The role of lipocalin-2 in age-related macular degeneration (AMD).

Lipocalins are a family of secreted adipokines which play important roles in various biological processes. Lipocalin-2 (LCN-2) has been shown to be involved in acute and chronic inflammation. This particular protein is critical in the pathogenesis of several diseases including cancer, diabetes, obesity, and multiple sclerosis. Herein, we discuss the general molecular basis for the involvement of LCN-2 in acute infections and chronic disease progression and also ascertain the probable role of LCN-2 in ocular diseases, particularly in age-related macular degeneration (AMD). We elaborate on the signaling cascades which trigger LCN-2 upregulation in AMD and suggest therapeutic strategies for targeting such pathways.

Neutrophils homing into the retina trigger pathology in early age-related macular degeneration.

Age-related macular degeneration (AMD) is an expanding problem as longevity increases worldwide. While inflammation clearly contributes to vision loss in AMD, the mechanism remains controversial. Here we show that neutrophils are important in this inflammatory process. In the retinas of both early AMD patients and in a mouse model with an early AMD-like phenotype, we show neutrophil infiltration. Such infiltration was confirmed experimentally using ribbon-scanning confocal microscopy (RSCM) and IFNλ- activated dye labeled normal neutrophils. With neutrophils lacking lipocalin-2 (LCN-2), infiltration was greatly reduced. Further, increased levels of IFNλ in early AMD trigger neutrophil activation and LCN-2 upregulation. LCN-2 promotes inflammation by modulating integrin ß1 levels to stimulate adhesion and transmigration of activated neutrophils into the retina. We show that in the mouse model, inhibiting AKT2 neutralizes IFNλ inflammatory signals, reduces LCN-2-mediated neutrophil infiltration, and reverses early AMD-like phenotype changes. Thus, AKT2 inhibitors may have therapeutic potential in early, dry AMD.

Targeting the Temporal Dynamics of Hypoxia-Induced Tumor-Secreted Factors Halts Tumor Migration.

Targeting microenvironmental factors that foster migratory cell phenotypes is a promising strategy for halting tumor migration. However, lack of mechanistic understanding of the emergence of migratory phenotypes impedes pharmaceutical drug development. Using our three-dimensional microtumor model with tight control over tumor size, we recapitulated the tumor size-induced hypoxic microenvironment and emergence of migratory phenotypes in microtumors from epithelial breast cells and patient-derived primary metastatic breast cancer cells, mesothelioma cells, and lung cancer xenograft cells. The microtumor models from various patient-derived tumor cells and patient-derived xenograft cells revealed upregulation of tumor-secreted factors, including matrix metalloproteinase-9 (MMP9), fibronectin (FN), and soluble E-cadherin, consistent with clinically reported elevated levels of FN and MMP9 in patient breast tumors compared with healthy mammary glands. Secreted factors in the conditioned media of large microtumors induced a migratory phenotype in nonhypoxic, nonmigratory small microtumors. Subsequent mathematical analyses identified a two-stage microtumor progression and migration mechanism whereby hypoxia induces a migratory phenotype in the initialization stage, which then becomes self-sustained through a positive feedback loop established among the tumor-secreted factors. Computational and experimental studies showed that inhibition of tumor-secreted factors effectively halts microtumor migration despite tumor-to-tumor variation in migration kinetics, while inhibition of hypoxia is effective only within a time window and is compromised by tumor-to-tumor variation, supporting our notion that hypoxia initiates migratory phenotypes but does not sustain it. In summary, we show that targeting temporal dynamics of evolving microenvironments, especially tumor-secreted factors during tumor progression, can halt tumor migration. SIGNIFICANCE: This study uses state-of-the-art three-dimensional microtumor models and computational approaches to highlight the temporal dynamics of tumor-secreted microenvironmental factors in inducing tumor migration.

The Efficacy of the Interferon Alpha/Beta Response versus Arboviruses Is Temperature Dependent.

