Identification of redundancy between human FcεRIß and MS4A6A proteins points toward additional complex mechanisms for FcεRI trafficking and signaling.

BACKGROUND: Allergic diseases are triggered by signaling through the high-affinity IgE receptor, FcεRI. In both mast cells (MCs) and basophils, FcεRI is a tetrameric receptor complex comprising a ligand-binding α subunit (FcεRIα), a tetraspan ß subunit (FcεRIß, MS4A2) responsible for trafficking and signal amplification, and a signal transducing dimer of single transmembrane γ subunits (FcεRIγ). However, FcεRI also exists as presumed trimeric complexes that lack FcεRIß and are expressed on several cell types outside the MC and basophil lineages. Despite known differences between humans and mice in the presence of the trimeric FcεRI complex, questions remain as to how it traffics and whether it signals in the absence of FcεRIß. We have previously reported that targeting FcεRIß with exon-skipping oligonucleotides eliminates IgE-mediated degranulation in mouse MCs, but equivalent targeting in human MCs was not effective at reducing degranulation. RESULTS: Here, we report that the FcεRIß-like protein MS4A6A exists in human MCs and compensates for FcεRIß in FcεRI trafficking and signaling. Human MS4A6A promotes surface expression of FcεRI complexes and facilitates degranulation. MS4A6A and FcεRIß are encoded by highly related genes within the MS4A gene family that cluster within the human gene loci 11q12-q13, a region linked to allergy and asthma susceptibility. CONCLUSIONS: Our data suggest the presence of either FcεRIß or MS4A6A is sufficient for degranulation, indicating that MS4A6A could be an elusive FcεRIß-like protein in human MCs that performs compensatory functions in allergic disease.

Targeting KIT by frameshifting mRNA transcripts as a therapeutic strategy for aggressive mast cell neoplasms.

Activating mutations in c-KIT are associated with the mast cell (MC) clonal disorders cutaneous mastocytosis and systemic mastocytosis and its variants, including aggressive systemic mastocytosis, MC leukemia, and MC sarcoma. Currently, therapies inhibiting KIT signaling are a leading strategy to treat MC proliferative disorders. However, these approaches may have off-target effects, and in some patients, complete remission or improved survival time cannot be achieved. These limitations led us to develop an approach using chemically stable exon skipping oligonucleotides (ESOs) that induce exon skipping of precursor (pre-)mRNA to alter gene splicing and introduce a frameshift into mature KIT mRNA transcripts. The result of this alternate approach results in marked downregulation of KIT expression, diminished KIT signaling, inhibition of MC proliferation, and rapid induction of apoptosis in neoplastic HMC-1.2 MCs. We demonstrate that in vivo administration of KIT targeting ESOs significantly inhibits tumor growth and systemic organ infiltration using both an allograft mastocytosis model and a humanized xenograft MC tumor model. We propose that our innovative approach, which employs well-tolerated, chemically stable oligonucleotides to target KIT expression through unconventional pathways, has potential as a KIT-targeted therapeutic alone, or in combination with agents that target KIT signaling, in the treatment of KIT-associated malignancies.

A truncated splice-variant of the FcεRIß receptor subunit is critical for microtubule formation and degranulation in mast cells.

Human linkage analyses have implicated the MS4A2-containing gene locus (encoding FcεRIß) as a candidate for allergy susceptibility. We have identified a truncation of FcεRIß (t-FcεRIß) in humans that contains a putative calmodulin-binding domain and thus, we sought to identify the role of this variant in mast cell function. We determined that t-FcεRIß is critical for microtubule formation and degranulation and that it may perform this function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals. Mutagenesis studies suggest that calmodulin binding to t-FcεRIß in the presence of Ca2+ could be critical for t-FcεRIß function. In addition, gene targeting of t-FcεRIß attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals. Therefore, t-FcεRIß mediates Ca2+ -dependent microtubule formation, which promotes degranulation and cytokine release. Because t-FcεRIß has this critical function, it represents a therapeutic target for the downregulation of allergic inflammation.

Regulation of Trafficking and Signaling of the High Affinity IgE Receptor by FcεRIß and the Potential Impact of FcεRIß Splicing in Allergic Inflammation.

