Extensive acute and sustained changes to neutrophil proteomes post-SARS-CoV-2 infection.

BACKGROUND: Neutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19), but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery. METHODS: Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96 h of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale. RESULTS: Neutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified, with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature, but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism, immunosuppression and pattern recognition, while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins, chemokine and leukotriene receptors, integrins and inhibitory receptors. CONCLUSIONS: SARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines.

Pregnancy Zone Protein Is Associated with Airway Infection, Neutrophil Extracellular Trap Formation, and Disease Severity in Bronchiectasis.

Rationale: PZP (pregnancy zone protein) is a broad-spectrum immunosuppressive protein believed to suppress T-cell function during pregnancy to prevent fetal rejection. It has not previously been reported in the airway.Objectives: To characterize PZP in the bronchiectasis airway, including its relationship with disease severity.Methods: Label-free liquid chromatography/mass spectrometry was performed for sputum protein profiling of patients with bronchiectasis confirmed by high-resolution computed tomography. Results for patients with and without Pseudomonas aeruginosa infection were compared. Sputum and serum PZP was measured by validated ELISA. Airway infection status was established by culture and 16S ribosomal RNA sequencing. Immunofluorescence, ELISA, and electron microscopy were used to identify the cellular source of PZP in neutrophils treated with multiple stimuli.Measurements and Main Results: Elevated PZP was identified by label-free liquid chromatography/mass spectrometry as being associated with P. aeruginosa infection. In a validation study of 124 patients, sputum but not serum concentrations of PZP were significantly associated with the Bronchiectasis Severity Index, the frequency of exacerbations, and symptoms. Airway infection with Proteobacteria such as P. aeruginosa was associated with higher concentrations of PZP. PZP in sputum was directly related to airway bacterial load. Neutrophils induced to form neutrophil extracellular traps (NETs) with phorbol myristate acetate released high concentrations of PZP in vitro, and fluorescence microscopy confirmed the presence of PZP in NETs, whereas fluorescence and electron microscopy localized PZP to the cytoplasm and nuclei of neutrophils. Effective antibiotic therapy reduced sputum PZP.Conclusions: PZP is released into NETs. We report a novel link between airway infection, NET formation, and disease severity in bronchiectasis during chronic airway inflammation.

Antimicrobial peptides, disease severity and exacerbations in bronchiectasis.

RATIONALE: Recently a frequent exacerbator phenotype has been described in bronchiectasis, but the underlying biological mechanisms are unknown. Antimicrobial peptides (AMPs) are important in host defence against microbes but can be proinflammatory in chronic lung disease. OBJECTIVES: To determine pulmonary and systemic levels of AMP and their relationship with disease severity and future risk of exacerbations in bronchiectasis. METHODS: A total of 135 adults with bronchiectasis were prospectively enrolled at three European centres. Levels of cathelicidin LL-37, lactoferrin, lysozyme and secretory leucocyte protease inhibitor (SLPI) in serum and sputum were determined at baseline by ELISA. Patients were followed up for 12 months. We examined the ability of sputum AMP to predict future exacerbation risk. MEASUREMENTS AND MAIN RESULTS: AMP levels were higher in sputum than in serum, suggesting local AMP release. Patients with more severe disease at baseline had dysregulation of airway AMP. Higher LL-37 and lower SLPI levels were associated with Bronchiectasis Severity Index, lower FEV1 (forced expiratory volume in 1 s) and Pseudomonas aeruginosa infection. Low SLPI levels were also associated with the exacerbation frequency at baseline. During follow-up, higher LL-37 and lower SLPI levels were associated with a shorter time to the next exacerbation, whereas LL-37 alone predicted exacerbation frequency over the next 12 months. CONCLUSIONS: Patients with bronchiectasis showed dysregulated sputum AMP levels, characterised by elevated LL-37 and reduced SLPI levels in the frequent exacerbator phenotype. Elevated LL-37 and reduced SLPI levels are associated with Pseudomonas aeruginosa infection and can predict future risk of exacerbations in bronchiectasis.