Interferon alpha/beta (IFN-α/ß) is a critical mediator of protection against most viruses, with host survival frequently impossible in its absence. Many studies have investigated the pathways involved in the induction of IFN-α/ß after virus infection and the resultant upregulation of antiviral IFN-stimulated genes (ISGs) through IFN-α/ß receptor complex signaling. However, other than examining the effects of genetic deletion of induction or effector pathway components, little is known regarding the functionality of these responses in intact hosts and whether host genetic or environmental factors might influence their potency. Here, we demonstrate that the IFN-α/ß response against multiple arthropod-vectored viruses, which replicate over a wide temperature range, is extremely sensitive to fluctuations in temperature, exhibiting reduced antiviral efficacy at subnormal cellular temperatures and increased efficacy at supranormal temperatures. The effect involves both IFN-α/ß and ISG upregulation pathways with a major aspect of altered potency reflecting highly temperature-dependent transcription of IFN response genes that leads to altered IFN-α/ß and ISG protein levels. Discordantly, signaling steps prior to transcription that were examined showed the opposite effect from gene transcription, with potentiation at low temperature and inhibition at high temperature. Finally, we demonstrate that by lowering the temperature of mice, chikungunya arbovirus replication and disease are exacerbated in an IFN-α/ß-dependent manner. This finding raises the potential for use of hyperthermia as a therapeutic modality for viral infections and in other contexts such as antitumor therapy. The increased IFN-α/ß efficacy at high temperatures may also reflect an innate immune-relevant aspect of the febrile response.IMPORTANCE The interferon alpha/beta (IFN-α/ß) response is a first-line innate defense against arthropod-borne viruses (arboviruses). Arboviruses, such as chikungunya virus (CHIKV), can infect cells and replicate across a wide temperature range due to their replication in both mammalian/avian and arthropod hosts. Accordingly, these viruses can cause human disease in tissues regularly exposed to temperatures below the normal mammalian core temperature, 37°C. We questioned whether temperature variation could affect the efficacy of IFN-α/ß responses against these viruses and help to explain some aspects of human disease manifestations. We observed that IFN-α/ß efficacy was dramatically lower at subnormal temperatures and modestly enhanced at febrile temperatures, with the effects involving altered IFN-α/ß response gene transcription but not IFN-α/ß pathway signaling. These results provide insight into the functioning of the IFN-α/ß response in vivo and suggest that temperature elevation may represent an immune-enhancing therapeutic modality for a wide variety of IFN-α/ß-sensitive infections and pathologies.

MUC5B expression and location in surfactant protein C mutations in children.

BACKGROUND: Mutations in Surfactant Protein C (SFTPC) can lead to fibrotic interstitial lung disease (ILD) with variable phenotypes, especially in children. The sources of phenotype variability are incompletely understood. A common MUC5B promoter variant rs35705950 is associated with adult Idiopathic Pulmonary Fibrosis (IPF). We examined whether MUC5B is similarly linked to ILD secondary to SFTPC mutations. METHODS: MUC5B concentration in bronchoalveolar lavage fluid (BALF) was measured in six pediatric patients with SFTPC mutations and diseased controls. Immunohistochemical localization of MUC5B was studied in fixed lung tissues in patients with SFTPC mutations, ABCA3 mutations, and controls. Genotyping for the MUC5B promoter variant rs35705950 was attempted in all samples. RESULTS: MUC5B glycoprotein was increased in BALF of patients with SFTPC mutations compared to diseased controls (P = 0.04). MUC5B was unexpectedly present in cells morphologically consistent with alveolar epithelial type II cells in patients with SFTPC mutations in the BRICHOS domain. Genotyping for the MUC5B promoter variant was successful in 18/27 patients, and there was no significant relationship between the MUC5B promoter variant and the BALF or MUC5B localization. CONCLUSION: MUC5B may play a role in the development of fibrosis in patients with SFTPC mutations, especially in patients with BRICHOS mutations. Understanding the role of MUC5B in adult and pediatric lung diseases may lead to a better understanding of the etiology of fibrotic lung disease as well as development of novel therapies.

AKT network of genes and impaired myocardial contractility during murine acute Chagasic myocarditis.

