Lung Cell Populations Research Articles

Single-cell analysis reveals fibroblast heterogeneity and myofibroblasts in systemic sclerosis-associated interstitial lung disease

ObjectivesMyofibroblasts are key effector cells in the extracellular matrix remodelling of systemic sclerosis-associated interstitial lung disease (SSc-ILD); however, the diversity of fibroblast populations present in the healthy and SSc-ILD lung...

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.

Synthetic CpG-ODN rapidly enriches immune compartments in neonatal chicks to induce protective immunity against bacterial infections

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain of Escherichia coli (E. coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3–4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4+ and CD8+ T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4+ and CD8+ T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks against E. coli infection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.

Flow Cytometry-Based Cell Type-Specific Assessment of Target Regulation by Pulmonary siRNA Delivery.

Pulmonary siRNA delivery has attracted strong interest and has been reported to successfully mediate target gene knockdown in a number of disease models. However, the nature of the epithelial cells that eventually take up siRNA and the question if other lung cell types may also be transfected has so far been neglected. Therefore, we describe here a flow cytometry-based method using transgenic enhanced green fluorescence protein (EGFP) expressing mice for the differentiation of transfected lung cell populations based on their antigen expression.

Cellular Metabolism in Lung Health and Disease.

The lung is often overlooked as a metabolically active organ, yet biochemical studies have long demonstrated that glucose utilization surpasses that of many other organs, including the heart, kidney, and brain. For most cells in the lung, energy consumption is relegated to performing common cellular tasks, like mRNA transcription and protein translation. However, certain lung cell populations engage in more specialized types of energy-consuming behaviors, such as the beating of cilia or the production of surfactant. While many extrapulmonary diseases are now linked to abnormalities in cellular metabolism, the pulmonary community has only recently embraced the concept of metabolic dysfunction as a driver of respiratory pathology. Herein, we provide an overview of the major metabolic pathways in the lung and discuss how cells sense and adapt to low-energy states. Moreover, we review some of the emerging evidence that links alterations in cellular metabolism to the pathobiology of several common respiratory diseases.

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues.

Human lung morphogenesis begins by embryonic life and continues after birth into early childhood to form a complex organ with numerous morphologically and functionally distinct cell types. Pulmonary organogenesis involves dynamic changes in cell proliferation, differentiation, and migration of specialized cells derived from diverse embryonic lineages. Studying the molecular and cellular processes underlying formation of the fully functional lung requires isolating distinct pulmonary cell populations during development. We now report novel methods to isolate four major pulmonary cell populations from pediatric human lung simultaneously. Cells were dissociated by protease digestion of neonatal and pediatric lung and isolated on the basis of unique cell membrane protein expression patterns. Epithelial, endothelial, nonendothelial mesenchymal, and immune cells were enriched by fluorescence-activated cell sorting. Dead cells and erythrocytes were excluded by 7-aminoactinomycin D uptake and glycophorin-A (CD235a) expression, respectively. Leukocytes were identified by membrane CD45 (protein tyrosine phosphatase, receptor type C), endothelial cells by platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (CD144), and both were isolated. Thereafter, epithelial cell adhesion molecule (CD326)-expressing cells were isolated from the endothelial- and immune cell-depleted population to enrich epithelial cells. Cells lacking these membrane markers were collected as "nonendothelial mesenchymal" cells. Quantitative RT-PCR and RNA sequencing analyses of population specific transcriptomes demonstrate the purity of the subpopulations of isolated cells. The method efficiently isolates major human lung cell populations that we announce are now available through the National Heart, Lung, and Blood Institute Lung Molecular Atlas Program (LungMAP) for their further study.

PO-081 Notch signalling pathway plays a crucial role in maintaining the cancer stem cell population in lung and colorectal cancer

