PDE3 Inhibition Reduces Epithelial Mast Cell Numbers in Allergic Airway Inflammation and Attenuates Degranulation of Basophils and Mast Cells

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

Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling

Background: 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. Method: 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. Results: 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. Conclusion: Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation

Exploring the Modulatory Effect of High-Fat Nutrition on Lipopolysaccharide-Induced Acute Lung Injury in Vagotomized Rats and the Role of the Vagus Nerve

During esophagectomy, the vagus nerve is transected, which may add to the development of postoperative complications. The vagus nerve has been shown to attenuate inflammation and can be activated by a high-fat nutrition via the release of acetylcholine. This binds to α7 nicotinic acetylcholine receptors (α7nAChR) and inhibits α7nAChR-expressing inflammatory cells. This study investigates the role of the vagus nerve and the effect of high-fat nutrition on lipopolysaccharide (LPS)-induced lung injury in rats. Firstly, 48 rats were randomized in 4 groups as follows: sham (sparing vagus nerve), abdominal (selective) vagotomy, cervical vagotomy and cervical vagotomy with an α7nAChR-agonist. Secondly, 24 rats were randomized in 3 groups as follows: sham, sham with an α7nAChR-antagonist and cervical vagotomy with an α7nAChR-antagonist. Finally, 24 rats were randomized in 3 groups as follows: fasting, high-fat nutrition before sham and high-fat nutrition before selective vagotomy. Abdominal (selective) vagotomy did not impact histopathological lung injury (LIS) compared with the control (sham) group (p > 0.999). There was a trend in aggravation of LIS after cervical vagotomy (p = 0.051), even after an α7nAChR-agonist (p = 0.090). Cervical vagotomy with an α7nAChR-antagonist aggravated lung injury (p = 0.004). Furthermore, cervical vagotomy increased macrophages in bronchoalveolar lavage (BAL) fluid and negatively impacted pulmonary function. Other inflammatory cells, TNF-α and IL-6, in the BALF and serum were unaffected. High-fat nutrition reduced LIS after sham (p = 0.012) and selective vagotomy (p = 0.002) compared to fasting. vagotomy. This study underlines the role of the vagus nerve in lung injury and shows that vagus nerve stimulation using high-fat nutrition is effective in reducing lung injury, even after selective vagotomy

Regulation of human mast cell Activation

Mast cell-mediated diseases, such as allergies and asthma, affect a growing percentage of the population, with significant unmet medical needs. As we slowly untangle the regulatory mechanisms that modulate these complex genetic and environment-influenced diseases, new opportunities in an area of potentially highly specific and effective therapeutic interventions are emerging. In this thesis, we aimed to a) investigate how the activation of a prime effector cell in allergy - the mast cell - can be regulated by dietary compounds such as short-chain fatty acids (SCFAs), b) study the transcriptional response in human mast cells in response to various stimuli to better understand endogenous feedback loops and c) develop a new technology platform to identify and validate novel regulators of human mast cell degranulation. We summarized the known effects of dietary fiber and its metabolites on immune and non-immune cells (Chapter 2.1), and more specifically, in the context of mast cell-mediated disease (Chapter 2.2). Given the plausible correlation between dietary fiber intake and mast cell–mediated pathology, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on human mast cell activation, including the molecular mechanisms involved. We showed that butyrate and propionate, but not acetate, potently inhibited human mast cell activation via inhibition of histone deacetylase (HDAC) activity (Chapter 3). Interestingly, butyrate downregulated expression of key signaling molecules involved in IgE-mediated mast cell activation (e.g., BTK, SYK, and LAT), which coincided with deacetylation near promoter regions of such genes. To investigate potential mechanisms underlying gene expression changes in response to butyrate-induced HDAC inhibition, we integrated RNA-Seq and time-course ChIP-Seq data from butyrate-treated primary human mast cells (Chapter 4). Although butyrate evoked broad histone acetylation, our data indicated that butyrate selectively regulated gene transcription in primary human mast cells and had stronger modulatory effects on only a small subset of chromatin regions. Next, we sought to map the transcriptional landscape of activated mast cells using a variety of stimuli that target either the high-affinity IgE receptor (FcεRI)- or Mas-Related G Protein-Coupled Receptor-X2 (MRGPRX2) receptor, and found that mast cells tightly control their own activity by regulating the expression of both positive and negative regulators of (mast) cell activation (Chapter 5). Our understanding of the molecules that regulate mast cell activation and degranulation is primarily based on evidence obtained in (mast cell deficient) mouse models. Unfortunately, the follow-up validation of the exact roles of these regulators in mediating degranulation in human mast cells has been hampered by (i) the limited availability of primary human mast cells and (ii) the lack of suitable methodology to functionally interrogate the putative roles of such regulators of degranulation using small numbers of primary cells. To this end, we developed a novel technology platform that allows for rapid identification of regulators of human mast cell degranulation using functional genomics coupled to high-resolution confocal microscopy (Chapter 6). In Chapter 7 we discuss the interpretations and implications of our main findings, as well as possible limitations of the study. Furthermore, suggestions for future research directions are proposed, in addition to novel therapeutic strategies for targeting mast functions. In this thesis, we uncovered molecular mechanisms that have evolved to control human mast cell activation. Future studies are bound to discover additional (deficiencies in the) regulatory mechanisms that control mast cell function

Effect of Dietary Fiber and Metabolites on Mast Cell Activation and Mast Cell-Associated Diseases

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

Microbiota-dependent and -independent effects of dietary fibre on human health

Dietary fiber, such as indigestible oligo- and polysaccharides, occurs in many foods and has gained considerable interest related to its beneficial effects on host health and specific diseases. Dietary fiber is neither digested nor absorbed in the small intestine and modulates the composition of the gut microbiota. New evidence indicates that dietary fiber also directly interacts with the epithelium and immune cells throughout the gastrointestinal tract by microbiota-independent effects. This review provides a focused overview of how dietary fiber improve human health and how these reported health benefits are connected to molecular pathways, in (1) a microbiota-independent manner, via interaction with specific surface receptors on epithelial and immune cells regulating intestinal barrier and immune function, (2) 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 fiber-derived microbial short-chain fatty acid production

Microbiota-dependent and -independent effects of dietary fiber on human health

Dietary fiber, such as indigestible oligo- and polysaccharides, occurs in many foods and has gained considerable interest related to its beneficial effects on host health and specific diseases. Dietary fiber is neither digested nor absorbed in the small intestine and modulates the composition of the gut microbiota. New evidence indicates that dietary fiber also directly interacts with the epithelium and immune cells throughout the gastrointestinal tract by microbiota-independent effects. This review provides a focused overview of how dietary fiber improve human health and how these reported health benefits are connected to molecular pathways, in (1) a microbiota-independent manner, via interaction with specific surface receptors on epithelial and immune cells regulating intestinal barrier and immune function, (2) 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 fiber-derived microbial short-chain fatty acid production