures derived from quantitative computed tomography.

OBJECTIVE: Quantitative computed tomography (qCT) is being increasingly incorporated in research studies and clinical trials aimed at understanding lung disease risk, progression, exacerbations, and intervention response. Menstrual cycle-based changes in lung function are recognized; however, the impact on qCT measures is currently unknown. We hypothesize that the menstrual cycle impacts qCT-derived measures of lung structure in healthy women and that the degree of measurement change may be mitigated in subjects on cyclic hormonal birth control. METHODS: Thirty-one non-smoking, healthy women with regular menstrual cycles (16 of which were on cyclic hormonal birth control) underwent pulmonary function testing and qCT imaging at both menses and early luteal phase time points. Data were evaluated to identify lung measurements which changed significantly across the two key time points and to compare degree of change across metrics for the sub-cohort with versus without birth control. RESULTS: The segmental airway measurements were larger and mean lung density was higher at menses compared to the early luteal phase. The sub-cohort with cyclic hormonal birth control did not have less evidence of measurement difference over the menstrual cycle compared to the sub-cohort without hormonal birth control. CONCLUSIONS: This study provides evidence that qCT-derived measures from the lung are impacted by the female menstrual cycle. This indicates studies seeking to use qCT as a more sensitive measure of cross-sectional differences or longitudinal changes in these derived lung measurements should consider acquiring data at a consistent time in the menstrual cycle for pre-menopausal women and warrants further exploration. KEY POINTS: • Lung measurements from chest computed tomography are used in multicenter studies exploring lung disease progression and treatment response. • The menstrual cycle impacts lung structure measurements. • Cyclic variability should be considered when evaluating longitudinal change with CT in menstruating women.

Cutting Edge: Steroid Responsiveness in Foxp3+ Regulatory T Cells Determines Steroid Sensitivity during Allergic Airway Inflammation in Mice.

Glucocorticoids are a highly effective first-line treatment option for many inflammatory diseases, including asthma. Some patients develop a steroid-resistant condition, yet, the cellular and molecular mechanisms underlying steroid resistance remain largely unknown. In this study, we used a murine model of steroid-resistant airway inflammation and report that combining systemic dexamethasone and intranasal IL-27 is able to reverse the inflammation. Foxp3+ regulatory T cells (Tregs) were required during dexamethasone/IL-27 treatment of steroid-resistant allergic inflammation, and importantly, direct stimulation of Tregs via glucocorticoid or IL-27 receptors was essential. Mechanistically, IL-27 stimulation in Tregs enhanced expression of the agonistic glucocorticoid receptor-α isoform. Overexpression of inhibitory glucocorticoid receptor-ß isoform in Tregs alone was sufficient to elicit steroid resistance in a steroid-sensitive allergic inflammation model. Taken together, our results demonstrate for the first time, to our knowledge, that Tregs are instrumental during steroid resistance and that manipulating steroid responsiveness in Tregs may represent a novel strategy to treat steroid refractory asthma.

Single-cell transcriptomic profile of human pulmonary artery endothelial cells in health and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is an insidious disease characterized by severe remodeling of the pulmonary vasculature caused in part by pathologic changes of endothelial cell functions. Although heterogeneity of endothelial cells across various vascular beds is well known, the diversity among endothelial cells in the healthy pulmonary vascular bed and the pathologic diversity among pulmonary arterial endothelial cells (PAEC) in PAH is unknown and previously unexplored. Here single-cell RNA sequencing technology was used to decipher the cellular heterogeneity among PAEC in the human pulmonary arteries isolated from explanted lungs from three patients with PAH undergoing lung transplantation and three healthy donor lungs not utilized for transplantation. Datasets of 36,368 PAH individual endothelial cells and 36,086 healthy cells were analyzed using the SeqGeq bioinformatics program. Total population differential gene expression analyses identified 629 differentially expressed genes between PAH and controls. Gene Ontology and Canonical Ingenuity analysis revealed pathways that are known to be involved in pathogenesis, as well as unique new pathways. At the individual cell level, dimensionality reduction followed by density based clustering revealed the presence of eight unique PAEC clusters that were typified by proliferative, angiogenic or quiescent phenotypes. While control and PAH harbored many similar subgroups of endothelial cells, PAH had greater proportions of angiogenic and proliferative subsets. These findings identify that only specific subgroups of PAH PAEC have gene expression different than healthy PAEC, and suggest these subpopulations lead to the pathologic functions leading to remodeling.

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.

An inherent dysfunction in soluble guanylyl cyclase is present in the airway of severe asthmatics and is associated with aberrant redox enzyme expression and compromised NO-cGMP signaling.

A subset of asthmatics develop a severe form of the disease whose etiology involves airway inflammation along with inherent drivers that remain ill-defined. To address this, we studied human airway smooth muscle cells (HASMC), whose relaxation drives airway bronchodilation and whose dysfunction contributes to airway obstruction and hypersensitivity in severe asthma. Because HASMC relaxation can be driven by the NO-soluble guanylyl cyclase (sGC)-cGMP signaling pathway, we questioned if HASMC from severe asthma donors might possess inherent defects in their sGC or in redox enzymes that support sGC function. We analyzed HASMC primary lines derived from 17 severe asthma and 16 normal donors and corresponding lung tissue samples regarding sGC activation by NO or by pharmacologic agonists, and also determined expression levels of sGC α1 and ß1 subunits, supporting redox enzymes, and related proteins. We found a majority of the severe asthma donor HASMC (12/17) and lung samples primarily expressed a dysfunctional sGC that was NO-unresponsive and had low heterodimer content and high Hsp90 association. This sGC phenotype correlated with lower expression levels of the supporting redox enzymes cytochrome b5 reductase, catalase, and thioredoxin-1, and higher expression of heme oxygenases 1 and 2. Together, our work reveals that severe asthmatics are predisposed toward defective NO-sGC-cGMP signaling in their airway smooth muscle due to an inherent sGC dysfunction, which in turn is associated with inherent changes in the cell redox enzymes that impact sGC maturation and function.

Optimized protocol for the preparation of single cells from cutaneous wounds for flow cytometric cell sorting and analysis of macrophages.

The incidence of chronic, non-healing skin wounds is accelerating, largely due to the epidemic of obesity-related Type 2 diabetes. Abnormal inflammation in wounds contributes to delayed healing. During wound repair, blood monocytes are recruited into the wound bed where they differentiate into macrophages that secrete cytokines and regulate subsequent repair events. Because the study of wound macrophages via immunohistochemistry is often unsatisfactory due to nonspecific antibody staining, the ability to isolate and analyze single cells is important for determining the phenotypes of the wound macrophages. In this article, we have expanded upon a protocol originally described by Wilson et al, 2002 [1], and optimized it for isolation of large numbers of viable macrophages from murine skin wounds that are suitable for flow cytometric cell sorting or analysis. Several parameters were found to be critical for improved macrophage yields, including: (1) The proper amount of starting material (skin tissue); (2) The optimal time for addition of Brefeldin A during enzymatic digestion; (3) Revamped guidelines for centrifugation to maximize cell pellet recovery. This optimized protocol could be further modified to perform cell sorting and flow-based immunophenotyping of any cell type involved in wound healing and inflammation.

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.