Stephen Galli, Postdoctoral Faculty Sponsor
PDE3 Inhibition Reduces Epithelial Mast Cell Numbers in Allergic Airway Inflammation and Attenuates Degranulation of Basophils and Mast Cells
FRONTIERS IN PHARMACOLOGY
2020; 11: 470
Epithelial mast cells are generally present in the airways of patients with allergic asthma that are inadequately controlled. Airway mast cells (MCs) are critically involved in allergic airway inflammation and contribute directly to the main symptoms of allergic patients. Phosphodiesterase 3 (PDE3) tailors signaling of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are critical intracellular second messenger molecules in various signaling pathways. This paper investigates the pathophysiological role and disease-modifying effects of PDE3 in mouse bone marrow-derived MCs (bmMCs), human LAD2- and HMC1 mast cell lines, human blood basophils, and peripheral blood-derived primary human MCs (HuMCs). In a chronic house dust mite (HDM)-driven allergic airway inflammation mouse model, we observed that PDE3 deficiency or PDE3 inhibition (PDE3i) therapy reduced the numbers of epithelial MCs, when compared to control mice. Mouse bone marrow-derived MCs (bmMCs) and the human HMC1 and LAD2 cell lines predominantly expressed PDE3B and PDE4A. BmMCs from Pde3-/- mice showed reduced loss of the degranulation marker CD107b compared with wild-type BmMCs, when stimulated in an immunoglobulin E (IgE)-dependent manner. Following both IgE-mediated and substance P-mediated activation, PDE3i-pretreated basophils, LAD2 cells, and HuMCs, showed less degranulation than diluent controls, as measured by surface CD63 expression. MCs lacking PDE3 or treated with the PDE3i enoximone exhibited a lower calcium flux upon stimulation with ionomycine. In conclusion PDE3 plays a critical role in basophil and mast cell degranulation and therefore its inhibition may be a treatment option in allergic disease.
View details for DOI 10.3389/fphar.2020.00470
View details for Web of Science ID 000536058800001
View details for PubMedID 32425769
View details for PubMedCentralID PMC7206980
Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy.
Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR-Cas9 gene editing. By using high-resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the alteration of functional responses, i.e., degranulation, in single human mast cells (10-12 weeks old). The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures, and the brief hands-on time of the functional analysis step enables high-throughput screening of shRNA or CRISPR-Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyze single cells substantially reduces the total number of cells required and enables the parallel visualization of the degranulation profiles of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference (RNAi) and CRISPR-Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data and image analysis can be completed within 5 d, requiring only standard laboratory equipment and expertise.
View details for DOI 10.1038/s41596-019-0288-6
View details for PubMedID 32060492
Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling.
Short-chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health and suppress mast cell-mediated diseases. However, the effects of SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs acetate, propionate and butyrate on mast cell-mediated pathology and human mast cell activation, including the molecular mechanisms involved.Precision-cut lung slices (PCLS) of allergen-exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co-cultured with SCFAs and assessed for degranulation after IgE- or non-IgE-mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays.Butyrate treatment inhibited allergen-induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE and non-IgE-mediated human or mouse mast cell degranulation in a concentration-dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43 or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate-induced downregulation of the tyrosine kinases BTK, SYK and LAT, critical transducers of FcεRI-mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK and LAT promoter regions.Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.
View details for DOI 10.1111/all.14254
View details for PubMedID 32112426
Microbiota-dependent and -independent effects of dietary fibre on human health
BRITISH JOURNAL OF PHARMACOLOGY
2020; 177 (6): 1363-1381
Dietary fibre, such as indigestible oligosaccharides and polysaccharides, occurs in many foods and has gained considerable importance related to its beneficial effects on host health and specific diseases. Dietary fibre is neither digested nor absorbed in the small intestine and modulates the composition of the gut microbiota. New evidence indicates that dietary fibre also interacts directly with the epithelium and immune cells throughout the gastrointestinal tract by microbiota-independent effects. This review focuses on how dietary fibre improves human health and the reported health benefits that are connected to molecular pathways, in (a) a microbiota-independent manner, via interaction with specific surface receptors on epithelial and immune cells regulating intestinal barrier and immune function, and (b) a microbiota-dependent manner via maintaining intestinal homeostasis by promoting beneficial microbes, including Bifidobacteria and Lactobacilli, limiting the growth, adhesion, and cytotoxicity of pathogenic microbes, as well as stimulating fibre-derived microbial short-chain fatty acid production. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.
View details for DOI 10.1111/bph.14871
View details for Web of Science ID 000502313600001
View details for PubMedID 31663129
View details for PubMedCentralID PMC7056464
Effect of Dietary Fiber and Metabolites on Mast Cell Activation and Mast Cell-Associated Diseases.
Frontiers in immunology
2018; 9: 1067
Many mast cell-associated diseases, including allergies and asthma, have seen a strong increase in prevalence during the past decades, especially in Western(ized) countries. It has been suggested that a Western diet may contribute to the prevalence and manifestation of allergies and asthma through reduced intake of dietary fiber and the subsequent production of their metabolites. Indeed, dietary fiber and its metabolites have been shown to positively influence the development of immune disorders via changes in microbiota composition and the regulation of B- and T-cell activation. However, the effects of these dietary components on the activation of mast cells, key effector cells of the inflammatory response in allergies and asthma, remain poorly characterized. Due to their location in the gut and vascularized tissues, mast cells are exposed to high concentrations of dietary fiber and/or its metabolites. Here, we provide a focused overview of current findings regarding the direct effects of dietary fiber and its various metabolites on the regulation of mast cell activity and the pathophysiology of mast cell-associated diseases.
View details for DOI 10.3389/fimmu.2018.01067
View details for PubMedID 29910798
View details for PubMedCentralID PMC5992428