Taylor A. Doherty

Lung type 2 innate lymphoid cells express cysteinyl leukotriene receptor 1, which regulates TH2 cytokine production.

BACKGROUND Cysteinyl leukotrienes (CysLTs) contribute to asthma pathogenesis, in part through cysteinyl leukotriene receptor 1 (CysLT1R). Recently discovered lineage-negative type 2 innate lymphoid cells (ILC2s) potently produce IL-5 and IL-13. OBJECTIVES We hypothesized that lung ILC2s might be activated by leukotrienes through CysLT1R. METHODS ILC2s (Thy1.2(+) lineage-negative lymphocytes) and CysLT1R were detected in the lungs of wild-type, signal transducer and activator of transcription 6-deficient (STAT6(-/-)), and recombination-activating gene 2-deficient (RAG2(-/-)) mice by means of flow cytometry. T(H)2 cytokine levels were measured in purified lung ILC2s stimulated with leukotriene D₄ (LTD₄) in the presence or absence of the CysLT1R antagonist montelukast. Calcium influx was measured by using Fluo-4 intensity. Intranasal leukotriene C₄, D₄, and E₄ were administered to naive mice, and levels of ILC2 IL-5 production were determined. Finally, LTD₄ was coadministered with Alternaria species repetitively to RAG2(-/-) mice (with ILC2s) and IL-7 receptor-deficient mice (lack ILC2s), and total ILC2 numbers, proliferation (Ki-67(+)), and bronchoalveolar lavage fluid eosinophil numbers were measured. RESULTS CysLT1R was expressed on lung ILC2s from wild-type, RAG2(-/-), and STAT6(-/-) naive and Alternaria species-challenged mice. In vitro LTD₄ induced ILC2s to rapidly generate high levels of IL-5 and IL-13 within 6 hours of stimulation. Interestingly, LTD4, but not IL-33, induced high levels of IL-4 by ILC2s. LTD₄ administered in vivo rapidly induced ILC2 IL-5 production that was significantly reduced by montelukast before treatment. Finally, LTD₄ potentiated Alternaria species-induced eosinophilia, as well as ILC2 accumulation and proliferation. CONCLUSIONS We present novel data that CysLT1R is expressed on ILC2s and LTD₄ potently induces CysLT1R-dependent ILC2 production of IL-4, IL-5, and IL-13. Additionally, LTD₄ potentiates Alternaria species-induced eosinophilia and ILC2 proliferation and accumulation.

Th9 and allergic disease

Helper CD4+ T‐cell subsets have improved our understanding of adaptive immunity in humans and in animal models of disease. These include T helper type 1 (Th1), Th2 and the interleukin‐17 (IL‐17) ‐producing population ‘Th17’. Th2 cells have been described as orchestrating the immune response in allergic disease based on studies with patient samples and animal models. The cytokine IL‐9 has largely been regarded as a Th2 cytokine that makes multifocal contributions to allergic disease. Recent data suggest that under certain conditions relevant to chronic disease (IL‐4 and transforming growth factor‐β), a distinct population of IL‐9‐producing ‘Th9’ helper T cells can exist. The contribution of Th9 cells in allergic disease is currently unknown, and this review will propose a model for how these cells may regulate chronic allergic inflammation.

Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance

Lung-resident antigen-presenting macrophages promote tolerance to inhaled antigens via the induction of regulatory T cells.

The tumor necrosis factor family member LIGHT is a target for asthmatic airway remodeling

Individuals with chronic asthma show a progressive decline in lung function that is thought to be due to structural remodeling of the airways characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we show that the tumor necrosis factor (TNF) family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin β receptor (LTβR) reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung transforming growth factor-β (TGF-β) and interleukin-13 (IL-13), cytokines implicated in remodeling in humans, whereas exogenous administration of LIGHT to the airways induces fibrosis and smooth muscle hyperplasia, Thus, LIGHT may be targeted to prevent asthma-related airway remodeling.

ORMDL3 is an inducible lung epithelial gene regulating metalloproteases, chemokines, OAS, and ATF6

Orosomucoid like 3 (ORMDL3) has been strongly linked with asthma in genetic association studies, but its function in asthma is unknown. We demonstrate that in mice ORMDL3 is an allergen and cytokine (IL-4 or IL-13) inducible endoplasmic reticulum (ER) gene expressed predominantly in airway epithelial cells. Allergen challenge induces a 127-fold increase in ORMDL3 mRNA in bronchial epithelium in WT mice, with lesser 15-fold increases in ORMDL-2 and no changes in ORMDL-1. Studies of STAT-6–deficient mice demonstrated that ORMDL3 mRNA induction highly depends on STAT-6. Transfection of ORMDL3 in human bronchial epithelial cells in vitro induced expression of metalloproteases (MMP-9, ADAM-8), CC chemokines (CCL-20), CXC chemokines (IL-8, CXCL-10, CXCL-11), oligoadenylate synthetases (OAS) genes, and selectively activated activating transcription factor 6 (ATF6), an unfolded protein response (UPR) pathway transcription factor. siRNA knockdown of ATF-6α in lung epithelial cells inhibited expression of SERCA2b, which has been implicated in airway remodeling in asthma. In addition, transfection of ORMDL3 in lung epithelial cells activated ATF6α and induced SERCA2b. These studies provide evidence of the inducible nature of ORMDL3 ER expression in particular in bronchial epithelial cells and suggest an ER UPR pathway through which ORMDL3 may be linked to asthma.

