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.

Interstitial macrophage phenotypes in Schistosoma-induced pulmonary hypertension.

Background: Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-ß, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1. Methods: Mice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression. Results: Flow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation. Conclusion: Schistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-ß and causes PH.

Experimental Schistosoma haematobium pulmonary hypertension.

Whether all Schistosoma species cause pulmonary hypertension (PH) is unclear. Experimentally exposing mice to Schistosoma haematobium eggs caused PH, which was less severe than that induced by S. mansoni exposure. These findings align with the relatively uncommon reports of pulmonary arterial hypertension associated with S. haematobium.

Is pulmonary arterial hypertension associated with schistosomiasis distinct from pulmonary arterial hypertension associated with portal hypertension?

Pulmonary arterial hypertension associated with schistosomiasis (SchPAH) and pulmonary arterial hypertension associated with portal hypertension (PoPAH) are lung diseases that develop in the presence of liver diseases. However, mechanistic pathways by which the underlying liver conditions and other drivers contribute to the development and progression of pulmonary arterial hypertension (PAH) are unclear for both etiologies. In turn, these unknowns limit certainty of strategies to prevent, diagnose, and reverse the resultant PAH. Here we consider specific mechanisms that contribute to SchPAH and PoPAH, identifying those that may be shared and those that appear to be unique to each etiology, in the hope that this exploration will both highlight known causal drivers and identify knowledge gaps appropriate for future research. Overall, the key pathophysiologic differences that we identify between SchPAH and PoPAH suggest that they are not variants of a single condition.

Dexamethasone prophylaxis protects from acute high-altitude illness by modifying the peripheral blood mononuclear cell inflammatory transcriptome.

Acute high-altitude (HA) exposure can induce several pathologies. Dexamethasone (DEX) can be taken prophylactically to prevent HA disease, but the mechanism by which it acts in this setting is unclear. We studied the transcriptome of peripheral blood mononuclear cells (PBMCs) from 16 subjects at low altitude (LA, 225 m) and then 3 days after acute travel to HA (3500 m) during the India-Leh-Dexamethasone-Expedition-2020 (INDEX2020). Half of the participants received oral DEX prophylaxis 4 mg twice daily in an unblinded manner, starting 1 day prior to travel to HA, and 12 h prior to the first PBMC collection. PBMC transcriptome data were obtained from 16 subjects, half of whom received DEX. The principal component analysis demonstrated a clear separation of the groups by altitude and treatment. HA exposure resulted in a large number of gene expression changes, particularly in pathways of inflammation or the regulation of cell division, translation, or transcription. DEX prophylaxis resulted in changes in fewer genes, particularly in immune pathways. The gene sets modulated by HA and DEX were distinct. Deconvolution analysis to assess PBMC subpopulations suggested changes in B-cell, T-cell, dendritic cell, and myeloid cell numbers with HA and DEX exposures. Acute HA travel and DEX prophylaxis induce significant changes in the PBMC transcriptome. The observed benefit of DEX prophylaxis against HA disease may be mediated by suppression of inflammatory pathways and changing leukocyte population distributions.

Repetitive schistosoma exposure causes perivascular lung fibrosis and persistent pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) can occur as a complication of schistosomiasis. In humans, schistosomiasis-PH persists despite antihelminthic therapy and parasite eradication. We hypothesized that persistent disease arises as a consequence of exposure repetition. METHODS: Following intraperitoneal sensitization, mice were experimentally exposed to Schistosoma eggs by intravenous injection, either once or three times repeatedly. The phenotype was characterized by right heart catheterization and tissue analysis. RESULTS: Following intraperitoneal sensitization, a single intravenous Schistosoma egg exposure resulted in a PH phenotype that peaked at 7-14 days, followed by spontaneous resolution. Three sequential exposures resulted in a persistent PH phenotype. Inflammatory cytokines were not significantly different between mice exposed to one or three egg doses, but there was an increase in perivascular fibrosis in those who received three egg doses. Significant perivascular fibrosis was also observed in autopsy specimens from patients who died of this condition. CONCLUSIONS: Repeatedly exposing mice to schistosomiasis causes a persistent PH phenotype, accompanied by perivascular fibrosis. Perivascular fibrosis may contribute to the persistent schistosomiasis-PH observed in humans with this disease.

