Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 — rs1420101 and rs11685480 — are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33–driven type 2 inflammation.
The molecular mechanism underlying IL1RL1 genetic asthma risk variants and their relationship to type 2 inflammation in the airway are demonstrated.
Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. Here, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived antigens by these cells supported recall T cell responses in the fat and also generated antigen-bearing DCs for emigration into adjacent lymph nodes. Enhanced recruitment of DCs to inflammation-reactive lymph nodes significantly relied on adipose tissue DCs to maintain sufficient numbers of antigen-bearing DCs as the lymph node expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for antigen transport into the adjacent lymph node.
Many adolescents have poor mental health literacy, stigmatising attitudes towards people with mental illness, and lack skills in providing optimal Mental Health First Aid to peers. These could be improved with training to facilitate better social support and increase appropriate help-seeking among adolescents with emerging mental health problems. teen Mental Health First Aid (teen MHFA), a new initiative of Mental Health First Aid International, is a 3 × 75 min classroom based training program for students aged 15–18 years.
An uncontrolled pilot of the teen MHFA course was undertaken to examine the feasibility of providing the program in Australian secondary schools, to test relevant measures of student knowledge, attitudes and behaviours, and to provide initial evidence of program effects.
Across four schools, 988 students received the teen MHFA program. 520 students with a mean age of 16 years completed the baseline questionnaire, 345 completed the post-test and 241 completed the three-month follow-up. Statistically significant improvements were found in mental health literacy, confidence in providing Mental Health First Aid to a peer, help-seeking intentions and student mental health, while stigmatising attitudes significantly reduced.
teen MHFA appears to be an effective and feasible program for training high school students in Mental Health First Aid techniques. Further research is required with a randomized controlled design to elucidate the causal role of the program in the changes observed.
Electronic supplementary material
The online version of this article (doi:10.1186/s13033-016-0034-1) contains supplementary material, which is available to authorized users.
Mental Health First Aid; Mental health literacy; Stigma; Adolescents; Secondary school
Alveolar type II (ATII) cells remain differentiated and express surfactant
proteins when cultured at an air-liquid (A/L) interface. When cultured under submerged
conditions, ATII cells dedifferentiate and change their gene expression profile. We have
previously shown that gene expression under submerged conditions is regulated by hypoxia
inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism.
Herein, we sought to further define gene expression changes in ATII cells cultured under
submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII
cells cultured under submerged conditions for 24–48 h after switching from an A/L
interface. We found significant alterations in gene expression, including upregulation of
the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2,
and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by
RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with
anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely
expressed in the lung, we further explored the potential functions of STC1 in the alveolar
epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this
induction occurred rapidly and reversibly. We also showed that recombinant human STC1
(rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar
epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS.
We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial
cells. In this study, we have found that several HIF target genes including STC1 are
upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF,
that this regulation is rapidly and reversibly, and that STC1 enhances wound healing
moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs
stimulated by LPS in vitro. Therefore, alterations in gene expression by
ATII cells under submerged conditions including STC1 were largely induced by hypoxia and
HIF, which may be relevant to our understanding of the pathogenesis of various lung
diseases in which the alveolar epithelium is exposed to relative hypoxia.
HIF; STC1; Gene expression; Alveolar epithelial cells
Motivation: Non–small-cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. Targeted tyrosine kinase inhibitors (TKIs) directed against the epidermal growth factor receptor (EGFR) have been widely and successfully used in treating NSCLC patients with activating EGFR mutations. Unfortunately, the duration of response is short-lived, and all patients eventually relapse by acquiring resistance mechanisms.
Result: We performed an integrative systems biology approach to determine essential kinases that drive EGFR-TKI resistance in cancer cell lines. We used a series of bioinformatics methods to analyze and integrate the functional genetics screen and RNA-seq data to identify a set of kinases that are critical in survival and proliferation in these TKI-resistant lines. By connecting the essential kinases to compounds using a novel kinase connectivity map (K-Map), we identified and validated bosutinib as an effective compound that could inhibit proliferation and induce apoptosis in TKI-resistant lines. A rational combination of bosutinib and gefitinib showed additive and synergistic effects in cancer cell lines resistant to EGFR TKI alone.