CFTR mutations altering CFTR fragmentation.

Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.

Dipeptidyl peptidase-1 inhibition in patients hospitalised with COVID-19: a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial.

BACKGROUND: Neutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19. METHODS: In a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012. FINDINGS: Between June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57-0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug. INTERPRETATION: Brensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19. FUNDING: Sponsored by the University of Dundee and supported through an Investigator Initiated Research award from Insmed, Bridgewater, NJ; STOP-COVID19 trial.

The protective effect of SARS-CoV-2 antibodies in Scottish healthcare workers.

BACKGROUND: Healthcare workers (HCWs) are believed to be at increased risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. It is not known to what extent the natural production of antibodies to SARS-CoV-2 is protective against re-infection. METHODS: A prospective observational study of HCWs in Scotland (UK) from May to September 2020 was performed. The Siemens SARS-CoV-2 total antibody assay was used to establish seroprevalence in this cohort. Controls, matched for age and sex to the general local population, were studied for comparison. New infections (up to 2 December 2020) post antibody testing were recorded to determine whether the presence of SARS-CoV-2 antibodies protects against re-infection. RESULTS: A total of 2063 health and social care workers were recruited for this study. At enrolment, 300 HCWs had a positive antibody test (14.5%). 11 out of 231 control sera tested positive (4.8%). HCWs therefore had an increased likelihood of a positive test (OR 3.4, 95% CI 1.85-6.16; p<0.0001). Dentists were most likely to test positive. 97.3% of patients who had previously tested positive for SARS-CoV-2 by reverse transcriptase (RT)-PCR had positive antibodies. 18.7% had an asymptomatic infection. There were 38 new infections with SARS-CoV-2 in HCWs who were previously antibody negative, and one symptomatic RT-PCR-positive re-infection. The presence of antibodies was therefore associated with an 85% reduced risk of re-infection with SARS-CoV-2 (hazard ratio 0.15, 95% CI 0.06-0.35; p=0.026). CONCLUSION: HCWs were three times more likely to test positive for SARS-CoV-2 than the general population. Almost all infected individuals developed an antibody response, which was 85% effective in protecting against re-infection with SARS-CoV-2.

Metformin treatment of diabetes mellitus increases the risk for pancreatitis in patients bearing the CFTR-mutation S573C.

Metformin use in diabetes can cause acidosis and might be linked to pancreatitis. Here, we mechanistically focus on this relationship via a point mutation in the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7). CFTR is an ATP-hydrolyzing, cAMP/PKA-activated anion channel regulating pancreatic bicarbonate/chloride secretion across duct-facing apical membranes in epithelia. CFTR has two nucleotide binding domains (NBD1/2) which clamp two ATP molecules across their opposed, inverted interfacial surfaces which generates anion-conductance after ATP hydrolysis. Notably, CFTR mutations not causal for classical cystic fibrosis segregate with unexplained pancreatitis and one of these lies in NBD1 near its ATP-clamp (S573C; close to the Walker B aspartate D572). We recently showed that after raising [cAMP], wt-CFTR chloride-conductance, when expressed in Xenopus oocytes, remains elevated despite the presence of metformin. Yet here, we find that S573C-CFTR manifests a metformin-inhibitable whole cell chloride-conductance after cAMP elevation. In the absence of metformin, cAMP-activated S573C-CFTR also displays a reduced anion-conductance relative to wt-CFTR. Furthermore, intra-oocyte acidification inhibited wt-CFTR and abolished S573C-CFTR conductance. We conclude that defective S573C-CFTR remains both poorly conducting and inhibited by metformin and intracellular acidosis. This might explain the propensity to pancreatitis with this rare CF mutation.

Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR.

