Circuits between infected macrophages and T cells in SARS-CoV-2 pneumonia.

Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses.

The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.

Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

The PYRIN Domain-only Protein POP1 Inhibits Inflammasome Assembly and Ameliorates Inflammatory Disease.

In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1ß and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial for the maintenance of homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1ß and IL-18 release, pyroptosis, and the release of ASC particles. There is no mouse ortholog for POP1, but transgenic expression of human POP1 in monocytes, macrophages, and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation, and ASC danger particles. POP1 expression was regulated by TLR and IL-1R signaling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.

Obesity Is Associated with Attenuated Tissue Immunity in COVID-19.

Rationale: Obesity affects 40% of U.S. adults, is associated with a proinflammatory state, and presents a significant risk factor for the development of severe coronavirus disease (COVID-19). To date, there is limited information on how obesity might affect immune cell responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: To determine the impact of obesity on respiratory tract immunity in COVID-19 across the human lifespan. Methods: We analyzed single-cell transcriptomes from BAL in three ventilated adult cohorts with (n = 24) or without (n = 9) COVID-19 from nasal immune cells in children with (n = 14) or without (n = 19) COVID-19, and from peripheral blood mononuclear cells in an independent adult COVID-19 cohort (n = 42), comparing obese and nonobese subjects. Measurements and Main Results: Surprisingly, we found that obese adult subjects had attenuated lung immune or inflammatory responses in SARS-CoV-2 infection, with decreased expression of IFN-α, IFN-γ, and TNF-α (tumor necrosis factor α) response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Peripheral blood immune cells in an independent adult cohort showed a similar but less marked reduction in type-I IFN and IFNγ response genes, as well as decreased serum IFNα, in obese patients with SARS-CoV-2. Nasal immune cells from obese children with COVID-19 also showed reduced enrichment of IFN-α and IFN-γ response genes. Conclusions: These findings show blunted tissue immune responses in obese patients with COVID-19, with implications for treatment stratification, supporting the specific application of inhaled recombinant type-I IFNs in this vulnerable subset.

Resetting proteostasis with ISRIB promotes epithelial differentiation to attenuate pulmonary fibrosis.

Pulmonary fibrosis is a relentlessly progressive and often fatal disease with a paucity of available therapies. Genetic evidence implicates disordered epithelial repair, which is normally achieved by the differentiation of small cuboidal alveolar type 2 (AT2) cells into large, flattened alveolar type 1 (AT1) cells as an initiating event in pulmonary fibrosis pathogenesis. Using models of pulmonary fibrosis in young adult and old mice and a model of adult alveologenesis after pneumonectomy, we show that administration of ISRIB, a small molecule that restores protein translation by EIF2B during activation of the integrated stress response (ISR), accelerated the differentiation of AT2 into AT1 cells. Accelerated epithelial repair reduced the recruitment of profibrotic monocyte-derived alveolar macrophages and ameliorated lung fibrosis. These findings suggest a dysfunctional role for the ISR in regeneration of the alveolar epithelium after injury with implications for therapy.

A Novel MIP-1-Expressing Macrophage Subtype in BAL Fluid from Healthy Volunteers.

Tissue availability remains an important limitation of single-cell genomic technologies for investigating cellular heterogeneity in human health and disease. BAL represents a minimally invasive approach to assessing an individual's lung cellular environment for diagnosis and research. However, the lack of high-quality, healthy lung reference data is a major obstacle to using single-cell approaches to study a plethora of lung diseases. Here, we performed single-cell RNA sequencing on over 40,000 cells isolated from the BAL of four healthy volunteers. Of the six cell types or lineages we identified, macrophages were consistently the most numerous across individuals. Our analysis confirmed the expression of marker genes defining cell types despite background signals because of the ambient RNA found in many single-cell studies. We assessed the variability of gene expression across macrophages and defined a distinct subpopulation of cells expressing a set of genes associated with Macrophage Inflammatory Protein 1 (MIP-1). RNA in situ hybridization and reanalysis of published lung single-cell data validated the presence of this macrophage subpopulation. Thus, our study characterizes lung macrophage heterogeneity in healthy individuals and provides a valuable resource for future studies to understand the lung environment in health and disease.