Conclusions: We have demonstrated a bioinformatics-driven discovery roadmap for drug repurposing and development in overcoming resistance in EGFR-mutant NSCLC, which could be generalized to other cancer types in the era of personalized medicine.
Availability and implementation: K-Map can be accessible at: http://tanlab.ucdenver.edu/kMap.
firstname.lastname@example.org or email@example.com
Supplementary data are available at Bioinformatics online.
Severe acute respiratory syndrome (SARS)–coronavirus (CoV) produces a devastating primary viral pneumonia with diffuse alveolar damage and a marked increase in circulating cytokines. One of the major cell types to be infected is the alveolar type II cell. However, the innate immune response of primary human alveolar epithelial cells infected with SARS-CoV has not been defined. Our objectives included developing a culture system permissive for SARS-CoV infection in primary human type II cells and defining their innate immune response. Culturing primary human alveolar type II cells at an air–liquid interface (A/L) improved their differentiation and greatly increased their susceptibility to infection, allowing us to define their primary interferon and chemokine responses. Viral antigens were detected in the cytoplasm of infected type II cells, electron micrographs demonstrated secretory vesicles filled with virions, virus RNA concentrations increased with time, and infectious virions were released by exocytosis from the apical surface of polarized type II cells. A marked increase was evident in the mRNA concentrations of interferon–β and interferon–λ (IL-29) and in a large number of proinflammatory cytokines and chemokines. A surprising finding involved the variability of expression of angiotensin-converting enzyme–2, the SARS-CoV receptor, in type II cells from different donors. In conclusion, the cultivation of alveolar type II cells at an air–liquid interface provides primary cultures in which to study the pulmonary innate immune responses to infection with SARS-CoV, and to explore possible therapeutic approaches to modulating these innate immune responses.
lung innate immune response; cytokine responses to SARS coronavirus; lung cell differentiation; air–liquid interface cultures
The most severe complication of influenza is viral pneumonia, which can lead to the acute respiratory distress syndrome. Alveolar epithelial cells (AECs) are the first cells that influenza virus encounters upon entering the alveolus. Infected epithelial cells produce cytokines that attract and activate neutrophils and macrophages, which in turn induce damage to the epithelial-endothelial barrier. Hepatocyte growth factor (HGF)/c-Met and transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGFR) are well known to regulate repair of damaged alveolar epithelium by stimulating cell migration and proliferation. Recently, TGF-α/EGFR signaling has also been shown to regulate innate immune responses in bronchial epithelial cells. However, little is known about whether HGF/c-Met signaling alters the innate immune responses and whether the innate immune responses in AECs are regulated by HGF/c-Met and TGF-α/EGFR. We hypothesized that HGF/c-Met and TGF-α/EGFR would regulate innate immune responses to influenza A virus infection in human AECs. We found that recombinant human HGF (rhHGF) and rhTGF-α stimulated primary human AECs to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly and IL-6 and monocyte chemotactic protein 1 moderately. Influenza infection stimulated the secretion of IL-8 and GM-CSF by AECs plated on rat-tail collagen through EGFR activation likely by TGF-α released from AECs and through c-Met activated by HGF secreted from lung fibroblasts. HGF secretion by fibroblasts was stimulated by AEC production of prostaglandin E2 during influenza infection. We conclude that HGF/c-Met and TGF-α/EGFR signaling enhances the innate immune responses by human AECs during influenza infections.
alveolar epithelial cells; influenza; innate immune responses; hepatocyte growth factor/c-Met; transforming growth factor-α/epidermal growth factor receptor
Human coronavirus strain 229E (HCoV-229E) commonly causes upper respiratory tract infections. However, lower respiratory tract infections can occur in some individuals, indicating that cells in the distal lung are susceptible to HCoV-229E. This study determined the virus susceptibility of primary cultures of human alveolar epithelial cells and alveolar macrophages (AMs). Fluorescent antibody staining indicated that HCoV-229E could readily infect AMs, but no evidence was found for infection in differentiated alveolar epithelial type II cells and only a very low level of infection in type II cells transitioning to the type I-like cell phenotype. However, a human bronchial epithelial cell line (16HBE) was readily infected. The innate immune response of AMs to HCoV-229E infection was evaluated for cytokine production and interferon (IFN) gene expression. AMs secreted significant amounts of tumour necrosis factor alpha (TNF-α), regulated on activation normal T-cell expressed and secreted (RANTES/CCL5) and macrophage inflammatory protein 1β (MIP-1β/CCL4) in response to HCoV-229E infection, but these cells exhibited no detectable increase in IFN-β or interleukin-29 in mRNA levels. AMs from smokers had reduced secretion of TNF-α compared with non-smokers in response to HCoV-229E infection. Surfactant protein A (SP-A) and SP-D are part of the innate immune system in the distal lung. Both surfactant proteins bound to HCoV-229E, and pre-treatment of HCoV-229E with SP-A or SP-D inhibited infection of 16HBE cells. In contrast, there was a modest reduction in infection in AMs by SP-A, but not by SP-D. In summary, AMs are an important target for HCoV-229E, and they can mount a pro-inflammatory innate immune response to infection.