Chagasic disease is associated with high morbidity in Latin America. Acute Chagasic myocarditis is consistently found in acute infections, but little is known about its contribution to chronic cardiomyopathy. The aim of the study was to phenotypically characterize two strains of mice with differential Chagas infection susceptibility and correlate strain myocarditis phenotypes with heart tissue gene expression. C57BL/6J and Balb/c mice were injected intraperitoneally with 0 or 150-200 tissue-derived trypomastigotes (Tulahuen strain). Echocardiograms, brain natriuretic peptide, and troponin were measured. Heart tissue was harvested for histopathological analysis and gene expression profiling on microarrays. Genes differently expressed between infected Balb/c and C57BL/6J mice were identified. Echocardiograms showed differences in Balb/c versus C57BL/6J infected mice in heart rate (413 versus 476 beats per minute; P = 0.0001), stroke volume (31.9 ± 9.3 versus 39.2 ± 5.5 µL; P = 0.03), and cardiac output (13.1 ± 3.5 versus 18.7 ± 3.2 µL/min; P = 0.002). Gene expression at 4 weeks analysis showed 32 statistically significant (q value < 0.05) differentially expressed genes between infected Balb/c and C57BL/6J mice that were enriched for genes related to the protein kinase B (AKT) pathway. These specific phenotypic features of cardiac response during acute Chagasic myocarditis may, in part, be related to host AKT network regulation.

Mitotic asynchrony induces transforming growth factor-ß1 secretion from airway epithelium.

We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to proinflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that mitotic asynchrony is associated with proinflammatory/fibrotic cytokine secretion (e.g., transforming growth factor [TGF]-ß1), here we provide evidence supporting cause-and-effect. Under normal conditions, primary airway epithelial basal cell populations undergo mitosis synchronously and do not secrete proinflammatory or profibrotic cytokines. However, when pairs of nonasthmatic cultures were mitotically synchronized at 12 hours off-set and then combined, the mixed cell populations secreted elevated levels of TGF-ß1. This shows that mitotic asynchrony is not only associated with but is also causative of TGF-ß1 secretion. The secreted cytokines and other mediators from asthmatic cells were not the cause of asynchronous regeneration; synchronously mitotic nonasthmatic epithelia exposed to conditioned media from asthmatic cells did not show changes in mitotic synchrony. We also tested if resynchronization of regenerating asthmatic airway epithelia reduces TGF-ß1 secretion and found that pulse-dosed dexamethasone, simvastatin, and aphidicolin were all effective. We therefore propose a new model for chronic inflammatory and fibrotic conditions where an underlying factor is mitotic asynchrony.

IL-1ß induction of MUC5AC gene expression is mediated by CREB and NF-κB and repressed by dexamethasone.

Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the proinflammatory cytokine interleukin-1 ß (IL-1ß) via activation of cAMP response element-binding protein (CREB) in the NCI-H292 cancer cell line and nuclear factor-κB (NF-κB) in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NF-κB subunits to the IL-1ß-induced upregulation of MUC5AC. Data show that ligand binding of IL-1ß to the IL-1ß receptor is required to increase MUC5AC mRNA abundance. Chromatin immunoprecipitation analyses show direct binding of CREB to the previously identified cAMP response element site and binding of p65 and p50 subunits to a novel NF-κB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NF-κB site in the distal promoter. P50 binds to both NF-κB sites at 1 h following IL-1ß exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus IL-1ß activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL-1ß-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to a honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.

Muc5b is required for airway defence.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

VBP15, a glucocorticoid analogue, is effective at reducing allergic lung inflammation in mice.

