Recent studies suggest that high expression of the pro-inflammatory cytokine interleukin-6 (IL-6) is associated with poor survival of lung cancer patients. Accordingly, IL-6 has been a target of great interest for lung cancer therapy. However, the role of IL-6 in lung cancer has not been determined yet. Here, we demonstrate that IL-6 plays opposite roles in the initiation and growth of lung cancer in a mouse model of lung cancer induced by the K-Ras oncogene. We find that compared to wild type mice, IL-6 deficient mice developed much more lung tumors after an activating mutant of K-Ras was induced in the lungs. However, lung tumors developed in IL-6 deficient mice were significantly smaller. Notably, both the lung tumor-suppressing and -promoting functions of IL-6 involve its ability in activating the transcription factor STAT3. IL-6/STAT3 signaling suppressed lung cancer initiation through maintaining lung homeostasis, regulating lung macrophages and activating cytotoxic CD8 T cells under K-Ras oncogenic stress, whereas it promoted lung cancer cell growth through inducing the cell proliferation regulator Cyclin D1. These studies reveal a previously unexplored role of IL-6/STAT3 signaling in maintaining lung homeostasis and suppressing lung cancer induction. These studies also significantly improve our understanding of lung cancer and provide a molecular basis for designing IL-6/STAT3-targeted therapies for this deadliest human cancer.
IL-6; lung cancer; Ras; STAT3; tumor initiation; tumor progression
Macrophage elastase (MMP12) is a key mediator of cigarette smoke (CS)-induced emphysema, yet its role in other smoking related pathologies remains unclear. The weight suppressing effects of smoking are a major hindrance to cessation efforts, and MMP12 is known to suppress the vascularization on which adipose tissue growth depends by catalyzing the formation of antiangiogenic peptides endostatin and angiostatin. The goal of this study was to determine the role of MMP12 in adipose tissue growth and smoking-related suppression of weight gain. Whole body weights and white adipose depots from wild-type and Mmp12-deficient mice were collected during early postnatal development and after chronic CS exposure. Adipose tissue specimens were analyzed for angiogenic and adipocytic markers and for content of the antiangiogenic peptides endostatin and angiostatin. Cultured 3T3-L1 adipocytes were treated with adipose tissue homogenate to examine its effects on vascular endothelial growth factor (VEGF) expression and secretion. MMP12 content and activity were increased in the adipose tissue of wild-type mice at 2 weeks of age, leading to elevated endostatin production, inhibition of VEGF secretion, and decreased adipose tissue vascularity. By 8 weeks of age, adipose MMP12 levels subsided, and the protein was no longer detectable. However, chronic CS exposure led to macrophage accumulation and restored adipose MMP12 activity, thereby suppressing adipose tissue mass and vascularity. Our results reveal a novel systemic role for MMP12 in postnatal adipose tissue expansion and smoking-associated weight loss by suppressing vascularity within the white adipose tissue depots.
proteases; obesity; angiogenesis
Cigarette smoke is the most common cause of pulmonary emphysema, which results in an irreversible loss of lung structure and function. Th1 and Th17 immune responses have been implicated in emphysema pathogenesis; however, the drivers of emphysema-associated immune dysfunction are not fully understood. In this issue of the JCI, Shan and colleagues found that peroxisome proliferator–activated receptor γ (PPARγ) is downregulated in APCs isolated from the lungs of emphysematous chronic smokers and mice exposed to cigarette smoke. Furthermore, treatment with a PPARγ agonist prevented emphysema development and appeared to reduce emphysema-associated lung volume expansion in mice exposed to cigarette smoke. Further work will need to be done to evaluate the potential of PPARγ agonists to restore lung capacity in emphysematous patients.
Multiple organ failure, wasting, increased morbidity, and mortality following acute illness complicates the health span of patients surviving sepsis. Persistent inflammation has been implicated, and it is proposed that insulin signaling contributes to persistent inflammatory signaling during the recovery phase after sepsis. However, mechanisms are unknown and suitable pre-clinical models are lacking. We therefore developed a novel Drosophila melanogaster model of sepsis to recapitulate the clinical course of sepsis, explored inflammation over time, and its relation to impaired mobility, metabolic disturbance, and changes in lifespan.