Alveolar type II epithelial cells (ATIIs) are one of the primary targets for influenza A pneumonia. The lack of a culture system for maintaining differentiated ATIIs hinders our understanding of pulmonary innate immunity during viral infection. We studied influenza A virus (IAV)-induced innate immune responses in differentiated primary human ATIIs and alveolar macrophages (AMs). Our results indicate that ATIIs, but not AMs, support productive IAV infection. Viral infection elicited strong inflammatory chemokine and cytokine responses in ATIIs, including secretion of IL-8, IL-6, MCP-1, RANTES, and MIP-1β, but not TNF-α, whereas AMs secreted TNF-α as well as other cytokines in response to infection. Wild-type virus A/PR/8/34 induced a greater cytokine response than reassortant PR/8 virus, A/Phil/82, despite similar levels of replication. IAV infection increased mRNA expression of IFN genes IFN-β, IL-29 (IFN-λ1), and IL-28A (IFN-λ2). The major IFN protein secreted by type II cells was IL-29 and ATIIs appear to be a major resource for production of IL-29. Administration of IL-29 and IFN-β before infection significantly reduced the release of infectious viral particles and CXC and CC chemokines. IL-29 treatment of type II cells induced mRNA expression of antiviral genes MX1, OAS, and ISG56 but not IFN-β. IL-29 induced a dose-dependent decrease of viral nucleoprotein and an increase of antiviral genes but not IFN-β. These results suggest that IL-29 exerts IFN-β-independent protection in type II cells through direct activation of antiviral genes during IAV infection.
Emphysema is caused by the cigarette smoke (CS)–induced destruction of alveolar wall septa, and CS is the main risk factor for chronic obstructive pulmonary disease (COPD). To study the mechanisms of response to this insult, we focused on oxidant-induced lung injury and the potential role of nuclear erythroid 2–related factor–2 (Nrf2), which is a key regulator of the antioxidant defense system. We studied the protective role of N-acetylcysteine (NAC) against the injury of alveolar type II (ATII) cells induced by CS in vivo and in vitro. ATII cells were isolated and purified using magnetic MicroBeads (Miltenyi Biotec, Auburn, CA) from Nrf2−/− mice and wild-type mice. We analyzed pulmonary injury, inflammation, glutathione (GSH) concentrations, the expression of glutathione cysteine ligase catalytic subunit mRNA, glutathione cysteine ligase modifier subunit mRNA, and glutathione reductase mRNA, and Nrf2, heme oxygenase–1, and nicotinamide adenine dinucleotide phosphate–reduced:quinone oxireductase levels by Western blotting, TUNEL assay, and immunocytofluorescence for 4-hydroxynonenal as a marker of oxidative stress. We found that CS induced greater injury in ATII cells obtained from Nrf2−/− mice than from wild-type mice. Furthermore, NAC attenuated the injuries by CS in ATII cells obtained from wild-type mice both in vivo and in vitro. Moreover, NAC decreased the injury of ATII cells obtained from Nrf2−/− mice. Our results suggest that Nrf2–GSH signaling is important for the protective activity of NAC. In addition, in ATII cells deficient in Nrf2, this compound can provide partial protection through its reactive oxygen species–scavenging activities. Targeting the antioxidant system regulated by Nrf2 may provide an effective strategy against lung injury in COPD.
murine alveolar type II cells; lung; Nrf2; NAC; cigarette smoke
The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6–8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host.
