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.

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.

Epithelial RABGEF1 deficiency promotes intestinal inflammation by dysregulating intrinsic MYD88-dependent innate signaling.

Intestinal epithelial cells (IECs) contribute to the regulation of intestinal homeostasis and inflammation through their interactions with the environment and host immune responses. Yet our understanding of IEC-intrinsic regulatory pathways remains incomplete. Here, we identify the guanine nucleotide exchange factor RABGEF1 as a regulator of intestinal homeostasis and innate pathways dependent on IECs. Mice with IEC-specific Rabgef1 deletion (called Rabgef1IEC-KO mice) developed a delayed spontaneous colitis associated with the local upregulation of IEC chemokine expression. In mouse models of colitis based on Interleukin-10 deficiency or dextran sodium sulfate (DSS) exposure, we found that IEC-intrinsic RABGEF1 deficiency exacerbated development of intestinal pathology and dysregulated IEC innate pathways and chemokine expression. Mechanistically, we showed that RABGEF1 deficiency in mouse IECs in vitro was associated with an impairment of early endocytic events, an increased activation of the p38 mitogen-activated protein kinase (MAPK)-dependent pathway, and increased chemokine secretion. Moreover, we provided evidence that the development of spontaneous colitis was dependent on microbiota-derived signals and intrinsic MYD88-dependent pathways in vivo. Our study identifies mouse RABGEF1 as an important regulator of intestinal inflammation, MYD88-dependent IEC-intrinsic signaling, and chemokine production. This suggests that RABGEF1-dependent pathways represent interesting therapeutic targets for inflammatory conditions in the gut.

Degradation of Monocyte Chemoattractant Protein-1 by Tryptase Co-Released in Immunoglobulin E-Dependent Activation of Primary Human Cultured Mast Cells.

BACKGROUND: Mast cells are key immune effector cells which release chemokines, proteases, and other inflammatory mediators upon activation by immunological stimuli. The aim of this study was to investigate the effects of co-releasing proteases on the kinetics of release of the chemokine monocyte chemoattractant protein-1 (MCP-1) in immunoglobulin E (IgE)-mediated activation of human mast cells. METHODS: Homogenous populations of mature and functional primary human mast cells were generated from CD34+ progenitors originated from buffy coats of healthy adult donors. The releases of MCP-1 from human mast cells in basal conditions and in response to FcεRI cross-linking were assessed at different time points. The effects of different types of protease inhibitors on MCP-1 release from these mast cells under stimulated or unstimulated conditions were also investigated. RESULTS: Cultured human mast cells released MCP-1 in basal conditions and its levels increased in a time-dependent manner. When stimulated by FcεRI cross-linking, the levels of MCP-1 detected in the medium gradually decreased over time after the initial peak induction. Such a decline in MCP-1 levels after IgE-dependent activation was completely prevented by pretreatment with a cocktail of protease inhibitors or the specific tryptase inhibitor APC366. CONCLUSIONS: Direct regulation of MCP-1 expression by co-release of tryptase in cultured human mast cells upon IgE-dependent activation demonstrates a role of the serglycin:serine protease axis in modulation of inflammatory reactions through proteolytic degradation of mediators such as chemokines.

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.

Author Correction: Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats.

The online version of the original article can.

Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats.

OBJECTIVE: The aim of this study was to develop a novel protocol for generating large populations of fully mature and functional human mast cells (HMC) from CD34+ hematopoietic stem cells which require less culturing time than previously reported methods. METHODS: CD34+ cells isolated from fresh human buffy coats were sequentially cultured with different combinations of SCF, IL-6, IL-3, IL-9 and IL-4 under selected culturing conditions and time periods. Cells were then harvested for immunohistochemical characterization of morphological phenotypes and were functionally characterized by assessing their responses to IgE-dependent and -independent stimuli by measuring the release of inflammatory mediators and cytokines. Moreover, the pharmacological profiles of several classes of anti-inflammatory drugs in inhibiting the activation of these HMC were also characterized. RESULTS: We have developed a novel protocol that can generate large homogenous populations of mature and functional HMC in 6 weeks. These cells expressed both tryptase and chymase and were activated by anti-IgE, cationic peptides and calcium ionophores. Moreover, IgE-dependent activation of these cells was significantly inhibited by anti-inflammatory drugs. The morphological and functional characteristics of these mast cells resembled those of MCTC type or connective tissue-type HMC. DISCUSSION: Our protocol represents a novel time-saving and economical approach for generating large numbers of primary HMC for functional studies of mast cell biology and for profiling novel anti-inflammatory therapeutic agents with mast cell-inhibitory properties in humans.

Guanine nucleotide exchange factor RABGEF1 regulates keratinocyte-intrinsic signaling to maintain skin homeostasis.

Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target.

RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells.

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

Thymic stromal lymphopoietin contributes to myeloid hyperplasia and increased immunoglobulins, but not epidermal hyperplasia, in RabGEF1-deficient mice.