Asthma is a chronic inflammatory condition of the lower respiratory tract associated with airway hyperreactivity and mucus obstruction in which a majority of cases are due to an allergic response to environmental allergens. Glucocorticoids such as prednisone have been standard treatment for many inflammatory diseases for the past 60 years. However, despite their effectiveness, long-term treatment is often limited by adverse side effects believed to be caused by glucocorticoid receptor-mediated gene transcription. This has led to the pursuit of compounds that retain the anti-inflammatory properties yet lack the adverse side effects associated with traditional glucocorticoids. We have developed a novel series of steroidal analogues (VBP compounds) that have been previously shown to maintain anti-inflammatory properties such as NFκB-inhibition without inducing glucocorticoid receptor-mediated gene transcription. This study was undertaken to determine the effectiveness of the lead compound, VBP15, in a mouse model of allergic lung inflammation. We show that VBP15 is as effective as the traditional glucocorticoid, prednisolone, at reducing three major hallmarks of lung inflammation--NFκB activity, leukocyte degranulation, and pro-inflammatory cytokine release from human bronchial epithelial cells obtained from patients with asthma. Moreover, we found that VBP15 is capable of reducing inflammation of the lung in vivo to an extent similar to that of prednisone. We found that prednisolone--but not VBP15 shortens the tibia in mice upon a 5 week treatment regimen suggesting effective dissociation of side effects from efficacy. These findings suggest that VBP15 may represent a potent and safer alternative to traditional glucocorticoids in the treatment of asthma and other inflammatory diseases.

Modification of a tumor antigen determinant to improve peptide/MHC stability is associated with increased immunogenicity and cross-priming a larger fraction of CD8+ T cells.

Altered peptide ligands (APLs) with enhanced binding to MHC class I can increase the CD8(+) T cell response to native Ags, including tumor Ags. In this study, we investigate the influence of peptide-MHC (pMHC) stability on recruitment of tumor Ag-specific CD8(+) T cells through cross-priming. Among the four known H-2(b)-restricted CD8(+) T cell determinants within SV40 large tumor Ag (TAg), the site V determinant ((489)QGINNLDNL(497)) forms relatively low-stability pMHC and is characteristically immunorecessive. Absence of detectable site V-specific CD8(+) T cells following immunization with wild-type TAg is due in part to inefficient cross-priming. We mutated nonanchor residues within the TAg site V determinant that increased pMHC stability but preserved recognition by both TCR-transgenic and polyclonal endogenous T cells. Using a novel approach to quantify the fraction of naive T cells triggered through cross-priming in vivo, we show that immunization with TAg variants expressing higher-stability determinants increased the fraction of site V-specific T cells cross-primed and effectively overcame the immunorecessive phenotype. In addition, using MHC class I tetramer-based enrichment, we demonstrate for the first time, to our knowledge, that endogenous site V-specific T cells are primed following wild-type TAg immunization despite their low initial frequency, but that the magnitude of T cell accumulation is enhanced following immunization with a site V variant TAg. Our results demonstrate that site V APLs cross-prime a higher fraction of available T cells, providing a potential mechanism for high-stability APLs to enhance immunogenicity and accumulation of T cells specific for the native determinant.

Glucocorticoid receptor and histone deacetylase-2 mediate dexamethasone-induced repression of MUC5AC gene expression.

Airway occlusion in obstructive airway diseases is caused in part by the overproduction of secretory mucin glycoproteins through the up-regulation of mucin (MUC) genes by inflammatory mediators. Some pharmacological agents, including the glucocorticoid dexamethasone (Dex), repress mucin concentrations in lung epithelial cancer cells. Here, we show that Dex reduces the expression of MUC5AC, a major airway mucin gene, in primary differentiated normal human bronchial epithelial (NHBE) cells in a dose-dependent and time-dependent manner, and that the Dex-induced repression is mediated by the glucocorticoid receptor (GR) and two glucocorticoid response elements (GREs) in the MUC5AC promoter. The pre-exposure of cells to RU486, a GR antagonist, and mutations in either the GRE3 or GRE5 cis-sites abolished the Dex-induced repression. Chromatin immunoprecipitation (ChIP) assays showed a rapid temporal recruitment of GR to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in NHBE and in A549 cells. Immunofluorescence showed nuclear colocalization of GR and histone deacetylase-2 (HDAC2) in MUC5AC-expressing NHBE cells. ChIP also showed a rapid temporal recruitment of HDAC2 to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in both cell types. The knockdown of HDAC2 by HDAC2-specific short interfering RNA prevented the Dex-induced repression of MUC5AC in NHBE and A549 cells. These data demonstrate that GR and HDAC2 are recruited to the GRE3 and GRE5 cis-sites in the MUC5AC promoter and mediate the Dex-induced cis repression of MUC5AC gene expression. A better understanding of the mechanisms whereby glucocorticoids repress MUC5AC gene expression may be useful in formulating therapeutic interventions in chronic lung diseases.

