Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro

BACKGROUND: Mucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Expression of two mucin genes, MUC2 and MUC5AC, and their protein products (mucins), is modulated in certain disease states. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways. METHODS: To study the differential expression of Muc2 and Muc5ac, a novel monoclonal antibody recognizing guinea pig Muc2 and a commercially-available antibody against human MUC5AC were optimized for recognition of specific guinea pig mucins by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). These antibodies were then used to analyze expression of Muc2 and another mucin subtype (likely Muc5ac) in guinea pig tracheal epithelial (GPTE) cells stimulated with a mixture of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interferon- γ (IFN-γ)]. RESULTS: The anti-Muc2 (C4) and anti-MUC5AC (45M1) monoclonal antibodies specifically recognized proteins located in Muc2-dominant small intestinal and Muc5ac-dominant stomach mucosae, respectively, in both Western and ELISA experimental protocols. IHC protocols confirmed that C4 recognizes murine small intestine mucosal proteins while 45M1 does not react. C4 and 45M1 also stained specific epithelial cells in guinea pig lung sections. In the resting state, Muc2 was recognized as a highly expressed intracellular mucin in GPTE cells in vitro. Following cytokine exposure, secretion of Muc2, but not the mucin recognized by the 45M1 antibody (likely Muc5ac), was increased from the GPTE cells, with a concomitant increase in intracellular expression of both mucins. CONCLUSION: Given the tissue specificity in IHC and the differential hybridization to high molecular weight proteins by Western blot, we conclude that the antibodies used in this study can recognize specific mucin subtypes in guinea pig airway epithelium and in proteins from GPTE cells. In addition, Muc2 is highly expressed constitutively, modulated by inflammation, and secreted differentially (as compared to Muc5ac) in GPTE cells. This finding contrasts with expression patterns in the airway epithelium of a variety of mammalian species in which only Muc5ac predominates

Crystal Structure of a Charge Engineered Human Lysozyme Having Enhanced Bactericidal Activity

Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers

Resolution of LPS-induced airway inflammation and goblet cell hyperplasia is independent of IL-18

BACKGROUND: The resolution of inflammatory responses in the lung has not been described in detail and the role of specific cytokines influencing the resolution process is largely unknown. METHODS: The present study was designed to describe the resolution of inflammation from 3 h through 90 d following an acute injury by a single intratracheal instillation of F344/N rats with LPS. We documented the inflammatory cell types and cytokines found in the bronchoalveolar lavage fluid (BALF), and epithelial changes in the axial airway and investigated whether IL-18 may play a role in the resolution process by reducing its levels with anti-IL-18 antibodies. RESULTS: Three major stages of inflammation and resolution were observed in the BALF during the resolution. The first stage was characterized by PMNs that increased over 3 h to 1 d and decreased to background levels by d 6–8. The second stage of inflammation was characterized by macrophage influx reaching maximum numbers at d 6 and decreasing to background levels by d 40. A third stage of inflammation was observed for lymphocytes which were elevated over d 3–6. Interestingly, IL-18 and IL-9 levels in the BALF showed a cyclic pattern with peak levels at d 4, 8, and 16 while decreasing to background levels at d 1–2, 6, and 12. Depletion of IL-18 caused decreased PMN numbers at d 2, but no changes in inflammatory cell number or type at later time points. CONCLUSION: These data suggest that IL-18 plays a role in enhancing the LPS-induced neutrophilic inflammation of the lung, but does not affect the resolution of inflammation

Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection

Mucin Dynamics in Intestinal Bacterial Infection

Bacterial gastroenteritis causes morbidity and mortality in humans worldwide. Murine Citrobacter rodentium infection is a model for gastroenteritis caused by the human pathogens enteropathogenic Escherichia coli and enterohaemorrhagic E. coli. Mucin glycoproteins are the main component of the first barrier that bacteria encounter in the intestinal tract.Using Immunohistochemistry, we investigated intestinal expression of mucins (Alcian blue/PAS, Muc1, Muc2, Muc4, Muc5AC, Muc13 and Muc3/17) in healthy and C. rodentium infected mice. The majority of the C. rodentium infected mice developed systemic infection and colitis in the mid and distal colon by day 12. C. rodentium bound to the major secreted mucin, Muc2, in vitro, and high numbers of bacteria were found in secreted MUC2 in infected animals in vivo, indicating that mucins may limit bacterial access to the epithelial surface. In the small intestine, caecum and proximal colon, the mucin expression was similar in infected and non-infected animals. In the distal colonic epithelium, all secreted and cell surface mucins decreased with the exception of the Muc1 cell surface mucin which increased after infection (p<0.05). Similarly, during human infection Salmonella St Paul, Campylobacter jejuni and Clostridium difficile induced MUC1 in the colon.Major changes in both the cell-surface and secreted mucins occur in response to intestinal infection

Mucin Variable Number Tandem Repeat Polymorphisms and Severity of Cystic Fibrosis Lung Disease: Significant Association with MUC5AC

