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Innate immune responses to innocuous Ags can either prevent or facilitate adaptive type 2 allergic inflammation, but the mechanisms are incompletely understood. We now demonstrate that macrophage UDP-specific type 6 purinergic (P2Y6) receptors selectively activate NFATC2, a member of the NFAT family, to drive an innate IL-12/IFN-γ axis that prevents type 2 allergic inflammation. UDP priming potentiated IL-12p40 production in bone marrow–derived macrophages (BMMs) stimulated by the house dust mite Dermatophagoides farinae (Df) in a P2Y6-dependent manner. Inhibitions of phospholipase C, calcium increase, and calcineurin eliminated UDP-potentiated Df-induced IL-12p40 production. UDP specifically induced nuclear translocation of NFATC2, but not NFATC1 and NFATC3, in BMMs in a P2Y6-dependent manner. UDP-potentiated IL-12p40 production by BMMs and Df-induced IL-12p40 gene expression by alveolar macrophages were abrogated in cells from Nfatc2 knockout mice. Pulmonary transplantation of wild-type but not Nfatc2 knockout macrophages increased Df-induced IL-12 production and IFN-γ expression in P2ry6 fl/fl/Cre/+ recipient mice. Finally, Nfatc2 knockout mice showed significantly increased indices of type 2 immunopathology in response to Df challenge, similar to P2ry6 fl/fl/Cre/+ mice. Thus, macrophage P2Y6 receptor signaling selectively utilizes NFATC2 to potentiate an innate IL-12/IFN-γ axis, a potential mechanism that protects against inappropriate type 2 immune responses.

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.

BACKGROUND: Mas-related G protein-couple receptor x2 (Mrgprx2) activation underlies many common non-IgE-mediated adverse drug reactions, yet the features of patients with reactions to Mrgprx2-activating drugs are unknown. OBJECTIVE: To characterize the patient-specific comorbidities and laboratory characteristics associated with listed reactions to Mrgprx2-activating drugs, including fluoroquinolones, morphine, neuromuscular blockade agents, vancomycin and leuprolide. METHODS: We used a retrospective, observational cohort study design, using electronic health record data from adults with an Mrgprx2-activating drug exposure recorded within a hospital system clinical Biobank. Odds ratios and incidence rate ratios for clinical characteristics associated with adverse drug reactions (ADR), including immediate hypersensitivity reactions, were calculated using multivariable logistic regression. RESULTS: Among 59,763 patients exposed to Mrgprx2-activating drugs, 4,846 had a listed ADR. Female sex, white race, asthma [OR 1.81, 95% CI (1.68 to 1.94)], chronic urticaria [OR 1.73, 95% CI (1.46 to 2.05)], and mastocytosis [OR 12.79, 95% CI (5.98 to 27.02)] were associated with increased odds of a reaction. Overall, patients with allergic disease had 1.21 times the rate of an ADR compared to patients without allergic disease. Elevated absolute eosinophil count was inversely associated with reactions and there was no association with elevated total IgE. Observed associations were similar in a patient subgroup with immediate type hypersensitivity reactions. CONCLUSION: Specific allergic diseases and common allergic biomarkers are differentially associated with ADRs to Mrgprx2-activating drugs. These findings from a large, “real world” drug-exposed population highlight clinical factors that may contribute to non-IgE mediated drug allergy.