BACKGROUND: Deletion of phenylalanine-508 (DeltaF508) from the first nucleotide-binding domain (NBD1) in the wild-type cystic fibrosis (CF) transmembrane-conductance regulator (wtCFTR) causes CF. However, the mechanistic relationship between DeltaF508-CFTR and the diversity of CF disease is unexplained. The surface location of F508 on NBD1 creates the potential for protein-protein interactions and nearby, lies a consensus sequence (SYDE) reported to control the pleiotropic protein kinase CK2. METHODS: Electrophysiology, immunofluorescence and biochemistry applied to CFTR-expressing cells, Xenopus oocytes, pancreatic ducts and patient biopsies. RESULTS: Irrespective of PKA activation, CK2 inhibition (ducts, oocytes, cells) attenuates CFTR-dependent Cl(-) transport, closing wtCFTR in cell-attached membrane patches. CK2 and wtCFTR co-precipitate and CK2 co-localized with wtCFTR (but not DeltaF508-CFTR) in apical membranes of human airway biopsies. Comparing wild-type and DeltaF508CFTR expressing oocytes, only DeltaF508-CFTR Cl(-) currents were insensitive to two CK2 inhibitors. Furthermore, wtCFTR was inhibited by injecting a peptide mimicking the F508 region, whereas the DeltaF508-equivalent peptide had no effect. CONCLUSIONS: CK2 controls wtCFTR, but not DeltaF508-CFTR. Others find that peptides from the F508 region of NBD1 allosterically control CK2, acting through F508. Hence, disruption of CK2-CFTR interaction by DeltaF508-CFTR might disrupt multiple, membrane-associated, CK2-dependent pathways, creating a new molecular disease paradigm for deleted F508 in CFTR.

Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. METHODS: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. RESULTS: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. CONCLUSIONS: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance.

BACKGROUND: Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial 'set-point' may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the 'reference range of normality' remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport. METHODOLOGY/PRINCIPAL FINDINGS: Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm2), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 µA/cm2; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport. CONCLUSIONS/SIGNIFICANCE: Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine.

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

Understanding protein kinase CK2 mis-regulation upon F508del CFTR expression.

We review areas of overlap between nucleoside diphosphate kinase (NDPK; nm23) and two proteins manifesting an equivalent diversity of action, each with many thousands of publications. The first is a constitutively active protein kinase, CK2 (formerly casein kinase 2), that includes NDPK amongst its hundreds of targets. The second is an enigmatic member of the ATP-binding cassette (ABC) family of membrane pumps that normally hydrolyse ATP to transport substrates. Yet our unusual family member (ABCC7) is not a pump but, uniquely, acts as a regulated anion channel. ABCC7 is the cystic fibrosis transmembrane conductance regulator (CFTR), and we discuss the highly prevalent CFTR mutation (F508del CFTR) in terms of the uncertainties surrounding the molecular basis of cystic fibrosis that cloud approaches to corrective therapy. Using lysates from cells stably expressing either wild-type or F508del CFTR, incubated with the CK2 substrate GTP, we show that the phosphoproteome of F508del CFTR-expressing cells both differs from wild-type CFTR-expressing cells and is significantly enhanced in intensity by ∼1.5-fold (p < 0.05, paired t test with Bonferroni correction, n = 4). Phosphorylation is about 50% attenuated with a specific CK2 inhibitor. We propose that a new function may exist for the CFTR region that is commonly mutated, noting that its sequence (PGTIKENIIF(508)GVSYDEYRYR) is not only highly conserved within the C sub-family of ABC proteins but also a related sequence is found in NDPK. We conclude that a latent path may exist between mutation of this conserved sequence, CK2 hyperactivity and disease pathogenesis that might also explain the heterozygote advantage for the common F508del CFTR mutant.

The post-mortem relationship between beta-hydroxybutyrate (BHB), acetone and ethanol in ketoacidosis.