The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidased lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxide formed by the direct effect of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5β,6β-epoxycholesterol (β-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and β-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2 (Nrf2), heat shock protein 70 (Hsp70) and Fos-related antigen 1 (Fra1) protein expressions. Furthermore, we found higher sensitivity of ATI-like cells than ATII cells exposed to ozone or treated with β-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. The importance of understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases.
alveolar type I-like cells; ozone; oxysterols; apoptosis
In order to obtain a global picture of how alveolar macrophages respond to influenza A virus (IAV) infection, we used a quantitative proteomics method to systematically examine protein expression in the IAV-infected primary human alveolar macrophages. Of the 1214 proteins identified, 43 were significantly up-regulated and 63 significantly down-regulated at > 95% confidence. The expression of an array of interferon (IFN)-induced proteins was significantly increased in the IAV-infected macrophages. The protein with the greatest expression increase was ISG15, an IFN-induced protein that has been shown to play an important role in antiviral defense. Concomitantly, quantitative real time PCR analysis revealed that the gene expression of type I IFNs increased substantially following virus infection. Our results are consistent with the notion that type I IFNs play a vital role in the response of human alveolar macrophages to IAV infection. In addition to the IFN-mediated responses, inflammatory response, apoptosis and redox state rebalancing appeared also to be major pathways that were affected by IAV infection. Furthermore, our data suggest that alveolar macrophages may play a crucial role in regenerating alveolar epithelium during IAV infection.
Influenza A virus; IFN-α/β; quantitative proteomics; primary alveolar macrophage; protein expression; LC-MS/MS; SILAC
Because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. Research on HCoV-HKU1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. Here we demonstrate that HCoV-HKU1 can infect and be serially propagated in primary human alveolar type II cells at the air-liquid interface. We were not able to infect alveolar type I-like cells or alveolar macrophages. Type II alveolar cells infected with HCoV-HKU1 demonstrated formation of large syncytium. At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ. These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. Furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease.
The 2009/2010 pandemic influenza virus (H1N1pdm) contains an avian-lineage PB2 gene that lacks E627K and D701N substitutions important in the pathogenesis and transmission of avian-origin viruses in humans or other mammals. Previous studies have shown that PB2-627K is not necessary because of a compensatory Q591R substitution. The role that PB2-701N plays in the H1N1pdm phenotype is not well understood. Therefore, PB2-D701N was introduced into an H1N1pdm virus (A/New York/1682/2009 (NY1682)) and analyzed in vitro and in vivo. Mini-genome replication assay, in vitro replication characteristics in cell lines, and analysis in the mouse and ferret models demonstrated that PB2-D701N increased virus replication rates and resulted in more severe pathogenicity in mice and more efficient transmission in ferrets. In addition, compared to the NY1682-WT virus, the NY1682-D701N mutant virus induced less IFN-λ and replicated to a higher titer in primary human alveolar epithelial cells. These findings suggest that the acquisition of the PB2-701N substitution by H1N1pdm viruses may result in more severe disease or increase transmission in humans.
Ozone is known to produce an acute influx of neutrophils, and alveolar epithelial cells can secrete chemokines and modulate inflammatory processes. However, direct exposure of alveolar epithelial cells and macrophages to ozone (O3) produces little chemokine response. To determine if cell–cell interactions might be responsible, we investigated the effect of alveolar macrophage–conditioned media after ozone exposure (MO3CM) on alveolar epithelial cell chemokine production. Serum-free media were conditioned by exposing a rat alveolar macrophage cell line NR8383 to ozone for 1 hour. Ozone stimulated secretion of IL-1α, IL-1β, and IL-18 from NR8383 cells, but there was no secretion of chemokines or TNF-α. Freshly isolated type II cells were cultured, so as to express the biological markers of type I cells, and these cells are referred to as type I–like cells. Type I–like cells were exposed to diluted MO3CM for 24 hours, and this conditioned medium stimulated secretion of cytokine-induced neutrophil chemattractant-1 (CXCL1) and monocyte chemoattractant protein-1 (CCL2). Secretion of these chemokines was inhibited by the IL-1 receptor antagonist. Although both recombinant IL-1α and IL-1β stimulated alveolar epithelial cells to secrete chemokines, recombinant IL-1α was 100-fold more potent than IL-1β. Furthermore, neutralizing anti-rat IL-1α antibodies inhibited the secretion of chemokines by alveolar epithelial cells, whereas neutralizing anti-rat IL-1β antibodies had no effect. These observations indicate that secretion of IL-1α from macrophages stimulates alveolar epithelial cells to secrete chemokines that can elicit an inflammatory response.
alveolar epithelial cells; cytokine-induced neutrophil chemoattractant; monocyte chemoattractant protein-1; IL-1α; cell–cell interactions
Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 hours and peaked at approximately 105 pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.
