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Key points Exposure of human bronchial epithelial (HBE) cells to fungal aeroallergens derived from Alternaria alternata stimulates Ca2+-dependent and Ca2+-independent ATP release across the apical membrane., The Ca2+-dependent component was blocked by inhibitors of both ATP uptake and transport of exocytotic vesicles to the plasma membrane., Treatment with inhibitors that target cysteine proteases significantly blocked Ca2+-dependent ATP release evoked by Alternaria in normal HBE cells, but not in cells derived from asthmatic patients., The magnitude of ATP release and associated intracellular Ca2+ mobilization was significantly greater in bronchial epithelial cells obtained from patients with asthma., These findings establish a novel role for ATP release as a mechanism underlying Alternaria aeroallergen activation of airway mucosal immunity and that cells derived from patients with asthma exhibit greater responsiveness to these allergens., Abstract Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation. [ABSTRACT FROM AUTHOR]

Calu-3 cells are a human airway epithelial cell line that exhibits secretory characteristics similar to airway gland epithelial cells when grown on Transwell membrane filters. QRT-PCR analysis revealed that Calu-3 cells express mRNA for multiple Toll receptor (TLR) subtypes including TLR1, TLR2, TLR3, TLR4 and TLR6. In addition, Calu-3 cells express MyD88, MD2 and CD14 mRNAs which are critical for downstream TLR signaling. Protein expression for TLR2 and TLR4 was confirmed by western blot analysis. Treatment of cells for 24 hours with the TLR-4 agonist LPS produced a 2 fold increase in TLR2 mRNA expression as measured by a shift in the threshold cycle number. In contrast, stimulation of TLR2 (or TLR2/TLR6) receptors with Zymosan did not alter the mRNA expression of other TLR subtypes. Acute apical treatment with 1 µg/ml LPS for 20 minutes enhanced the peak and plateau short circuit current (Isc) responses to basolateral PGE2 stimulation. In contrast, acute exposure to Zymosan had no effect on PGE2 stimulated Isc. LPS or Zymosan stimulation was also found to increase the basolateral release of several pro-inflammatory cytokines including IL-6, IL-8, GROα and MCP-1 from Calu-3 cells. Cytokine release in response to LPS was potentiated if cells were pre-treated with Zymosan for 24 hours, suggesting that TLR2 receptor pre-stimulation could increase the LPS responsiveness of the epithelium. The results of these experiments demonstrate that TLR activation in an airway gland epithelial cell model can alter anion and cytokine secretion properties of the epithelium and that TLR-2 activation enhances the expression of TLR4. [ABSTRACT FROM AUTHOR]

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.