Mast cells (MCs) are pleiotropic cells that accumulate in the esophagus of patients with eosinophilic esophagitis (EoE) and are thought to contribute to disease pathogenesis, yet their properties and ...functions in this organ are largely unknown.
This study aimed to perform a comprehensive molecular and spatial characterization of esophageal MCs in EoE.
Esophageal biopsies obtained from patients with active EoE, patients with EoE in histologic remission, and individuals with histologically normal esophageal biopsies and no history of esophageal disease (ie, control individuals) were subject to single-cell RNA sequencing, flow cytometry, and immunofluorescence analyses.
This study probed 39,562 single esophageal cells by single-cell RNA sequencing; approximately 5% of these cells were MCs. Dynamic MC expansion was identified across disease states. During homeostasis, TPSAB1highAREGhigh resident MCs were mainly detected in the lamina propria and exhibited a quiescent phenotype. In patients with active EoE, resident MCs assumed an activated phenotype, and 2 additional proinflammatory MC populations emerged in the intraepithelial compartment, each linked to a proliferating MKI67high cluster. One proinflammatory activated MC population, marked as KIThighIL1RL1highFCER1Alow, was not detected in disease remission (termed “transient MC”), whereas the other population, marked as CMA1highCTSGhigh, was detected in disease remission where it maintained an activated state (termed “persistent MC”). MCs were prominent producers of esophageal IL-13 mRNA and protein, a key therapeutic target in EoE.
Esophageal MCs comprise heterogeneous populations with transcriptional signatures associated with distinct spatial compartmentalization and EoE disease status. In active EoE, they assume a proinflammatory state and locally proliferate, and they remain activated and poised to reinitiate inflammation even during disease remission.
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Eosinophilic asthma and nasal polyposis are hallmarks of aspirin-exacerbated respiratory disease (AERD), and IL-5 inhibition has been shown to provide therapeutic benefit. However, IL-5Rα is ...expressed on many cells in addition to eosinophils, and the mechanisms by which IL-5 inhibition leads to clinical benefit in eosinophilic asthma and nasal polyposis are unlikely to be due exclusively to antieosinophil effects.
We sought to identify the mechanisms by which anti–IL-5 treatment with mepolizumab improves respiratory inflammation in AERD.
The clinical characteristics, circulating granulocytes, nasal scraping transcripts, eosinophilic cationic protein, tryptase, and antibody levels, and urinary and nasal eicosanoid levels were measured for 18 subjects with AERD who were taking mepolizumab and compared with those of 18 matched subjects with AERD who were not taking mepolizumab.
Subjects taking mepolizumab had significantly fewer peripheral blood eosinophils and basophils, and those cells that remained had higher surface CRTH2 expression than did the cells from subjects not taking mepolizumab. Nasal prostaglandin F2α, prostaglandin D2 metabolites, leukotriene B4, and thromboxane levels were lower in subjects taking mepolizumab, as were urinary levels of tetranor–prostaglandin D2 and leukotriene E4. The nasal epithelial cell transcripts that were overexpressed among subjects with AERD who were taking mepolizumab were enriched for genes involved in tight junction formation and cilium organization. Nasal and urinary prostaglandin E2, tryptase, and antibody levels were not different between the 2 groups.
IL-5 inhibition in AERD decreases production of inflammatory eicosanoids and upregulates tight junction–associated nasal epithelial cell transcripts, likely due to decreased IL-5 signaling on tissue mast cells, eosinophils, and epithelial cells. These direct effects on multiple relevant immune cells contribute to the mechanism of benefit afforded by mepolizumab.
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The development of biomolecular devices that interface with biological systems to reveal new insights and produce novel functions is one of the defining goals of synthetic biology. Our lab previously ...described a synthetic, riboregulator system that affords for modular, tunable, and tight control of gene expression in vivo. Here we highlight several experimental advantages unique to this RNA-based system, including physiologically relevant protein production, component modularity, leakage minimization, rapid response time, tunable gene expression, and independent regulation of multiple genes. We demonstrate this utility in four sets of in vivo experiments with various microbial systems. Specifically, we show that the synthetic riboregulator is well suited for GFP fusion protein tracking in wild-type cells, tight regulation of toxic protein expression, and sensitive perturbation of stress response networks. We also show that the system can be used for logic-based computing of multiple, orthogonal inputs, resulting in the development of a programmable kill switch for bacteria. This work establishes a broad, easy-to-use synthetic biology platform for microbiology experiments and biotechnology applications.
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.
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•PGE2 is necessary to elevate sST2 levels in the lung with type 2 lung inflammation•Intraepithelial mast cells are the principal source of PGE2-driven lung sST2 production•Deficiency of mast-cell-derived sST2 exaggerates type 2 lung inflammation•PGE2 alters the mast cell transcriptome and promotes short Il1rl1 expression
Limiting IL-33-driven lung inflammation is a promising means of controlling asthma. Alhallak et al. demonstrate that prostaglandin E2 modulates airway mucosal mast cells and enhances their production of soluble ST2 (IL-33 receptor) to limit asthma-associated lung inflammation. These findings may also help explain the severity of PGE2-low respiratory disease.