The role of macrophages in right ventricular remodeling in experimental pulmonary hypertension.

Right ventricular (RV) failure is the primary cause of death in pulmonary hypertension (PH), but the mechanisms of RV failure are not well understood. We hypothesized macrophages in the RV contribute to the RV response in PH. We induced PH in mice with hypoxia (FiO2 10%) and Schistosoma mansoni exposure, and in rats with SU5416-hypoxia. We quantified cardiac macrophages in mice using flow cytometry. Parabiosis between congenic CD45.1/.2 mice or Cx3cr1-green fluorescent protein and wild-type mice was used to quantify circulation-derived macrophages in experimental PH conditions. We administered clodronate liposomes to Sugen hypoxia (SU-Hx) exposed rats to deplete macrophages and evaluated the effect on the extracellular matrix (ECM) and capillary network in the RV. In hypoxia exposed mice, the overall number of macrophages did not significantly change but two macrophage subpopulations increased. Parabiosis identified populations of RV macrophages that at steady state is derived from the circulation, with one subpopulation that significantly increased with PH stimuli. Clodronate treatment of SU-Hx rats resulted in a change in the RV ECM, without altering the RV vasculature, and correlated with improved RV function. Populations of RV macrophages increase and contribute to RV remodeling in PH, including through regulation of the RV ECM.

Contribution of fatty acid oxidation to the pathogenesis of pulmonary hypertension.

Dysregulated metabolism characterizes both animal and human forms of pulmonary hypertension (PH). Enzymes involved in fatty acid metabolism have previously not been assessed in human pulmonary arteries affected by pulmonary arterial hypertension (PAH), and how inhibition of fatty acid oxidation (FAO) may attenuate PH remains unclear. Fatty acid metabolism gene transcription was quantified in laser-dissected pulmonary arteries from 10 explanted lungs with advanced PAH (5 idiopathic, 5 associated with systemic sclerosis), and 5 donors without lung diseases. Effects of oxfenicine, a FAO inhibitor, on female Sugen 5416-chronic hypoxia (SuHx) rats were studied in vivo using right heart catheterization, and ex vivo using perfused lungs and pulmonary artery ring segments. The impact of pharmacologic (oxfenicine) and genetic (carnitine palmitoyltransferase 1a heterozygosity) FAO suppression was additionally probed in mouse models of Schistosoma and hypoxia-induced PH. Potential mechanisms underlying FAO-induced PH pathogenesis were examined by quantifying ATP and mitochondrial mass in oxfenicine-treated SuHx pulmonary arterial cells, and by assessing pulmonary arterial macrophage infiltration with immunohistochemistry. We found upregulated pulmonary arterial transcription of 26 and 13 FAO genes in idiopathic and systemic sclerosis-associated PAH, respectively. In addition to promoting de-remodeling of pulmonary arteries in SuHx rats, oxfenicine attenuated endothelin-1-induced vasoconstriction. FAO inhibition also conferred modest benefit in the two mouse models of PH. Oxfenicine increased mitochondrial mass in cultured rat pulmonary arterial cells, and decreased the density of perivascular macrophage infiltration in pulmonary arteries of treated SuHx rats. In summary, FAO inhibition attenuated experimental PH, and may be beneficial in human PAH.

Single-cell RNA sequencing and binary hierarchical clustering define lung interstitial macrophage heterogeneity in response to hypoxia.