We used wild-type (WT), Drosomycin-green fluorescent protein (GFP), and NF-κB-luc reporter male Drosophila melanogaster 4–5 days of age (unmanipulated). We infected Drosophila with Staphylococcus aureus (infected without treatment) or pricked with aseptic needles (sham). Subsets of insects were treated with oral linezolid after the infection (infected with antibiotics). We assessed rapid iterative negative geotaxis (RING) in all the groups as a surrogate for neuromuscular functional outcome up to 96 h following infection. We harvested the flies over the 7-day course to evaluate bacterial burden, inflammatory and metabolic pathway gene expression patterns, NF-κB translation, and metabolic reserve. We also followed the lifespan of the flies.
Our results showed that when treated with antibiotics, flies had improved survival compared to infected without treatment flies in the early phase of sepsis up to 1 week (81 %, p = 0.001). However, the lifespan of infected with antibiotics flies was significantly shorter than that of sham controls (p = 0.001). Among infected with antibiotic sepsis survivors, we observed persistent elevation of NF-κB in the absence of any obvious infection as shown by culturing flies surviving sepsis. In the same group, geotaxis had an early (18 h) and sustained decline compared to its baseline. Geotaxis in infected with antibiotics sepsis survivors was significantly lower than that in sham and age-matched unmanipulated flies at 18 and 48 h. Expression of antimicrobial peptides (AMP) remained significantly elevated over the course of 7 days after sepsis, especially drosomycin (5.7-fold, p = 0.0145) on day 7 compared to that of sham flies. Infected with antibiotics flies had a trend towards decreased Akt activation, yet their glucose stores were significantly lower than those of sham flies (p = 0.001). Sepsis survivors had increased lactate levels and LDH activity by 1 week, whereas ATP and pyruvate content was similar to that of the sham group.
In summary, our model mimics human survivors of sepsis with persistent inflammation, impaired motility, dysregulated glucose metabolism, and shortened lifespan.
Electronic supplementary material
The online version of this article (doi:10.1186/s40635-016-0075-4) contains supplementary material, which is available to authorized users.
Drosophila; Sepsis; Recovery; Drosomycin; Insulin; Akt
Signal transducer and activator of transcription 3 (STAT3) is linked to multiple cancers, including pulmonary adenocarcinoma. However, the role of STAT3 in lung cancer pathogenesis has not been determined. Using lung epithelial-specific inducible knockout strategies, we demonstrate that STAT3 plays contrasting roles in the initiation and growth of both chemically and genetically induced lung cancers. Selective deletion of lung epithelial STAT3 in mice prior to cancer induction by the smoke carcinogen, urethane, resulted in increased lung tissue damage and inflammation, K-Ras oncogenic mutations, and tumorigenesis. Deletion of lung epithelial STAT3 after establishment of lung cancer inhibited cancer cell proliferation. Simultaneous deletion of STAT3 and expression of oncogenic K-Ras in mouse lung elevated pulmonary injury, inflammation, and tumorigenesis, but reduced tumor growth. These studies indicate that STAT3 prevents lung cancer initiation by maintaining pulmonary homeostasis under oncogenic stress, whereas it facilitates lung cancer progression by promoting cancer cell growth. These studies also provide a mechanistic basis for targeting STAT3 to lung cancer therapy.
lung cancer; lung injury; lung inflammation; lung homeostasis; Ras; STAT3
Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between the stem/progenitor cells that proliferate or differentiate and the somatic cells of lung, we utilized a novel ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, β3, and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conserved population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. Over 50% of the AIC population was cKit+ or double-positive for CD45+ and CD11b+ antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. AICs undergo remodeling to form a cellular lining at the air/gel interface, and TGFβ1 treatment modifies protein expression, implying direct-differentiation of this population. These data confirm the active participation of clonogenic hematopietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which embrace sustained cell viability, proliferation, and differentiation, for use in studies of compensatory pulmonary growth.