IntroductionThere is increasing evidence that cancer stem cells (CSCs) could mediate resistance to chemo- and radiotherapy, metastasis and recurrence in a wide range of solid tumours. Notch signalling pathway is involved in CSCs maintenance. Notch influences intra- and intercellular communications of various cell types in embryogenesis and adult organs. The oncogenic role of Notch signalling in survival, proliferation, self-renewal, differentiation and migration of cancer cells is demonstrated in most solid tumours and leukaemia. This study aims to explore the effect of Notch1 suppression on CSCs in human lung (A549) and colorectal (HCT116) adenocarcinoma cell lines.Material and methodsKnockdown of Notch1 by shRNA was confirmed by PCR and western-blot analyses. PCR was used for estimation of stemness, epithelia-to-mesenchyma transition (EMT) markers and proto-oncogene c-MYC expression. The proportion of CD133+ cells in different subpopulations was identified by Flow cytometry. Cell migration potential was examined by Boyden chamber assay. The effect of Notch1 inhibition on CSCs was established using classical detection methods of CSCs in heterogeneous cancer cell cultures: tumourisphere formation, tumourigenicity in vivo and quantitative assessment of ABC-transporter activity assays.Results and discussionsNotch1 inhibition resulted in stemness (SOX2, OCT3/4, NES), EMT (TWIST1, HES1, SNAIL1), CSCs (CD133) markers, c-MYC expression decrease and CDH1 (E-cadherin) expression increase suggesting that Notch pathway is involved in EMT. Loss of Notch1 did not influence cell proliferation in vitro but reduced migration potential. It was confirmed that Notch signalling interruption leads to reduction of stemness features: decreased drug resistance (A549 but not HCT116), ability to form tumourispheres and tumourigenic potential in vivo. ABC-transporter activity in HCT116 could be upregulated by TAZ/YAP signalling pathway and not by Notch. Moreover, reduced Notch1 expression increased the minimum inoculum cell dose for xenograft formation. The xenograft vascularisation assay revealed that Notch1 inhibition is not necessary for neovascularizaton but it is essential for angiogenesis but in tumour.ConclusionThe Notch signalling pathway is important for maintaining the CSCs population, stemness and migration so Notch1 could be considered as a potential therapeutic target in lung and colorectal cancer. The study was supported Russian Science Foundation (RSCF), grant No. 14-15-00467.

PO-272 Leukemia-associated NPM mutations promote quiescence of hematopoietic stem cells and prevent their functional exhaustion upon oncogene-induced hyper-proliferation

IntroductionIntroduction: Lung neuroendocrine tumours (LNETs) arise from a rare lung cell population, called pulmonary neuroendocrine cells (PNECs), and account for 20%–25% of all lung cancers. Despite the involvement of PNECs in LNETs and a number of other respiratory diseases, relatively little is known about their normal function or contribution to cancer development and progression. To date, the study of PNECs has been limited by difficulties in isolating and culturing this cell population.Material and methodsBuilding on methods developed in our lab for the isolation and long-term expansion of healthy and diseased human airway cells, we have generated lung organoids derived from embryonic lung tissue. We have identified cultures conditions that allow for the enrichment of PNECs in embryonic lung organoids. In parallel, we have begun to generate a biobank of both low- and high-grade LNET organoids.ResultsPNEC-enriched embryonic lung organoids can be expanded indefinitely over multiple passages. Preliminary analysis by qPCR and whole mount immunofluorescence shows that PNEC-enriched organoids express a number of PNEC lineage markers including ASCL1, NEUROD1, and UCHL1. PNEC-enriched lung organoids also express PNEC-associated bioactive compounds such as SST and CGRP.ConclusionsWe have generated human embryonic lung organoids that contain significant numbers of PNECs. Applying the same principles, we have established organoid cultures of human LNETs. PNEC-enriched organoids and LNET organoids are currently being used for further molecular and genetic analyses.

The Lymphangioleiomyomatosis Lung Cell and Its Human Cell Models.

Lymphangioleiomyomatosis (LAM) is a multisystem disease of women, affecting lungs, kidneys, and lymphatics. It is caused by the proliferation of abnormal smooth muscle-like LAM cells, with mutations and loss of heterozygosity in the TSC1 or, more frequently, TSC2 genes. Isolated pulmonary LAM cells have been difficult to maintain in culture, and most studies of LAM lung cells involve mixtures of TSC2 wild-type and TSC2-null cells. A clonal population of LAM lung cells has not been established, making analysis of the cells challenging. Cell lines have been established from angiomyolipomas, a common manifestation of LAM, and from tumors from patients with TSC. Circulating LAM cells have also been isolated from blood and other body fluids. LAM cells may also be identified in clusters apparently derived from lymphatic vessels. Genetics, patterns of antigen expression, and signaling pathways have been studied in LAM lung tissue and in LAM cell models, although rarely all in the same study. We show here that LAM cells manifest differences in these characteristics, depending on the source investigated, suggesting further studies.