SARS; lung; alveolar macrophage; ACE2
Cultures of differentiating fetal human type II cells have been available for many years. However, studies with differentiated adult human type II cells are limited. We used a published method for type II cell isolation and developed primary culture systems for maintenance of differentiated adult human alveolar epithelial cells for in vitro studies. Human type II cells cultured on Matrigel (basolateral access) or a mixture of Matrigel and rat tail collagen (apical access) in the presence of keratinocyte growth factor, isobutylmethylxanthine, 8-bromo-cyclicAMP, and dexamethasone (KIAD) expressed the differentiated type II cell phenotype as measured by the expression of surfactant protein (SP)-A, SP-B, SP-C, and fatty acid synthase and their morphologic appearance. These cells contain lamellar inclusion bodies and have apical microvilli. In both systems the cells appear well differentiated. In the apical access system, type II cell differentiation markers initially decreased and then recovered over 6 d in culture. Lipid synthesis was also increased by the addition of KIAD. In contrast, type II cells cultured on rat tail collagen (or tissue culture plastic) slowly lose their lamellar inclusions and expression of the surfactant proteins and increase the expression of type I cell markers. The expression of the phenotypes is regulated by the culture conditions and is, in part, reversible in vitro.
type I cell; type II cell; surfactant; lipogenesis
Alveolar type II (ATII) cells cultured at an air–liquid (A/L) interface maintain differentiation, but they lose these properties when they are submerged. Others showed that an oxygen tension gradient develops in the culture medium as ATII cells consume oxygen. Therefore, we wondered whether hypoxia inducible factor (HIF) signaling could explain differences in the phenotypes of ATII cells cultured under A/L interface or submerged conditions. ATII cells were isolated from male Sprague-Dawley rats and cultured on inserts coated with a mixture of rat-tail collagen and Matrigel, in medium including 5% rat serum and 10 ng/ml keratinocyte growth factor, with their apical surfaces either exposed to air or submerged. The A/L interface condition maintained the expression of surfactant proteins, whereas that expression was down-regulated under the submerged condition, and the effect was rapid and reversible. Under submerged conditions, there was an increase in HIF1α and HIF2α in nuclear extracts, mRNA levels of HIF inducible genes, vascular endothelial growth factor, glucose transporter–1 (GLUT1), and the protein level of pyruvate dehydrogenase kinase isozyme–1. The expression of surfactant proteins was suppressed and GLUT1 mRNA levels were induced when cells were cultured with 1 mM dimethyloxalyl glycine. The expression of surfactant proteins was restored under submerged conditions with supplemented 60% oxygen. HIF signaling and oxygen tension at the surface of cells appears to be important in regulating the phenotype of rat ATII cells.
HIF; ATII cells; surfactant proteins; VEGF; GLUT1
During the past ten years, functions of alveolar type II cells have been well characterized with isolated cells in vitro. Some of the functions were well known from studies in vivo, but others such as transepithelial sodium transport were unsuspected. A better understanding of this important pulmonary cell type improves our knowledge of the pathophysiology of adult respiratory distress syndrome and may in time lead to new therapeutic strategies.
Alveolar Type II (ATII) cells are important targets for seasonal and pandemic influenza. To investigate the influenza-induced innate immune response in those cells, we measured the global gene expression profile of highly differentiated ATII cells infected with the influenza A virus at a multiplicity of infection of 0.5 at 4 hours and 24 hours after inoculation. Infection with influenza stimulated a significant increase in the mRNA concentrations of many host defense–related genes, including pattern/pathogen recognition receptors, IFN, and IFN-induced genes, chemokines, and suppressors of cytokine signaling. We verified these changes by quantitative real-time RT-PCR. At the protein level, we detected a robust virus-induced secretion of the three glutamic acid-leucine-arginine (ELR)-negative chemokines CXCL9, CXCL10, and CXCL11, according to ELISA. The ultraviolet inactivation of virus abolished the chemokine and cytokine response. Viral infection did not appear to alter the differentiation of ATII cells, as measured by cellular mRNA and concentrations of surfactant proteins. However, viral infection significantly reduced the secretion of surfactant protein (SP)–A and SP–D. In addition, influenza A virus triggered a time-dependent activation of phosphatidylinositol 3–kinase signaling in ATII cells. The inhibition of this pathway significantly decreased the release of infectious virus and the chemokine response, but did not alter virus-induced cell death. This study provides insights into influenza-induced innate immunity in differentiated human ATII cells, and demonstrates that the alveolar epithelium is a critical part of the initial innate immune response to influenza.
