Cystic fibrosis (CF) airways disease produces a mucoobstructive lung phenotype characterized by airways mucus plugging, epithelial mucous cell metaplasia/hyperplasia, chronic infection, and ...inflammation. Simultaneous biochemical and functional in vivo studies of mucin synthesis and secretion from CF airways are not available. In vitro translational models may quantitate differential CF versus normal mucin and fluid secretory responses to infectious/inflammatory stimuli.
We tested the hypothesis that CF airways exhibit defective epithelial fluid, but not mucin, secretory responses to bacterial/inflammatory host products.
Well-differentiated primary human bronchial epithelial cultures were exposed to supernatant from mucopurulent material (SMM) from human CF airways as a test of bacterial/inflammatory host product stimulus. Human bronchial epithelia (HBE) with normal CF transmembrane conductance regulator function were compared with ΔF508/ΔF508 CF HBE.
Acute (up to 60 min) SMM exposure promoted mucin secretion, but mucins were degraded by the proteolytic enzymes present in SMM. Chronic SMM exposure induced upregulation of mucin synthesis and storage and generated absolute increases in basal and stimulated mucin release in normal and CF cultures. These responses were similar in normal and CF cultures. In contrast, SMM produced a coordinated CF transmembrane conductance regulator-mediated Cl
secretory response in normal HBE, but not in CF HBE. The absence of the fluid secretory response in CF produced quantitatively more dehydrated mucus.
Our study reveals the interplay between regulation of mucin and fluid secretion rates in inflamed versus noninflamed conditions and why a hyperconcentrated mucus is produced in CF airways.
The multi-organ disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (
) that lead to diminished transepithelial anion transport. CF lungs are ...characterised by airway surface liquid (ASL) dehydration, chronic infection/inflammation and neutrophilia. Dysfunctional CFTR may upregulate the epithelial Na
channel (ENaC), further exacerbating dehydration. We previously demonstrated that short palate lung and nasal epithelial clone 1 (SPLUNC1) negatively regulates ENaC in normal airway epithelia.Here, we used pulmonary tissue samples, sputum and human bronchial epithelial cells (HBECs) to determine whether SPLUNC1 could regulate ENaC in a CF-like environment.We found reduced endogenous SPLUNC1 in CF secretions, and rapid degradation of recombinant SPLUNC1 (rSPLUNC1) by CF secretions. Normal sputum, containing SPLUNC1 and SPLUNC1-derived peptides, inhibited ENaC in both normal and CF HBECs. Conversely, CF sputum activated ENaC, and rSPLUNC1 could not reverse this phenomenon. Additionally, we observed upregulation of ENaC protein levels in human CF bronchi. Unlike SPLUNC1, the novel SPLUNC1-derived peptide SPX-101 resisted protease degradation, bound apically to HBECs, inhibited ENaC and prevented ASL dehydration following extended pre-incubation with CF sputum.Our data indicate that CF mucosal secretions drive ASL hyperabsorption and that protease-resistant peptides,
SPX-101, can reverse this effect to rehydrate CF ASL.
We investigated the role of the TLR2 receptor in the recognition of ligands from Gram-positive bacteria in fish. Comparative sequence analysis showed a highly conserved Toll/IL-1 receptor domain. ...Although the leucine-rich repeat domain was less conserved, the position of the critical peptidoglycan (PGN)-binding residues in the leucine-rich repeat domain of carp TLR2 were conserved. Transfection of human embryonic kidney 293 cells with TLR2 corroborated the ability of carp TLR2 to bind the prototypical mammalian vertebrate TLR2 ligands lipoteichoic acid (LTA) and PGN from Staphylococcus aureus. The synthethic triacylated lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-Cys-(S)-Ser-(S)-Lys(4) trihydrochloride (Pam(3)CSK(4)) but not the diacylated lipopeptide macrophage-activating lipopeptide-2 (MALP-2) also activated TLR2 transfected human cells. We identified clear differences between the mammalian vertebrates and carp TLR2-mediated response. The use of the same ligands on carp macrophages indicated that fish cells require high concentrations of ligands from Gram-positive bacteria (LTA, PGN) for activation and signal transduction, react less strongly (Pam(3)CSK(4)) or do not react at all (MALP-2). Overexpression of TLR2 in carp macrophages confirmed TLR2 reactivity of the response to LTA and PGN, low-responsiveness to Pam(3)CSK(4) and nonresponsiveness to MALP-2. A putative relation with the apparent absence of accessory proteins such as CD14 from the fish TLR2-containing receptor complex is discussed. Moreover, activation of carp macrophages by PGN resulted in increased TLR2 gene expression and enhanced TLR2 mRNA stability, MAPK-p38 phosphorylation and increased radical production. Finally, we could show that NADPH oxidase-derived radicals and MAPK-p38 activation cooperatively determine the level of PGN-induced TLR2 gene expression. We propose that the H(2)O(2)-MAPK-p38-dependent axis is crucial for regulation of TLR2 gene expression in fish macrophages.