BackgroundChronic rhinosinusitis with nasal polyposis (CRSwNP) is a type 2 (T2) inflammatory disease associated with an increased number of airway basal epithelial cells (BCs). Recent studies have identified transcriptionally distinct BCs, but functional data are lacking and the molecular pathways that support or inhibit human BC proliferation and differentiation are largely unknown.ObjectiveTo determine the role of T2 cytokines in regulating airway BCsMethodsSingle cell and bulk RNA-sequencing of sinus and lung airway epithelial cells was analyzed. Human sinus BCs were stimulated with IL-4 and IL-13 in the presence and absence of IL4R inhibitors. Confocal analysis of human sinus tissue and murine airway was performed. Murine BC subsets were sorted for RNA sequencing and functional assays. Fate labeling was performed in a murine model of tracheal injury and repair.ResultsHere we find two subsets of BCs in human and murine respiratory mucosa distinguished by the expression of BC adhesion molecule (BCAM). BCAM expression identifies airway stem cells among P63+KRT5+NGFR+ BCs. In the sinonasal mucosa, BCAMhiBCs expressingTSLP,IL33,CCL26,and the canonical BC transcription factorTP63are increased in patients with CRSwNP. In cultured BCs, IL-4/13 increases expression ofBCAMandTP63through an Insulin Receptor Substrate (IRS)-dependent signaling pathway that is increased in CRSwNP.ConclusionsThese findings establish BCAM as a marker of airway stem cells among the BC pool and demonstrate that airway epithelial remodeling in T2 inflammation extends beyond goblet cell metaplasia to the support of a BC stem state poised to perpetuate inflammation.CAPSULE SUMMARYType 2 cytokines drive an airway stem cell program through IRS signalingKEY MESSAGESTwo subsets of airway BCs have distinct transcriptional signatures and functionHigh levels of BCAM expression mark the earliest BC progenitorIL-4 and IL-13 upregulate BCAM and P63 in an IRS-dependent fashion which prevents BC differentiation to secretory epithelial cellsBCAMhiBCs are increased in CRSwNP

The olfactory neuroepithelium serves as a sensory organ for odors and is part of the nasal mucosal barrier. Olfactory sensory neurons are surrounded and supported by epithelial cells. A subset of these, microvillous cells (MVCs), are strategically positioned at the apical surface but their specific functions are still enigmatic and their relationship to the rest of the solitary chemosensory cell family is unclear. Here, we establish that the larger family of MVCs comprises tuft cells and ionocytes in both mice and humans. Olfactory TRPM5+tuft-MVCs share a core transcriptional profile with the chemosensory tuft family, prominently including the machinery for lipid mediator generation. Integrating analysis of the respiratory and olfactory epithelium, we define the unique receptor expression of TRPM5+tuft-MVC compared to the Gɑ-gustducin+respiratory tuft cells and characterize a new population of glandular DCLK1+tuft cells. To establish how allergen sensing by tuft-MVCs might direct olfactory mucosal responses, we employed an integrated single-cell transcriptional and protein analysis. We defined a remodeling olfactory epithelial switch pathway with induction ofChil4and a distinct pathway of proliferation of the quiescent olfactory horizontal basal stem cell (HBC), both triggered in the absence of significant olfactory apoptosis. While theChil4pathway was dependent on STAT6 signaling and innate lymphocytes, neither were required for HBC proliferation. HBC proliferation was dependent on tuft-MVCs, establishing these specialized epithelial cells as both sensors for allergens and regulators of olfactory stem cell responses. Together our data provide high resolution characterization of the nasal tuft cell heterogeneity and uncover a novel mechanism by which TRPM5+tuft cells direct the olfactory mucosal response to allergens.One Sentence SummaryWe identify the enigmatic TRPM5+olfactory microvillous cells as tuft cells, and show their functional role as regulators of olfactory stem cell proliferation in response to environmental signals.

Cysteinyl-maresins, also known as maresin-conjugates in tissue regeneration (MCTRs), are recently discovered lipid mediators proposed to reduce airway inflammation.To investigate the influence of MCTRs on IL-13-induced airway hyperresponsiveness in isolated human and mice airways.Before responsiveness to contractile agonists were assessed in myographs, human small bronchi were cultured for 2 days and mouse tracheas were cultured for 1-4 days. During the culture procedure airways were exposed to interleukin (IL)-13 in the presence or absence of MCTRs. Signalling mechanisms were explored using pharmacologic agonists and antagonists, and genetically modified mice.IL-13 treatment increased contractions to histamine, carbachol and leukotriene DMCTR3, but not LTD

Aspirin-exacerbated respiratory disease has fascinated and frustrated specialists in allergy/immunology, pulmonology, and otorhinolaryngology for decades. It generally develops in previously healthy young adults and is unremitting and challenging to treat. The classical triad of asthma, nasal polyposis, and pathognomonic respiratory reactions to aspirin and other cyclooxygenase-1 inhibitors is accompanied by high levels of mast cell activation, cysteinyl leukotriene production, platelet activation, and severe type 2 respiratory inflammation. The "unbraking" of mast cell activation and further cysteinyl leukotriene generation induced by cyclooxygenase-1 inhibition reflect an idiosyncratic dependency on cyclooxygenase-1-derived products, likely prostaglandin E