Mast cells are tissue-resident immune cells that function in both innate and adaptive immunity through the release of both preformed granule-stored mediators, and newly generated proinflammatory mediators that contribute to the generation of both the early and late phases of the allergic inflammatory response. Although mast cells can be activated by a vast array of mediators to contribute to homeostasis and pathophysiology in diverse settings and contexts, in this review, we will focus on the canonical setting of IgE-mediated activation and allergic inflammation. IgE-dependent activation of mast cells occurs through the high affinity IgE receptor, FcεRI, which is a multimeric receptor complex that, once crosslinked by antigen, triggers a cascade of signaling to generate a robust response in mast cells. Here, we discuss FcεRI structure and function, and describe established and emerging roles of the ß subunit of FcεRI (FcεRIß) in regulating mast cell function and FcεRI trafficking and signaling. We discuss current approaches to target IgE and FcεRI signaling and emerging approaches that could target FcεRIß specifically. We examine how alternative splicing of FcεRIß alters protein function and how manipulation of splicing could be employed as a therapeutic approach. Targeting FcεRI directly and/or IgE binding to FcεRI are promising approaches to therapeutics for allergic inflammation. The characteristic role of FcεRIß in both trafficking and signaling of the FcεRI receptor complex, the specificity to IgE-mediated activation pathways, and the preferential expression in mast cells and basophils, makes FcεRIß an excellent, but challenging, candidate for therapeutic strategies in allergy and asthma, if targeting can be realized.

Vibratory Urticaria Associated with a Missense Variant in ADGRE2.

Patients with autosomal dominant vibratory urticaria have localized hives and systemic manifestations in response to dermal vibration, with coincident degranulation of mast cells and increased histamine levels in serum. We identified a previously unknown missense substitution in ADGRE2 (also known as EMR2), which was predicted to result in the replacement of cysteine with tyrosine at amino acid position 492 (p.C492Y), as the only nonsynonymous variant cosegregating with vibratory urticaria in two large kindreds. The ADGRE2 receptor undergoes autocatalytic cleavage, producing an extracellular subunit that noncovalently binds a transmembrane subunit. We showed that the variant probably destabilizes an autoinhibitory subunit interaction, sensitizing mast cells to IgE-independent vibration-induced degranulation. (Funded by the National Institutes of Health.).

Exon skipping of FcεRIß eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy.

Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the ß-subunit of the high-affinity IgE receptor (FcεRIß) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIß expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIß is a potential approach for mast cell-specific treatment of allergic diseases.

Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation.

Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (FcεRI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on FcεRI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (∼50-fold) of cells to FcεRI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLCγ1. In contrast, degranulation and calcium responses elicited by the G protein-coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed FcεRI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate FcεRI-mediated degranulation independently of mTORC2.

Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions.

BACKGROUND: Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS: We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS: Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS: Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation.

BACKGROUND: Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied. OBJECTIVE: Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease. METHODS: We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking. RESULTS: Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation. CONCLUSION: This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others.

The exon-skipping oligonucleotide, KitStop, depletes tissue-resident mast cells in vivo to ameliorate anaphylaxis.

Introduction: Anaphylaxis represents the most extreme and life-threatening form of allergic disease and is considered a medical emergency requiring immediate intervention. Additionally, some people with mastocytosis experience recurrent episodes of anaphylaxis during normal daily activities without exposure to known triggers. While acute therapy consists primarily of epinephrine and supportive care, chronic therapy relies mostly on desensitization and immunotherapy against the offending allergen, which is a time-consuming and sometimes unsuccessful process. These treatments also necessitate identification of the triggering allergen which is not always possible, and thus highlighting a need for alternative treatments for mast cell-mediated diseases. Methods: The exon-skipping oligonucleotide KitStop was administered to mice intradermally, intraperitoneally, or systemically at a dose of 12.5 mg/kg. Local mast cell numbers were enumerated via peritoneal lavage or skin histology, and passive systemic anaphylaxis was induced to evaluate KitStop's global systemic effect. A complete blood count and biochemistry panel were performed to assess the risk of acute toxicity following KitStop administration. Results: Here, we report the use of an exon-skipping oligonucleotide, which we have previously termed KitStop, to safely reduce the severity and duration of the anaphylactic response via mast cell depopulation in tissues. KitStop administration results in the integration of a premature stop codon within the mRNA transcript of the KIT receptor-a receptor tyrosine kinase found primarily on mast cells and whose gain-of-function mutation can lead to systemic mastocytosis. Following either local or systemic KitStop treatment, mice had significantly reduced mast cell numbers in the skin and peritoneum. In addition, KitStop-treated mice experienced a significantly diminished anaphylactic response using a model of passive systemic anaphylaxis when compared with control mice. Discussion: KitStop treatment results in a significant reduction in systemic mast cell responses, thus offering the potential to serve as a powerful additional treatment modality for patients that suffer from anaphylaxis.