Few studies have examined lung interstitial macrophage (IM) molecular phenotypes after being exposed to hypoxia in vivo at the single-cell level, even though macrophages contribute to hypoxic pulmonary hypertension (PH). We aimed to determine IM diversity and its association with hypoxia-induced PH. We hypothesized that integrating single-cell RNA sequencing (scRNAseq) and binary hierarchal clustering (BHC) could resolve IM heterogeneity under normal homeostatic conditions and changes induced by hypoxia exposure. Cx3cr1GFP/+ reporter mice were exposed to normoxic conditions (∼21% [Formula: see text]) or exposed to 1 day (D1) or 7 days (D7) of hypoxia (∼10% [Formula: see text]). We used flow cytometry to isolate Cx3cr1+ IMs and the 10X Genomics platform for scRNAseq, Cell Ranger, Seurat, ClusterMap, monocle, ingenuity pathway analysis, and Fisher's exact test (q value < 0.05) for functional investigations. n = 374 (normoxia), n = 2,526 (D1), and n = 1,211 (D7) IMs were included in the analyses. We identified three normoxia-related cell types, five hypoxia-associated cell types that emerged at D1, and three that appeared at D7. We describe the existence of a putative resident trained innate IM, which is present in normoxia, transiently depleted at D1, and recovered after 7 days of sustained hypoxia. We also define a rare putative pathogenic population associated with transcripts implicated in PH development that emerges at D7. In closing, we describe the successful integration of BHC with scRNAseq to determine IM heterogeneity and its association with PH. These results shed light on how resident-trained innate IMs become more heterogeneous but ultimately accustomed to hypoxia.

Experimental Schistosoma japonicum-induced pulmonary hypertension.

BACKGROUND: Schistosomiasis, a major cause of pulmonary arterial hypertension (PAH) worldwide, is most clearly described complicating infection by one species, Schistosoma mansoni. Controlled exposure of mice can be used to induce Type 2 inflammation-dependent S. mansoni pulmonary hypertension (PH). We sought to determine if another common species, S. japonicum, can also cause experimental PH. METHODS: Schistosome eggs were obtained from infected mice, and administered by intraperitoneal sensitization followed by intravenous challenge to experimental mice, which underwent right heart catheterization and tissue analysis. RESULTS: S. japonicum sensitized and challenged mice developed PH, which was milder than that following S. mansoni sensitization and challenge. The degree of pulmonary vascular remodeling and Type 2 inflammation in the lungs was similarly proportionate. Cross-sensitization revealed that antigens from either species are sufficient to sensitize for intravenous challenge with either egg, and the degree of PH severity depended on primarily the species used for intravenous challenge. Compared to a relatively uniform distribution of S. mansoni eggs, S. japonicum eggs were observed in clusters in the lungs. CONCLUSIONS: S. japonicum can induce experimental PH, which is milder than that resulting from comparable S. mansoni exposure. This difference may result from the distribution of eggs in the lungs, and is independent of which species is used for sensitization. This result is consistent with the clearer association between S. mansoni infection and the development of schistosomiasis-associated PAH in humans.

Endothelial cell PHD2-HIF1α-PFKFB3 contributes to right ventricle vascular adaptation in pulmonary hypertension.

Humans and animals with pulmonary hypertension (PH) show right ventricular (RV) capillary growth, which positively correlates with overall RV hypertrophy. However, molecular drivers of RV vascular augmentation in PH are unknown. Prolyl hydroxylase (PHD2) is a regulator of hypoxia-inducible factors (HIFs), which transcriptionally activates several proangiogenic genes, including the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We hypothesized that a signaling axis of PHD2-HIF1α-PFKFB3 contributes to adaptive coupling between the RV vasculature and tissue volume to maintain appropriate vascular density in PH. We used design-based stereology to analyze endothelial cell (EC) proliferation and the absolute length of the vascular network in the RV free wall, relative to the tissue volume in mice challenged with hypoxic PH. We observed increased RV EC proliferation starting after 6 h of hypoxia challenge. Using parabiotic mice, we found no evidence for a contribution of circulating EC precursors to the RV vascular network. Mice with transgenic deletion or pharmacological inhibition of PHD2, HIF1α, or PFKFB3 all had evidence of impaired RV vascular adaptation following hypoxia PH challenge. PHD2-HIF1α-PFKFB3 contributes to structural coupling between the RV vascular length and tissue volume in hypoxic mice, consistent with homeostatic mechanisms that maintain appropriate vascular density. Activating this pathway could help augment the RV vasculature and preserve RV substrate delivery in PH, as an approach to promote RV function.