Aeroallergen sensing by airway epithelial cells can trigger pathogenic immune responses leading to chronic type 2 inflammation, the hallmark of airway diseases such as asthma. Airway tuft cells are specialized chemosensory epithelial cells and the dominant source of the epithelial cytokine IL-25 in the trachea and of cysteinyl leukotrienes (CysLTs) in the naïve murine nasal mucosa. The interaction of IL-25 and CysLTs and the contribution of tuft cell-derived CysLTs to the development of allergen-triggered inflammation in the airways has not been clarified. Here we show that inhalation of LTC4 in combination with a subthreshold dose of IL-25 leads to dramatic synergistic induction of type 2 inflammation throughout the lungs, causing rapid eosinophilia, dendritic cell (DC) and inflammatory type 2 innate lymphoid cell (ILC2) expansion, and goblet cell metaplasia. While lung eosinophilia is dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells require signaling through both CysLT1R and CysLT2R. Tuft cell-specific deletion of the terminal enzyme requisite for CysLT production, Ltc4s, was sufficient to reduce both the innate immune response in the lung – eosinophilia, KLRG1+ ILC2 activation and DC recruitment – and the systemic immune response in the draining lymph nodes after inhalation of the mold aeroallergen Alternaria. Our findings identify surprisingly potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs.One Sentence SummaryTuft cells produce two highly synergistic mediators: LTC4 and IL-25 to cooperatively induce allergen-driven airway inflammation.

Rationale: Daily high-dose aspirin therapy benefits many patients with aspirin-exacerbated respiratory disease but provides no benefit for aspirin-tolerant patients with asthma. Type 2 inflammation...

Cysteinyl leukotrienes (cysLTs) facilitate eosinophilic mucosal type 2 immunopathology, especially in aspirin-exacerbated respiratory disease (AERD), by incompletely understood mechanisms. We now demonstrate that platelets, activated through the type 2 cysLT receptor (CysLT2R), cause IL-33-dependent immunopathology through a rapidly inducible mechanism requiring the actions of high mobility box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE). Leukotriene C4 (LTC4) induces surface HMGB1 expression by mouse platelets in a CysLT2R-dependent manner. Blockade of RAGE and neutralization of HMGB1 prevent LTC4-induced platelet activation. Challenges of AERD-like Ptges−/− mice with inhaled lysine aspirin (Lys-ASA) elicit LTC4 synthesis and cause rapid intrapulmonary recruitment of platelets with adherent granulocytes, along with platelet- and CysLT2R-mediated increases in lung IL-33, IL-5, IL-13, and bronchoalveolar lavage fluid HMGB1. The intrapulmonary administration of exogenous LTC4 mimics these effects. Platelet depletion, HMGB1 neutralization, and pharmacologic blockade of RAGE eliminate all manifestations of Lys-ASA challenges, including increase in IL-33, mast cell activation, and changes in airway resistance. Thus, CysLT2R signaling on platelets prominently utilizes RAGE/HMGB1 as a link to downstream type 2 respiratory immunopathology and IL-33-dependent mast cell activation typical of AERD. Antagonists of HMGB1 or RAGE may be useful to treat AERD and other disorders associated with type 2 immunopathology.