Individual meropenem epithelial lining fluid and plasma PK/PD target attainment.

It is unclear whether plasma is a reliable surrogate for target attainment in the epithelial lining fluid (ELF). The objective of this study was to characterize meropenem target attainment in plasma and ELF using prospective samples. The first 24-hour T>MIC was evaluated vs 1xMIC and 4xMIC targets at the patient (i.e., fixed MIC of 2 mg/L) and population [i.e., cumulative fraction of response (CFR) according to EUCAST MIC distributions] levels for both plasma and ELF. Among 65 patients receiving ≥24 hours of treatment, 40% of patients failed to achieve >50% T>4xMIC in plasma and ELF, and 30% of patients who achieved >50% T>4xMIC in plasma had <50% T>4xMIC in ELF. At 1xMIC and 4xMIC targets, 3% and 25% of patients with >95% T>MIC in plasma had <50% T>MIC in ELF, respectively. Those with a CRCL >115 mL/min were less likely to achieve >50%T>4xMIC in ELF (P < 0.025). In the population, CFR for Escherichia coli at 1xMIC and 4xMIC was >97%. For Pseudomonas aeruginosa, CFR was ≥90% in plasma and ranged 80%-85% in ELF at 1xMIC when a loading dose was applied. CFR was reduced in plasma (range: 75%-81%) and ELF (range: 44%-60%) in the absence of a loading dose at 1xMIC. At 4xMIC, CFR for P. aeruginosa was 60%-86% with a loading dose and 18%-62% without a loading dose. We found that plasma overestimated ELF target attainment inup to 30% of meropenem-treated patients, CRCL >115 mL/min decreased target attainment in ELF, and loading doses increased CFR in the population.

Regulator of Cell Cycle Protein (RGCC/RGC-32) Protects against Pulmonary Fibrosis.

Some previous studies in tissue fibrosis have suggested a profibrotic contribution from elevated expression of a protein termed either RGCC (regulator of cell cycle) or RGC-32 (response gene to complement 32 protein). Our analysis of public gene expression datasets, by contrast, revealed a consistent decrease in RGCC mRNA levels in association with pulmonary fibrosis. Consistent with this observation, we found that stimulating primary adult human lung fibroblasts with transforming growth factor (TGF)-ß in cell cultures elevated collagen expression and simultaneously attenuated RGCC mRNA and protein levels. Moreover, overexpression of RGCC in cultured lung fibroblasts attenuated the stimulating effect of TGF-ß on collagen levels. Similar to humans with pulmonary fibrosis, the levels of RGCC were also decreased in vivo in lung tissues of wild-type mice challenged with bleomycin in both acute and chronic models. Mice with constitutive RGCC gene deletion accumulated more collagen in their lungs in response to chronic bleomycin challenge than did wild-type mice. RNA-Seq analyses of lung fibroblasts revealed that RGCC overexpression alone had a modest transcriptomic effect, but in combination with TGF-ß stimulation, induced notable transcriptomic changes that negated the effects of TGF-ß, including on extracellular matrix-related genes. At the level of intracellular signaling, RGCC overexpression delayed early TGF-ß-induced Smad2/3 phosphorylation, elevated the expression of total and phosphorylated antifibrotic mediator STAT1, and attenuated the expression of a profibrotic mediator STAT3. We conclude that RGCC plays a protective role in pulmonary fibrosis and that its decline permits collagen accumulation. Restoration of RGCC expression may have therapeutic potential in pulmonary fibrosis.

Lung Injury Induces Alveolar Type 2 Cell Hypertrophy and Polyploidy with Implications for Repair and Regeneration.