Low-dose intrapulmonary drug delivery device for studies on next-generation therapeutics in mice.

Although nebulizers have been developed for delivery of small molecules in human patients, no tunable device has been purpose-built for targeted delivery of modern large molecule and temperature-sensitive therapeutics to mice. Mice are used most of all species in biomedical research and have the highest number of induced models for human-relevant diseases and transgene models. Regulatory approval of large molecule therapeutics, including antibody therapies and modified RNA highlight the need for quantifiable dose delivery in mice to model human delivery, proof-of-concept studies, efficacy, and dose-response. To this end, we developed and characterized a tunable nebulization system composed of an ultrasonic transducer equipped with a mesh nebulizer fitted with a silicone restrictor plate modification to control the nebulization rate. We have identified the elements of design that influence the most critical factors to targeted delivery to the deep lungs of BALB/c mice. By comparing an in silico model of the mouse lung with experimental data, we were able to optimize and confirm the targeted delivery of over 99% of the initial volume to the deep portions of the mouse lung. The resulting nebulizer system provides targeted lung delivery efficiency far exceeding conventional nebulizers preventing waste of expensive biologics and large molecules during proof-of-concept and pre-clinical experiments involving mice. (Word Count =207).

Human lung mast cells mediate pneumococcal cell death in response to activation by pneumolysin.

Mast cells are emerging as contributors to innate immunity. Mouse mast cells have a pivotal role in protection against bacterial infection, and human cord blood-derived mast cells reduce bacterial viability in culture. The objectives of this study were to determine whether human lung mast cells (HLMCs) might be protective against pneumococcal lung infection through direct antimicrobial activity. Tissue-derived HLMCs and the human mast cell lines HMC-1 and LAD2 were cocultured with wild-type and mutant pneumococci, and viability and functional assays were performed. Mast cells were also stimulated with purified pneumolysin. HLMCs killed wild-type serotype-2 (D39) pneumococci in coculture but had no effect on an isogenic pneumolysin-deficient (PLN-A) pneumococcus. D39 wild-type, but not PLN-A pneumococci, induced the release of leukotriene C4 from human mast cells in a dose-dependent manner, which was not accompanied by histamine release. Stimulation of mast cells with sublytic concentrations of purified pneumolysin replicated this effect. Furthermore, pneumolysin induced the release of the cathelicidin LL-37 from HLMCs, purified LL-37 reduced pneumococcal viability, and neutralizing Ab to LL-37 attenuated mast cell-dependent pneumococcal killing. In addition, at high concentrations, all pneumococcal strains tested reduced HLMC viability through a combination of pneumolysin and H2O2-dependent mechanisms. HLMCs exhibit direct antimicrobial activity to pneumococci through their activation by pneumolysin. This antimicrobial activity is mediated, in part, by the release of LL-37 from HLMCs. This suggests that mast cells provide an early warning system and potentially limit pneumococcal dissemination early in the course of invasive pulmonary pneumococcal disease.

Functional KCa3.1 K+ channels are required for human fibrocyte migration.