Mast cells (MCs) are key effector cells in the development of type 2 inflammation, such as asthma and nasal polyposis. Intraepithelial MCs are phenotypically distinct from those found within the subepithelium and preferentially expand during mucosal inflammation, yet the mechanism driving their development and their relative contribution to inflammation is not clear. We previously found that TGF-β directed the murine intraepithelial airway MC phenotype. Here, we investigate whether TGF-β has a similar effect on human MCs. Using single cell RNA-sequencing of primary nasal polyp MCs and bulk sequencing of in vitro peripheral blood-derived MCs (PB-MCs) stimulated with TGF-β, we identified a shared signature of TGF-β within human intraepithelial MCs. This included downregulation of subepithelial MC-associated granule components (NDST2, CMA1, and CTSG), differential regulation of lipid mediator biosynthetic enzymes (PTGS1, HPGDS), and upregulation of transcription factors (SKIL, FOXP1). TGF-β suppressed incorporation of the proteases chymase and cathepsin G into PB-MC granules in vitro as part of a broader intraepithelial MC phenotype, allowing direct assessment of the differential role of MC subsets during inflammation. Through this approach, we found that TGF-β modifies MC production of inflammatory mediators in response to stimulation, enhancing MC production of the lipid mediators, PGD2 and CysLTs, in response to IgE-crosslinking; selectively decreasing IL-13, TNF-α, and IL-6; and enhancing chemokine production (IL-8 and MIP-1β). Thus, we identify a central role for TGF-β signaling in driving the human intraepithelial MC phenotype and re-shaping their response to activation. Supported by NIH (K22 AI146281, U19AI070535)

There is an acute need for advances in pharmacologic therapies and a better understanding of novel drug targets for severe asthma. Imatinib, a tyrosine kinase inhibitor, has been shown to improve forced expiratory volume in 1 s (FEV1) in a clinical trial of patients with severe asthma. In a pilot study, we applied systems biology approaches to epithelium gene expression from these clinical trial patients treated with imatinib to better understand lung function response with imatinib treatment. Bronchial brushings from ten imatinib-treated patient samples and 14 placebo-treated patient samples were analyzed. We used personalized perturbation profiles (PEEPs) to characterize gene expression patterns at the individual patient level. We found that strong responders—patients with greater than 20% increase in FEV1—uniquely shared multiple downregulated mitochondrial-related pathways. In comparison, weak responders (5–10% FEV1 increase), and non-responders to imatinib shared none of these pathways. The use of PEEP highlights its potential for application as a systems biology tool to develop individual-level approaches to predicting disease phenotypes and response to treatment in populations needing innovative therapies. These results support a role for mitochondrial pathways in airflow limitation in severe asthma and as potential therapeutic targets in larger clinical trials.

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(TC)) and epithelial MCs expressing tryptase without chymase (MC(T)). 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(T) and MC(TC), which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We discover a subepithelial population of CD38(high)CD117(high) MCs that is markedly expanded during T2 inflammation. These CD38(high)CD117(high) MCs exhibit an intermediate phenotype relative to the expanded MC(T) and MC(TC) subsets. CD38(high)CD117(high) MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease (AERD), a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MC(T)-like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MC(TC) in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.

Mast cells (MCs) comprise two lineages that differ in tissue localization and ontologic origin. Derakhshan et al. define the transcriptomes of these lineages during airway inflammation, identifying distinct programs associated with each lineage and a role for TGF-β signaling in inducible MC development., 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 β7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. β7High MCs expand and mature during lung inflammation as part of a TGF-β–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 TGF-β–mediated development and found that epithelial cells directly elicit TGF-β–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.

© 2020 American Academy of Allergy, Asthma & Immunology Background: The cause of severe nasal polyposis in aspirin-exacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but upstream drivers of local antibody production in nasal polyps are undetermined. Objective: We sought to identify upstream drivers and phenotypic properties of local antibody-expressing cells in nasal polyps from subjects with AERD. Methods: Sinus tissue was obtained from subjects with AERD, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps, and controls without CRS. Tissue antibody levels were quantified via ELISA and immunohistochemistry and were correlated with disease severity. Antibody-expressing cells were profiled with single-cell RNA sequencing, flow cytometry, and immunofluorescence, with IL-5Rα function determined through IL-5 stimulation and subsequent RNA sequencing and quantitative PCR. Results: Tissue IgE and IgG4 levels were elevated in AERD compared with in controls (P