Histologic characteristics and mucin immunohistochemistry of cystic fibrosis sinus mucosa.

OBJECTIVES: To evaluate the histologic characteristics of paranasal sinus mucosa of a disease control population and children with chronic rhinosinusitis and cystic fibrosis (CRS/CF) (1) to determine whether goblet cell (GC) hyperplasia and/or submucosal gland (SMG) hyperplasia occur in pediatric CRS/CF and (2) to compare expression and localization of MUC5AC and MUC5B mucins in the sinus mucosa of both cohorts. DESIGN: Histologic and morphometric analyses of paranasal sinus mucosa were used to quantify the number of GCs and mucin-expressing cells. Digital imaging was used to evaluate the SMG area. Immunohistochemistry was performed to identify the cellular localization of MUC5AC and MUC5B mucins, and confocal microscopy was used to determine whether MUC5AC and MUC5B mucins were expressed in the same secretory cells. SETTING: Children's National Medical Center, Washington, DC. PARTICIPANTS: Twenty-one children with CRS/CF who underwent endoscopic sinus surgical procedures and 18 children who underwent craniofacial resection or neurosurgical procedures for abnormalities other than sinusitis. RESULTS: A statistically significant increased area (4.4-fold) of SMGs was detected in the sinus mucosa of patients with CRS/CF compared with the controls (P = .02). Neither GC hyperplasia nor increased expression of MUC5AC was observed in the CRS/CF group. MUC5AC was expressed only in a subpopulation of GCs in both cohorts, and MUC5B was expressed in a subpopulation of GCs as well as in SMGs. There was a positive trend toward increased glandular MUC5B expression in the CRS/CF cohort. Colocalization of MUC5AC and MUC5B expression was observed in a subset of GCs. CONCLUSIONS: Significant SMG hyperplasia and a trend toward increased glandular MUC5B expression exist in children with CRS/CF. This suggests that SMG hyperplasia and glandular MUC5B mucin contribute to mucus overproduction in the sinus mucosa of this population.

Asthmatic airway epithelium is intrinsically inflammatory and mitotically dyssynchronous.

Asthma is an inflammatory condition for which anti-inflammatory glucocorticoids are the standard of care. However, similar efficacy has not been shown for agents targeting inflammatory cells and pathways. This suggests a noninflammatory cell contributor (e.g., epithelium) to asthmatic inflammation. Herein, we sought to define the intrinsic and glucocorticoid-affected properties of asthmatic airway epithelium compared with normal epithelium. Human primary differentiated normal and asthmatic airway epithelia were cultured in glucocorticoid-free medium beginning at -48 hours. They were pulsed with dexamethasone (20 nM) or vehicle for 2 hours at -26, -2, +22, and +46 hours. Cultures were mechanically scrape-wounded at 0 hours and exposed continuously to bromodeoxyuridine (BrdU). Cytokine secretions were analyzed using cytometric bead assays. Wound regeneration/mitosis was analyzed by microscopy and flow cytometry. Quiescent normal (n = 3) and asthmatic (n = 6) epithelia showed similar minimal inflammatory cytokine secretion and mitotic indices. After wounding, asthmatic epithelia secreted more basolateral TGF-ß1, IL-10, IL-13, and IL-1ß (P < 0.05) and regenerated less efficiently than normal epithelia (+48 h wound area reduction = [mean ± SEM] 50.2 ± 7.5% versus 78.6 ± 7.7%; P = 0.02). Asthmatic epithelia showed 40% fewer BrdU(+) cells at +48 hours (0.32 ± 0.05% versus 0.56 ± 0.07% of total cells; P = 0.03), and those cells were more dyssynchronously distributed along the cell cycle (52 ± 10, 25 ± 4, 23 ± 7% for G1/G0, S, and G2/M, respectively) than normal epithelia (71 ± 1, 12 ± 2, and 17 ± 2% for G1/G0, S, and G2/M, respectively). Dexamethasone pulses improved asthmatic epithelial inflammation and regeneration/mitosis. In summary, we show that inflammatory/fibrogenic cytokine secretions are correlated with dyssynchronous mitosis upon injury. Intermittent glucocorticoids simultaneously decreased epithelial cytokine secretions and resynchronized mitosis. These data, generated in an airway model lacking inflammatory cells, support the concept that epithelium contributes to asthmatic inflammation.