PO-271 Using human lung organoids to study pulmonary neuroendocrine cells

Introduction Introduction: Lung neuroendocrine tumours (LNETs) arise from a rare lung cell population, called pulmonary neuroendocrine cells (PNECs), and account for 20%–25% of all lung cancers. Despite the involvement of PNECs in LNETs and a number of other respiratory diseases, relatively little is known about their normal function or contribution to cancer development and progression. To date, the study of PNECs has been limited by difficulties in isolating and culturing this cell population. Material and methods Building on methods developed in our lab for the isolation and long-term expansion of healthy and diseased human airway cells, we have generated lung organoids derived from embryonic lung tissue. We have identified cultures conditions that allow for the enrichment of PNECs in embryonic lung organoids. In parallel, we have begun to generate a biobank of both low- and high-grade LNET organoids. Results PNEC-enriched embryonic lung organoids can be expanded indefinitely over multiple passages. Preliminary analysis by qPCR and whole mount immunofluorescence shows that PNEC-enriched organoids express a number of PNEC lineage markers including ASCL1, NEUROD1, and UCHL1. PNEC-enriched lung organoids also express PNEC-associated bioactive compounds such as SST and CGRP. Conclusions We have generated human embryonic lung organoids that contain significant numbers of PNECs. Applying the same principles, we have established organoid cultures of human LNETs. PNEC-enriched organoids and LNET organoids are currently being used for further molecular and genetic analyses.

IL-1 cytokines promote ADAMTS-mediated remodeling of the lung extracellular matrix during severe influenza A infection in mice

Abstract The lung extracellular matrix (ECM) is extensively remodeled following respiratory viral infection. This remodeling occurs during inflammation when immune cells migrate to the site of infection and during repair as the ECM is modified to regenerate damaged tissue. Matrix proteases, including the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) gene family, play a critical role in these remodeling processes. The immune mechanisms that regulate matrix protease activity and their effect on host responses to lung infection have not been well characterized. Previously, we found that ADAMTS-4 knockout mice exhibited improved survival following lethal influenza A (IAV) challenge and signs of reduced immunopathology, including fewer numbers of inflammatory T cells. Here, by flow cytometric sorting of immune and stromal cell populations, we identified fibroblasts as the main producers of ADAMTS-4 in the lung during severe IAV infection, with ~50-fold higher levels of mRNA compared to other lung cell populations. Stimulation of primary lung fibroblasts in vitro with a wide range of inflammatory cytokines demonstrated that only IL-1β and IL-1α induced expression of ADAMTS-4. Co-stimulation of lung fibroblasts with IL-1 cytokines and TNF-α resulted in a synergistic effect and an increase of ADAMTS-4 expression to greater than 6-fold above baseline levels. IL-1 treatment of lung fibroblasts also induced expression of the lung ECM proteoglycan versican (VCAN), a major enzymatic target of ADAMTS-4. These results suggest a role for IL-1 cytokines in regulating lung ECM remodeling, and highlight the potential of modulating the IL-1/ADAMTS-4 pathway to reduce immunopathology following severe respiratory infection.

Novel role of CREB in regulating maturation of alveolar macrophages and thereby in maintaining lung‐fluid balance

Alveolar macrophages (AM) represent a specialized subpopulation of lung resident macrophages responsible for coordinating both the initiation and resolution of inflammatory lung injury. Analysis of the dynamics of lung resident macrophage populations using LyzM‐GFP mice (green myeloid cells) over the course of five days post‐injury by nebulized LPS showed a gradual decline in AM during injury phase but AM expanded and returned to baseline levels by 5th day post injury. While evidence suggests that recruited bone marrow monocytes can adopt AM phenotype following their differentiation into interstitial macrophage mechanisms reconstituting AM remains elusive. CREB, a cAMP‐Response Element Binding protein, is known to transcribe genes which regulate several cellular processes including cell differentiation, angiogenesis and anti‐inflammatory signaling. Mice lacking CREB rapidly die after birth primarily due to impairment of lung function. Thus, we generated mice lacking CREB in myeloid cells (CREBΔMye) by crossing CREBfl/fl mice with LyzM‐Cre mice to test the hypothesis that CREB regulates AM generation and thereby maintains lung‐fluid homeostasis. We show that in contrast to CREBfl/fl mice, CREBΔMye lungs developed lung edema basally. LPS induced significantly more edema formation at 4h in CREBΔMye than control mice. Whereas the edema resolved fully in control mice at 24h CREBΔMye lungs remained edematous. FACS analysis showed ~50% reduction in AM in CREBΔMye lungs (SiglecF+/CD11b‐/CD11c+ cells) basally and at 24 h following LPS‐induced lung injury. Intriguingly, we found increase in MHCII+/Siglec+/CD11clo lung cell population in CREBΔMye lungs suggesting loss of CREB in myeloid cells led to generation of incompetent pre‐AM/interstitial phenotype. Bone marrow derived macrophages from floxed and CREB lacking mice indicated dysregulation of several glycolytic enzymes, including hexokinase and pyruvate dehydrogenase. Further studies are being carried out to fully characterize the transcriptome of CREB lacking lung macrophages and whether CREB regulation of metabolic reprogramming is required for full differentiation of Pre‐AM/interstitial macrophages into AMs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of Human Lung Mast Cell Apoptosis by Granule Permeabilization: A Novel Approach for Targeting Mast Cells.