Variability in cystic fibrosis (CF) lung disease is partially due to non-CFTR genetic modifiers. Mucin genes are very polymorphic, and mucins play a key role in the pathogenesis of CF lung disease; therefore, mucin genes are strong candidates as genetic modifiers. DNA from CF patients recruited for extremes of lung phenotype was analyzed by Southern blot or PCR to define variable number tandem repeat (VNTR) length polymorphisms for MUC1, MUC2, MUC5AC, and MUC7. VNTR length polymorphisms were tested for association with lung disease severity and for linkage disequilibrium (LD) with flanking single nucleotide polymorphisms (SNPs). No strong associations were found for MUC1, MUC2, or MUC7. A significant association was found between the overall distribution of MUC5AC VNTR length and CF lung disease severity (p = 0.025; n = 468 patients); plus, there was robust association of the specific 6.4 kb HinfI VNTR fragment with severity of lung disease (p = 6.2 x 10(-4) after Bonferroni correction). There was strong LD between MUC5AC VNTR length modes and flanking SNPs. The severity-associated 6.4 kb VNTR allele of MUC5AC was confirmed to be genetically distinct from the 6.3 kb allele, as it showed significantly stronger association with nearby SNPs. These data provide detailed respiratory mucin gene VNTR allele distributions in CF patients. Our data also show a novel link between the MUC5AC 6.4 kb VNTR allele and severity of CF lung disease. The LD pattern with surrounding SNPs suggests that the 6.4 kb allele contains, or is linked to, important functional genetic variation

Blood neutrophil activation markers in severe asthma: lack of inhibition by prednisolone therapy

BACKGROUND: Neutrophils are increased in the airways and in induced sputum of severe asthma patients. We determined the expression of activation markers from circulating neutrophils in severe asthma, and their supressibility by corticosteroids. METHODS: We compared blood neutrophils from mild, moderate-to-severe and severe steroid-dependent asthma, and non-asthmatics (n = 10 each). We examined the effect of adding or increasing oral prednisolone (30 mg/day;1 week). RESULTS: Flow cytometric expression of CD35 and CD11b, but not of CD62L or CD18, was increased in severe asthma. F-met-leu-phe increased CD11b, CD35 and CD18 and decreased CD62L expression in all groups, with a greater CD35 increase in severe asthma. In severe steroid-dependent asthma, an increase in prednisolone dose had no effect on neutrophil markers particularly CD62L, but reduced CD11b and CD62L on eosinophils. Phorbol myristate acetate-stimulated oxidative burst and IL-8 release by IL-1β, lipopolysaccharide and GM-CSF in whole blood from mild but not severe asthmatics were inhibited after prednisolone. There were no differences in myeloperoxidase or neutrophil elastase release from purified neutrophils. CONCLUSION: Because blood neutrophils in severe asthma are activated and are not inhibited by oral corticosteroids, they may be important in the pathogenesis of severe asthma

Common Gene Therapy Viral Vectors Do Not Efficiently Penetrate Sputum from Cystic Fibrosis Patients

Norwalk virus and human papilloma virus, two viruses that infect humans at mucosal surfaces, have been found capable of rapidly penetrating human mucus secretions. Viral vectors for gene therapy of Cystic Fibrosis (CF) must similarly penetrate purulent lung airway mucus (sputum) to deliver DNA to airway epithelial cells. However, surprisingly little is known about the rates at which gene delivery vehicles penetrate sputum, including viral vectors used in clinical trials for CF gene therapy. We find that sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus (d∼80 nm) and adeno-associated virus (AAV serotype 5; d∼20 nm), leading to average effective diffusivities that are ∼3,000-fold and 12,000-fold slower than their theoretical speeds in water, respectively. Both viral vectors are slowed by adhesion, as engineered muco-inert nanoparticles with diameters as large as 200 nm penetrate the same sputum samples at rates only ∼40-fold reduced compared to in pure water. A limited fraction of AAV exhibit sufficiently fast mobility to penetrate physiologically thick sputum layers, likely because of the lower viscous drag and smaller surface area for adhesion to sputum constituents. Nevertheless, poor penetration of CF sputum is likely a major contributor to the ineffectiveness of viral vector based gene therapy in the lungs of CF patients observed to date

Mucin Secretion Induced by Titanium Dioxide Nanoparticles

Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO2), one of the PM10 components, is widely utilized in the nanoindustry for manufacturing and processing of various commercial products. Although TiO2 NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO2 NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO2 NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca2+ signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca2+ concentration were monitored with Rhod-2, a fluorescent Ca2+ dye. We found that TiO2 NP-evoked mucin secretion was a function of increasing intracellular Ca2+ concentration resulting from an extracellular Ca2+ influx via membrane Ca2+ channels and cytosolic ER Ca2+ release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca2+ signal and in sustaining a cytosolic Ca2+ increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion