During recent years chemosensory cells in extraoral tissues have been established as mediators for the detection and regulation of innate immune processes in response to pathogens. Under ...physiological conditions, chemosensory cells are present throughout the respiratory epithelium of the upper and lower airways as well as in the main olfactory epithelium. Additionally, they emerge in the alveolar region of the lung upon viral infections. Chemosensory cells in the upper and the lower airways detect signalling molecules from gram‐positive and gram‐negative bacteria as well as aeroallergens and fungi. Upon stimulation they release multiple molecules, such as the transmitter acetylcholine, the cysteinyl leukotriene E4 and the cytokine interleukin‐25, which act as autocrine and paracrine signals and thereby orchestrate the innate immune responses in the respiratory system. Activation of chemosensory cells stimulates various immune cells, e.g. type 2 innate lymphoid cells, modulates mucociliary clearance and induces a protective neurogenic inflammation. This review compiles and discusses recent findings regarding chemosensory cell function in the respiratory tract.
figure legend Chemosensory cells that are characterised by the expression of a functional bitter taste signalling cascade are widely expressed in tissues outside of taste buds in the tongue. In the respiratory system they are known under diverse names: microvillous cells in the main olfactory epithelium, solitary chemosensory cells in the upper airways, brush cells in the lower airways and tuft cells in the lung. In recent literature, some authors refer to these chemosensory cells generally as tuft cells, regardless of the organ they are expressed in. While little is known about the function of microvillous cells and lung tuft cells, a clear role has been established for solitary chemosensory cells and brush cells in eliciting important innate immune functions after stimulation by metabolites from pathogens. This review discusses recent findings on the function of these chemosensory cells in the respiratory tract.
Synthesis of DNA fragments based on gene sequences that are available in public resources has become an efficient and affordable method that has gradually replaced traditional cloning efforts such as ...PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterisation of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: (1) Not every gene has yet been annotated; (2) not all gene annotations are necessarily correct; (3) transcripts may contain automated corrections; (4) there are mismatches between gene, mRNA and protein sequences; and (5) splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems.
figure legend Projects involving heterologous gene expression are often characterised by similar steps. Initially, database research (A) is necessary to retrieve information of full or partial sequences of a gene of interest. A multitude of genome assemblies are annotated and deposited in public databases or are available for refined search options using individual sequence information. The search results need to be scrutinised and compared with already available information (B). Once the sequence has been determined, DNA synthesis (C) by PCR or commercial synthesis is necessary for further cloning procedures (D). Eventually, the DNA needs to be transfected (E) and expressed in, for example, eukaryotic cells (F). Finally, the expression of the gene of interest needs to be documented and its function analysed (G).
For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally ...released bacterial bitter “taste” substances are most probably initiated by tracheal brush cells (BC). Our single‐cell RNA‐seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in Ca2+i in BC and subsequent ACh‐release. ACh‐release is regulated in an autocrine manner. While the muscarinic ACh‐receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased Ca2+i in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5‐ and M3R‐mediated. We show that ACh‐release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.
Background and Purpose
Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of ...the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown.
Experimental Approach
Transepithelial short circuit currents (ISC) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in Ca2+i were studied on freshly dissociated mouse tracheal epithelial cells.
Key Results
Apical application of the nAChR agonist nicotine transiently increased ISC. The nicotine effect was abolished by the nAChR antagonist mecamylamine. α‐Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3β2, α4β2, α4β4 and α3β4) and A‐85380 (α4β2 and α3β4) increased ISC. The antagonists dihydro‐β‐erythroidine (α4β2, α3β2, α4β4 and α3β4), α‐conotoxin MII (α3β2) and α‐conotoxin PnIA (α3β2) reduced the nicotine effect. Nicotine‐ and epibatidine‐induced currents were unaltered in β2−/−mice, but in β4−/− mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+‐ATPase inhibitor) or the ryanodine receptor antagonists JTV‐519 and dantrolene there was a reduction in the nicotine‐effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H‐89 and the TMEM16A (Ca2+‐activated chloride channel) inhibitor T16Ainh‐A01 significantly reduced the nicotine‐effect.
Conclusion and Implications
α3β4 nAChRs are responsible for the nicotine‐induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.
Nicotinic acetylcholine receptors (nAChR) are widely distributed in neuronal and non-neuronal tissues, where they play diverse physiological roles. In this review, we highlight the recent findings ...regarding the role of nAChR in the respiratory tract with a special focus on the involvement of nAChR in the regulation of multiple processes in health and disease. We discuss the role of nAChR in mucociliary clearance, inflammation, and infection and in airway diseases such as asthma, chronic obstructive pulmonary disease, and cancer. The subtype diversity of nAChR enables differential regulation, making them a suitable pharmaceutical target in many diseases. The stimulation of the α3β4 nAChR could be beneficial in diseases accompanied by impaired mucociliary clearance, and the anti-inflammatory effect due to an α7 nAChR stimulation could alleviate symptoms in diseases with chronic inflammation such as chronic obstructive pulmonary disease and asthma, while the inhibition of the α5 nAChR could potentially be applied in non-small cell lung cancer treatment. However, while clinical studies targeting nAChR in the airways are still lacking, we suggest that more detailed research into this topic and possible pharmaceutical applications could represent a valuable tool to alleviate the symptoms of diverse airway diseases.
Neuroimmune interaction has long been discussed in the pathogenesis of allergic airway diseases, such as allergic asthma. Mediators released during inflammation can alter the function of both sensory ...and parasympathetic neurons innervating the airways. Evidence has been provided that the inflammatory response can be altered by various mediators that are released by sensory and parasympathetic neurons and vice versa. Our aim is to demonstrate recent developments in the reciprocal neuroimmune interaction and to include, if available, data from in vivo and clinical studies.
