Hyperoxia prevents the dynamic neonatal increases in lung mesenchymal cell diversity.

Rapid expansion of the pulmonary microvasculature through angiogenesis drives alveolarization, the final stage of lung development that occurs postnatally and dramatically increases lung gas-exchange surface area. Disruption of pulmonary angiogenesis induces long-term structural and physiologic lung abnormalities, including bronchopulmonary dysplasia, a disease characterized by compromised alveolarization. Although endothelial cells are primary determinants of pulmonary angiogenesis, mesenchymal cells (MC) play a critical and dual role in angiogenesis and alveolarization. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung to profile mesenchymal cells during alveolarization and in the context of lung injury. Specific mesenchymal cell subtypes were present at birth with increasing diversity during alveolarization even while expressing a distinct transcriptomic profile from more mature correlates. Hyperoxia arrested the transcriptomic progression of the MC, revealed differential cell subtype vulnerability with pericytes and myofibroblasts most affected, altered cell to cell communication, and led to the emergence of Acta1 expressing cells. These insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world.

Transforming Growth Factor-induced Protein Promotes NF-κB-mediated Angiogenesis during Postnatal Lung Development.

Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-κB promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-κB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-κB signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis. NF-κB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-ß-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and Tgfbi null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Secreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PECs. Proteomic analysis identified TGFBI as one protein highly expressed by the early alveolar lung that promoted PEC migration by activating NF-κB via αvß3 integrins. RNA sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PECs. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. Our studies identify TGFBI as a developmentally regulated protein that promotes NF-κB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

Diverse homeostatic and immunomodulatory roles of immune cells in the developing mouse lung at single cell resolution.

At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.

S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis.

Neutrophil accumulation is associated with lung pathology during active tuberculosis (ATB). However, the molecular mechanism or mechanisms by which neutrophils accumulate in the lung and contribute to TB immunopathology are not fully delineated. Using the well-established mouse model of TB, our new data provide evidence that the alarmin S100A8/A9 mediates neutrophil accumulation during progression to chronic TB. Depletion of neutrophils or S100A8/A9 deficiency resulted in improved Mycobacterium tuberculosis (Mtb) control during chronic but not acute TB. Mechanistically, we demonstrate that, following Mtb infection, S100A8/A9 expression is required for upregulation of the integrin molecule CD11b specifically on neutrophils, mediating their accumulation during chronic TB disease. These findings are further substantiated by increased expression of S100A8 and S100A9 mRNA in whole blood in human TB progressors when compared with nonprogressors and rapidly decreased S100A8/A9 protein levels in the serum upon TB treatment. Furthermore, we demonstrate that S100A8/A9 serum levels along with chemokines are useful in distinguishing between ATB and asymptomatic Mtb-infected latent individuals. Thus, our results support targeting S100A8/A9 pathways as host-directed therapy for TB.

Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.

Tuberculosis is the leading cause of death by an infectious disease worldwide1. However, the involvement of innate lymphoid cells (ILCs) in immune responses to infection with Mycobacterium tuberculosis (Mtb) is unknown. Here we show that circulating subsets of ILCs are depleted from the blood of participants with pulmonary tuberculosis and restored upon treatment. Tuberculosis increased accumulation of ILC subsets in the human lung, coinciding with a robust transcriptional response to infection, including a role in orchestrating the recruitment of immune subsets. Using mouse models, we show that group 3 ILCs (ILC3s) accumulated rapidly in Mtb-infected lungs and coincided with the accumulation of alveolar macrophages. Notably, mice that lacked ILC3s exhibited a reduction in the accumulation of early alveolar macrophages and decreased Mtb control. We show that the C-X-C motif chemokine receptor 5 (CXCR5)-C-X-C motif chemokine ligand 13 (CXCL13) axis is involved in Mtb control, as infection upregulates CXCR5 on circulating ILC3s and increases plasma levels of its ligand, CXCL13, in humans. Moreover, interleukin-23-dependent expansion of ILC3s in mice and production of interleukin-17 and interleukin-22 were found to be critical inducers of lung CXCL13, early innate immunity and the formation of protective lymphoid follicles within granulomas. Thus, we demonstrate an early protective role for ILC3s in immunity to Mtb infection.

Interleukin-17 limits hypoxia-inducible factor 1α and development of hypoxic granulomas during tuberculosis.

