Respiratory viruses: New frontiers-a Keystone Symposia report.

Respiratory viruses are a common cause of morbidity and mortality around the world. Viruses like influenza, RSV, and most recently SARS-CoV-2 can rapidly spread through a population, causing acute infection and, in vulnerable populations, severe or chronic disease. Developing effective treatment and prevention strategies often becomes a race against ever-evolving viruses that develop resistance, leaving therapy efficacy either short-lived or relevant for specific viral strains. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Respiratory Viruses: New Frontiers." Researchers presented new insights into viral biology and virus-host interactions to understand the mechanisms of disease and identify novel treatment and prevention approaches that are effective, durable, and broad.

Unusual X chromosome inactivation maintenance in female alveolar type 2 cells is correlated with increased numbers of X-linked escape genes and sex-biased gene expression.

Sex differences exist for many lung pathologies, including COVID-19 and pulmonary fibrosis, but the mechanistic basis for this remains unclear. Alveolar type 2 cells (AT2s), which play a key role in alveolar lung regeneration, express the X-linked Ace2 gene that has roles in lung repair and SARS-CoV-2 pathogenesis, suggesting that X chromosome inactivation (XCI) in AT2s might impact sex-biased lung pathology. Here we investigate XCI maintenance and sex-specific gene expression profiles using male and female AT2s. Remarkably, the inactive X chromosome (Xi) lacks robust canonical Xist RNA "clouds" and less enrichment of heterochromatic modifications in human and mouse AT2s. We demonstrate that about 68% of expressed X-linked genes in mouse AT2s, including Ace2, escape XCI. There are genome-wide expression differences between male and female AT2s, likely influencing both lung physiology and pathophysiologic responses. These studies support a renewed focus on AT2s as a potential contributor to sex-biased differences in lung disease.

Stem cells, cell therapies, and bioengineering in lung biology and disease 2021.

The 9th biennial conference titled "Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases" was hosted virtually, due to the ongoing COVID-19 pandemic, in collaboration with the University of Vermont Larner College of Medicine, the National Heart, Lung, and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, and the International Society for Cell & Gene Therapy. The event was held from July 12th through 15th, 2021 with a pre-conference workshop held on July 9th. As in previous years, the objectives remained to review and discuss the status of active research areas involving stem cells (SCs), cellular therapeutics, and bioengineering as they relate to the human lung. Topics included 1) technological advancements in the in situ analysis of lung tissues, 2) new insights into stem cell signaling and plasticity in lung remodeling and regeneration, 3) the impact of extracellular matrix in stem cell regulation and airway engineering in lung regeneration, 4) differentiating and delivering stem cell therapeutics to the lung, 5) regeneration in response to viral infection, and 6) ethical development of cell-based treatments for lung diseases. This selection of topics represents some of the most dynamic and current research areas in lung biology. The virtual workshop included active discussion on state-of-the-art methods relating to the core features of the 2021 conference, including in situ proteomics, lung-on-chip, induced pluripotent stem cell (iPSC)-airway differentiation, and light sheet microscopy. The conference concluded with an open discussion to suggest funding priorities and recommendations for future research directions in basic and translational lung biology.

DNA binding to TLR9 expressed by red blood cells promotes innate immune activation and anemia.

Red blood cells (RBCs) are essential for aerobic respiration through delivery of oxygen to distant tissues. However, RBCs are currently considered immunologically inert, and few, if any, secondary functions of RBCs have been identified. Here, we showed that RBCs serve as critical immune sensors through surface expression of the nucleic acid­sensing Toll-like receptor 9 (TLR9). Mammalian RBCs expressed TLR9 on their surface and bound CpG-containing DNA derived from bacteria, plasmodia, and mitochondria. RBC-bound mitochondrial DNA was increased during human and murine sepsis and pneumonia. In vivo, CpG-carrying RBCs drove accelerated erythrophagocytosis and innate immune activation characterized by increased interferon signaling. Erythroid-specific deletion of TLR9 abrogated erythrophagocytosis and decreased local and systemic cytokine production during CpG-induced inflammation and polymicrobial sepsis. Thus, detection and capture of nucleic acid by TLR9-expressing RBCs regulated red cell clearance and inflammatory cytokine production, demonstrating that RBCs function as immune sentinels during pathologic states. Consistent with these findings, RBC-bound mitochondrial DNA was elevated in individuals with viral pneumonia and sepsis secondary to coronavirus disease 2019 (COVID-19) and associated with anemia and severity of disease. These findings uncover a previously unappreciated role of RBCs as critical players in inflammation distinct from their function in gas transport.

Regeneration of the pulmonary vascular endothelium after viral pneumonia requires COUP-TF2.

Acute respiratory distress syndrome is associated with a robust inflammatory response that damages the vascular endothelium, impairing gas exchange. While restoration of microcapillaries is critical to avoid mortality, therapeutic targeting of this process requires a greater understanding of endothelial repair mechanisms. Here, we demonstrate that lung endothelium possesses substantial regenerative capacity and lineage tracing reveals that native endothelium is the source of vascular repair after influenza injury. Ablation of chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TF2) (Nr2f2), a transcription factor implicated in developmental angiogenesis, reduced endothelial proliferation, exacerbating viral lung injury in vivo. In vitro, COUP-TF2 regulates proliferation and migration through activation of cyclin D1 and neuropilin 1. Upon influenza injury, nuclear factor κB suppresses COUP-TF2, but surviving endothelial cells ultimately reestablish vascular homeostasis dependent on restoration of COUP-TF2. Therefore, stabilization of COUP-TF2 may represent a therapeutic strategy to enhance recovery from pathogens, including H1N1 influenza and SARS-CoV-2.

Basal-like Progenitor Cells: A Review of Dysplastic Alveolar Regeneration and Remodeling in Lung Repair.

Despite the central importance of the respiratory system, the exact mechanisms governing lung repair after severe injury remain unclear. The notion that alveolar type 2 cells (AT2s) self-renew and differentiate into alveolar type 1 cells (AT1s) does not fully encompass scenarios where these progenitors are severely affected by disease, e.g., H1N1 influenza or SARS-CoV-2 (COVID-19). Intrapulmonary p63+ progenitor cells, a rare cell type in mice but potentially encompassing more numerous classic basal cells in humans, are activated in such severe injury settings, proliferating and migrating into the injured alveolar parenchyma, providing a short-term "emergency" benefit. While the fate of these cells is controversial, most studies indicate that they represent a maladaptive repair pathway with a fate restriction toward airway cell types, rarely differentiating into AT2 or AT1 cells. Here, we discuss the role of intrapulmonary basal-like p63+ cells in alveolar regeneration and suggest a unified model to guide future studies.