Direct presentation regulates the magnitude of the CD8+ T cell response to cell-associated antigen through prolonged T cell proliferation.

The magnitude and complexity of Ag-specific CD8(+) T cell responses is determined by intrinsic properties of the immune system and extrinsic factors, such as vaccination. We evaluated mechanisms that regulate the CD8(+) T cell response to two distinct determinants derived from the same protein Ag, SV40 T Ag (T Ag), following immunization of C57BL/6 mice with T Ag-transformed cells. The results show that direct presentation of T cell determinants by T Ag-transformed cells regulates the magnitude of the CD8(+) T cell response in vivo but not the immunodominance hierarchy. The immunodominance hierarchy was reversed in a dose-dependent manner by addition of excess naive T cells targeting the subdominant determinant. However, T cell competition played only a minor role in limiting T cell accumulation under physiological conditions. We found that the magnitude of the T cell response was regulated by the ability of T Ag-transformed cells to directly present the T Ag determinants. The hierarchy of the CD8(+) T cell response was maintained when Ag presentation in vivo was restricted to cross-presentation, but the presence of T Ag-transformed cells capable of direct presentation dramatically enhanced T cell accumulation at the peak of the response. This enhancement was due to a prolonged period of T cell proliferation, resulting in a delay in T cell contraction. Our findings reveal that direct presentation by nonprofessional APCs can dramatically enhance accumulation of CD8(+) T cells during the primary response, revealing a potential strategy to enhance vaccination approaches.

Cigarette smoke alters tissue inhibitor of metalloproteinase 1 and matrix metalloproteinase 9 levels in the basolateral secretions of human asthmatic bronchial epithelium in vitro.

BACKGROUND: Asthma, a major cause of chronic lung disease worldwide, has increased in prevalence in all age and ethnic groups, particularly in urban areas where cigarette smoking is common. Cigarette smoke (CS) significantly impacts the development of asthma and is strongly associated with increased asthma-related morbidity. PURPOSE: To evaluate bioinformatic analyses predicting that CS would alter expression of tissue inhibitor of metalloproteinase (TIMP) 1 and matrix metalloproteinase (MMP) 9 in asthmatic epithelium. METHODS: Primary differentiated normal (n = 4) and asthmatic (n = 4) human respiratory epithelia on collagen-coated Transwells at air-liquid interface were exposed for 1 hour to CS condensate (CSC) or hydrogen peroxide (H2O2). Tissue inhibitor of metalloproteinase 1 and MMP-9 protein levels were measured at 24 hours by enzyme-linked immunosorbent assay in cell lysates and in apical and basolateral secretions. RESULTS: Tissue inhibitor of metalloproteinase 1 and MMP-9 levels in the apical secretions of normal and asthmatic epithelia were unchanged after exposure to CSC and H2O2. However, CSC increased TIMP-1 levels in the basolateral secretions of both normal and asthmatic epithelia, but decreased MMP-9 levels only in asthmatic basolateral secretions, resulting in a 2.5-fold lower MMP-9/TIMP-1 ratio that corresponded to decreased MMP-9 activity in CS-exposed asthmatic basolateral secretions. CONCLUSIONS: These data validate our prior bioinformatic analyses predicting that TIMP-1 plays a role in the stress response to CS and indicate that asthmatics exposed to CS may be more susceptible to MMP-9-mediated airway remodeling. This is in agreement with the current paradigm that a reduction in the MMP-9/TIMP-1 ratio is a milieu that favors subepithelial airway remodeling in chronic asthma.