BACKGROUND: Fibrocytes are bone marrow-derived CD34(+) collagen I-positive cells present in peripheral blood that develop α-smooth muscle actin expression and contractile activity in tissue culture. They are implicated in the pathogenesis of tissue remodeling and fibrosis in both patients with asthma and those with idiopathic pulmonary fibrosis. Targeting fibrocyte migration might therefore offer a new approach for the treatment of these diseases. Ion channels play key roles in cell function, but the ion-channel repertoire of human fibrocytes is unknown. OBJECTIVE: We sought to examine whether human fibrocytes express the K(Ca)3.1 K(+) channel and to determine its role in cell differentiation, survival, and migration. METHODS: Fibrocytes were cultured from the peripheral blood of healthy subjects and patients with asthma. Whole-cell patch-clamp electrophysiology was used for the measurement of ion currents, whereas mRNA and protein were examined to confirm channel expression. Fibrocyte migration and proliferation assays were performed in the presence of K(Ca)3.1 ion-channel blockers. RESULTS: Human fibrocytes cultured from the peripheral blood of both healthy control subjects and asthmatic patients expressed robust K(Ca)3.1 ion currents together with K(Ca)3.1 mRNA and protein. Two specific and distinct K(Ca)3.1 blockers (TRAM-34 and ICA-17043) markedly inhibited fibrocyte migration in transwell migration assays. Channel blockers had no effect on fibrocyte growth, apoptosis, or differentiation in cell culture. CONCLUSIONS: The K(+) channel K(Ca)3.1 plays a key role in human fibrocyte migration. Currently available K(Ca)3.1-channel blockers might therefore attenuate tissue fibrosis and remodeling in patients with diseases such as idiopathic pulmonary fibrosis and asthma through the inhibition of fibrocyte recruitment.

Immunologic and pathologic characterization of a novel swine biomedical research model for eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergy-mediated condition with an increasing incidence in both children and adults. Despite EoE's strong impact on human health and welfare, there is a large unmet need for treatments with only one recently FDA-approved medication for EoE. The goal of this study was to establish swine as a relevant large animal model for translational biomedical research in EoE with the potential to facilitate development of therapeutics. We recently showed that after intraperitoneal sensitization and oral challenge with the food allergen hen egg white protein (HEWP), swine develop esophageal eosinophilia-a hallmark of human EoE. Herein, we used a similar sensitization and challenge treatment and evaluated immunological and pathological markers associated with human EoE. Our data demonstrate that the incorporated sensitization and challenge treatment induces (i) a systemic T-helper 2 and IgE response, (ii) a local expression of eotaxin-1 and other allergy-related immune markers, (iii) esophageal eosinophilia (>15 eosinophils/0.24 mm2), and (iv) esophageal endoscopic findings including linear furrows and white exudates. Thereby, we demonstrate that our sensitization and oral challenge protocol not only induces the underlying immune markers but also the micro- and macro-pathological hallmarks of human EoE. This swine model for EoE represents a novel relevant large animal model that can drive translational biomedical research to develop urgently needed treatment strategies for EoE.

A novel FcεRIß-chain truncation regulates human mast cell proliferation and survival.

Mast cells contribute to allergy through IgE-dependent activation via the high-affinity IgE receptor FcεRI. The role of the FcεRIß chain (MS4A2) in mast cell function is not understood fully, although it serves to amplify FcεRI-dependent signaling. We demonstrate the expression of a novel MS4A2 truncation lacking exon 3 in human mast cells termed MS4A2(trunc). MS4A2(trunc) gene expression was regulated negatively by the mast cell growth factor stem cell factor (SCF), and its expression was not detected in the SCF receptor gain-of-function human mast cell line HMC-1. Unlike MS4A2, MS4A2(trunc) did not traffic to the cytoplasmic membrane but instead was associated with the nuclear membrane. Overexpression of MS4A2(trunc) induced human lung mast cell death and profoundly inhibited HMC-1 cell proliferation by inducing G(2)-phase cell cycle arrest and apoptosis. Thus, we have identified a novel splice variant of MS4A2 that might be important in the regulation of human mast cell proliferation and survival. This finding demonstrates that the MS4A2 gene has multiple roles, extending beyond the regulation of acute allergic responses. By understanding the mechanisms regulating its function, it might be possible to induce its expression in mast cells in vivo, which could lead to better treatments for diseases such as mastocytosis and asthma.