Mycobacterium tuberculosis (Mtb) is a global health threat, compounded by the emergence of drug-resistant strains. A hallmark of pulmonary tuberculosis (TB) is the formation of hypoxic necrotic granulomas, which upon disintegration, release infectious Mtb. Furthermore, hypoxic necrotic granulomas are associated with increased disease severity and provide a niche for drug-resistant Mtb. However, the host immune responses that promote the development of hypoxic TB granulomas are not well described. Using a necrotic Mtb mouse model, we show that loss of Mtb virulence factors, such as phenolic glycolipids, decreases the production of the proinflammatory cytokine IL-17 (also referred to as IL-17A). IL-17 production negatively regulates the development of hypoxic TB granulomas by limiting the expression of the transcription factor hypoxia-inducible factor 1α (HIF1α). In human TB patients, HIF1α mRNA expression is increased. Through genotyping and association analyses in human samples, we identified a link between the single nucleotide polymorphism rs2275913 in the IL-17 promoter (-197G/G), which is associated with decreased IL-17 production upon stimulation with Mtb cell wall. Together, our data highlight a potentially novel role for IL-17 in limiting the development of hypoxic necrotic granulomas and reducing disease severity in TB.

Inhibition of Neutrophil Extracellular Trap Formation after Stem Cell Transplant by Prostaglandin E2.

RATIONALE: Autologous and allogeneic hematopoietic stem cell transplant (HSCT) patients are susceptible to pulmonary infections, including bacterial pathogens, even after hematopoietic reconstitution. We previously reported that murine bone marrow transplant (BMT) neutrophils overexpress cyclooxygenase-2, overproduce prostaglandin E2 (PGE2), and exhibit defective intracellular bacterial killing. Neutrophil extracellular traps (NETs) are DNA structures that capture and kill extracellular bacteria and other pathogens. OBJECTIVES: To determine whether NETosis was defective after transplant and if so, whether this was regulated by PGE2 signaling. METHODS: Neutrophils isolated from mice and humans (both control and HSCT subjects) were analyzed for NETosis in response to various stimuli in the presence or absence of PGE2 signaling modifiers. MEASUREMENTS AND MAIN RESULTS: NETs were visualized by immunofluorescence or quantified by Sytox Green fluorescence. Treatment of BMT or HSCT neutrophils with phorbol 12-myristate 13-acetate or rapamycin resulted in reduced NET formation relative to control cells. NET formation after BMT was rescued both in vitro and in vivo with cyclooxygenase inhibitors. Additionally, the EP2 receptor antagonist (PF-04418948) or the EP4 antagonist (AE3-208) restored NET formation in neutrophils isolated from BMT mice or HSCT patients. Exogenous PGE2 treatment limited NETosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by cAMP- and protein kinase A-dependent manner. CONCLUSIONS: Our results suggest blockade of the PGE2-EP2 or EP4 signaling pathway restores NETosis after transplantation. Furthermore, these data provide the first description of a physiologic inhibitor of NETosis.

Transforming growth factor-ß induces microRNA-29b to promote murine alveolar macrophage dysfunction after bone marrow transplantation.

Hematopoietic stem cell transplantation (HSCT) is complicated by pulmonary infections that manifest posttransplantation. Despite engraftment, susceptibility to infections persists long after reconstitution. Previous work using a murine bone marrow transplant (BMT) model implicated increased cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in promoting impaired alveolar macrophage (AM) responses. However, mechanisms driving COX-2 overexpression remained elusive. Previously, transforming growth factor-ß (TGF-ß) signaling after BMT was shown to promote hypomethylation of the COX-2 gene. Here, we provide mechanistic insight into how this occurs and show that TGF-ß induces microRNA (miR)-29b while decreasing DNA methyltransferases (DNMT)1, DNMT3a, and DNMT3b in AMs after BMT. De novo DNMT3a and DNMT3b were decreased upon transient transfection of miR-29b, resulting in decreased methylation of the COX-2 promoter and induction of COX-2. As a consequence, miR-29b-driven upregulation of COX-2 promoted AM dysfunction, and transfection of BMT AMs with a miR-29b inhibitor rescued the bacterial-killing defect. MiR-29b-mediated defects in BMT AMs were dependent on increased levels of PGE2, as miR-29b-transfected AMs treated with a novel E prostanoid receptor 2 antagonist abrogated the impaired bacterial killing. We also demonstrate that patients that have undergone HSCT exhibit increased miR-29b; thus these studies highlight miR-29b in driving defective AM responses and identify this miRNA as a potential therapeutic target.

Innate Immunity Post-Hematopoietic Stem Cell Transplantation: Focus on Epigenetics.

Epigenetic regulation of gene expression is important for normal biological processes like immune cell development, immune responses, and differentiation from hematopoietic stem cells. Furthermore, it is well understood that epigenetic mechanisms can include methylation, histone modification, and more recently, microRNAs. Interestingly, aberrant epigenetic modification can also promote pathology in many diseases like cancer. The effects of methylation on gene expression and its resulting phenotype have been extensively studied. In this review, we discuss the inhibition of innate immunity that occurs in humans and animal models post-stem cell transplant. In addition, we highlight the changes methylation and microRNA profiles have on regulating pulmonary innate immune responses in the context of hematopoietic stem cell transplantation in experimental animal models.

Defective pulmonary innate immune responses post-stem cell transplantation; review and results from one model system.