Anchorage-independent cells; Suspension culture; CD45/CD11b positive cells; Lung progenitors
Several recent clinical studies have implied a role for the receptor for advanced glycation end products (RAGE) and its variants in chronic obstructive pulmonary disease (COPD). In this study we have defined a role for RAGE in the pathogenesis of emphysema in mice. RAGE deficient mice (RAGE-/-) exposed to chronic cigarette smoke were significantly protected from smoke induced emphysema as determined by airspace enlargement and had no significant reduction in lung tissue elastance when compared to their air exposed controls contrary to their wild type littermates. The progression of emphysema has been largely attributed to an increased inflammatory cell-mediated elastolysis. Acute cigarette smoke exposure in RAGE-/- mice revealed an impaired early recruitment of neutrophils, approximately a 6-fold decrease compared to wild type mice. Hence, impaired neutrophil recruitment with continued cigarette smoke exposure reduces elastolysis and consequent emphysema.
We aimed to identify peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes in idiopathic pulmonary fibrosis (IPF) by performing microarray experiments of PBMCs in discovery and replication cohorts of IPF patients. Microarray analyses identified 52 genes associated with transplant-free survival (TFS) in the discovery cohort. Clustering the microarray samples of the replication cohort using the 52-gene outcome-predictive signature distinguished two patient groups with significant differences in TFS. We studied the pathways associated with TFS in each independent microarray cohort and identified decreased expression of “The costimulatory signal during T cell activation” Biocarta pathway and, in particular, the genes CD28, ICOS, LCK, and ITK, results confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A proportional hazards model, including the qRT-PCR expression of CD28, ICOS, LCK, and ITK along with patient’s age, gender, and percent predicted forced vital capacity (FVC%), demonstrated an area under the receiver operating characteristic curve of 78.5% at 2.4 months for death and lung transplant prediction in the replication cohort. To evaluate the potential cellular source of CD28, ICOS, LCK, and ITK expression, we analyzed and found significant correlation of these genes with the PBMC percentage of CD4+CD28+ T cells in the replication cohort. Our results suggest that CD28, ICOS, LCK, and ITK are potential outcome biomarkers in IPF and should be further evaluated for patient prioritization for lung transplantation and stratification in drug studies.
Sepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis.
We performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE−/−) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE−/− mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation.
ApoE−/− mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (−6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05).
Using a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13054-014-0469-1) contains supplementary material, which is available to authorized users.
Theories of emphysema traditionally revolved around proteolytic destruction of extracellular matrix. Models have recently been developed that show airspace enlargement with the induction of pulmonary cell apoptosis. The purpose of this study was to determine the mechanism by which a model of epithelial cell apoptosis caused airspace enlargement. Mice were treated with either intratracheal microcystin (MC) to induce apoptosis, intratracheal porcine pancreatic elastase (PPE), or their respective vehicles. Mice from all groups were inflated and morphometry was measured at various time points. Physiology measurements were performed for airway resistance, tissue elastance, and lung volumes. The groups were further analyzed by air–saline quasistatic measurements, surfactant staining, and surfactant functional studies. Mice treated with MC showed evidence of reversible airspace enlargement. In contrast, PPE-treated mice showed irreversible airspace enlargement. The airspace enlargement in MC-treated mice was associated with an increase in elastic recoil due to an increase in alveolar surface tension. PPE-treated mice showed a loss of lung elastic recoil and normal alveolar surface tension, a pattern more consistent with human emphysema. Airspace enlargement that occurs with the MC model of pulmonary epithelial cell apoptosis displays physiology distinct from human emphysema. Reversibility, restrictive physiology due to changes in surface tension, and alveolar enlargement associated with heterogeneous alveolar collapse are most consistent with a mild acute lung injury. Inflation near total lung capacity gives the appearance of enlarged alveoli as neighboring collapsed alveoli exert tethering forces.
