Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been ...described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.
Obesity and elevated circulating cholesterol are risk factors for breast cancer recurrence, while the use of statins, cholesterol biosynthesis inhibitors widely used for treating ...hypercholesterolemia, is associated with improved disease-free survival. Here, we show that cholesterol mediates the metastatic effects of a high-fat diet via its oxysterol metabolite, 27-hydroxycholesterol. Ablation or inhibition of CYP27A1, the enzyme responsible for the rate-limiting step in 27-hydroxycholesterol biosynthesis, significantly reduces metastasis in relevant animal models of cancer. The robust effects of 27-hydroxycholesterol on metastasis requires myeloid immune cell function, and it was found that this oxysterol increases the number of polymorphonuclear-neutrophils and γδ-T cells at distal metastatic sites. The pro-metastatic actions of 27-hydroxycholesterol requires both polymorphonuclear-neutrophils and γδ-T cells, and 27-hydroxycholesterol treatment results in a decreased number of cytotoxic CD8
T lymphocytes. Therefore, through its actions on γδ-T cells and polymorphonuclear-neutrophils, 27-hydroxycholesterol functions as a biochemical mediator of the metastatic effects of hypercholesterolemia.High cholesterol is a risk factor for breast cancer recurrence. Here the authors show that cholesterol promotes breast cancer metastasis via its metabolite 27-hydroxycholesterol (27HC) that acts on immune myeloid cells residing at the distal metastatic sites, thus promoting an immune suppressive environment.
Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage ...(BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry-based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206(+)) subsets and other CD206(-) leukocytes. The CD206(-) cells include: (1) three monocyte (CD14(+)) subsets, (2) CD11c(+) dendritic cells (CD14(-), CD11c(+), HLA-DR(+)), (3) plasmacytoid dendritic cells (CD14(-), CD11c(-), HLA-DR(+), CD123(+)), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169(+) and CD169(-) macrophages. In lung tissue, CD169(-) macrophages were a prominent cell type. Using confocal microscopy, CD169(+) macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169(-) macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.
An increasing body of evidence suggests that bone marrow-derived myeloid cells play a critical role in the pathophysiology of pulmonary hypertension (PH). However, the true requirement for myeloid ...cells in PH development has not been demonstrated, and a specific disease-promoting myeloid cell population has not been identified. Using bone marrow chimeras, lineage labeling, and proliferation studies, we determined that, in murine hypoxia-induced PH, Ly6C
nonclassical monocytes are recruited to small pulmonary arteries and differentiate into pulmonary interstitial macrophages. Accumulation of these nonclassical monocyte-derived pulmonary interstitial macrophages around pulmonary vasculature is associated with increased muscularization of small pulmonary arteries and disease severity. To determine if the sensing of hypoxia by nonclassical monocytes contributes to the development of PH, mice lacking expression of hypoxia-inducible factor-1α in the Ly6C
monocyte lineage were exposed to hypoxia. In these mice, vascular remodeling and PH severity were significantly reduced. Transcriptome analyses suggest that the Ly6C
monocyte lineage regulates PH through complement, phagocytosis, Ag presentation, and chemokine/cytokine pathways. Consistent with these murine findings, relative to controls, lungs from pulmonary arterial hypertension patients displayed a significant increase in the frequency of nonclassical monocytes. Taken together, these findings show that, in response to hypoxia, nonclassical monocytes in the lung sense hypoxia, infiltrate small pulmonary arteries, and promote vascular remodeling and development of PH. Our results demonstrate that myeloid cells, specifically cells of the nonclassical monocyte lineage, play a direct role in the pathogenesis of PH.