BACKGROUND: The 3 cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4) (LTC(4)), LTD(4), and LTE(4), have different biologic half-lives, cellular targets, and receptor specificities. CysLT(2)R binds LTC(4) and LTD(4) in vitro with similar affinities, but it displays a marked selectivity for LTC(4) in vivo. LTC(4), but not LTD(4), strongly potentiates allergen-induced pulmonary eosinophilia in mice through a CysLT(2)R-mediated, platelet-and IL-33–dependent pathway. OBJECTIVE: We sought to determine whether LTD(4) functionally antagonizes LTC(4) signaling at CysLT(2)R. METHODS: We used 2 different in vivo models of CysLT(2)R-dependent immunopathology, as well as ex vivo activation of mouse and human platelets. RESULTS: LTC(4)-induced CD62P expression; HMGB1 release; and secretions of thromboxane A(2), CXCL7, and IL-33 by mouse platelets were all were blocked by a selective CysLT(2)R antagonist and inhibited by LTD(4). These effects did not depend on CysLT(1)R. Inhaled LTD(4) blocked LTC(4)-mediated potentiation of ovalbumin-induced eosinophilic inflammation; recruitment of platelet-adherent eosinophils; and increases in IL-33, IL-4, IL-5, and IL-13 levels in lung tissue. In contrast, the effect of administration of LTE(4), the preferred ligand for CysLT(3)R, was additive with LTC(4). The administration of LTD(4) to Ptges(−/−) mice, which display enhanced LTC(4) synthesis similar to that in aspirin-exacerbated respiratory disease, completely blocked the physiologic response to subsequent lysine-aspirin inhalation challenges, as well as increases in levels of IL-33, type 2 cytokines, and biochemical markers of mast cell and platelet activation. CONCLUSION: The conversion of LTC(4) to LTD(4) may limit the duration and extent of potentially deleterious signaling through CysLT(2)R, and it may contribute to the therapeutic properties of desensitization to aspirin in aspirin-exacerbated respiratory disease.

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases1. Specialized subsets of epithelial cells—including secretory and ciliated cells—differentiate from basal stem cells to collectively protect the upper airway2–4. Allergic inflammation can develop from persistent activation5 of type 2 immunity6 in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps7. Basal cell hyperplasia is a hallmark of severe disease7–9, but it is not known how these progenitor cells2,10,11 contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing12, report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic13, epigenetic14,15 and extrinsic factors11,16,17 that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories. Single-cell RNA sequencing is used to characterize cell types in nasal tissues from human patients with chronic rhinosinusitis, revealing a role for tissue stem cells in allergic inflammatory memory.

Type 2 immunopathology is a cardinal feature of allergic diseases and involves cooperation between adaptive immunity and innate effector responses. Virtually all cell types relevant to this pathology generate leukotriene and/or prostaglandin mediators that derive from arachidonic acid, express receptors for such mediators, or both. Recent studies highlight prominent functions for these mediators in communication between the innate and adaptive immune systems, as well as amplification or suppression of type 2 effector responses. This review focuses on recent advances and insights, and highlights existing and potential therapeutic applications of drugs that target these mediators or their receptors, with a special emphasis on their regulation of the innate and adaptive lymphocytes relevant to type 2 immunopathology.

Background The 3 cysteinyl leukotrienes (cysLTs), leukotriene (LT) C4 (LTC4), LTD4, and LTE4, have different biologic half-lives, cellular targets, and receptor specificities. CysLT2R binds LTC4 and LTD4 in vitro with similar affinities, but it displays a marked selectivity for LTC4 in vivo. LTC4, but not LTD4, strongly potentiates allergen-induced pulmonary eosinophilia in mice through a CysLT2R-mediated, platelet- and IL-33–dependent pathway. Objective We sought to determine whether LTD4 functionally antagonizes LTC4 signaling at CysLT2R. Methods We used 2 different in vivo models of CysLT2R-dependent immunopathology, as well as ex vivo activation of mouse and human platelets. Results LTC4-induced CD62P expression; HMGB1 release; and secretions of thromboxane A2, CXCL7, and IL-33 by mouse platelets were all were blocked by a selective CysLT2R antagonist and inhibited by LTD4. These effects did not depend on CysLT1R. Inhaled LTD4 blocked LTC4-mediated potentiation of ovalbumin-induced eosinophilic inflammation; recruitment of platelet-adherent eosinophils; and increases in IL-33, IL-4, IL-5, and IL-13 levels in lung tissue. In contrast, the effect of administration of LTE4, the preferred ligand for CysLT3R, was additive with LTC4. The administration of LTD4 to Ptges–/– mice, which display enhanced LTC4 synthesis similar to that in aspirin-exacerbated respiratory disease, completely blocked the physiologic response to subsequent lysine-aspirin inhalation challenges, as well as increases in levels of IL-33, type 2 cytokines, and biochemical markers of mast cell and platelet activation. Conclusion The conversion of LTC4 to LTD4 may limit the duration and extent of potentially deleterious signaling through CysLT2R, and it may contribute to the therapeutic properties of desensitization to aspirin in aspirin-exacerbated respiratory disease.

