Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Introduction: 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. Methods: 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. Results: 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: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi 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. Discussion: 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.

Characterization of pulmonary arterial stiffness using cardiac MRI.

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.

Immunotoxin-αCD40 therapy activates innate and adaptive immunity and generates a durable antitumor response in glioblastoma models.

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 CD40 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.

Allergic Asthma Responses Are Dependent on Macrophage Ontogeny.

The ontogenetic composition of tissue-resident macrophages following injury, environmental exposure, or experimental depletion can be altered upon re-establishment of homeostasis. However, the impact of altered resident macrophage ontogenetic milieu on subsequent immune responses is poorly understood. Hence, we assessed the effect of macrophage ontogeny alteration following return to homeostasis on subsequent allergic airway responses to house dust mites (HDM). Using lineage tracing, we confirmed alveolar and interstitial macrophage ontogeny and their replacement by bone marrow-derived macrophages following LPS exposure. This alteration in macrophage ontogenetic milieu reduced allergic airway responses to HDM challenge. In addition, we defined a distinct population of resident-derived interstitial macrophages expressing allergic airway disease genes, located adjacent to terminal bronchi, and reduced by prior LPS exposure. These findings support that the ontogenetic milieu of pulmonary macrophages is a central factor in allergic airway responses and has implications for how prior environmental exposures impact subsequent immune responses and the development of allergy.

Tensions in Taxonomies: Current Understanding and Future Directions in the Pathobiologic Basis and Treatment of Group 1 and Group 3 Pulmonary Hypertension.

In the over 100 years since the recognition of pulmonary hypertension (PH), immense progress and significant achievements have been made with regard to understanding the pathophysiology of the disease and its treatment. These advances have been mostly in idiopathic pulmonary arterial hypertension (IPAH), which was classified as Group 1 Pulmonary Hypertension (PH) at the Second World Symposia on PH in 1998. However, the pathobiology of PH due to chronic lung disease, classified as Group 3 PH, remains poorly understood and its treatments thus remain limited. We review the history of the classification of the five groups of PH and aim to provide a state-of-the-art review of the understanding of the pathogenesis of Group 1 PH and Group 3 PH including insights gained from novel high-throughput omics technologies that have revealed heterogeneities within these categories as well as similarities between them. Leveraging the substantial gains made in understanding the genomics, epigenomics, proteomics, and metabolomics of PAH to understand the full spectrum of the complex, heterogeneous disease of PH is needed. Multimodal omics data as well as supervised and unbiased machine learning approaches after careful consideration of the powerful advantages as well as of the limitations and pitfalls of these technologies could lead to earlier diagnosis, more precise risk stratification, better predictions of disease response, new sub-phenotype groupings within types of PH, and identification of shared pathways between PAH and other types of PH that could lead to new treatment targets. © 2023 American Physiological Society. Compr Physiol 13:4295-4319, 2023.

The Pathobiology of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension is characterized by obliteration and obstruction of the pulmonary arterioles that in turn results in high right ventricular afterload and right heart failure. The pathobiology of pulmonary arterial hypertension is complex, with contributions from multiple pathophysiologic processes that are regulated by a variety of molecular mechanisms. This nature likely explains the limited efficacy of our current therapies, which only target a small portion of the pathobiological mechanisms that underlie advanced disease. Here we review the pathobiology of pulmonary arterial hypertension, focusing on the systemic, cellular, and molecular mechanisms that underlie the disease.

Nonclassical Monocytes Sense Hypoxia, Regulate Pulmonary Vascular Remodeling, and Promote Pulmonary Hypertension.

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, Ly6Clo 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 Ly6Clo monocyte lineage were exposed to hypoxia. In these mice, vascular remodeling and PH severity were significantly reduced. Transcriptome analyses suggest that the Ly6Clo 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.

Isolation and Characterization of Human Lung Myeloid Cells.

Multiparameter flow cytometry of human lungs allows for characterization, isolation, and examination of human pulmonary immune cell composition, phenotype, and function. Here we describe an approach to process lung tissues and then utilize a base antibody panel to define all of the major immune cell types in a single staining condition. This base antibody panel can also be used to identify major immune cell types in human blood and bronchoalveolar lavage (BAL) fluid.