We report here senescent changes in the structure and organization of the mucociliary pseudostratified epithelium of the mouse trachea and main stem bronchi. We confirm previous reports of the ...gradual appearance of age-related, gland-like structures (ARGLS) in the submucosa, especially in the intercartilage regions and carina. Immunohistochemistry shows these structures contain ciliated and secretory cells and Krt5+ basal cells, but not the myoepithelial cells or ciliated ducts typical of normal submucosal glands. Data suggest they arise de novo by budding from the surface epithelium rather than by delayed growth of rudimentary or cryptic submucosal glands. In old mice the surface epithelium contains fewer cells per unit length than in young mice and the proportion of Krt5+, p63+ basal cells is reduced in both males and females. However, there appears to be no significant difference in the ability of basal stem cells isolated from individual young and old mice to form clonal tracheospheres in culture or in the ability of the epithelium to repair after damage by inhaled sulfur dioxide. Gene expression analysis by Affymetrix microarray and quantitative PCR, as well as immunohistochemistry and flow sorting studies, are consistent with low-grade chronic inflammation in the tracheas of old versus young mice and an increase in the number of immune cells. The significance of these changes for ARGL formation are not clear since several treatments that induce acute inflammation in young mice did not result in budding of the surface epithelium.
Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal ...roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development.
We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia.
Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCII
CCR2
EAR2
cells characterized the acute inflammatory phase, while TLF
VCAM1
cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways.
Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.
D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a ...subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8
T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6
CD8
T cells with a progenitor phenotype and decreased terminally exhausted CD8
T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8
T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established
up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.
In pulmonary arterial hypertension (PAH), there is overexpression of the chemokine, C-C chemokine ligand type 2 (CCL2), and infiltration of myeloid cells into the pulmonary vasculature. Inhibition of ...CCL2 in animals decreases PAH, suggesting that the CCL2 receptor (CCR2) plays a role in PAH development. To test this hypothesis, we exposed wild-type (WT) and CCR2-deficient (Ccr2(-/-)) mice to chronic hypobaric hypoxia to induce PAH. After hypoxic stress, Ccr2(-/-) mice displayed a more severe PAH phenotype, as demonstrated by increased right ventricular (RV) systolic pressures, RV hypertrophy, and tachycardia relative to WT mice. However, these mice also exhibited increased RV systolic pressures and increased pulmonary artery muscularization under normoxic conditions. Moreover, Ccr2(-/-) mice displayed decreased pulmonary vascular branching at 3 weeks of age and increased vascular muscularization at birth, suggesting that an abnormality in pulmonary vascular development leads to spontaneous PAH in these animals. No significant differences in cytokine responses were observed between WT and Ccr2(-/-) mice during either normoxia or hypoxia. However, Ccr2(-/-) mice displayed increased Notch-3 signaling and dysregulated Notch ligand expression, suggesting a possible cause for their abnormal pulmonary vascular development. Our findings imply that CCR2 does not directly contribute to the development of PAH, but does play a previously unrecognized role in pulmonary vasculature development and remodeling wherein the absence of CCR2 results in spontaneous PAH, most likely via dysregulation of Notch signaling. Our results demonstrate that CCR2 has impacts beyond leukocyte recruitment, and is required for the proper expression of Notch signaling molecules.
Pulmonary arterial stiffness (PAS) is a pathologic hallmark of all types of pulmonary hypertension (PH). Cardiac MRI (CMR), a gold-standard imaging modality for the evaluation of pulmonary flow, ...biventricular morphology and function has been historically reserved for the longitudinal clinical follow-up, PH phenotyping purposes, right ventricular evaluation, and research purposes. Over the last two decades, numerous indices combining invasive catheterization and non-invasive CMR have been utilized to phenotype the character and severity of PAS in different types of PH and to assess its clinically prognostic potential with encouraging results. Many recent studies have demonstrated a strong role of CMR derived PAS markers in predicting long-term clinical outcomes and improving currently gold standard risk assessment provided by the REVEAL calculator. With the utilization of a machine learning strategies, strong diagnostic and prognostic performance of CMR reported in multicenter studies, and ability to detect PH at early stages, the non-invasive assessment of PAS is on verge of routine clinical utilization. In this review, we focus on appraising important CMR studies interrogating PAS over the last 20 years, describing the benefits and limitations of different PAS indices, and their pathophysiologic relevance to pulmonary vascular remodeling. We also discuss the role of CMR and PAS in clinical surveillance and phenotyping of PH, and the long-term future goal to utilize PAS as a biomarker to aid with more targeted therapeutic management.
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