apoptosis; emphysema; physiology; murine model
Rtp801, a stress – related protein triggered by adverse environmental conditions, inhibits mTOR and enhances oxidative stress – dependent cell death. We postulated that Rtp801 acts as potential amplifying switch in the development of cigarette smoke – induced lung injury, leading to emphysema. Rtp801 was overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The upregulation of Rtp801 expression by cigarette smoke in the lung relied on oxidative stress – dependent activation of the CCAAT response element. Rtp801 was necessary and sufficient for NF – κ B activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF – kB activation, alveolar inflammation, oxidative stress, and apoptosis of alveolar septal cells. On the other hand, Rtp801 − / − mice were markedly protected against acute cigarette smoke – induced lung injury, partly via increased mTOR signaling, and, when exposed chronically, against emphysema. Our data support the notion that Rtp801 may represent an important molecular sensor and mediator of lung injury to cigarette smoke.
Rtp801; cigarette smoke; oxidative stress; apoptosis; inflammation; NF –κB; rapamycin
Increased numbers of macrophages are found in the lungs of smokers and those with chronic obstructive pulmonary disease. Experimental evidence shows the central role of macrophages in elaboration of inflammatory mediators such as TNF-a and the progression toward cigarette smoke-induced emphysema. We investigated the role of CX3CR1 in recruitment of mononuclear phagocytes, inflammatory cytokine responses, and tissue destruction in the lungs after cigarette smoke exposure. Using mice in which egfp is expressed at the locus of the cx3cr1 gene, we show that alveolar macrophages increased transmembrane ligand CX3CL1 expression and soluble CX3CL1 was detectable in the airspaces, but cx3cr1GFP/GFP and cx3cr1GFP/+ mice failed to show recruitment of CX3CR1+ cells into the airspaces with cigarette smoke. In contrast, cigarette smoke increased the accumulation of CX3CR1+CD11b+ mononuclear phagocytes that were spatially confined to the lung interstitium and heterogenous in their expression of CD11c, MHC class II, and autofluorescent property. Although an intact CX3CL1–CX3CR1 pathway amplified the percentage of CX3CR1+CD11b+ mononuclear phagocytes in the lungs, it was not essential for recruitment. Rather, functional CX3CR1 was required for a subset of tissue-bound mononuclear phagocytes to produce TNF-α and IL-6 in response to cigarette smoke, and the absence of functional CX3CR1 protected mice from developing tissue-destructive emphysema. Thus, CX3CR1+ “tissue resident” mononuclear phagocytes initiate an innate immune response to cigarette smoke by producing TNF-α and IL-6 and are capable of promoting emphysema.
Excess collagen deposition occurs in pulmonary fibrosis. A new study suggests that collagen overproduction may originate from cells derived from bone marrow precursors rather than parenchymal lung fibroblasts .
Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the hemidesmosomal proteins BP180 and BP230. In the IgG passive transfer model of BP, blister formation is triggered by anti-BP180 IgG and depends on complement activation, mast cell degranulation, and neutrophil recruitment. Mice lacking neutrophil elastase (NE) do not develop experimental BP. Here, we demonstrated that NE degrades recombinant mouse BP180 within the immunodominant extracellular domain at amino acid positions 506 and 561, generating peptide p561 and peptide p506. Peptide p561 is chemotactic for neutrophils both in vitro and in vivo. Local injection of NE into B6 mice recruits neutrophils to the skin, and neutrophil infiltration is completely blocked by co-injection with the NE inhibitor α1-proteinase inhibitor. More importantly, NE directly cleaves BP180 in mouse and human skin, as well as the native human BP180 trimer molecule. These results demonstrate that (i) NE directly damages the extracellular matrix and (ii) NE degradation of mouse BP180 generates neutrophil chemotactic peptides that amplify disease severity at the early stage of the disease.
bullous pemphigoid; chemotaxis; basement membrane; neutrophil elastase; BP180; autoimmune mouse model
Since the discovery of alpha-1 antitrypsin in the early 1960s, several new genes have been suggested to play a role in chronic obstructive pulmonary disease (COPD) pathogenesis. Yet, in spite of those advances, much about the genetic basis of COPD still remains to be discovered. Unbiased approaches, such as genome-wide association (GWA) studies, are critical to identify genes and pathways and to verify suggested genetic variants. Indeed, most of our current understanding about COPD candidate genes originates from GWA studies. Experiments in form of cross-study replications and advanced meta-analyses have propelled the field towards unravelling details about COPD's pathogenesis. Here, we review the discovery of genetic variants in association with COPD phenotypes by discussing the available approaches and current findings. Limitations of current studies are considered and future directions provided.