Delineation of a gene network underlying the pulmonary response to oxidative stress in asthma.

Cigarette smoke exposure induces a respiratory epithelial response that is mediated in part by oxidative stress. The contribution of oxidative stress to cigarette smoke-induced responses in asthmatic respiratory epithelium is not well understood. We sought to increase this understanding by employing data integration and systems biology approaches to publicly available microarray data deposited over the last several years. In this study, we analyzed 14 publicly available asthma- or tobacco-relevant data series and found 4 (2 mice and 2 human) that fulfilled adequate signal/noise thresholds using unsupervised clustering and F test statistics. Using significance filters and a 4-way Venn diagram approach, we identified 26 overlapping genes in the epithelial transcriptional stress response to cigarette smoke and asthma. This test set corresponded to a 26-member gene/protein network containing 18 members that were highly regulated in a fifth data series of direct lung oxidative stress. Of those network members, 2 stood out (ie, tissue inhibitor of metalloproteinase 1 and thrombospondin 1) owing to central location within the network and marked up-regulation sustained at later times in response to oxidative stress. These analyses identified key relationships and primary hypothetical targets for future studies of cigarette smoke-induced oxidative stress in asthma.

IL-8 regulates mucin gene expression at the posttranscriptional level in lung epithelial cells.

Airway inflammation and mucus hypersecretion/overproduction/obstruction are pathophysiological characteristics of cystic fibrosis, asthma, and chronic obstructive pulmonary disease. Up-regulation of airway mucin genes by inflammatory/immune response mediators is one of the major contributors to mucin overproduction. IL-8, a potent proinflammatory mediator and neutrophil chemoattractant, is present at high levels in the airway secretions of such patients. In this study, the effects of IL-8 on expression of two major airway mucin genes, MUC5AC and MUC5B, were evaluated. IL-8 increased the mRNA abundance of both mucin genes in two human respiratory tract-derived cell lines (A549 and NCI-H292) in a time- and concentration-dependent manner. IL-8 also increased MUC5AC and MUC5B mRNA levels in primary normal differentiated human bronchial epithelial cells, with a high concentration of IL-8 required to increase MUC5B mRNA levels. IL-8 did not transcriptionally up-regulate MUC5AC gene expression, but rather increased the stability of the MUC5AC transcript, suggesting regulation at the posttranscriptional level. In addition, IL-8 altered the levels of RNA-binding proteins to specific domains in the 3'-untranslated region of the MUC5AC transcript. Taken together, these data indicate that the IL-8-induced binding of RNA-binding proteins to the 3'-untranslated region of MUC5AC is a potential mechanism for regulating MUC5AC gene expression at the posttranscriptional level, thus suggesting a new role whereby IL-8 sustains mucin gene expression in inflamed airways.

An SV40 VP1-derived epitope recognized by CD8+ T cells is naturally processed and presented by HLA-A*0201 and cross-reactive with human polyomavirus determinants.

The CD8+ T cell responses directed toward the VP1 antigens of human polyomaviruses JC and BK recently were shown to be cross-reactive. Two HLA-A0201-restricted determinants from each virus have been defined and include JCp100-108 (ILMWEAVTL) and BKp108-116 (LLMWEAVTV) as well as JCp36-44 (SITEVECFL) and BKp44-52 (AITEVECFL). We asked whether VP1 from the related SV40 contains similar HLA-A0201-restricted determinants. In this study, we demonstrate that CD8+ T cells specific for SV40 VP1 p110-118 (ILMWEAVTV), but not p46-54 (SFTEVECFL), can be induced in HLA-A0201-transgenic mice and that these CD8+ T cells cross-react with the corresponding determinants from JC and BK virus. The SV40 p110 determinant was found to be processed and presented in SV40-infected cells. These results indicate that the JCp36/BKp44 determinants are distinctive for the human polyomaviruses while the JCp100/BKp108/SVp110 determinants are shared by all three viruses, providing a target for CD8+ T cell cross-reactivity.