Mast cells control lung type 2 inflammation via prostaglandin E2-driven soluble ST2.

Severe asthma and sinus disease are consequences of type 2 inflammation (T2I), mediated by interleukin (IL)-33 signaling through its membrane-bound receptor, ST2. Soluble (s)ST2 reduces available IL-33 and limits T2I, but little is known about its regulation. We demonstrate that prostaglandin E2 (PGE2) drives production of sST2 to limit features of lung T2I. PGE2-deficient mice display diminished sST2. In humans with severe respiratory T2I, urinary PGE2 metabolites correlate with serum sST2. In mice, PGE2 enhanced sST2 secretion by mast cells (MCs). Mice lacking MCs, ST2 expression by MCs, or E prostanoid (EP)2 receptors by MCs showed reduced sST2 lung concentrations and strong T2I. Recombinant sST2 reduced T2I in mice lacking PGE2 or ST2 expression by MCs back to control levels. PGE2 deficiency also reversed the hyperinflammatory phenotype in mice lacking ST2 expression by MCs. PGE2 thus suppresses T2I through MC-derived sST2, explaining the severe T2I observed in low PGE2 states.

IL-4Rα signaling promotes barrier-altering oncostatin M and IL-6 production in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is a severe disease involving dysregulated type 2 inflammation. However, the role other inflammatory pathways play in AERD is poorly understood. OBJECTIVE: We sought to broadly define the inflammatory milieu of the upper respiratory tract in AERD and to determine the effects of IL-4Rα inhibition on mediators of nasal inflammation. METHODS: Twenty-two AERD patients treated with dupilumab for 3 months were followed over 3 visits and compared to 10 healthy controls. Nasal fluid was assessed for 45 cytokines and chemokines using Olink Target 48. Blood neutrophils and cultured human mast cells, monocytes/macrophages, and nasal fibroblasts were assessed for response to IL-4/13 stimulation in vitro. RESULTS: Of the nasal fluid cytokines measured, nearly one third were higher in AERD patients compared to healthy controls, including IL-6 and the IL-6 family-related cytokine oncostatin M (OSM), both of which correlated with nasal albumin levels, a marker of epithelial barrier dysregulation. Dupilumab significantly decreased many nasal mediators, including OSM and IL-6. IL-4 stimulation induced OSM production from mast cells and macrophages but not from neutrophils, and OSM and IL-13 stimulation induced IL-6 production from nasal fibroblasts. CONCLUSION: In addition to type 2 inflammation, innate and IL-6-related cytokines are also elevated in the respiratory tract in AERD. Both OSM and IL-6 are locally produced in nasal polyps and likely promote pathology by negatively affecting epithelial barrier function. IL-4Rα blockade, although seemingly directed at type 2 inflammation, also decreases mediators of innate inflammation and epithelial dysregulation, which may contribute to dupilumab's therapeutic efficacy in AERD.

IgG:FcγRIIb signals block effector programs of IgE:FcεRI-activated mast cells but spare survival pathways.

BACKGROUND: IgE-induced mast cell (MC) degranulation can be inhibited by IgG antibodies, signaling via FcγRIIb, but the effects of IgG on IgE-induced MC transcription are unknown. OBJECTIVE: We sought to assess inhibitory IgG:FcγRIIb effects on MC responses to IgE using complementary transcriptomic and functional approaches. METHODS: RNA sequencing was performed on bone marrow-derived MCs from wild-type and FcγRIIb-deficient mice to identify genes activated following IgE receptor crosslinking that were further modulated in the presence of antigen-specific IgG in an FcγRIIb-dependent fashion. Parallel analyses of signaling pathways and allergic responses in vivo were performed to assess the impact of these changes in gene expression. RESULTS: Rapid changes in the transcription of 879 genes occurred in MCs activated by IgE, peaking at 1 hour. Surprisingly, only 12% of these were altered by IgG signaling via FcγRIIb, including numerous transcripts involved in orchestrating type 2 responses linked to spleen tyrosine kinase signaling. Consistent with this finding, IgG suppressed IgE-induced phospho-intermediates in the spleen tyrosine kinase signaling pathway. In vivo studies confirmed that the IgG-mediated suppression of both systemic anaphylaxis and MC-driven tissue recruitment of inflammatory cells following allergen challenge was dependent on FcγRIIb. In contrast, genes in the STAT5a cell survival pathway were unaltered by IgG, and STAT5a phosphorylation increased after IgE-induced MC activation but was unaffected by IgG. CONCLUSIONS: Our findings indicate that inhibitory IgG:FcγRIIb signals block an IgE-induced proallergic program but spare a prosurvival program.