Human immunodeficiency virus-1 (HIV-1) persists as a global health concern, with an incidence rate of approximately 2 million, and estimated global prevalence of over 35 million. Combination ...antiretroviral treatment is highly effective, but HIV-1 patients that have been treated still suffer from chronic inflammation and residual viral replication. It is therefore paramount to identify therapeutically efficacious strategies to eradicate viral reservoirs and ultimately develop a cure for HIV-1. It has been long accepted that the restriction factor tripartite motif protein 5 isoform alpha (TRIM5α) restricts HIV-1 infection in a species-specific manner, with rhesus macaque TRIM5α strongly restricting HIV-1, and human TRIM5α having a minimal restriction capacity. However, several recent studies underscore human TRIM5α as a cell-dependent HIV-1 restriction factor. Here, we present an overview of the latest research on human TRIM5α and propose a novel conceptualization of TRIM5α as a restriction factor with a varied portfolio of antiviral functions, including mediating HIV-1 degradation through autophagy- and proteasome-mediated mechanisms, and acting as a viral sensor and effector of antiviral signaling. We have also expanded on the protective antiviral roles of autophagy and outline the therapeutic potential of autophagy modulation to intervene in chronic HIV-1 infection.
Abstract
Introduction
Alveolar macrophages (AMs) are lung-resident immune cells that phagocytose inhaled particles and pathogens, and help coordinate the lung’s immune response to infection. Little ...is known about the impact of chronic e-cigarette use (ie, vaping) on this important pulmonary cell type. Thus, we determined the effect of vaping on AM phenotype and gene expression.
Aims and Methods
We recruited never-smokers, smokers, and e-cigarette users (vapers) and performed research bronchoscopies to isolate AMs from bronchoalveolar lavage fluid samples and epithelial cells from bronchial brushings. We then performed morphological analyses and used the Nanostring platform to look for changes in gene expression.
Results
AMs obtained from smokers and vapers were phenotypically distinct from those obtained from nonsmokers, and from each other. Immunocytochemistry revealed that vapers AMs had significantly elevated inducible nitric oxide synthase (M1) expression and significantly reduced CD301a (M2) expression compared with nonsmokers or smokers. Vapers’ AMs and bronchial epithelia exhibited unique changes in gene expression compared with nonsmokers or smokers. Moreover, vapers’ AMs were the most affected of all groups and had 124 genes uniquely downregulated. Gene ontology analysis revealed that vapers and smokers had opposing changes in biological processes.
Conclusions
These data indicate that vaping causes unique changes to AMs and bronchial epithelia compared with nonsmokers and smokers which may impact pulmonary host defense.
Implications
These data indicate that normal “healthy” vapers have altered AMs and may be at risk of developing abnormal immune responses to inflammatory stimuli.
In cystic fibrosis (CF), defective biogenesis and activity of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to airway dehydration and impaired mucociliary clearance, resulting ...in chronic airway infection and inflammation. The most common CFTR mutation, F508del, results in a processing defect in which the protein is retained in the endoplasmic reticulum and does not reach the apical surface. CFTR corrector compounds address this processing defect to promote mutant CFTR transfer to the apical membrane. When coupled with potentiators to increase CFTR channel activity, these drugs yield significant clinical benefits in CF patients carrying the F508del mutation. However, processing of CFTR and other proteins can be influenced by environmental factors such as inflammation, and the impact of airway inflammation on pharmacological activity of CFTR correctors is not established. The present study evaluated CFTR-rescuing therapies in inflamed CF airway epithelial cultures, utilizing models that mimic the inflammatory environment of CF airways. Primary bronchial epithelial cultures from F508del/F508del CF patients were inflamed by mucosal exposure to one of two inflammatory stimuli: 1) supernatant from mucopurulent material from CF airways with advanced lung disease, or 2) bronchoalveolar lavage fluid from pediatric CF patients. Cultures inflamed with either stimulus exhibited augmented F508del responses following therapy with correctors VX-809 or VX-661, and overcame the detrimental effects of chronic exposure to the CFTR potentiator VX-770. Remarkably, even the improved CFTR rescue responses resulting from a clinically effective triple therapy (VX-659/VX-661/VX-770) were enhanced by epithelial inflammation. Thus, the airway inflammatory milieu from late- and early-stage CF lung disease improves the efficacy of CFTR modulators, regardless of the combination therapy used. Our findings suggest that pre-clinical evaluation of CFTR corrector therapies should be performed under conditions mimicking the native inflammatory status of CF airways, and altering the inflammatory status of CF airways may change the efficacy of CFTR modulator therapies.