Mast cells are present in the airways of patients who have severe asthma despite glucocorticoid treatment; these cells are associated with disease characteristics including poor quality of life and inadequate asthma control. Stem cell factor and its receptor, KIT, are central to mast-cell homeostasis. We conducted a proof-of-principle trial to evaluate the effect of imatinib, a KIT inhibitor, on airway hyperresponsiveness, a physiological marker of severe asthma, as well as on airway mast-cell numbers and activation in patients with severe asthma.We conducted a randomized, double-blind, placebo-controlled, 24-week trial of imatinib in patients with poorly controlled severe asthma who had airway hyperresponsiveness despite receiving maximal medical therapy. The primary end point was the change in airway hyperresponsiveness, measured as the concentration of methacholine required to decrease the forced expiratory volume in 1 second by 20% (PCAmong the 62 patients who underwent randomization, imatinib treatment reduced airway hyperresponsiveness to a greater extent than did placebo. At 6 months, the methacholine PCIn patients with severe asthma, imatinib decreased airway hyperresponsiveness, mast-cell counts, and tryptase release. These results suggest that KIT-dependent processes and mast cells contribute to the pathobiologic basis of severe asthma. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01097694 .).

Antagonists of the type 1 cysteinyl leukotriene receptor (CysLT(1)R) are widely used to treat asthma and allergic rhinitis, with variable response rates. Alveolar macrophages express UDP-specific P2Y(6) receptors that can be blocked by off-target effects of CysLT(1)R antagonists. Sensitizing intranasal doses of an extract from the house dust mite Dermatophagoides farinae (Df) sharply increased the levels of UDP detected in bronchoalveolar lavage fluid of mice. Conditional deletion of P2Y(6) receptors before sensitization exacerbated eosinophilic lung inflammation and type 2 cytokine production in response to subsequent Df challenge. P2Y(6) receptor signaling was necessary for dectin-2–dependent production of protective IL-12p40 and Th1 chemokines by alveolar macrophages, leading to activation of NK cells to generate IFN-γ. Administration of CysLT(1)R antagonists during sensitization blocked UDP-elicited potentiation of IL-12p40 production by macrophages in vitro, suppressed the Df-induced production of IL-12p40 and IFN-γ in vivo, and suppressed type 2 inflammation only in P2Y(6)-deficient mice. Thus, P2Y(6) receptor signaling drives an innate macrophage/IL-12/NK cell/IFN-γ axis that prevents inappropriate allergic type 2 immune responses on respiratory allergen exposure and counteracts the Th2 priming effect of CysLT(1)R signaling at sensitization. Targeting P2Y(6) signaling might prove to be a potential additional treatment strategy for allergy.

The idiosyncratic activation of mast cells (MCs) in response to the administration of nonselective cyclooxygenase (COX) inhibitors is a cardinal feature of aspirin exacerbated respiratory disease (AERD). Older studies using mast cell stabilizing drugs support a critical role for MCs and their products in driving the severe eosinophilic inflammation and respiratory dysfunction that is typical of AERD. Since patients with AERD react to all nonselective COX inhibitors regardless of their chemical structure, the mechanism of MC activation is not due to classical, antigen-induced cross-linking of IgE receptors. Recent studies in both human subjects and animal models have revealed a complex and multifactorial process culminating in dysregulation of MC function, and an aberrant dependency on COX-1-derived prostaglandin E(2) to maintain a tenuous homeostasis. This article reviews the factors most likely to contribute to MC dysregulation in AERD, and the potential diagnostic and therapeutic implications.

