Inflammation-induced upregulation of P2X4 expression augments mucin secretion in airway epithelia.

Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion. Previous studies revealed that ATP acts via metabotropic P2Y2 purinoreceptors on goblet cells. Extracellular ATP, however, is also a potent agonist for ionotropic P2X purinoreceptors. Expression of several P2X isoforms has been reported in airways, but cell type-specific expression and the function thereof remained elusive. With this study, we now provide evidence that P2X4 is the predominant P2X isoform expressed in secretory airway epithelial cells. After IL-13 treatment of either human primary tracheal epithelial cells or mice, P2X4 expression is upregulated in vitro and in vivo under conditions of chronic inflammation, mucous metaplasia, and hyperplasia. Upregulation of P2X4 is strongest in MUC5AC-positive goblet cells. Moreover, activation of P2X4 by extracellular ATP augments intracellular Ca2+ signals and mucin secretion, whereas Ca2+ signals and mucin secretion are dampened by inhibition of P2X4 receptors. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo.

Glucocorticoids Regulate Tight Junction Permeability of Lung Epithelia by Modulating Claudin 8.

The lung epithelium constitutes a selective barrier that separates the airways from the aqueous interstitial compartment. Regulated barrier function controls water and ion transport across the epithelium and is essential for maintaining lung function. Tight junctions (TJs) seal the epithelial barrier and determine the paracellular transport. The properties of TJs depend especially on their claudin composition. Steroids are potent drugs used to treat a variety of airway diseases. Therefore, we addressed whether steroid hormones directly act on TJ properties in lung epithelia. Primary human tracheal epithelial cells and NCI-H441 cells, both cultivated under air-liquid interface conditions, were used as epithelial cell models. Our results demonstrate that glucocorticoids, but not mineralocorticoids, decreased paracellular permeability and shifted the ion permselectivity of TJs toward Cl(-). Glucocorticoids up-regulated claudin 8 (cldn8) expression via glucocorticoid receptors. Silencing experiments revealed that cldn8 is necessary to recruit occludin at the TJs. Immunohistochemistry on human lung tissue showed that cldn8 is specifically expressed in resorptive epithelia of the conducting and respiratory airways but not in the alveolar epithelium. We conclude that glucocorticoids enhance lung epithelia barrier function and increase paracellular Cl(-) selectivity via modulation of cldn8-dependent recruitment of occludin at the TJs. This mode of glucocorticoid action on lung epithelia might be beneficial to patients who suffer from impaired lung barrier function in various diseased conditions.

Correction: Ziemssen et al. Immune Response to Initial and Booster SARS-CoV-2 mRNA Vaccination in Patients Treated with Siponimod-Final Analysis of a Nonrandomized Controlled Clinical Trial (AMA-VACC). Vaccines 2023, 11, 1374.

The authors would like to make the following corrections to this published paper [...].

Immune Response to Initial and Booster SARS-CoV-2 mRNA Vaccination in Patients Treated with Siponimod-Final Analysis of a Nonrandomized Controlled Clinical Trial (AMA-VACC).

BACKGROUND: Evidence on SARS-CoV-2 mRNA vaccination under siponimod treatment is rare. METHODS: AMA-VACC is a prospective, open-label clinical study on SARS-CoV-2 mRNA vaccination during ongoing siponimod treatment (cohort 1), during siponimod interruption (cohort 2), or during treatment with other disease-modifying therapies or without therapy (cohort 3). SARS-CoV-2-specific antibodies and T-cell reactivity were measured six months after the initial vaccination and one month after the booster. RESULTS: 41 patients were recruited into cohort 1 (n = 17), cohort 2 (n = 4), and cohort 3 (n = 20). Seroconversion for SARS-CoV-2 neutralizing antibodies was reached by 50.0%, 100.0%, and 90.0% of patients at month 6 and by 81.3%, 100.0%, and 100.0% one month after booster (cohorts 1, 2, and 3, respectively). Antibody levels in cohort 1 increased after the booster compared to month 6 but remained lower compared to cohorts 2 and 3. T-cell responses were seen in 28.5%, 25.0%, and 73.7% at month 6 and in 28.6%, 50.0%, and 83.3% after the booster (cohorts 1, 2, and 3, respectively). In cohort 1, the extent of T-cell response was lower at month 6 compared to cohorts 2 and 3 but reached almost similar levels after the booster. CONCLUSIONS: The antibody and T-cell responses support SARS-CoV-2 (booster) vaccines in siponimod-treated patients.

Long-term real-world effectiveness and safety of fingolimod over 5 years in Germany.