Counterregulation of beta(2)-adrenoceptor function in human mast cells by stem cell factor.

BACKGROUND: Mast cells contribute to the pathophysiology of asthma with the sustained release of both preformed and newly generated mediators in response to allergens and other diverse stimuli. Stem cell factor (SCF) is the key human mast cell growth factor, but also primes mast cells for mediator release. SCF expression is markedly increased in asthmatic airways. Short-acting beta(2)-adrenoceptor drugs such as albuterol inhibit human lung mast cell (HLMC) degranulation in vitro in the absence of SCF, but their effect in the presence of SCF is not known. OBJECTIVE: The aim of this study was to elucidate the effects of albuterol on HLMC function in the presence of SCF. METHODS: Mediator release and K(Ca)3.1 ion channel activity were analyzed in purified HLMC. Intracellular signalling and beta(2)-adrenoceptor phosphorylation and internalization were analyzed in the HMC-1 human mast cell line. RESULTS: beta(2)-Adrenoceptor agonist-dependent inhibition of K(Ca)3.1 ion channels and HLMC mediator release was markedly attenuated in the presence of SCF. Remarkably, albuterol actually potentiated IgE-induced histamine release in a dose-dependent manner when both SCF and IgE were present. These effects were related to the SCF-dependent phosphorylation of Tyr350 on the beta(2)-adrenoceptor with immediate uncoupling of the receptor followed by receptor internalization. CONCLUSION: The potentially beneficial effects of beta(2)-adrenoceptor agonists in asthmatic airways may be blunted as a result of the high concentrations of SCF present.

Activation of human mast cells through the platelet-activating factor receptor.

BACKGROUND: In human subjects platelet-activating factor (PAF) concentrations are markedly increased in the plasma after anaphylactic reactions, and these correlate strongly with the severity of the response. The mechanism for the systemic spread of mast cell (MC) activation in anaphylaxis is often assumed to relate to the hematogenous spread of allergen, but this is implausible, and amplification mechanisms need to be considered. OBJECTIVE: We have investigated the ability of PAF to induce human MC degranulation using skin, lung, and peripheral blood (PB)-derived cultured MCs and the signaling pathways activated in PB-derived MCs in response to PAF. METHODS: The expression of PAF receptor was investigated by means of RT-PCR and Western blot analysis. Cell-signaling pathways in PB-derived MCs in response to PAF were investigated by analyzing the effect of various inhibitors and the silencing of phospholipase C (PLC) mRNA on PAF-mediated histamine release. RESULTS: We show for the first time that PAF induces histamine release from human lung MCs and PB-derived MCs but not skin MCs. Activation of PAF receptor-coupled G(alphai) leads to degranulation through PLCgamma1 and PLCbeta2 activation in human MCs. PAF-induced degranulation was rapid, being maximal at 5 seconds, and was partially dependent on extracellular Ca(2+). CONCLUSION: Our findings provide a mechanism whereby PAF mediates an amplification loop for MC activation in the generation of anaphylaxis.

Mast Cell-Biomaterial Interactions and Tissue Repair.

Tissue engineers often use biomaterials to provide structural support along with mechanical and chemical signals to modulate the wound healing process. Biomaterials that are implanted into the body interact with a heterogeneous and dynamic inflammatory environment that is present at the site of injury. Whether synthetically derived, naturally derived, or a combination of both, it is important to assess biomaterials for their ability to modulate inflammation to understand their potential clinical use. One important, but underexplored cell in the context of biomaterials is the mast cell (MC). MCs are granulocytic leukocytes that engage in a variety of events in both the innate and adaptive immune systems. Although highly recognized for their roles in allergic reactions, MCs play an important role in wound healing by recognizing antigens through pattern recognition receptors and the high-affinity immunoglobulin E receptor (FceRI) and releasing granules that affect cell recruitment, fibrosis, extracellular matrix deposition, angiogenesis, and vasculogenesis. MCs also mediate the foreign body response, contributing to the incorporation or rejection of implants. Studies of MC-biomaterial interactions can aid in the elucidation of MC roles during the host tissue response and tissue repair. This review is designed for those in the tissue engineering and biomaterial fields who are interested in exploring the role MCs may play in wound-biomaterial interactions and wound healing. With this review, we hope to inspire more research in the MC-biomaterial space to accelerate the design and construction of optimized implants. Impact statement Mast cells (MCs) are highly specialized inflammatory cells that have crucial, but not fully understood, roles in wound healing and tissue repair. Upon stimulation, they recognize foreign antigens and release granules that help orchestrate the inflammatory response after tissue damage or biomaterial implantation. This review summarizes the current use of MCs in biomaterial research along with literature from the past decade focusing on MC interactions with materials used for tissue repair and regeneration. Studying MC-biomaterial interactions will help (i) further understand the process of inflammation and (ii) design biomaterials and tissue-engineered constructs for optimal repair and regeneration.