Mast cells are implicated as detrimental players in inflammatory lung diseases, particularly asthma. Mast cells respond to activating stimuli by releasing a wide panel of pro-inflammatory compounds that can contribute profoundly to the pathology, and there is currently an unmet need for strategies that efficiently ameliorate harmful effects of mast cells under such conditions. Here, we sought to evaluate a novel concept for targeting human lung mast cells, by assessing the possibility of selectively depleting the lung mast cells by induction of apoptosis. For this purpose, we used lysosomotropic agents, i.e., compounds that are known to permeabilize the secretory granules of mast cells, thereby releasing the contents of the granules into the cytosol. Either intact human lung tissue, purified human lung mast cells or mixed populations of human lung cells were incubated with the lysosomotropic agents mefloquine or siramesine, followed by measurement of apoptosis, reactive oxygen species (ROS) production, and release of cytokines. We show that human lung mast cells were highly susceptible to apoptosis induced by this strategy, whereas other cell populations of the lung were largely refractory. Moreover, we demonstrate that apoptosis induced by this mode is dependent on the production of ROS and that the treatment of lung tissue with lysosomotropic agents causes a decrease in the release of pathogenic cytokines. We conclude that selective apoptosis of human lung mast cells can be accomplished by administration of lysosomotropic agents, thus introducing the possibility of using such drugs as novel therapeutics in the treatment of inflammatory lung disorders such as asthma.

Mitogenic stimulation accelerates influenza-induced mortality by increasing susceptibility of alveolar type II cells to infection

Development of pneumonia is the most lethal consequence of influenza, increasing mortality more than 50-fold compared with uncomplicated infection. The spread of viral infection from conducting airways to the alveolar epithelium is therefore a pivotal event in influenza pathogenesis. We found that mitogenic stimulation with keratinocyte growth factor (KGF) markedly accelerated mortality after infectious challenge with influenza A virus (IAV). Coadministration of KGF with IAV markedly accelerated the spread of viral infection from the airways to alveoli compared with challenge with IAV alone, based on spatial and temporal analyses of viral nucleoprotein staining of lung tissue sections and dissociated lung cells. To better define the temporal relationship between KGF administration and susceptibility to IAV infection in vivo, we administered KGF 120, 48, 24, and 0 h before intrapulmonary IAV challenge and assessed the percentages of proliferating and IAV-infected, alveolar type II (AECII) cells in dispersed lung cell populations. Peak AECII infectivity coincided with the timing of KGF administration that also induced peak AECII proliferation. AECII from mice that were given intrapulmonary KGF before isolation and then infected with IAV ex vivo exhibited the same temporal pattern of proliferation and infectious susceptibility. KGF-induced increases in mortality, AECII proliferation, and enhanced IAV susceptibility were all reversed by pretreatment of the animals with the mTOR inhibitor rapamycin before mitogenic stimulation. Taken together, these data suggest mTOR signaling-dependent, mitogenic conditioning of AECII is a determinant of host susceptibility to infection with IAV.

Shared epithelial pathways to lung repair and disease.

