29 research outputs found
Chemosensory cells in the respiratory tract as crucial regulators of innate immune responses
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
Nicotinic Acetylcholine Receptors in the Respiratory Tract
Nicotinic acetylcholine receptors (nAChR) are widely distributed in neuronal and nonneuronal 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
α3ÎČ4 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
Pitfalls of using sequence databases for heterologous expression studies : a technical review
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
Novel human sex-typing strategies based on the autism candidate gene NLGN4X and its male-specific gametologue NLGN4Y
Background: Since the early days of PCR techniques, sex identification, âsex-typing,â of genomic DNA samples
has been a fundamental part of human forensic analysis but also in animal genetics aiming at strategic livestock
breeding. Most analyses are employing the AMELX/AMELY gene loci on the X and Y chromosomes present in most
mammals. We hypothesize that sex-typing in humans is also possible based on the genes NLGN4X and NLGN4Y,
which represent X and Y chromosome-specific copies of a common ancestral neuroligin-4 orthologue.
Methods: Genomic DNA was isolated from human blood and buccal cell samples (total n = 111) and submitted
to two different strategies: (a) a traditional two-primer PCR approach detecting an insertion/deletion (indel)
polymorphism immediately upstream of the translational start on exon 1 and (b) detection of a single nucleotide
polymorphism, SNP, on the translational stop carrying exon 7. The SNP detection was based on a quantitative PCR
approach (rhAMP genotyping) employing DNA/RNA hybrid oligonucleotides that were blocked and which could
only be activated upon perfect annealing to the target DNA sequence.
Results: All indel PCR-tested human DNA samples showed two bands for males representing X- and Y-specific
copies of NLGN4 and a single band for female samples, i.e., homozygosity of NLGN4X and absence of NLGN4Y, in
accordance with the self-reported sex of the donors. These results were in perfect agreement with the results of
the rhAMP-based SNP-detection method: all males were consequently positive for both alleles, representing either
SNP variant, and females were interpreted as homozygous regarding the SNP variant found in NLGN4X. Both
methods have shown reliable and consistent results that enabled us to infer the sex of donor DNA samples across
different ethnicities.
Conclusions: These results indicate that the detection of human NLGN4X/Y is a suitable alternative to previously
reported methods employing gene loci such as AMELX/Y. Furthermore, this is the first report applying successfully
the rhAMP-genotyping strategy as a means for SNP-based sex-typing, which consequently will be applicable to
other gene loci or different species as well
Caveolin-3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function
Nicotine stimulates ion transport via metabotropic ÎČ4 subunit containing nicotinic ACh receptors
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. αâBungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3ÎČ2, α4ÎČ2, α4ÎČ4 and α3ÎČ4) and Aâ85380 (α4ÎČ2 and α3ÎČ4) increased ISC. The antagonists dihydroâÎČâerythroidine (α4ÎČ2, α3ÎČ2, α4ÎČ4 and α3ÎČ4), αâconotoxin MII (α3ÎČ2) and αâconotoxin PnIA (α3ÎČ2) reduced the nicotine effect. Nicotineâ and epibatidineâinduced currents were unaltered in ÎČ2â/âmice, but in ÎČ4â/â mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+âATPase inhibitor) or the ryanodine receptor antagonists JTVâ519 and dantrolene there was a reduction in the nicotineâeffect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor Hâ89 and the TMEM16A (Ca2+âactivated chloride channel) inhibitor T16AinhâA01 significantly reduced the nicotineâeffect.
Conclusion and Implications
α3ÎČ4 nAChRs are responsible for the nicotineâinduced 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
Caveolin-1: functional insights into its role in muscarine- and serotonin-induced smooth muscle constriction in murine airways
Human airway tuft cells influence the mucociliary clearance through cholinergic signalling
Background Airway tuft cells, formerly called brush cells have long been described only morphologically in human
airways. More recent RNAseq studies described a chemosensory cell population, which includes tuft cells, by a distinct
gene transcription signature. Yet, until which level in the tracheobronchial tree in native human airway epithelium tuft
cells occur and if they function as regulators of innate immunity, e.g., by regulating mucociliary clearance, remained
largely elusive.
Methods We performed immunohistochemistry, RT-PCR and immunoblotting analyses for various tuft cell markers
to confirm the presence of this cell type in human tracheal samples. Immunohistochemistry was conducted to
study the distribution of tuft cells along the intrapulmonary airways in humans. We assessed the influence of bitter
substances and the taste transduction pathway on mucociliary clearance in mouse and human tracheal samples by
measuring particle transport speed.
Results Tuft cells identified by the expression of their well-established marker POU class 2 homeobox 3 (POU2F3)
were present from the trachea to the bronchioles. We identified choline acetyltransferase in POU2F3 expressing cells
as well as the transient receptor potential melastatin 5 (TRPM5) channel in a small population of tracheal epithelial
cells with morphological appearance of tuft cells. Application of bitter substances, such as denatonium, led to an
increase in mucociliary clearance in human tracheal preparations. This was dependent on activation of the TRPM5
channel and involved cholinergic and nitric oxide signalling, indicating a functional role for human tuft cells in the
regulation of mucociliary clearance. Conclusions We were able to detect tuft cells in the tracheobronchial tree down to the level of the bronchioles.
Moreover, taste transduction and cholinergic signalling occur in the same cells and regulate mucociliary clearance.
Thus, tuft cells are potentially involved in the regulation of innate immunity in human airways
Combining mass spectrometry and genetic labeling in mice to report TRP channel expression
Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice.
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We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution.
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We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice
The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation
Staphylococcus epidermidis is a common cause of device related infections on which pathogens
form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we
report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such
biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide
intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its
parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A
decreased biofilm formation capacity was also observed when 1457 âspoVG cells were co-cultured
with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while
the cis-complemented 1457 âspoVG::spoVG derivative formed biofilms comparable to the levels seen
with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly
altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of
the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic
mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic
region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression.
However, when mice were challenged with the 1457 âspoVG mutant in a foreign body infection
model, only marginal differences in biomasses produced on the infected catheter fragments between
the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the
PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable
for biofilm formation of this skin commensal under in vivo conditions