COPD; genes; genetics; genome-wide association studies; obstructive pulmonary disease
Acute exacerbations are a significant source of morbidity and mortality associated with chronic obstructive pulmonary disease. Among patients with COPD, some patients suffer an inordinate number of exacerbations while others remain relatively protected. We undertook a study to determine the clinical factors associated with "frequent exacerbator" status within a population of subjects with severe COPD.
Case-control cohort recruited from two Boston-area practices. All subjects had GOLD stage 3 or 4 (FEV1 ≤50% predicted) COPD. "Frequent exacerbators" (n=192) had an average of ≥2 moderate-to-severe exacerbations per year while "non-exacerbators" (n=153) had no exacerbations in the preceding 12 months. Multivariate logistic regression was performed to determine the significant clinical predictors of "frequent exacerbator" status.
Physician-diagnosed asthma was a significant predictor of frequent exacerbations. Within a subset of our cohort, the modified Medical Research Council dyspnea score and FEF25–75 % predicted were also significant clinical predictors of frequent exacerbator status (p<0.05). Differences in exacerbation frequency were not found to be due to increased current tobacco use or decreased rates of maintenance medication use.
Within our severe COPD cohort, a history of physician-diagnosed asthma was found to be a significant clinical predictor of frequent exacerbations. Although traditional risk factors such as decreased FEV1% predicted were not significantly associated with frequent exacerbator status, lower mid-expiratory flow rates, as assessed by FEF 25–75 % predicted, were significantly associated with frequent exacerbations in a subset of our cohort.
Exposure to cigarette smoke (CS) was shown to impair the capacity of macrophages to clear bacteria and apoptotic cells. Here, we show that both the exposure of macrophages to cigarette smoke extract (CSE) in vitro and an acute single exposure to CS in vivo impair the macrophage clearance of apoptotic polymorphonuclear leukocytes (PMNs). Upon longer periods of exposure to smoke in vivo (4–12 weeks), the impaired capacity of macrophages to clear apoptotic cells persisted after the cessation of smoking, with slow recovery to normality observed 4 weeks later. With respect to the mechanism by which CS impairs the macrophage uptake of apoptotic PMNs, we did not detect altered surface expression of receptors associated with apoptotic cell clearance. We did observe the impaired phosphorylation of the guanine nucleotide exchange factor Vav1 and the downstream inhibition of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Consistent with these findings, CS impaired the macrophage cytoskeletal changes observed after stimulation with apoptotic cells. A loss of actin occurred at the leading edge, manifested as impaired ruffling of the cell membrane and a decreased capacity to engulf apoptotic cells. The inability to clear PMNs would lead to a greater release of destructive PMN products, and would diminish the reparative phenotype induced by the macrophage clearance of apoptotic cells.
cigarette smoke; alveolar macrophage; phagocytosis and engulfment of apoptotic cells; actin rearrangements; small GTP protein Rac1
Chronic obstructive pulmonary disease (COPD) is characterized by alveolar destruction and abnormal inflammatory responses to noxious stimuli. Surfactant protein–D (SFTPD) is immunomodulatory and essential to host defense. We hypothesized that polymorphisms in SFTPD could influence the susceptibility to COPD. We genotyped six single-nucleotide polymorphisms (SNPs) in surfactant protein D in 389 patients with COPD in the National Emphysema Treatment Trial (NETT) and 472 smoking control subjects from the Normative Aging Study (NAS). Case-control association analysis was performed using Cochran–Armitage trend tests and multivariate logistic regression. The replication of significant associations was attempted in the Boston Early-Onset COPD Study, the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) Study, and the Bergen Cohort. We also correlated SFTPD genotypes with serum concentrations of surfactant protein–D (SP-D) in the ECLIPSE Study. In the NETT–NAS case-control analysis, four SFTPD SNPs were associated with susceptibility to COPD: rs2245121 (P = 0.01), rs911887 (P = 0.006), rs6413520 (P = 0.004), and rs721917 (P = 0.006). In the family-based analysis of the Boston Early-Onset COPD Study, rs911887 was associated with prebronchodilator and postbronchodilator FEV1 (P = 0.003 and P = 0.02, respectively). An intronic SNP in SFTPD, rs7078012, was associated with COPD in the ECLIPSE Study and the Bergen Cohort. Multiple SFTPD SNPs were associated with serum SP-D concentrations in the ECLIPSE Study. We demonstrated an association of polymorphisms in SFTPD with COPD in multiple populations. We demonstrated a correlation between SFTPD SNPs and SP-D protein concentrations. The SNPs associated with COPD and SP-D concentrations differed, suggesting distinct genetic influences on susceptibility to COPD and SP-D concentrations.