Defining mast cell differentiation and heterogeneity through single-cell transcriptomics analysis.

Mast cells (MCs) are widely recognized as central effector cells during type 2 inflammatory reactions and thought to also play a role in innate immune responses, wound healing, and potentially cancer. Circulating progenitor cells mature to MCs in peripheral tissues, where they exhibit phenotypic and functional heterogeneity. This diversity likely originates from differences in MC development imprinted by microenvironmental signals. The advent of single-cell transcriptomics reveals MC diversity beyond differences in proteases that were classically used to identify MC phenotypes. Here, we provide an overview of the current knowledge on MC progenitor differentiation and characteristics, and MC heterogeneity seen in health versus disease, that are drastically advanced through single-cell profiling technologies. This powerful approach can provide detailed cellular maps of tissues to decipher the complex cellular functions and interactions that may lead to identifying candidate factors to target in therapies.

Detection and Isolation of Airway Mast Cell Subsets in Mouse and Human.

Mast cells are heterogeneous, tissue-resident immune effector cells widely recognized to play pathobiologic roles in the development of mucosal type 2 inflammation. While mast cell progenitors can be found in peripheral blood, phenotypically mature mast cells can only be found within peripheral tissues. Here, we describe optimized tissue digestion protocols for obtaining mast cells from the murine lung and from human sinus tissue. Following tissue digestion, mast cells can be identified and sorted by flow cytometry using antibodies against defined surface markers. We also provide protocol for intracellular protease immunostaining, allowing for further characterization of mast cells.

Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation.

Murine mast cells (MCs) contain two lineages: inducible bone marrow-derived mucosal MCs (MMCs) and constitutive embryonic-derived connective tissue MCs (CTMCs). Here, we use RNA sequencing, flow cytometry, and genetic deletion in two allergic lung inflammation models to define these two lineages. We found that inducible MCs, marked by ß7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. ß7High MCs expand and mature during lung inflammation as part of a TGF-ß-inducible transcriptional program that includes the MMC-associated proteases Mcpt1 and Mcpt2, the basophil-associated protease Mcpt8, granule components, and the epithelial-binding αE integrin. In vitro studies using bone marrow-derived MCs (BMMCs) identified a requirement for SCF in this this TGF-ß-mediated development and found that epithelial cells directly elicit TGF-ß-dependent BMMC up-regulation of mMCP-1 and αE integrin. Thus, our findings characterize the expansion of a distinct inducible MC subset in C57BL/6 mice and highlight the potential for epithelium to direct MMC development.

Human airway mast cells proliferate and acquire distinct inflammation-driven phenotypes during type 2 inflammation.

Mast cells (MCs) play a pathobiologic role in type 2 (T2) allergic inflammatory diseases of the airway, including asthma and chronic rhinosinusitis with nasal polyposis (CRSwNP). Distinct MC subsets infiltrate the airway mucosa in T2 disease, including subepithelial MCs expressing the proteases tryptase and chymase (MCTC) and epithelial MCs expressing tryptase without chymase (MCT). However, mechanisms underlying MC expansion and the transcriptional programs underlying their heterogeneity are poorly understood. Here, we use flow cytometry and single-cell RNA-sequencing (scRNA-seq) to conduct a comprehensive analysis of human MC hyperplasia in CRSwNP, a T2 cytokine-mediated inflammatory disease. We link discrete cell surface phenotypes to the distinct transcriptomes of CRSwNP MCT and MCTC, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38highCD117high MCs that is markedly expanded during T2 inflammation. These CD38highCD117high MCs exhibit an intermediate phenotype relative to the expanded MCT and MCTC subsets. CD38highCD117high MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease, a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MCT-like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MCTC in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest that in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.