human Type II cell; influenza; chemokine; PI3k; differentiation
Keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis in alveolar type II cells in vitro. KGF stimulates lipogenic enzymes, including fatty acid synthase and stearyl-CoA desaturase-1, and transcription factors involved in lipogenesis, such as sterol regulatory element binding protein (SREBP)-1c and CCAAT/enhancer binding protein (C/EBP)α and C/EBPδ. To define the role of SREBP-1c on the induction of lipogenic genes and lipogenesis by KGF in primary cultures of rat type II cells, we used adenoviral vectors to alter levels of SREBP-1c. Overexpression of a dominant-negative form of SREBP-1 decreased lipogenesis and decreased the induction of fatty acid synthase and stearyl coenzyme A desaturase–1 by KGF. Conversely, adenovirus-mediated overexpression of a constitutively active form of SREBP-1c mimicked the effect of KGF on lipogenic enzymes and lipogenesis. These data indicate that SREBP-1c is required for the stimulation of lipogenesis by KGF in the alveolar type II cells and is a key regulator of lung lipid metabolism and that expression of SREBP-1c is sufficient to induce lipogenesis in rat type II cells.
adenovirus; fatty acid synthesis; surfactant
Ozone exposure produces acute inflammation and neutrophil influx in the distal lung. Alveolar epithelial cells cover a large surface area, secrete chemokines, and may initiate or modify the inflammatory response. The effect of ozone on chemokine production by these cells has not been defined. Isolated rat type II cells were cultured in different conditions to express the morphologic appearance and biochemical markers for the type I and the type II cell phenotypes. These cells were exposed to ozone at an air/liquid interface. The type I–like cells were more susceptible to injury than the type II cells and showed signs of injury at exposure levels of 100 ppb ozone for 60 min. Both phenotypes showed evidence of lipid peroxidation after ozone exposure as measured by 8-isoprostane production, but neither phenotype secreted increased amounts of MIP-2 (CXCL3), CINC-1 (CXCL1), or MCP-1 (CCL2) in response to ozone. Both cell phenotypes secreted MIP-2 and MCP-1 in response to IL-1β or lipopolysaccharide, but there was no priming or synergy with ozone. It is likely that the inflammatory response to ozone in the alveolar compartment is not due to the direct effect of ozone on epithelial cells.
alveolar epithelium; LPS; 8-isoprostane; MCP-1; MIP-2
Alveolar epithelial cells are among the first cells to encounter inhaled particles or organisms. These cells likely participate in the initiation and modulation of the inflammatory response by production of chemokines. However, there is little information on the extent or regulation of chemokine production by these cells. Rat type II cells were studied under differentiated and dedifferentiated conditions to determine their ability to express and secrete CXC chemokines. Both differentiated and dedifferentiated type II cells secreted MIP-2, MCP-1, and CINC-2 in response to a cytokine mixture of IL-1β, TNF-α, and IFN-γ or to IL-1β alone. The cytokine mixture also induced iNOS expression and nitrite secretion. Both differentiated and dedifferentiated type II cells expressed CINC-1 (GRO), CINC-2α, CINC-3 (MIP-2), and MCP-1 mRNA, and their expression was increased by the cytokine mixture or by IL-1β alone. However, CINC-2β, a splice variant of CINC-2, was only expressed under differentiated conditions stimulated by KGF and was not increased by the cytokine mixture or by IL-1β. In situ hybridization of normal lung and lung instilled with Ad-KGF demonstrated that CINC-2β was expressed by alveolar and bronchiolar epithelial cells in vivo. We conclude that CINC-2β is regulated differently from most other chemokines and that its expression is related to the state of alveolar type II cell differentiation.
CCL2; chemokines; CXCL1; inflammation; type II cells