OBJECTIVE: To evaluate the 5-year real-world benefit-risk profile of fingolimod in patients with relapsing-remitting MS (RRMS) in Germany. METHODS: Post-Authorization Non-interventional German sAfety study of GilEnyA (PANGAEA) is a non-interventional real-world study to prospectively assess the effectiveness and safety of fingolimod in routine clinical practice in Germany. The follow-up period comprised 5 years. Patients were included if they had been diagnosed with RRMS and had been prescribed fingolimod as part of clinical routine. There were no exclusion criteria except the contraindications for fingolimod as defined in the European label. The effectiveness and safety analysis set comprised 4032 and 4067 RRMS patients, respectively. RESULTS: At the time of the 5-year follow-up of PANGAEA, 66.57% of patients still continued fingolimod therapy. Annualized relapse rates decreased from baseline 1.5 ± 1.15 to 0.42 ± 0.734 at year 1 and 0.21 ± 0.483 at year 5, and the disability status remained stable, as demonstrated by the Expanded Disability Status Scale mean change from baseline (0.1 ± 2.51), the decrease of the Multiple Sclerosis Severity Score from 5.1 ± 2.59 at baseline to 3.9 ± 2.31 at the 60-months follow-up, and the percentage of patients with 'no change' in the Clinical Global Impression scale at the 60-months follow-up (78.11%). Adverse events (AE) occurring in 75.04% of patients were in line with the known safety profile of fingolimod and were mostly non-serious AE (33.62%) and non-serious adverse drug reactions (50.59%; serious AE 4.98%; serious ADR 10.82%). CONCLUSIONS: PANGAEA demonstrated the sustained beneficial effectiveness and safety of fingolimod in the long-term real-world treatment of patients with RRMS.

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo.

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.

Role of the C5a-C5a receptor axis in the inflammatory responses of the lungs after experimental polytrauma and hemorrhagic shock.

Singular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

A Novel Fibroblast Reporter Cell Line for in vitro Studies of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of the lower respiratory tract with restricted therapeutic options. Repetitive injury of the bronchoalveolar epithelium leads to activation of pulmonary fibroblasts, differentiation into myofibroblasts and excessive extracellular matrix (ECM) deposition resulting in aberrant wound repair. However, detailed molecular and cellular mechanisms underlying initiation and progression of fibrotic changes are still elusive. Here, we report the generation of a representative fibroblast reporter cell line (10-4A BFP ) to study pathophysiological mechanisms of IPF in high throughput or high resolution in vitro live cell assays. To this end, we immortalized primary fibroblasts isolated from the distal lung of Sprague-Dawley rats. Molecular and transcriptomic characterization identified clone 10-4A as a matrix fibroblast subpopulation. Mechanical or chemical stimulation induced a reversible fibrotic state comparable to effects observed in primary isolated fibroblasts. Finally, we generated a reporter cell line (10-4A BFP ) to express nuclear blue fluorescent protein (BFP) under the promotor of the myofibroblast marker alpha smooth muscle actin (Acta2) using CRISPR/Cas9 technology. We evaluated the suitability of 10-4A BFP as reporter tool in plate reader assays. In summary, the 10-4A BFP cell line provides a novel tool to study fibrotic processes in vitro to gain new insights into the cellular and molecular processes involved in fibrosis formation and propagation.

ATP is stored in lamellar bodies to activate vesicular P2X4 in an autocrine fashion upon exocytosis.

Vesicular P2X4 receptors are known to facilitate secretion and activation of pulmonary surfactant in the alveoli of the lungs. P2X4 receptors are expressed in the membrane of lamellar bodies (LBs), large secretory lysosomes that store lung surfactant in alveolar type II epithelial cells, and become inserted into the plasma membrane after exocytosis. Subsequent activation of P2X4 receptors by adenosine triphosphate (ATP) results in local fusion-activated cation entry (FACE), facilitating fusion pore dilation, surfactant secretion, and surfactant activation. Despite the importance of ATP in the alveoli, and hence lung function, the origin of ATP in the alveoli is still elusive. In this study, we demonstrate that ATP is stored within LBs themselves at a concentration of ∼1.9 mM. ATP is loaded into LBs by the vesicular nucleotide transporter but does not activate P2X4 receptors because of the low intraluminal pH (5.5). However, the rise in intravesicular pH after opening of the exocytic fusion pore results in immediate activation of vesicular P2X4 by vesicular ATP. Our data suggest a new model in which agonist (ATP) and receptor (P2X4) are located in the same intracellular compartment (LB), protected from premature degradation (ATP) and activation (P2X4), and ideally placed to ensure coordinated and timely receptor activation as soon as fusion occurs to facilitate surfactant secretion.