Congenital Zika Syndrome (CZS) is characterized by many impairments especially in the central nervous system, potentially compromising neurodevelopment and causing significant morbidity in affected ...children. The aim was to assess gross motor function in children with CZS. This was a cross-sectional investigation nested within a prospective cohort study of children with CZS based in a Brazilian referral hospital in Rio de Janeiro. Between March/2017 and February/2018, we performed gross motor function assessments using the Gross Motor Function Classification (GMFCS) and the Gross Motor Function Measure (GMFM), estimating the mean and standard deviation of GMFM scores among GMFCS groups. The study sample included 72 children, with a median age of 13 months (7–25). Of these, 63 (87.5%) had severe motor impairment, 3 (4%) had moderate impairment, and 6 (8%) had mild impairment. The mean GMFM score for each group was respectively 11.6, 26.1, and 81.6, with statistically significant differences (p-value < 0.001). Severely affected children only achieved head control in the sitting posture when supported. Children with milder forms were able to develop walking skills.
Conclusion:
Most children with CZS have major motor disabilities and a poor prognosis. Better understanding of limitations and functionality in children with CZS can serve as a prognostic guide in their management.
What is Known:
• Severe motor impairment was present in 63 (87.5%) children with CZS.
• The degree of neurological impairment was inversely associated with motor performance.
What is New:
• Microcephaly was more frequent among children with severe gross motor function impairment.
• Children with CZS have major motor disabilities and a poor prognosis.
Knowledge of how congenital Zika syndrome (CZS) impacts motor development of children longitudinally is important to guide management. The objective of the present study was to describe the evolution ...of gross motor function in children with CZS in a Rio de Janeiro hospital. In children with CZS without arthrogryposis or other congenital osteoarticular malformations who were followed in a prospective cohort study, motor performance was evaluated at two timepoints using the Gross Motor Function Classification System (GMFCS) and the Gross Motor Function Measurement test (GMFM-88). Among 74 children, at the baseline evaluation, the median age was 13 (8–24) months, and on follow-up, 28 (24–48) months. According to GMFCS at the second timepoint, 6 children were classified as mild, 11 as moderate, and 57 as severe. In the GMFM-88 assessment, children in the severe group had a median score of 10.05 in the baseline evaluation and a follow-up score of 12.40, the moderate group had median scores of 25.60 and 29.60, and the mild group had median scores of 82.60 and 91.00, respectively. Although a small developmental improvement was observed, the motor impairment of children was mainly consistent with severe cerebral palsy. Baseline motor function assessments were predictive of prognosis.
Human Langerhans cells (LCs) reside in foreskin and vaginal mucosa and are the first immune cells to interact with HIV-1 during sexual transmission. LCs capture HIV-1 through the C-type lectin ...receptor langerin, which routes the virus into Birbeck granules (BGs), thereby preventing HIV-1 infection. BGs are langerin-positive organelles exclusively present in LCs, however, their origin and function are unknown.
Here, we not only show that langerin and caveolin-1 co-localize at the cell membrane and in vesicles but also that BGs are langerin/caveolin-1-positive vesicles are linked to the lysosomal degradation pathway in LCs. Moreover, inhibition of caveolar endocytosis in primary LCs abrogated HIV-1 sequestering into langerin(+) caveolar structures. Notably, both inhibition of caveolar uptake and silencing of caveolar structure protein caveolin-1 resulted in increased HIV-1 integration and subsequent infection. In contrast, inhibition of clathrin-mediated endocytosis did not affect HIV-1 integration, even though HIV-1 uptake was decreased, suggesting that clathrin-mediated endocytosis is not involved in HIV-1 restriction in LCs.
Thus, our data strongly indicate that BGs belong to the caveolar endocytosis pathway and that caveolin-1 mediated HIV-1 uptake is an intrinsic restriction mechanism present in human LCs that prevents HIV-1 infection. Harnessing this particular internalization pathway has the potential to facilitate strategies to combat HIV-1 transmission.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Sexual transmission is the main route of HIV-1 infection and the CCR5-using (R5) HIV-1 is predominantly transmitted, even though CXCR4-using (X4) HIV-1 is often abundant in chronic HIV-1 patients. ...The mechanisms underlying this tropism selection are unclear. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 and here we investigated the role of LCs in selection of R5 HIV-1 using an ex vivo epidermal and vaginal transmission models.
Immature LCs were productively infected by X4 as well as R5 HIV-1. However, only R5 but not X4 viruses were selectively transmitted by immature LCs to T cells. Transmission of HIV-1 was depended on de novo production of HIV-1 in LCs, since it could be inhibited by CCR5 fusion inhibitors as well as reverse transcription inhibitors. Notably, the activation state of LCs affected the restriction in X4 HIV-1 transmission; immune activation by TNF facilitated transmission of X4 as well as R5 HIV-1.
These data suggest that LCs play a crucial role in R5 selection and that immature LCs effectively restrict X4 at the level of transmission.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