Abstract
Background
Caveolins are the principal structural components of plasma membrane caveolae. Dominant pathogenic mutations in the muscle‐specific caveolin‐3 (Cav3) gene isoform, such as the ...limb girdle muscular dystrophy type 1C (LGMD‐1C) P104L mutation, result in dramatic loss of the Cav3 protein and pathophysiological muscle weakness/wasting. We hypothesize that such muscle degeneration may be linked to disturbances in signalling events that impact protein turnover. Herein, we report studies assessing the effects of Cav3 deficiency on mammalian or mechanistic target of rapamycin complex 1 (mTORC1) signalling in skeletal muscle cells.
Methods
L6 myoblasts were stably transfected with Cav3
P104L
or expression of native Cav3 was abolished by CRISPR/Cas9 genome editing (Cav3 knockout Cav3KO) prior to performing subcellular fractionation and immunoblotting, analysis of real‐time mitochondrial respiration or fixed cell immunocytochemistry. Skeletal muscle from wild‐type and Cav3
−/−
mice was processed for immunoblot analysis of downstream mTORC1 substrate phosphorylation.
Results
Cav3 was detected in lysosomal‐enriched membranes isolated from L6 myoblasts and observed by confocal microscopy to co‐localize with lysosomal‐specific markers. Cav3
P104L
expression, which results in significant (~95%) loss of native Cav3, or CRISPR/Cas9‐mediated Cav3KO, reduced amino acid‐dependent mTORC1 activation. The decline in mTORC1‐directed signalling was detected by immunoblot analysis of L6 muscle cells and gastrocnemius Cav3
−/−
mouse muscle as judged by reduced phosphorylation of mTORC1 substrates that play key roles in the initiation of protein synthesis (4EBP1
S65
and S6K1
T389
). S6K1
T389
and 4EBP1
S65
phosphorylation reduced by over 75% and 80% in Cav3KO muscle cells and by over 90% and 30% in Cav3
−/−
mouse skeletal muscle, respectively. The reduction in protein synthetic capacity in L6 muscle cells was confirmed by analysis of puromycylated peptides using the SUnSET assay. Cav3 loss was also associated with a 26% increase in lysosomal cholesterol, and pharmacological manipulation of lysosomal cholesterol was effective in replicating the reduction in mTORC1 activity observed in Cav3KO cells. Notably, re‐expression of Cav3 in Cav3KO myoblasts normalized lysosomal cholesterol content, which coincided with a recovery in protein translation and an associated increase in mTORC1‐directed phosphorylation of downstream targets.
Conclusions
Our findings indicate that Cav3 can localize on lysosomal membranes and is a novel regulator of mTORC1 signalling in muscle. Cav3 deficiency associated with the Cav3
P104L
mutation impairs mTORC1 activation and protein synthetic capacity in skeletal muscle cells, which may be linked to disturbances in lysosomal cholesterol trafficking and contribute to the pathology of LGMD‐1C.
In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically ...similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signalling molecules (α-gustducin, phospholipase C β2 ) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.
Background
Caveolin‐3 (Cav3) is the principal structural component of caveolae in skeletal muscle. Dominant pathogenic mutations in the Cav3 gene, such as the Limb Girdle Muscular Dystrophy‐1C ...(LGMD1C) P104L mutation, result in substantial loss of Cav3 and myopathic changes characterized by muscle weakness and wasting. We hypothesize such myopathy may also be associated with disturbances in mitochondrial biology. Herein, we report studies assessing the effects of Cav3 deficiency on mitochondrial form and function in skeletal muscle cells.
Methods
L6 myoblasts were stably transfected with Cav3P104L or expression of native Cav3 repressed by shRNA or CRISPR/Cas9 genome editing prior to performing fixed/live cell imaging of mitochondrial morphology, subcellular fractionation and immunoblotting, or analysis of real time mitochondrial respiration. Skeletal muscle from wild‐type and Cav3−/− mice was processed for analysis of mitochondrial proteins by immunoblotting.
Results
Caveolin‐3 was detected in mitochondrial‐enriched membranes isolated from mouse gastrocnemius muscle and L6 myoblasts. Expression of Cav3P104L in L6 myoblasts led to its targeting to the Golgi and loss of native Cav3 (>95%), including that associated with mitochondrial membranes. Cav3P104L reduced mitochondrial mass and induced fragmentation of the mitochondrial network that was associated with significant loss of proteins involved in mitochondrial biogenesis, respiration, morphology, and redox function i.e. PGC1α, succinate dehyrdogenase (SDHA), ANT1, MFN2, OPA1, and MnSOD). Furthermore, Cav3P104L myoblasts exhibited increased mitochondrial cholesterol and loss of cardiolipin. Consistent with these changes, Cav3P104L expression reduced mitochondrial respiratory capacity and increased myocellular superoxide production. These morphological, biochemical, and functional mitochondrial changes were phenocopied in myoblasts in which Cav3 had been silenced/knocked‐out using shRNA or CRISPR. Reduced mitochondrial mass, PGC1α, SDHA, ANT1, and MnSOD were also demonstrable in Cav3−/− mouse gastrocnemius. Strikingly, Cav3 re‐expression in Cav3KO myoblasts restored its mitochondrial association and facilitated reformation of a tubular mitochondrial network. Significantly, re‐expression also mitigated changes in mitochondrial superoxide, cholesterol, and cardiolipin content and recovered cellular respiratory capacity.
Conclusions
Our results identify Cav3 as an important regulator of mitochondrial homeostasis and reveal that Cav3 deficiency in muscle cells associated with the Cav3P104L mutation invokes major disturbances in mitochondrial respiration and energy status that may contribute to the pathology of LGMD1C.
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