Human Mast Cell Development from Hematopoietic Stem Cells in a Connective Tissue-Equivalent Model.

Mast cells (MCs) play critical roles in the pathogenesis of IgE- and non-IgE-mediated immune responses, as well as host defense against parasites, bacteria, and viruses. Due to the effect of extracellular matrix components on tissue morphogenesis and cell behavior, utilizing a tissue model that mimics MC microenvironmental conditions in vivo has greater relevance for in vitro studies. For this work, MCs were developed within a connective tissue-equivalent model and cell function was examined in response to an allergen. MCs are located in proximity to fibroblasts and endothelial cells (ECs) that play a role in MC development and maturity. Accordingly, MC progenitors isolated from human peripheral blood were co-cultured with human primary fibroblasts in a 3D collagen matrix to represent the connective tissue. The matrix was coated with type IV collagen and fibronectin before seeding with primary human ECs, representing the capillary wall. The stem cell-derived cells demonstrated MC characteristics, including typical MC morphology, and the expression of cytoplasmic granules and phenotypic markers. Also, the generated cells released histamine in IgE-mediated reactions, showing typical MC functional phenotype in an immediate-type allergenic response. The created tissue model is applicable to a variety of research studies and allergy testing. Impact Statement Mast cells (MCs) are key effector and immunoregulatory cells in immune disorders; however, their role is not fully understood. Few studies have investigated human ex vivo MCs in culture, due to the difficulties in isolating large numbers. Our study demonstrates, for the first time, the generation of cells exhibiting MC phenotypic and functional characteristics from hematopoietic stem cells within a connective tissue-equivalent model with ancillary cells. Utilizing the 3D matrix-embedded cells can advance our understanding of MC biological profile and immunoregulatory roles. The tissue model can also be used for studying the mechanism of allergic diseases and other inflammatory disorders.

Development of Human Mast Cells from Hematopoietic Stem Cells within a 3D Collagen Matrix: Effect of Stem Cell Media on Mast Cell Generation.

Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues, large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells, the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs, fibroblasts, and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival, while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs.

A Three-Dimensional Human Tissue-Engineered Lung Model to Study Influenza A Infection.

Influenza A virus (IAV) claims ∼250,000-500,000 lives annually worldwide. Currently, there are a few in vitro models available to study IAV immunopathology. Monolayer cultures of cell lines and primary lung cells (two-dimensional [2D] cell culture) is the most commonly used tool, however, this system does not have the in vivo-like structure of the lung and immune responses to IAV as it lacks the three-dimensional (3D) tissue structure. To recapitulate the lung physiology in vitro, a system that contains multiple cell types within a 3D environment that allows cell movement and interaction would provide a critical tool. In this study, as a first step in designing a 3D-Human Tissue-Engineered Lung Model (3D-HTLM), we describe the 3D culture of primary human small airway epithelial cells (HSAEpCs) and determined the immunophenotype of this system in response to IAV infections. We constructed a 3D chitosan-collagen scaffold and cultured HSAEpCs on these scaffolds at air-liquid interface (ALI). These 3D cultures were compared with 2D-cultured HSAEpCs for viability, morphology, marker protein expression, and cell differentiation. Results showed that the 3D-cultured HSAEpCs at ALI yielded maximum viable cells and morphologically resembled the in vivo lower airway epithelium. There were also significant increases in aquaporin-5 and cytokeratin-14 expression for HSAEpCs cultured in 3D compared to 2D. The 3D culture system was used to study the infection of HSAEpCs with two major IAV strains, H1N1 and H3N2. The HSAEpCs showed distinct changes in marker protein expression, both at mRNA and protein levels, and the release of proinflammatory cytokines. This study is the first step in the development of the 3D-HTLM, which will have wide applicability in studying pulmonary pathophysiology and therapeutics development.