Autophagy in Cystic Fibrosis Pathogenesis and Treatment

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. In epithelial cells, the CFTR channel conducts anions and plays a critical role in regulating the volume and composition of airway surface liquid. This thin layer of aqueous fluid and mucus covering the airway surface facilitates mucociliary clearance, bacterial killing, and epithelial cell homeostasis. The importance of the CFTR channel in macrophages was revealed in recent work that demonstrated that defective CFTR function is accompanied by impaired innate immune responses to specific infections. Notably, most CF-associated infections are caused by microbes that are cleared by autophagy in healthy cells. Autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens, inflammatory molecules, and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their cells exhibit limited autophagy activity. The mechanisms linking a malfunctioning ion channel function to the defective autophagy remains unclear. In this chapter, we describe and discuss the recent findings indicating the presence of several mechanisms leading to defective autophagy in CF cells. Thus, these novel data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF

In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli

<p/> <p>Background</p> <p>Anthrax is caused by the bacterium <it>Bacillus anthracis </it>and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including <it>Streptococcus mutans, Porphyromonas gingivalis</it>, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen.</p> <p>Results</p> <p>The neutralising anti-PA scFv, 1H, was expressed in <it>L. paracasei </it>as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an <it>in vitro </it>cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, <it>in vivo</it>.</p> <p>Conclusion</p> <p>We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both <it>in vitro </it>and <it>in vivo</it>. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.</p

Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR

CFTR plays a key role in maintenance of lung fluid homeostasis. Cigarette smoke decreases CFTR expression in the lung but neither the mechanisms leading to CFTR loss, nor potential ways to prevent its loss have been identified to date

Aldehyde dehydrogenase-1a1 induces oncogene suppressor genes in B cell populations

AbstractThe deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic IgG1+/CD19− and IgG1+/CD19+ B cell populations. Aldh1a1 regulated transcription factors during B cell differentiation in a sequential manner: 1) retinoic acid receptor alpha (Rara) in IgG1+/CD19− and 2) zinc finger protein Zfp423 and peroxisome proliferator-activated receptor gamma (Pparg) in IgG1+/CD19+ splenocytes. In Aldh1a1−/− mice, splenic IgG1+/CD19− and IgG1+/CD19+ B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma B cell lines also lack Aldh1a1 expression; however, ectopic Aldh1a1 expression rescued Rara and Znf423 expressions in these cells. Our data highlight a mechanism by which an enzyme involved in vitamin A metabolism can improve B cell resistance to oncogenesis

Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Abstract Background The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation. Methods Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts. Results We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells. Conclusions CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD

Low Temperature and Chemical Rescue Affect Molecular Proximity of F508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Epithelial Sodium Channel (ENaC)

An imbalance of chloride and sodium ion transport in several epithelia is a feature of cystic fibrosis (CF), an inherited disease that is a consequence of mutations in the cftr gene. The cftr gene codes for a Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Some mutations in this gene cause the balance between Cl(-) secretion and Na(+) absorption to be disturbed in the airways; Cl(-) secretion is impaired, whereas Na(+) absorption is elevated. Enhanced Na(+) absorption through the epithelial sodium channel (ENaC) is attributed to the failure of mutated CFTR to restrict ENaC-mediated Na(+) transport. The mechanism of this regulation is controversial. Recently, we have found evidence for a close association of wild type (WT) CFTR and WT ENaC, further underscoring the role of ENaC along with CFTR in the pathophysiology of CF airway disease. In this study, we have examined the association of ENaC subunits with mutated ΔF508-CFTR, the most common mutation in CF. Deletion of phenylalanine at position 508 (ΔF508) prevents proper processing and targeting of CFTR to the plasma membrane. When ΔF508-CFTR and ENaC subunits were co-expressed in HEK293T cells, we found that individual ENaC subunits could be co-immunoprecipitated with ΔF508-CFTR, much like WT CFTR. However, when we evaluated the ΔF508-CFTR and ENaC association using fluorescence resonance energy transfer (FRET), FRET efficiencies were not significantly different from negative controls, suggesting that ΔF508-CFTR and ENaC are not in close proximity to each other under basal conditions. However, with partial correction of ΔF508-CFTR misprocessing by low temperature and chemical rescue, leading to surface expression as assessed by total internal reflection fluorescence (TIRF) microscopy, we observed a positive FRET signal. Our findings suggest that the ΔF508 mutation alters the close association of CFTR and ENaC

Infection with 2009 H1N1 influenza virus primes for immunological memory in human nose-associated lymphoid tissue, offering cross-reactive immunity to H1N1 and avian H5N1 viruses

Influenza is a highly contagious mucosal infection in the respiratory tract. 2009 pandemic H1N1 (pH1N1) virus infection resulted in substantial morbidity and mortality in humans. Little is known on whether immunological memory develops following pH1N1 infection and whether it provides protection against other virus subtypes. Enzyme-linked immunosorbent spot assay was used to analyze hemagglutinin (HA)-specific memory B cell responses after virus antigen stimulation in nasal-associated lymphoid tissues (NALT) from children and adults. Individuals with serological evidence of previous exposure to pH1N1 showed significant cross-reactive HA-specific memory B responses to pH1N1, seasonal H1N1(sH1N1) and avian H5N1(aH5N1) viruses upon pH1N1 virus stimulation. pH1N1 virus antigen elicited stronger cross-reactive memory B cell responses than sH1N1 virus. Intriguingly, aH5N1 virus also activated cross-reactive memory responses to sH1N1 and pH1N1 HAs in those who had previous pH1N1 exposure, and that correlated well with the memory response stimulated by pH1N1 virus antigen. These memory B cell responses resulted in cross-reactive neutralizing antibodies against sH1N1, 1918 H1N1 and aH5N1viruses. 2009 pH1N1 infection appeared to have primed human host with B cell memory in NALT that offers cross-protective mucosal immunity against not only H1N1 but also aH5N1 viruses. These findings may have important implications to future vaccination strategies against influenza. It will be important to induce and/or enhance such cross-protective mucosal memory B cells

Mucosal Targeting of a BoNT/A Subunit Vaccine Adjuvanted with a Mast Cell Activator Enhances Induction of BoNT/A Neutralizing Antibodies in Rabbits

We previously reported that the immunogenicity of Hcβtre, a botulinum neurotoxin A (BoNT/A) immunogen, was enhanced by fusion to an epithelial cell binding domain, Ad2F, when nasally delivered to mice with cholera toxin (CT). This study was performed to determine if Ad2F would enhance the nasal immunogenicity of Hcβtre in rabbits, an animal model with a nasal cavity anatomy similar to humans. Since CT is not safe for human use, we also tested the adjuvant activity of compound 48/80 (C48/80), a mast cell activating compound previously determined to safely exhibit nasal adjuvant activity in mice.New Zealand White or Dutch Belted rabbits were nasally immunized with Hcβtre or Hcβtre-Ad2F alone or combined with CT or C48/80, and serum samples were tested for the presence of Hcβtre-specific binding (ELISA) or BoNT/A neutralizing antibodies.Hcβtre-Ad2F nasally administered with CT induced serum anti-Hcβtre IgG ELISA and BoNT/A neutralizing antibody titers greater than those induced by Hcβtre + CT. C48/80 provided significant nasal adjuvant activity and induced BoNT/A-neutralizing antibodies similar to those induced by CT.Ad2F enhanced the nasal immunogenicity of Hcβtre, and the mast cell activator C48/80 was an effective adjuvant for nasal immunization in rabbits, an animal model with a nasal cavity anatomy similar to that in humans