Epithelial polyploidization after injury is a conserved phenomenon recently shown to improve barrier restoration during wound healing. Whether lung injury can induce alveolar epithelial polyploidy is not known. We show that bleomycin injury induces alveolar type 2 cell (AT2) hypertrophy and polyploidy. AT2 polyploidization is also seen in short term ex vivo cultures, where AT2-to-AT1 transdifferentiation is associated with substantial binucleation due to failed cytokinesis. Both hypertrophic and polyploid features of AT2 cells can be attenuated by inhibiting the integrated stress response using the small molecule ISRIB. These data suggest that AT2 hypertrophic growth and polyploidization may be a feature of alveolar epithelial injury. Because AT2 cells serve as facultative progenitors for the distal lung epithelium, a propensity for injury-induced binucleation has implications for AT2 self-renewal and regenerative potential upon reinjury, which may benefit from targeting the integrated stress response.

Age-related Differences in the Nasal Mucosal Immune Response to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 180 million people since the onset of the pandemic. Despite similar viral load and infectivity rates between children and adults, children rarely develop severe illness. Differences in the host response to the virus at the primary infection site are among the mechanisms proposed to account for this disparity. Our objective was to investigate the host response to SARS-CoV-2 in the nasal mucosa in children and adults and compare it with the host response to respiratory syncytial virus (RSV) and influenza virus. We analyzed clinical outcomes and gene expression in the nasal mucosa of 36 children with SARS-CoV-2, 24 children with RSV, 9 children with influenza virus, 16 adults with SARS-CoV-2, and 7 healthy pediatric and 13 healthy adult controls. In both children and adults, infection with SARS-CoV-2 led to an IFN response in the nasal mucosa. The magnitude of the IFN response correlated with the abundance of viral reads, not the severity of illness, and was comparable between children and adults infected with SARS-CoV-2 and children with severe RSV infection. Expression of ACE2 and TMPRSS2 did not correlate with age or presence of viral infection. SARS-CoV-2-infected adults had increased expression of genes involved in neutrophil activation and T-cell receptor signaling pathways compared with SARS-CoV-2-infected children, despite similar severity of illness and viral reads. Age-related differences in the immune response to SARS-CoV-2 may place adults at increased risk of developing severe illness.

The discovAIR project: a roadmap towards the Human Lung Cell Atlas.

The Human Cell Atlas (HCA) consortium aims to establish an atlas of all organs in the healthy human body at single-cell resolution to increase our understanding of basic biological processes that govern development, physiology and anatomy, and to accelerate diagnosis and treatment of disease. The Lung Biological Network of the HCA aims to generate the Human Lung Cell Atlas as a reference for the cellular repertoire, molecular cell states and phenotypes, and cell-cell interactions that characterise normal lung homeostasis in healthy lung tissue. Such a reference atlas of the healthy human lung will facilitate mapping the changes in the cellular landscape in disease. The discovAIR project is one of six pilot actions for the HCA funded by the European Commission in the context of the H2020 framework programme. discovAIR aims to establish the first draft of an integrated Human Lung Cell Atlas, combining single-cell transcriptional and epigenetic profiling with spatially resolving techniques on matched tissue samples, as well as including a number of chronic and infectious diseases of the lung. The integrated Human Lung Cell Atlas will be available as a resource for the wider respiratory community, including basic and translational scientists, clinical medicine, and the private sector, as well as for patients with lung disease and the interested lay public. We anticipate that the Human Lung Cell Atlas will be the founding stone for a more detailed understanding of the pathogenesis of lung diseases, guiding the design of novel diagnostics and preventive or curative interventions.

Individual target pharmacokinetic/pharmacodynamic attainment rates among meropenem-treated patients admitted to the ICU with hospital-acquired pneumonia.