The aim of this study was to assess if tactical and technical performance indicators (PIs) could be used in combination to model match outcomes in Australian Football (AF). A database of 101 ...technical PIs and 14 tactical PIs from every match in the 2009-2016 Australian Football League (AFL) seasons was merged. Two outcome measures Win-loss and Score margin were used as dependent variables. The top 45 ranked technical and tactical PIs from a feature selection process were used to model match outcome using decision tree and Generalised Linear Models (GLMs). Of the top 45 selected features, this included seven tactical PIs. The Win-loss-based Decision tree model achieved a classification accuracy of 89.0% and GLM 93.2%. A Score margin-based GLM achieved a root mean squared error (RMSE) of 6.9 points. A combined approach to the classification of match outcomes provided no improvement in model accuracy compared with previous literature. However, this study has established the relative importance of technical and tactical measures of performance in relation to successful team performance in AF.
The American Initiative in Mast Cell Diseases (AIM) held its inaugural investigator conference at Stanford University School of Medicine in May 2019. The overarching goal of this meeting was to ...establish a Pan-American organization of physicians and scientists with multidisciplinary expertise in mast cell disease. To serve this unmet need, AIM envisions a network where basic, translational, and clinical researchers could establish collaborations with both academia and biopharma to support the development of new diagnostic methods, enhanced understanding of the biology of mast cells in human health and disease, and the testing of novel therapies. In these AIM proceedings, we highlight selected topics relevant to mast cell biology and provide updates regarding the recently described hereditary alpha-tryptasemia. In addition, we discuss the evaluation and treatment of mast cell activation (syndromes), allergy and anaphylaxis in mast cell disorders, and the clinical and biologic heterogeneity of the more indolent forms of mastocytosis. Because mast cell disorders are relatively rare, AIM hopes to achieve a coordination of scientific efforts not only in the Americas but also in Europe by collaborating with the well-established European Competence Network on Mastocytosis.
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.
We sought to assess inhibitory ...IgG:FcγRIIb effects on MC responses to IgE using complementary transcriptomic and functional approaches.
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.
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.
Our findings indicate that inhibitory IgG:FcγRIIb signals block an IgE-induced proallergic program but spare a prosurvival program.
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 (MC
) and epithelial MCs expressing tryptase without chymase (MC
). 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 MC
and MC
, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38
CD117
MCs that is markedly expanded during T2 inflammation. These CD38
CD117
MCs exhibit an intermediate phenotype relative to the expanded MC
and MC
subsets. CD38
CD117
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 MC
-like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MC
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.
Macrophages expressing group V phospholipase A
(Pla2g5) release the free fatty acid (FFA) linoleic acid (LA), potentiating lung type 2 inflammation. Although Pla2g5 and LA increase in viral ...infections, their role remains obscure. We generated Pla2g5
mice, deleted Pla2g5 by using the Cx3cr1
transgene, and activated bone marrow-derived macrophages (BM-Macs) with poly:IC, a synthetic double-stranded RNA that triggers a viral-like immune response, known Pla2g5-dependent stimuli (IL-4, LPS + IFNγ, IL-33 + IL-4 + GM-CSF) and poly:IC + LA followed by lipidomic and transcriptomic analysis. Poly:IC-activated Pla2g5
;Cx3cr1
BM-Macs had downregulation of major bioactive lipids and critical enzymes producing those bioactive lipids. In addition, AKT phosphorylation was lower in poly:IC-stimulated Pla2g5
;Cx3cr1
BM-Macs, which was not restored by adding LA to poly:IC-stimulated BM-Macs. Consistently, Pla2g5
;Cx3cr1
mice had diminished poly:IC-induced lung inflammation, including inflammatory macrophage proliferation, while challenging Pla2g5
;Cx3cr1
mice with poly:IC + LA partially restored lung inflammation and inflammatory macrophage proliferation. Finally, mice lacking FFA receptor-1 (Ffar1)-null mice had reduced poly:IC-induced lung cell recruitment and tissue macrophage proliferation, not corrected by LA. Thus, Pla2g5 contributes to poly:IC-induced lung inflammation by regulating inflammatory macrophage proliferation and LA/Ffar1-mediated lung cell recruitment and tissue macrophage proliferation.
Recent studies have demonstrated the important enzymatic, structural and regulatory roles of RNA in the cell. Here we present a post-transcriptional regulation system in Escherichia coli that uses ...RNA to both silence and activate gene expression. We inserted a complementary cis sequence directly upstream of the ribosome binding site in a target gene. Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5'-untranslated region of the mRNA. The stem-loop structure interferes with ribosome binding, silencing gene expression. A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression. Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression.
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