BackgroundThe cause of nasal polyposis in aspirin-exacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but the upstream drivers and cellular mechanisms of local antibody production in AERD remain to be investigated.ObjectiveWe sought to identify the upstream drivers and phenotypic properties of local antibody-secreting cells in nasal polyps and to understand their clinical relevance in AERD.MethodsSinus tissue was obtained from subjects with AERD, aspirin-tolerant chronic rhinosinusitis with nasal polyps (CRSwNP), aspirin-tolerant chronic rhinosinusitis without nasal polyps (CRSsNP), and healthy controls. Tissue antibody levels were quantified via ELISA and immunohistochemistry, and were correlated with clinical markers of disease severity. Tissue cytokine mRNA levels were measured with quantitative PCR (qPCR). Antibody-secreting cells were profiled with a combination of single-cell RNA-sequencing (scRNA-seq), flow cytometry and immunofluorescence.ResultsTissue IgE and IgG4 were elevated in AERD compared to controls (pIL5RA, IGHG4, and IGHE in the antibody-associated cells of subjects with AERD compared to CRSwNP. Total plasma cells and IL-5Rα+ plasma cell numbers in the polyp tissue from AERD exceeded those in polyps from CRSwNP (p=0.0051 and p=0.026, respectively) by flow cytometry. With immunofluorescence, we determined that IL-5Rα and IgG4 are co-expressed in antibody-secreting cells in AERD.ConclusionsOur study identifies unique clusters of antibody-secreting cells in AERD defined by enrichment of transcripts encoding IL5RA, IGHG4 and IGHE. We confirm surface expression of IL-5Rα on these cells, and identify T cells as a unique transcriptional source of IL-5. Tissue antibody levels are elevated in AERD and correlate with disease severity. Our findings suggest a role for IL-5 in facilitating local antibody production that may drive features of severe sinus disease.Key MessagesIgG4 and IgE levels are markedly increased in nasal polyp tissue from subjects with AERD compared to aspirin-tolerant CRSwNP.Tissue IgG4 levels positively correlate with disease recurrence.IL-10 mRNA levels are significantly higher in AERD polyp tissue compared to CRSwNP tissue, but differences were not noted for type 2 cytokines or cytokines involved in class switch recombination.IL-5Rα transcript and protein surface expression is elevated in antibody-secreting cells from subjects with AERD and may play a role in facilitating class switching and/or survival of antibody-secreting cells.Capsule SummarySingle-cell RNA-sequencing (scRNA-seq) of whole nasal polyp tissue identified increased IL5RA, IGHE, and IGHG4 expression in the antibody-secreting cell compartment of subjects with aspirin-exacerbated respiratory disease (AERD) compared to aspirin-tolerant chronic rhinosinusitis with nasal polyps (CRSwNP). IgE and IgG4 levels are elevated in nasal polyp tissue from subjects with AERD compared to CRSwNP and correlate with disease recurrence.

Mast cells and basophils are functionally related effector cells of hematopoietic origin that are implicated in allergy, type 2 immune responses to parasites, and innate immunity. Both cell types derive from bone marrow progenitors and express high-affinity IgE Fc receptors (FceRI) on their surface. Both release histamine and generate a variety of other inflammatory mediators in response to both IgE- and non-IgE-mediated activation. However, there are important differences between mast cells and basophils in terms of their developmental origins, morphology, distribution, and mechanisms and responses to stimulation. Whereas mast cells are normal residents of all vascularized tissues, basophils are recruited to mucosal sites in response to inflammation. Both cells are important effectors of hypersensitivity responses, and both play roles in host defense as well as the pathophysiology of a variety of disease processes. Unchecked mast cell growth underlies mastocytosis, a group of disorders defined by a constellation of symptoms resulting from excessive mast cell products and infiltration into tissues.