OBJECTIVES: Critical illness reduces ß-lactam pharmacokinetic/pharmacodynamic (PK/PD) attainment. We sought to quantify PK/PD attainment in patients with hospital-acquired pneumonia. METHODS: Meropenem plasma PK data (n = 70 patients) were modelled, PK/PD attainment rates were calculated for empirical and definitive targets, and between-patient variability was quantified [as a coefficient of variation (CV%)]. RESULTS: Attainment of 100% T>4×MIC was variable for both empirical (CV% = 92) and directed (CV% = 33%) treatment. CONCLUSIONS: Individualization is required to achieve suggested PK/PD targets in critically ill patients.

Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1.

Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration-approved Ab antagonist to the IL-6R (tocilizumab) attenuated the severity of influenza A-induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, atrogin-1+/- and atrogin-1-/- mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome.

A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages.

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

Inflammatory Monocytes Drive Influenza A Virus-Mediated Lung Injury in Juvenile Mice.

Healthy children are more likely to die of influenza A virus (IAV) infection than healthy adults. However, little is known about the mechanisms underlying the impact of young age on the development of life-threatening IAV infection. We report increased mortality in juvenile mice compared with adult mice at each infectious dose of IAV. Juvenile mice had sustained elevation of type I IFNs and persistent NLRP3 inflammasome activation in the lungs, both of which were independent of viral titer. Juvenile mice, but not adult mice, had increased MCP-1 levels that remained high even after viral clearance. Importantly, continued production of MCP-1 was associated with persistent recruitment of monocytes to the lungs and prolonged elevation of inflammatory cytokines. Transcriptional signatures of recruited monocytes to the juvenile and adult IAV-infected lungs were assessed by RNA-seq. Genes associated with a proinflammatory signature were upregulated in the juvenile monocytes compared with adult monocytes. Depletion of monocytes with anti-CCR2 Ab decreased type I IFN secretion, NLRP3 inflammasome activation, and lung injury in juvenile mice. This suggests an exaggerated inflammatory response mediated by increased recruitment of monocytes to the lung, and not an inability to control viral replication, is responsible for severe IAV infection in juvenile mice. This study provides insight into severe IAV infection in juveniles and identifies key inflammatory monocytes that may be central to pediatric acute lung injury secondary to IAV.

Multidimensional Assessment of the Host Response in Mechanically Ventilated Patients with Suspected Pneumonia.

Rationale: The identification of informative elements of the host response to infection may improve the diagnosis and management of bacterial pneumonia. Objectives: To determine whether the absence of alveolar neutrophilia can exclude bacterial pneumonia in critically ill patients with suspected infection and to test whether signatures of bacterial pneumonia can be identified in the alveolar macrophage transcriptome. Methods: We determined the test characteristics of alveolar neutrophilia for the diagnosis of bacterial pneumonia in three cohorts of mechanically ventilated patients. In one cohort, we also isolated macrophages from alveolar lavage fluid and used the transcriptome to identify signatures of bacterial pneumonia. Finally, we developed a humanized mouse model of Pseudomonas aeruginosa pneumonia to determine if pathogen-specific signatures can be identified in human alveolar macrophages. Measurements and Main Results: An alveolar neutrophil percentage less than 50% had a negative predictive value of greater than 90% for bacterial pneumonia in both the retrospective (n = 851) and validation cohorts (n = 76 and n = 79). A transcriptional signature of bacterial pneumonia was present in both resident and recruited macrophages. Gene signatures from both cell types identified patients with bacterial pneumonia with test characteristics similar to alveolar neutrophilia. Conclusions: The absence of alveolar neutrophilia has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection. Macrophages can be isolated from alveolar lavage fluid obtained during routine care and used for RNA-Seq analysis. This novel approach may facilitate a longitudinal and multidimensional assessment of the host response to bacterial pneumonia.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

HIF and HOIL-1L-mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury.

Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α1-Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells (CreSPC/HOIL-1Lfl/fl ) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α1-Na,K-ATPase construct bearing an S18A (α1-S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α1-S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of CreSPC/HOIL-1Lfl/fl mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and lung injury.