A reduced blood pH (ketoacidosis) from the production of beta-oxidative ketone bodies as a result of alcoholism (alcoholic ketoacidosis, AKA) or diabetes (diabetic ketoacidosis, DKA) can feature in many fatalities and analytical evidence can be used to support a pathological diagnosis, or provide a possible cause of death in the absence of other pathologically significant findings. Existing beliefs concerning the relationship of BHB concentrations, acetone and ethanol have been re-examined by analysis of BHB, acetone and ethanol in over 350 fatalities grouped into alcoholics, diabetics, alcoholic diabetics, coupled with speculative cases and those with an alternative cause of death. Uniquely, the concentrations of BHB were measured in post-mortem blood, urine and vitreous humour using selective GC-MS. The results showed that existing beliefs need to be re-evaluated. Ethanol is not always low (<10mg/dL) or absent in cases of AKA. Also, the absence of acetone precludes [corrected] a high BHB (>250mg/L), therefore acetone can be used as an initial marker pathologically significant ketoacidosis. For blood and urine BHB concentrations the following interpretative ranges can be used (in mg/L); normal (<50mg/L), raised (51-249mg/L), high and pathologically significant (>250mg/L). Initial data suggest vitreous humour BHB could be a useful alternative in the absence of blood (same interpretative ranges may also apply). Analytical recommendation for investigation of post-mortem ketoacidosis is also presented.

Expression of wild-type CFTR suppresses NF-kappaB-driven inflammatory signalling.

BACKGROUND: Mutation of the cystic fibrosis transmembrane-conductance regulator (CFTR) causes cystic fibrosis (CF) but not all CF aspects can easily be explained by deficient ion transport. CF-inflammation provides one example but its pathogenesis remains controversial. Here, we tested the simple but fundamental hypothesis that wild-type CFTR is needed to suppress NF-kappaB activity. METHODOLOGY/PRINCIPAL FINDINGS: In lung epithelial (H441) and engineered (H57) cell lines; we report that inflammatory markers are significantly suppressed by wild-type CFTR. Transient-transfection of wild-type CFTR into CFTR-naïve H441 cells, dose-dependently down-regulates both basal and Tumour Necrosis Factor-alpha evoked NF-kappaB activity when compared to transfection with empty vector alone (p<0.01, n>5). This effect was also observed in CFTR-naïve H57-HeLa cells which stably express a reporter of NF-kappaB activity, confirming that the CFTR-mediated repression of inflammation was not due to variable reporter gene transfection efficiency. In contrast, H57 cells transfected with a control cyano-fluorescent protein show a significantly elevated basal level of NF-kappaB activity above control. Initial cell seeding density may be a critical factor in mediating the suppressive effects of CFTR on inflammation as only at a certain density (1x10(5) cells/well) did we observe the reduction in NF-kappaB activity. CFTR channel activity may be necessary for this suppression because the CFTR specific inhibitor CFTR(inh172) significantly stimulates NF-kappaB activity by approximately 30% in CFTR expressing 16HBE14o- cells whereas pharmacological elevation of cyclic-AMP depresses activity by approximately 25% below baseline. CONCLUSIONS/SIGNIFICANCE: These data indicate that CFTR has inherent anti-inflammatory properties. We propose that the hyper-inflammation found in CF may arise as a consequence of disrupted repression of NF-kappaB signalling which is normally mediated by CFTR. Our data therefore concur with in vivo and in vitro data from Vij and colleagues which highlights CFTR as a suppressor of basal inflammation acting through NF-kappaB, a central hub in inflammatory signalling.

Epithelial IgG and its relationship to the loss of F508 in the common mutant form of the cystic fibrosis transmembrane conductance regulator.

The most debilitating feature of cystic fibrosis (CF) disease is uncontrolled inflammation of respiratory epithelium. The relationship between the commonest mutated form of CFTR (F508del or DeltaF508) and inflammation has not yet been elucidated. Here, we present a new paradigm suggesting that CFTR can interact with intra-epithelial IgG, establishing a direct link between normal CFTR and the immune system. Further, our data show that the amino-acid sequence local to F508 can bind IgG with high affinity, dependent on F508, such that loss of F508 abolishes this link both in vitro and in the intact cell.

Mechanistic insight into control of CFTR by AMPK.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser(737) and Ser(768)) in the R domain, formerly identified as "inhibitory" PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser(768) having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.