Chronic lung diseases present tremendous health burdens and share a common pathobiology of dysfunctional epithelial repair. Lung adenocarcinoma, the leading cancer killer worldwide, is caused mainly by chemical carcinogens of tobacco smoke that induce mutations in pulmonary epithelial cells leading to uncontrolled epithelial proliferation. Lung epithelial cells that possess the capacity for self-renewal and regeneration of other lung cell types are believed to underlie the pathobiology of chronic obstructive, fibrotic and neoplastic lung disorders. However, the understanding of lung epithelial progenitor cell hierarchy and turnover is incomplete and a comprehensive model of the cellular and transcriptional events that underlie lung regeneration and carcinogenesis is missing. The mapping of these processes is extremely important, since their modulation would potentially allow effective cure and/or prevention of chronic lung diseases. In this review we describe current knowledge on cellular and molecular pathways at play during lung repair and carcinogenesis and summarise the critical lung cell populations with regenerative and cancerous potential.

Identification of a SOX2/SOX9 Double Positive Cells Progenitor Cell Population Required for Branching Morphogenesis in Human Lung

BackgroundTo date, the proximal‐distal (PD) axis of the lung has been distinguished by the localization of two main transcription factors: Sox2 and Sox9. In the developing mouse lung, distal tip epithelial progenitors express Sox9, whereas proximal progenitors in the stalk express Sox2. Moreover, in the mouse lung, Fgf10 controls the PD axis by preventing the differentiation of epithelial Sox9+ progenitors into Sox2‐expressing airway cells. Whereas lung branching has been extensively studied in animal models little is known about the mechanisms underlying human lung branching and whether findings from mouse apply to human. We aimed to determine the role of FGF signaling in human fetal lung.MethodsFresh human fetal lung tissue between gestational ages 10–16 weeks were collected and either stained or used for culture. Distal lung segments (1–3 mm3) with intact edges were cultured in air‐liquid interface for up to 72 hours. Explants were treated with different fibroblast growth factors (FGFs) known to alter PD epithelial cell differentiation as well as the tyrosine kinase inhibitor (SU5402).ResultsCareful examination of the human fetal lung showed that, similar to mouse, proximal progenitors express SOX2. However, the distal tip progenitors co‐expressed SOX2 and SOX9, a pattern never seen in mouse. This SOX2/SOX9+ progenitor population persists during the pseudoglandular stage up to around 16 weeks gestation. Moreover, we showed that in response to FGF7 and FGF9, branching was impaired accompanied by cysting of distal buds, decreased SOX2 expression, expansion of SOX9 with decreased of SOX2/SOX9 double positive cells. FGF10 did not promote branching in whole explant cultures or in epithelial bud cultures. A decline in SOX2/SOX9 double positive cells was also observed. In contrast, treatment of human lung explants with SU5402, a tyrosine kinase inhibitor, resulted in expansion of SOX2 and decrease of SOX9, resulting in the decline of SOX2/SOX9+ progenitor cell population.ConclusionIn conclusion, we identified the presence of a new progenitor cell population in the human lung co‐expressing SOX2 and SOX9, never seen in mouse. While FGF7 and FGF9 have similar effects on human and mouse lung branching, the effect of Fgf10 was different in that it failed to induce branching of human lung. We believe that this is mainly due to the alteration of the cells co‐expressing SOX2/SOX9+. We therefore conclude that the maintenance of this progenitor population is required for proper branching morphogenesis in human lung. However, we still need to identify the signals required for the maintenance of SOX2/SOX9 progenitor cell identity. Understanding the mechanisms driving airway branching will transform our concepts of human lung development and may allow for the discovery of possible therapeutic avenues to restore or enhance lung development for severely premature infants at increased risk for lung diseases.Support or Funding InformationAmerican Heart Association, Saban Research Institute

Calorie Restriction Attenuates Terminal Differentiation of Immune Cells.

Immune senescence is a natural consequence of aging and may contribute to frailty and loss of homeostasis in later life. Calorie restriction increases healthy life-span in C57BL/6J (but not DBA/2J) mice, but whether this is related to preservation of immune function, and how it interacts with aging, is unclear. We compared phenotypic and functional characteristics of natural killer (NK) cells and T cells, across the lifespan, of calorie-restricted (CR) and control C57BL/6 and DBA/2 mice. Calorie restriction preserves a naïve T cell phenotype and an immature NK cell phenotype as mice age. The splenic T cell populations of CR mice had higher proportions of CD11a−CD44lo cells, lower expression of TRAIL, KLRG1, and CXCR3, and higher expression of CD127, compared to control mice. Similarly, splenic NK cells from CR mice had higher proportions of less differentiated CD11b−CD27+ cells and correspondingly lower proportions of highly differentiated CD11b+CD27−NK cells. Within each of these subsets, cells from CR mice had higher expression of CD127, CD25, TRAIL, NKG2A/C/E, and CXCR3 and lower expression of KLRG1 and Ly49 receptors compared to controls. The effects of calorie restriction on lymphoid cell populations in lung, liver, and lymph nodes were identical to those seen in the spleen, indicating that this is a system-wide effect. The impact of calorie restriction on NK cell and T cell maturation is much more profound than the effect of aging and, indeed, calorie restriction attenuates these age-associated changes. Importantly, the effects of calorie restriction on lymphocyte maturation were more marked in C57BL/6 than in DBA/2J mice indicating that delayed lymphocyte maturation correlates with extended lifespan. These findings have implications for understanding the interaction between nutritional status, immunity, and healthy lifespan in aging populations.