COPD; surfactant protein–D; single-nucleotide polymorphisms; genetics
Chronic Obstructive Pulmonary Disease (COPD) is characterized by airspace enlargement and peribronchial lymphoid follicles; however, the immunological mechanisms leading to these pathologic changes remain undefined. Here we show that cigarette smoke is a selective adjuvant that augments in vitro and in vivo Th17, but not Th1, cell differentiation via the aryl hydrocarbon receptor. Smoke exposed IL-17RA−/− mice failed to induce CCL2 and MMP12 compared to WT mice. Remarkably, in contrast to WT mice, IL-17RA−/− mice failed to develop emphysema after 6 months of cigarette smoke exposure. Taken together, these data demonstrate that cigarette smoke is a potent Th17 adjuvant and that IL-17RA signaling is required for chemokine expression necessary for MMP12 induction and tissue emphysema.
To identify non-invasive gene expression markers for chronic obstructive pulmonary disease (COPD), we performed genome-wide expression profiling of peripheral blood samples from 12 subjects with significant airflow obstruction and an equal number of non-obstructed controls. RNA was isolated from Peripheral Blood Mononuclear Cells (PBMCs) and gene expression was assessed using Affymetrix U133 Plus 2.0 arrays.
Tests for gene expression changes that discriminate between COPD cases (FEV1< 70% predicted, FEV1/FVC < 0.7) and controls (FEV1> 80% predicted, FEV1/FVC > 0.7) were performed using Significance Analysis of Microarrays (SAM) and Bayesian Analysis of Differential Gene Expression (BADGE). Using either test at high stringency (SAM median FDR = 0 or BADGE p < 0.01) we identified differential expression for 45 known genes. Correlation of gene expression with lung function measurements (FEV1 & FEV1/FVC), using both Pearson and Spearman correlation coefficients (p < 0.05), identified a set of 86 genes. A total of 16 markers showed evidence of significant correlation (p < 0.05) with quantitative traits and differential expression between cases and controls. We further compared our peripheral gene expression markers with those we previously identified from lung tissue of the same cohort. Two genes, RP9and NAPE-PLD, were identified as decreased in COPD cases compared to controls in both lung tissue and blood. These results contribute to our understanding of gene expression changes in the peripheral blood of patients with COPD and may provide insight into potential mechanisms involved in the disease.
Microarray; Biomarkers; PBMC
Lung cancer is the leading cause of cancer death worldwide1. Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness2-3. To determine the role of neutrophil elastase (NE or Elane) on tumor progression, we utilized the LSL-K-ras model of murine lung adenocarcinoma4 to generate LSL-K-ras/Elane−/− mice. Tumor burden was markedly reduced in LSL-K-ras/Elane−/− mice at all time points following induction of mutant K-ras expression. Kaplan-Meier life survival analysis demonstrated that while 100% of LSL-K-ras/Elane+/+ mice died, none of the mice lacking NE died. NE directly induced tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells where it degraded insulin receptor substrate-1 (IRS1). Co-immunoprecipitation studies showed that as NE degraded IRS1, there was increased interaction between PI3K and the potent mitogen platelet derived growth factor receptor (PDGFR) thereby skewing the PI3K axis toward tumor cell proliferation. The inverse relationship identified between NE and IRS1 in LSL-K-ras mice was also identified in human lung adenocarcinomas, thus translating these findings to human disease. This study identifies IRS1 as a key regulator of PI3K within malignant cells. Additionally, this is the first description of a secreted proteinase gaining access to a cell beyond its plasma membrane and altering intracellular signaling.