Schistosomiasis Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a disease of the lung blood vessels that results in right heart failure. PAH is thought to occur in about 5% to 10% of patients with hepatosplenic schistosomiasis, particularly due to S. mansoni. The lung blood vessel injury may result from a combination of embolization of eggs through portocaval shunts into the lungs causing localized Type 2 inflammatory response and vessel remodeling, triggering of autonomous pathology that becomes independent of the antigen, and high cardiac output as seen in portopulmonary hypertension. The condition is likely underdiagnosed as there is little systematic screening, and risk factors for developing PAH are not known. Screening is done by echocardiography, and formal diagnosis requires invasive right heart catheterization. Patients with Schistosoma-associated PAH show reduced functional capacity and can be treated with pulmonary vasodilators, which improves symptoms and may improve survival. There are animal models of this disease that might help in understanding disease pathogenesis and identify novel targets to screen and treatment. Pathogenic mechanisms include Type 2 immunity and activation and signaling in the TGF-ß pathway. There are still major uncertainties regarding Schistosoma-associated PAH development, course and treatment.

Pathophysiology and potential future therapeutic targets using preclinical models of COVID-19.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) gains entry into the lung epithelial cells by binding to the surface protein angiotensin-converting enzyme 2. Severe SARS-CoV-2 infection, also known as coronavirus disease 2019 (COVID-19), can lead to death due to acute respiratory distress syndrome mediated by inflammatory immune cells and cytokines. In this review, we discuss the molecular and biochemical bases of the interaction between SARS-CoV-2 and human cells, and in doing so we highlight knowledge gaps currently precluding development of new effective therapies. In particular, discovery of novel treatment targets in COVID-19 will start from understanding pathologic changes based on a large number of autopsy lung tissue samples. Pathogenetic roles of potential molecular targets identified in human lung tissues must be validated in established animal models. Overall, this stepwise approach will enable appropriate selection of candidate therapeutic modalities targeting SARS-CoV2 and the host inflammatory response.

Publisher Correction: Stable isotope metabolomics of pulmonary artery smooth muscle and endothelial cells in pulmonary hypertension and with TGF-beta treatment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stable isotope metabolomics of pulmonary artery smooth muscle and endothelial cells in pulmonary hypertension and with TGF-beta treatment.

Altered metabolism in pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) contributes to the pathology of pulmonary hypertension (PH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human PASMCs and PAECs from PH versus control specimens, and that TGF-ß treatment would phenocopy these metabolic changes. We used 13C-labeled glucose, glutamine or a long-chain fatty acid mixture added to cell culture media, and mass spectrometry-based metabolomics to detect and quantify 13C-labeled metabolites. We found PH PASMCs had increased glucose uptake and utilization by glycolysis and the pentose shunt, but no changes in glutamine or fatty acid uptake or utilization. Diseased PAECs had increased proximate glycolysis pathway intermediates, less pentose shunt flux, increased anaplerosis from glutamine, and decreased fatty acid ß-oxidation. TGF-ß treatment increased glycolysis in PASMCs, but did not recapitulate the PAEC disease phenotype. In TGF-ß-treated PASMCs, glucose, glutamine and fatty acids all contributed carbons to the TCA cycle. In conclusion, PASMCs and PAECs collected from PH subjects have significant changes in metabolite uptake and utilization, partially recapitulated by TGF-ß treatment.

Interstitial macrophage-derived thrombospondin-1 contributes to hypoxia-induced pulmonary hypertension.

AIMS: Transforming growth factor-ß (TGF-ß) signalling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-ß by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signalling pathway that link activated TGF-ß to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages (IMs), are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-ß with increased Rho-kinase signalling, causing vasoconstriction. METHODS AND RESULTS: Flow cytometry revealed that a specific subset of IMs is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho-kinase-mediated vasoconstriction was a major downstream target of active TGF-ß. Thbs1 deficient bone marrow (BM) protected against hypoxic-PH by blocking TGF-ß activation and Rho-kinase-mediated vasoconstriction. CONCLUSION: In hypoxia-challenged mice, BM derived and circulating monocytes are recruited to become IMs which express TSP-1, resulting in TGF-ß activation and Rho-kinase-mediated vasoconstriction.