Phenotyping Tumor-Associated Macrophages.

Multi-parametric flow cytometry of tumor-bearing murine nonlymphoid tissue allows for characterization, isolation, and examination of immune cell composition, phenotype, and function. Here we describe an approach to process nonlymphoid tissues and then utilize a base antibody panel to define all of the major immune cell types in a single staining condition. This panel can be used to phenotype tumor-associated macrophages.

The cholesterol metabolite 27 hydroxycholesterol facilitates breast cancer metastasis through its actions on immune cells.

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.

A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.

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.

Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues.

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.

Age-related changes in the cellular composition and epithelial organization of the mouse trachea.

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.

CCR2 deficiency, dysregulation of Notch signaling, and spontaneous pulmonary arterial hypertension.

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.

CX3CR1 deficiency impairs dendritic cell accumulation in arterial intima and reduces atherosclerotic burden.

OBJECTIVE: Dendritic cells (DCs) have recently been found in atherosclerosis-predisposed regions of arteries and have been proposed to be causal in atherosclerosis. The chemokine receptor CX3CR1 is associated with arterial injury and atherosclerosis. We sought to determine whether a link exists between arterial DC accumulation, CX3CR1, and atherosclerosis. METHODS AND RESULTS: Mouse aortas were isolated and subjected to en face immunofluorescence analysis. We found that DCs were located predominantly in the intimal regions of arterial branch points and curvatures. Consistent with the increased accumulation of intimal DCs in aged and ApoE-/- aortas compared with young WT aortas (P=0.004 and 0.05, respectively), the incidence of atherosclerosis was 88.9% for aged WT and 100% for ApoE-/- mice compared with 0% for young WT mice. CX3CR1 was expressed on intimal DCs and DC numbers were decreased in CX3CR1-deficient aortas of young, aged, and ApoE-/- mice (P=0.0008, 0.013, and 0.0099). The reduced DC accumulation in CX3CR1-deficiency was also correlated with decreased atherosclerosis in these animals. CONCLUSIONS: The accumulation of intimal DC increases in aged and ApoE-/- aortas and correlates with the generation of atherosclerosis. CX3CR1-deficiency impairs the accumulation of DC in the aortic wall and markedly reduces the atherosclerotic burden.

Defective antitumor responses in CX3CR1-deficient mice.

Innate immunity is critically important for tumor surveillance and regulating tumor metastasis. Fractalkine (FKN, CX3CL1), operating through the receptor CX3CR1, is an effective chemoattractant and adhesion receptor for NK cells and monocytes, important constituents of the innate immune response. Previous studies have shown that over-expression of CX3CL1 by tumor cells enhances antitumor responses. However, since most tumors do not express CX3CL1, it remains unclear if CX3CL1/CX3CR1 has a role in tumor immunity in the absence of ligand over-expression. To determine the role of CX3CL1 and CX3CR1 in regulating antitumor immune responses, we tested the response of wildtype and CX3CR1-deficient animals to unmanipulated B16 melanoma that does not express CX3CL1. We studied the distribution and trafficking of mononuclear cells (MNC) under homeostatic conditions and in the presence of B16 metastatic melanoma, cytotoxic activity, and cytokine production in wild-type and CX3CR1-deficient animals. We found that B16-treated CX3CR1-/- mice had increased lung tumor burden and cachexia. There was a selective reduction of monocytes and NK cells in the lungs of CX3CR1-deficient animals under homeostatic conditions and in response to B16. CX3CR1-deficient NK cells effectively killed B16 cells in cytotoxicity assays. However, CX3CR1-deficient NK cells exhibited a tumorigenic cytokine production profile with defective IFN-gamma expression and enhanced IL-6 production in response to TLR3 activation with polyIC. Our studies indicate that CX3CR1 is an important contributor to innate immunity at multiple levels. Its role in tumor immunity is not limited by expression of CX3CL1 by tumor cells.

Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice.

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated ("desensitized") by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various beta-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of beta-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the beta-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from beta-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that beta-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.