Infectious pulmonary complications limit the success of hematopoietic stem cell transplantation (HSCT) as a therapy for malignant and non-malignant disorders. Susceptibility to pathogens in both autologous and allogeneic HSCT recipients persists despite successful immune reconstitution. As studying the causal effects of these immune defects in the human population can be limiting, a bone marrow transplant (BMT) mouse model can be used to understand the defect in mounting a productive innate immune response post-transplantation. When syngeneic BMT is performed, this system allows the study of BMT-induced alterations in innate immune cell function that are independent of the confounding effects of immunosuppressive therapy and graft-versus-host disease. Studies from several laboratories, including our own show that pulmonary susceptibility to bacterial infections post-BMT are largely due to alterations in the lung alveolar macrophages. Changes in these cells post-BMT include cytokine and eicosanoid dysregulations, scavenger receptor alterations, changes in micro RNA profiles, and alterations in intracellular signaling molecules that limit bacterial phagocytosis and killing. The changes that occur highlight mechanisms that promote susceptibility to infections commonly afflicting HSCT recipients and provide insight into therapeutic targets that may improve patient outcomes post-HSCT.

Prostaglandin E2-induced changes in alveolar macrophage scavenger receptor profiles differentially alter phagocytosis of Pseudomonas aeruginosa and Staphylococcus aureus post-bone marrow transplant.

The effectiveness of hematopoietic stem cell transplantation as a therapy for malignant and nonmalignant conditions is complicated by pulmonary infections. Using our syngeneic bone marrow transplant (BMT) mouse model, BMT mice with a reconstituted hematopoietic system displayed increased susceptibility to Pseudomonas aeruginosa and Staphylococcus aureus. BMT alveolar macrophages (AMs) exhibited a defect in P. aeruginosa phagocytosis, whereas S. aureus uptake was surprisingly enhanced. We hypothesized that the difference in phagocytosis was due to an altered scavenger receptor (SR) profile. Interestingly, MARCO expression was decreased, whereas SR-AI/II was increased. To understand how these dysregulated SR profiles might affect macrophage function, CHO cells were transfected with SR-AI/II, and phagocytosis assays revealed that SR-AI/II was important for S. aureus uptake but not for P. aeruginosa. Conversely, AMs treated in vitro with soluble MARCO exhibited similar defects in P. aeruginosa internalization as did BMT AMs. The 3'-untranslated region of SR-AI contains a putative target region for microRNA-155 (miR-155), and miR-155 expression is decreased post-BMT. Anti-miR-155-transfected AMs exhibited an increase in SR-AI/II expression and S. aureus phagocytosis. Elevated PGE2 has been implicated in driving an impaired innate immune response post-BMT. In vitro treatment of AMs with PGE2 increased SR-AI/II and decreased MARCO and miR-155. Despite a difference in phagocytic ability, BMT AMs harbor a killing defect to both P. aeruginosa and S. aureus. Thus, our data suggest that PGE2-driven alterations in SR and miR-155 expression account for the differential phagocytosis of P. aeruginosa and S. aureus, but impaired killing ultimately confers increased susceptibility to pulmonary infection.

COX-2 expression is upregulated by DNA hypomethylation after hematopoietic stem cell transplantation.

Hematopoietic stem cell transplant therapy is limited by pulmonary infections. Mice with fully reconstituted hematopoietic compartments, including alveolar macrophages (AMs), after bone marrow transplantation (BMT) have impaired host defense against Gram-negative Pseudomonas aeruginosa. Impaired innate immunity is related to increased production of PGE(2) by AMs. Cyclooxygenase (COX)-2 is the rate-limiting enzyme for synthesis of PGE(2) from arachidonic acid, and COX-2 expression is elevated in AMs post-BMT. We hypothesized that epigenetic mechanisms may be responsible for upregulation of COX-2 in AMs. Using bisulfite sequencing, we observed the 5'-untranslated region and exon 1 of the COX-2 gene is hypomethylated in the AMs of BMT mice compared with control. COX-2 expression was increased in primary AMs and in the AM cell line (MHS) after treatment with 5-aza-2'-deoxycytidine (a methyltransferase inhibitor). Methylation by SssI methyltransferase of a 698-bp region of the COX-2 promoter including the beginning of exon 1 driving a luciferase reporter silenced luciferase expression. Because TGF-ß1 is elevated in lungs post-BMT, we tested whether TGF-ß1 could promote expression of COX-2 in a hypermethylated COX-2 vector, and observed TGF-ß1-induced modest expression of COX-2, suggesting an ability to demethylate the promoter. Finally, BMTs performed with marrow from mice expressing a dominant-negative form of the TGF-ßRII on CD11c-expressing cells (which includes AMs) demonstrated improved host defense and AM function. Our findings suggest impaired innate immunity and PGE(2) elevation post-BMT are due to hypomethylation of the COX-2 gene, which is at least partly regulated by TGF-ß1.