Mice overexpressing the proallergic cytokine thymic stromal lymphopoietin (TSLP) in the skin develop a pathology resembling atopic dermatitis. RabGEF1, a guanine nucleotide exchange factor for Rab5 GTPase, is a negative regulator of IgE-dependent mast cell activation, and Rabgef1-/- and TSLP transgenic mice share many similar phenotypic characteristics, including elevated serum IgE levels and severe skin inflammation, with infiltrates of both lymphocytes and eosinophils. We report here that Rabgef1-/- mice also develop splenomegaly, lymphadenopathy, myeloid hyperplasia, and high levels of TSLP. Rabgef1-/-TSLPR-/- mice, which lack TSLP/TSLP receptor (TSLPR) signaling, had levels of blood neutrophils, spleen myeloid cells, and serum IL-4, IgG1, and IgE levels that were significantly reduced compared with those in Rabgef1-/-TSLPR+/+ mice. However, Rabgef1-/-TSLPR-/- mice, like Rag1- or eosinophil-deficient Rabgef1-/- mice, developed cutaneous inflammation and epidermal hyperplasia. Therefore, in Rabgef1-/- mice, TSLP/TSLPR interactions are not required for the development of epidermal hyperplasia but contribute to the striking myeloid hyperplasia and overproduction of immunoglobulins observed in these animals. Our study shows that RabGEF1 can negatively regulate TSLP production in vivo and that excessive production of TSLP contributes to many of the phenotypic abnormalities in Rabgef1-/- mice. However, the marked epidermal hyperplasia, cutaneous inflammation, and increased numbers of dermal mast cells associated with RabGEF1 deficiency can develop via a TSLPR-independent pathway, as well as in the absence of Rag1 or eosinophils.

Roles of RabGEF1/Rabex-5 domains in regulating Fc epsilon RI surface expression and Fc epsilon RI-dependent responses in mast cells.

RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (-/-) mast cells and examined Fc epsilon RI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in -/- mast cells, including decreased basal Fc epsilon RI expression, slowed Fc epsilon RI internalization, elevated IgE + Ag-induced degranulation and IL-6 production, and the decreased ability of -/- cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface Fc epsilon RI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen-activated -/- mast cells. By contrast, correction of these -/- phenotypes required a functional Vps9 domain. Thus, Fc epsilon RI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.

Mast cells can promote the development of multiple features of chronic asthma in mice.

Bronchial asthma, the most prevalent cause of significant respiratory morbidity in the developed world, typically is a chronic disorder associated with long-term changes in the airways. We developed a mouse model of chronic asthma that results in markedly increased numbers of airway mast cells, enhanced airway responses to methacholine or antigen, chronic inflammation including infiltration with eosinophils and lymphocytes, airway epithelial goblet cell hyperplasia, enhanced expression of the mucin genes Muc5ac and Muc5b, and increased levels of lung collagen. Using mast cell-deficient (Kit(W-sh/W-sh) and/or Kit(W/W-v)) mice engrafted with FcRgamma+/+ or FcRgamma-/- mast cells, we found that mast cells were required for the full development of each of these features of the model. However, some features also were expressed, although usually at less than wild-type levels, in mice whose mast cells lacked FcRgamma and therefore could not be activated by either antigen- and IgE-dependent aggregation of Fc epsilonRI or the binding of antigen-IgG1 immune complexes to Fc gammaRIII. These findings demonstrate that mast cells can contribute to the development of multiple features of chronic asthma in mice and identify both Fc Rgamma-dependent and Fc Rgamma-independent pathways of mast cell activation as important for the expression of key features of this asthma model.

RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells.

We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (Fc epsilonRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (-/-) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1-/- BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1-/- BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1-/- BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef1-/- BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells.

Using mast cell knock-in mice to analyze the roles of mast cells in allergic responses in vivo.

It is well established that mast cells are important effector cells mediating the acute phase of IgE-associated allergic disorders, but their roles in late phase reactions and chronic allergic inflammation are not well defined. Here we describe an experimental approach for analyzing mast cell functions in vivo by comparing the biological responses in wild-type mice, genetically mast cell-deficient mice, and 'mast cell knock-in mice' (mast cell-deficient mice selectively repaired of their mast cell deficiency). Studies using 'mast cell knock-in mice' have indicated that mast cells can contribute importantly to IgE-associated late phase reactions and to chronic allergic inflammation. Moreover, 'mast cell knock-in mice' containing adoptive-transferred mast cell populations with defined alterations in the expression of specific mast cell products can be used to characterize the mechanisms by which mast cells contribute to the expression of the response of interest.

Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.