Dermal Extracellular Matrix-Derived Hydrogels as an In Vitro Substrate to Study Mast Cell Maturation.

Mast cells (MCs) are pro-inflammatory tissue-resident immune cells that play a key role in inflammation. MCs circulate in peripheral blood as progenitors and undergo terminal differentiation in the tissue microenvironment where they can remain for many years. This in situ maturation results in tissue- and species-specific MC phenotypes, culminating in significant variability in response to environmental stimuli. There are many challenges associated with studying mature tissue-derived MCs, particularly in humans. In cases where cultured MCs are able to differentiate in two-dimensional in vitro cultures, there remains an inability for full maturation. Extracellular matrix (ECM) scaffolds provide for a more physiologically relevant environment for cells in vitro and have been shown to modulate the response of other immune cells such as T cells, monocytes, and macrophages. To improve current in vitro testing platforms of MCs and to assess future use of ECM scaffolds for MC regulation, we studied the in vitro response of human MCs cultured on decellularized porcine dermis hydrogels (dermis extracellular matrix hydrogel [dECM-H]). This study investigated the effect of dECM-H on cellular metabolic activity, cell viability, and receptor expression compared to collagen type I hydrogel (Collagen-H). Human MCs showed different metabolic activity when cultured in the dECM-H and also upregulated immunoglobulin E (IgE) receptors associated with MC maturation/activation compared to collagen type I. These results suggest an overall benefit in the long-term culture of human MCs in the dECM-H compared to Collagen-H providing important steps toward a model that is more representative of in vivo conditions. Graphical abstract [Formula: see text] Impact statement Mast cells (MCs) are difficult to culture in vitro as current culture conditions and substrates fail to promote similar phenotypic features observed in vivo. Extracellular matrix (ECM)-based biomaterials offer three-dimensional, tissue-specific environments that more closely resemble in vivo conditions. Our study explores the use of dermal ECM hydrogels for MC culture and shows significant upregulation of metabolic activity, cell viability, and gene expression of markers associated with MC maturation or activation compared to collagen type I-hydrogel and tissue culture plastic controls at 7 days. These results are among the first to describe MC behavior in response to ECM hydrogels.

Identification of an ATP/P2X7/mast cell pathway mediating ozone-induced bronchial hyperresponsiveness.

Ozone is a highly reactive environmental pollutant with well-recognized adverse effects on lung health. Bronchial hyperresponsiveness (BHR) is one consequence of ozone exposure, particularly for individuals with underlying lung disease. Our data demonstrated that ozone induced substantial ATP release from human airway epithelia in vitro and into the airways of mice in vivo and that ATP served as a potent inducer of mast cell degranulation and BHR, acting through P2X7 receptors on mast cells. Both mast cell-deficient and P2X7 receptor-deficient (P2X7-/-) mice demonstrated markedly attenuated BHR to ozone. Reconstitution of mast cell-deficient mice with WT mast cells and P2X7-/- mast cells restored ozone-induced BHR. Despite equal numbers of mast cells in reconstituted mouse lungs, mice reconstituted with P2X7-/- mast cells demonstrated significantly less robust BHR than mice reconstituted with WT mast cells. These results support a model where P2X7 on mast cells and other cell types contribute to ozone-induced BHR.