Genetic variants influencing lung function in children and adults may ultimately lead to the development of chronic obstructive pulmonary disease (COPD), particularly in high-risk groups.
We tested for an association between single-nucleotide polymorphisms (SNPs) in the gene encoding matrix metalloproteinase 12 (MMP12) and a measure of lung function (prebronchodilator forced expiratory volume in 1 second [FEV1]) in more than 8300 subjects in seven cohorts that included children and adults. Within the Normative Aging Study (NAS), a cohort of initially healthy adult men, we tested for an association between SNPs that were associated with FEV1 and the time to the onset of COPD. We then examined the relationship between MMP12 SNPs and COPD in two cohorts of adults with COPD or at risk for COPD.
The minor allele (G) of a functional variant in the promoter region of MMP12 (rs2276109 [−82A→G]) was positively associated with FEV1 in a combined analysis of children with asthma and adult former and current smokers in all cohorts (P=2×10−6). This allele was also associated with a reduced risk of the onset of COPD in the NAS cohort (hazard ratio, 0.65; 95% confidence interval [CI], 0.46 to 0.92; P = 0.02) and with a reduced risk of COPD in a cohort of smokers (odds ratio, 0.63; 95% CI, 0.45 to 0.88; P = 0.005) and among participants in a family-based study of early-onset COPD (P = 0.006).
The minor allele of a SNP in MMP12 (rs2276109) is associated with a positive effect on lung function in children with asthma and in adults who smoke. This allele is also associated with a reduced risk of COPD in adult smokers.
Airway hyper-responsiveness (AHR) is a critical phenotype of human asthma and animal models of asthma. Other studies have measured AHR in nine mouse strains, but only six strains have been used to identify genetic loci underlying AHR. Our goals were to increase the genetic diversity of available strains by surveying 27 additional strains, to apply haplotype association mapping to the 36-strain survey, and to identify new genetic determinants for AHR. We derived AHR from the increase in airway resistance in females subjected to increasing levels of methacholine concentrations. We used haplotype association mapping to identify associations between AHR and haplotypes on chromosomes 3, 5, 8, 12, 13, and 14. And we used bioinformatics techniques to narrow the identified region on chromosome 13, reducing the region to 29 candidate genes, with 11 of considerable interest. Our combined use of haplotype association mapping with bioinformatics tools is the first study of its kind for AHR on these 36 strains of mice. Our analyses have narrowed the possible QTL genes and will facilitate the discovery of novel genes that regulate AHR in mice.
Mice; Genetics; Asthma
Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented1,2. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins3–9. We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages10 and is implicated in several disease processes, including emphysema11. Physiological functions for MMP12 have not been described. Here we show that Mmp12−/− mice exhibit impaired bacterial clearance and increased mortality when challenged with both Gram-negative and Gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed β loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.
Respiratory tract dendritic cells (DCs) are juxtaposed to directly sample inhaled environmental particles. Processing and presentation of these airborne Ags could result in either the development of immunity or tolerance. The purpose of this study was to determine the consequences of cigarette smoke exposure on DC function in mice. We demonstrate that while cigarette smoke exposure decreased the number of DCs in the lungs, Ag-induced DC migration to the regional thoracic lymph nodes was unaffected. However, cigarette smoking suppressed DC maturation within the lymph nodes as demonstrated by reduced cell surface expression of MHC class II and the costimulatory molecules CD80 and CD86. Consequently, DCs from cigarette smoke-exposed animals had a diminished capacity to induce IL-2 production by T cells that was associated with diminished Ag-specific T cell proliferation in vivo. Smoke-induced defects in DC function leading to impaired CD4+ T cell function could inhibit tumor surveillance and predispose patients with chronic obstructive pulmonary disease to infections and exacerbations.