Mice carrying certain mutations in the white spotting (W) locus (ie, c-kit) exhibit reduced c-kit tyrosine kinase-dependent signaling that results in mast cell deficiency and other phenotypic abnormalities. The c-kit mutations in Kit(W/W-v) mice impair melanogenesis and result in anemia, sterility, and markedly reduced levels of tissue mast cells. In contrast, Kit(W-sh/W-sh) mice, bearing the W-sash (W(sh)) inversion mutation, have mast cell deficiency but lack anemia and sterility. We report that adult Kit(W-sh/W-sh) mice had a profound deficiency in mast cells in all tissues examined but normal levels of major classes of other differentiated hematopoietic and lymphoid cells. Unlike Kit(W/W-v) mice, Kit(W-sh/W-sh) mice had normal numbers of TCR gammadelta intraepithelial lymphocytes in the intestines and did not exhibit a high incidence of idiopathic dermatitis, ulcers, or squamous papillomas of the stomach, but like Kit(W/W-v) mice, they lacked interstitial cells of Cajal in the gut and exhibited bile reflux into the stomach. Systemic or local reconstitution of mast cell populations was achieved in nonirradiated adult Kit(W-sh/W-sh) mice by intravenous, intraperitoneal, or intradermal injection of wild-type bone marrow-derived cultured mast cells but not by transplantation of wild-type bone marrow cells. Thus, Kit(W-sh/W-sh) mice represent a useful model for mast cell research, especially for analyzing mast cell function in vivo.

RabGEF1, a negative regulator of Ras signalling, mast cell activation and skin inflammation.

Mast cell activation induced by the aggregation of FcepsilonRI with IgE and antigen is mediated through the activation of multiple protein kinase cascades. This process induces mast cells to undergo degranulation, to synthesize and release lipid mediators, and to secrete multiple cytokines, chemokines and growth factors. We found that RabGEF1 (Rabex-5) binds to Ras and negatively regulates Ras activation and downstream effector pathways during FcepsilonRI-dependent mouse mast cell activation. Mast cells derived from RabGEF1-deficient mice exhibit significantly enhanced levels of degranulation, release of lipid mediators and secretion of cytokines in response to FcepsilonRI aggregation. RabGEF1 knockout mice have increased perinatal mortality and the mice that do survive develop severe skin inflammation and increased numbers of mast cells in the dermis, some of which exhibit morphological evidence of degranulation. These mice also show elevated concentrations of serum histamine and IgE. Thus, RabGEF1 is a negative regulator of Ras signalling and FcepsilonRI-dependent mast cell activation in vitro, and a lack of RabGEF1 results in the development of elevated numbers of mast cells in the skin and severe skin inflammation in vivo.

RabGEF1 is a negative regulator of mast cell activation and skin inflammation.

Mast cell activation induced by aggregation of Fc epsilon RI receptors with immunoglobulin E and antigen is mediated through the activation of multiple protein kinase cascades. Here we report that the regulatory protein RabGEF1 bound to Ras and negatively regulated Ras activation and its 'downstream' effector pathways in Fc epsilon RI-dependent mast cell activation. RabGEF1-deficient mast cells showed enhanced degranulation and release of lipid mediators and cytokines in response to Fc epsilon RI aggregation. RabGEF1-deficient mice developed severe skin inflammation and had increased numbers of mast cells. Thus, RabGEF1 is a negative regulator of Fc epsilon RI-dependent mast cell activation, and a lack of RabGEF1 results in the development of skin inflammation in vivo.

Transcriptional response of human mast cells stimulated via the Fc(epsilon)RI and identification of mast cells as a source of IL-11.

BACKGROUND: In asthma and other allergic disorders, the activation of mast cells by IgE and antigen induces the cells to release histamine and other mediators of inflammation, as well as to produce certain cytokines and chemokines. To search for new mast cell products, we used complementary DNA microarrays to analyze gene expression in human umbilical cord blood-derived mast cells stimulated via the high-affinity IgE receptor (Fc(epsilon)RI). RESULTS: One to two hours after Fc(epsilon)RI-dependent stimulation, more than 2,400 genes (about half of which are of unknown function) exhibited 2-200 fold changes in expression. The transcriptional program included changes in the expression of IL-11 and at least 30 other cytokines and chemokines. Human mast cells secreted 130-529 pg of IL-11/106 cells by 6 h after stimulation with anti-IgE. CONCLUSION: Our initial analysis of the transcriptional program induced in in vitro-derived human mast cells stimulated via the Fc(epsilon)RI has identified many products that heretofore have not been associated with this cell type, but which may significantly influence mast cell function in IgE-associated host responses. We also have demonstrated that mast cells stimulated via the Fc(epsilon)RI can secrete IL-11. Based on the previously reported biological effects of IL-11, our results suggest that production of IL-11 may represent one link between IgE-dependent mast cell activation in subjects with allergic asthma and the development of a spectrum of structural changes in the airways of these individuals; such changes, collectively termed "airway remodeling," can constitute an important long term consequence of asthma.

Mast cells derived from embryonic stem cells: a model system for studying the effects of genetic manipulations on mast cell development, phenotype, and function in vitro and in vivo.

Large quantities of highly enriched populations of mast cells can be generated from mouse embryonic stem (ES) cells using an in vitro differentiation system. These embryonic stem cell-derived mast cells (ESMCs) exhibit many similarities to mouse bone marrow-derived cultured mast cells (BMCMCs), including the abilities to survive and to orchestrate immunologically specific immunoglobulin E (IgE)-dependent reactions in vivo after transplantation into genetically mast cell-deficient KitW/KitW-v mice. Coupled with the current spectrum of techniques for genetically manipulating ES cells, ESMCs represent a unique model system to analyze the effects of specific alterations in gene structure, expression, or function, including embryonic lethal mutations, on mast cell development, phenotype, and function in vitro and in vivo.