Exposure Memory and Lung Regeneration.

Many acute and chronic lung diseases could benefit from improved regeneration therapy. In development and throughout life, genetically encoded exposure memory systems allow environmental exposures, diet, and infectious agents to direct subsequent phenotypic adaptation and responses. The impact of such memory systems on lung regeneration is currently unknown. This article provides a brief overview of advances in redox biology and medicine as a framework for elucidating exposure memory and delineating spatiotemporal responses in lung regeneration. New imaging and omics methods enable precise definition to advance knowledge of development and the cumulative changes in lung biochemistry, structure, and cell populations occurring from prior and ongoing exposures. Importantly, conditioning steps may be needed to reverse exposure memory and enable effective regeneration. Thus, to complement developmental biology and regenerative medicine, research programs are needed to gain systematic knowledge of how lifelong exposures impact lung biology and support transition of lung regeneration from hypothetical to practical medicine.

Involvement of the different lung compartments in the pathogenesis of pH1N1 influenza virus infection in ferrets.

Severe cases after pH1N1 infection are consequence of interstitial pneumonia triggered by alveolar viral replication and an exacerbated host immune response, characterized by the up-regulation of pro-inflammatory cytokines and the influx of inflammatory leukocytes to the lungs. Different lung cell populations have been suggested as culprits in the unregulated innate immune responses observed in these cases. This study aims to clarify this question by studying the different induction of innate immune molecules by the distinct lung anatomic compartments (vascular, alveolar and bronchiolar) of ferrets intratracheally infected with a human pH1N1 viral isolate, by means of laser microdissection techniques. The obtained results were then analysed in relation to viral quantification in the different anatomic areas and the histopathological lesions observed. More severe lung lesions were observed at 24 h post infection (hpi) correlating with viral antigen detection in bronchiolar and alveolar epithelial cells. However, high levels of viral RNA were detected in all anatomic compartments throughout infection. Bronchiolar areas were the first source of IFN-α and most pro-inflammatory cytokines, through the activation of RIG-I. In contrast, vascular areas contributed with the highest induction of CCL2 and other pro-inflammatory cytokines, through the activation of TLR3.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0395-0) contains supplementary material, which is available to authorized users.

Human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional CD69−CD56dim cells

In contrast to the extensive knowledge about human natural killer (NK) cells in peripheral blood, relatively little is known about NK cells in the human lung. Knowledge about the composition, differentiation, and function of human lung NK cells is critical to better understand their role in diseases affecting the lung, including asthma, chronic obstructive pulmonary disease, infections, and cancer. We sought to analyze and compare the phenotypic and functional characteristics of NK cells in the human lung and peripheral blood at the single-cell level. NK cells in human lung tissue and matched peripheral blood from 132 subjects were analyzed by using 16-color flow cytometry and confocal microscopy. CD56dimCD16+ NK cells made up the vast majority of NK cells in human lungs, had a more differentiated phenotype, and more frequently expressed educating killer cell immunoglobulin-like receptors compared with NK cells in peripheral blood. Despite this, human lung NK cells were hyporesponsive toward target cell stimulation, even after priming with IFN-α. Furthermore, we detected a small subset of NK cells expressing CD69, a marker of tissue residency. These CD69+ NK cells in the lung consisted predominantly of immature CD56brightCD16- NK cells and less differentiated CD56dimCD16+ NK cells. Here, we characterize the major NK cell populations in the human lung. Our data suggest a model in which the majority of NK cells in the human lung dynamically move between blood and the lung rather than residing in the lung as bona fide tissue-resident CD69+ NK cells.