Cysteinyl leukotrienes (cysLTs), leukotriene C4 (LTC4), LTD4, and LTE4 are proinflammatory lipid mediators with pathobiologic function in asthma. LTE4, the stable cysLT, is a weak agonist for the type 1 and type 2 cysLT receptors (CysLTRs), which constrict airway smooth muscle, but elicits airflow obstruction and pulmonary inflammation in patients with asthma. We recently identified GPR99 as a high-affinity receptor for LTE4 that mediates cutaneous vascular permeability. Here we demonstrate that a single intranasal exposure to extract from the respiratory pathogen Alternaria alternata elicits profound epithelial cell (EpC) mucin release and submucosal swelling in the nasal mucosa of mice that depends on cysLTs, as it is absent in mice deficient in the terminal enzyme for cysLT biosynthesis, LTC4 synthase (LTC4S). These mucosal changes are associated with mast cell (MC) activation and absent in MC-deficient mice, suggesting a role for MCs in control of EpC function. Of the three CysLTRs, only GPR99-deficient mice are fully protected from EpC mucin release and swelling elicited by Alternaria or by intranasal LTE4 GPR99 expression is detected on lung and nasal EpCs, which release mucin to doses of LTE4 one log lower than that required to elicit submucosal swelling. Finally, mice deficient in MCs, LTC4S, or GPR99 have reduced baseline numbers of goblet cells, indicating an additional function in regulating EpC homeostasis. These results demonstrate a novel role for GPR99 among CysLTRs in control of respiratory EpC function and suggest that inhibition of LTE4 and of GPR99 may have therapeutic benefits in asthma.

Summary There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection., Graphical Abstract, Highlights • Meta-analysis of human, non-human primate, and mouse single-cell RNA-seq datasets for putative SARS-CoV-2 targets • Type II pneumocytes, nasal secretory cells, and absorptive enterocytes are ACE2 + TMPRSS2 + • Interferon and influenza increase ACE2 in human nasal epithelia and lung tissue • Mouse Ace2 is not upregulated by interferon, raising implications for disease modeling, Analysis of single-cell RNA-seq datasets from human, non-human primate, and mouse barrier tissues identifies putative cellular targets of SARS-CoV-2 on the basis of ACE2 and TMPRSS2 expression. ACE2 represents a previously unappreciated interferon-stimulated gene in human, but not mouse, epithelial tissues, identifying anti-viral induction of a host tissue-protective mechanism, but also a potential means for viral exploitation of the host response.

Plasma tryptase elevation during aspirin-induced respiratory reactions occurs in 50% of reactions despite CysLT(1)R antagonist prophylaxis and identifies lung function decline and extra-respiratory symptoms in aspirin-exacerbated respiratory disease (AERD).

Tissue barrier dysfunction is a poorly defined feature hypothesized to drive chronic human inflammatory disease1,2. The epithelium of the upper respiratory tract represents one such barrier, responsible for separating inhaled agents, such as pathogens and allergens, from the underlying submucosa. Specialized epithelial subsets—including secretory, glandular, and ciliated cells—differentiate from basal progenitors to collectively realize this role3-5. Allergic inflammation in the upper airway barrier can develop from persistent activation of Type 2 immunity (T2I), resulting in the disease spectrum known as chronic rhinosinusitis (CRS), ranging from rhinitis to severe nasal polyps6-8. Whether recently identified epithelial progenitor subsets, and their differentiation trajectory, contribute to the clinical presentation and barrier dysfunction in T2I-mediated disease in humans remains unexplored3,9,10. Profiling twelve primary human samples spanning the range of clinical severity with the Seq-Well platform11 for massively-parallel single-cell RNA-sequencing (scRNA-seq), we report the first single-cell transcriptomes for human respiratory epithelial cell subsets, immune cells, and parenchymal cells (18,036 total cells) from a T2I inflammatory disease, and map key mediators. We find striking differences between non-polyp and polyp tissues within the epithelial compartments of human T2I cellular ecosystems. More specifically, across 10,383 epithelial cells, we identify a global reduction in epithelial diversity in polyps characterized by basal cell hyperplasia, a concomitant decrease in glandular and ciliated cells, and phenotypic shifts in secretory cell function. We validate these findings through flow cytometry, histology, and bulk tissue RNA-seq of an independent cohort. Furthermore, we detect an aberrant basal progenitor differentiation trajectory in polyps, and uncover cell-intrinsic and extrinsic factors that may lock polyp basal cells into an uncommitted state. Overall, our data define severe T2I barrier dysfunction as a reduction in epithelial diversity, characterized by profound functional shifts stemming from basal cell defects, and nominate a cellular mechanism for the persistence and chronicity of severe human respiratory disease.