STAT6 regulates natural helper cell proliferation during lung inflammation initiated by Alternaria

Asthma exacerbations can be caused by a number of factors, including the fungal allergen Alternaria, which is specifically associated with severe and near-fatal attacks. The mechanisms that trigger lung responses are unclear and might vary between allergens. A comparison between Alternaria, Aspergillus, Candida, and house dust mite, all allergens in humans, showed that only Alternaria promoted immediate innate airway eosinophilia within 12 h of inhalation in nonsensitized mice. Alternaria, but not the other allergens, induced a rapid increase in airway levels of IL-33, accompanied by IL-33 receptor (IL-33R)-positive natural helper cell (NHC) production of IL-5 and IL-13. NHCs in the lung and bone marrow constitutively expressed transcription factors [GATA-3 and E26 transformation-specific sequence-1 (ETS-1)] that could allow for rapid induction of T helper type 2 (Th2) cytokines. Lung NHC numbers and proliferation (%Ki-67), but not IL-5 or GATA-3 expression, were significantly reduced in STAT6-deficient mice 3 days after one challenge with Alternaria. Alternaria induced NHC expression of the EGF receptor ligand amphiregulin (partially dependent on STAT6), as well as EGF receptor signaling in the airway epithelium. Finally, human peripheral blood NHCs (CRTH2(+)CD127(+) lineage-negative lymphocytes) from allergic individuals highly expressed GATA-3 and ETS-1, similar to lung NHCs in mice. In summary, Alternaria-induced lung NHC proliferation and expression of amphiregulin are regulated by STAT6. In addition, NHCs in mouse and humans are primed to express Th2 cytokines through constitutive expression of GATA-3 and ETS-1. Thus several transcription factor pathways (STAT6, GATA-3, and ETS-1) may contribute to NHC proliferation and Th2-type responses in Alternaria-induced asthma.

Prostaglandin D2 regulates human type 2 innate lymphoid cell chemotaxis.

To the Editor: Type 2 innate lymphoid cells (ILC2) were identified in 2010 and include natural helper cells, nuocytes, and innate type 2 helper cells.1–3 ILC2 do not express known T-cell, B-cell, or natural killer–cell lineage markers (lineage-negative) and produce large amounts of IL-5 and IL-13 in response to cytokines IL-33, IL-25, or both.1–3 Importantly, ILC2 have been shown to contribute to airway hyperresponsiveness and type 2 lung inflammatory responses in mice infected with influenza virus, and after challenge with multiple allergens including Alternaria, papain, house dust mite, and ovalbumin, suggesting a potential role for ILC2 in the pathogenesis of allergic inflammation and asthma (reviewed elsewhere4). Studies of ILC2 in humans have demonstrated their presence in peripheral blood, the gastrointestinal tract, lung, bronchoalveolar lavage (BAL), and nasal polyps.5–7 Our group7 and others8 have reported that ILC2 in human peripheral blood highly express the master TH2 cytokine transcription factor GATA-3, suggesting that ILC2 are primed for rapid and robust TH2 cytokine production in humans. ILC2 are cells derived from the bone marrow that circulate in the blood and localize to tissues relevant to allergic inflammation including the lung, BAL, and nasal polyps. Although recent studies have provided an insight into ILC2 development and cytokine production, mechanisms that regulate ILC2 recruitment to tissues have not previously been reported. As human ILC2 express the chemokine receptor CRTH2 (the chemoattractant receptor homologous molecule expressed on TH2 lymphocytes) that binds to prostaglandin D2 (PGD2), we hypothesized that PGD2 may promote one of the steps of human peripheral blood recruitment to tissues, namely, chemotaxis in mucosal sites in which PGD2 is highly expressed. Because PGD2 is released by several cell types important to allergic inflammation including mast cells, macrophages, and eosinophils, activation of these cell types at mucosal sites in the upper or lower airway could promote PGD2-mediated chemotaxis in tissues. To determine whether PGD2 induced chemotaxis of human peripheral blood ILC2, we recruited 4 atopic subjects (immediate hypersensitivity skin test positive to dust mite, cat, or grass pollen), aged 34.3 ± 18.4 years (3 males and 1 female), and 6 nonatopic healthy volunteers aged 33.3 ± 6.6 years (3 males and 3 females), who each donated blood in a protocol approved by the University of California San Diego Human Subjects Protection Committee. PBMCs were isolated by using density gradient centrifugation in Vacutainer Cell Preparation Tubes with sodium citrate (BD, Franklin Lakes, NJ). PBMCs (1 × 106) in 200 µL RPMI media were applied in duplicate to the upper chamber of a 5-µm pore-size 24-well plate (BD). The lower chambers contained varying concentrations of PGD2 (0, 5, or 25 nM) (Cayman Chemical, Ann Arbor, Mich) each dissolved in 5% dimethyl sulfoxide in PBS. After incubation of PGD2 in the lower chamber for 90 minutes at 37°C, the 5-µm pore-size filter and any remaining cells on the filter were discarded. The plate was then placed on ice for 30 minutes, and cells in the lower chamber were collected from the plate by washing followed by staining for fluorescence-activated cell sorting analysis to detect the number of ILC2 that had chemotaxed into the lower chamber. To detect ILC2, cells in the lower chamber were stained with a fluorescein isothiocyanate lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56; BD), TCRgd (BD), CD4, CD11b, CD235a, and FceRI (eBioscience, San Diego, Calif), Alexa-647 conjugated anti-CD127 (eBioscience), and biotin-conjugated CRTH2 (Miltenyi, Auburn, Calif) followed by streptavidin phycoerythrin as previously described in this laboratory to detect human peripheral blood ILC2.7 This staining protocol defines ILC2 as lineage-negative lymphocytes (do not express T-cell, B-cell, natural killer–cell, mast-cell, and basophil markers) that do express CRTH2 as well as the IL-7 receptor CD127 (Fig 1). Flow cytometry was performed by using the BD Accuri FACS machine. ILC2 represent approximately 1% to 20% of lineage-negative lymphocytes and 0.01% to 0.04% of total lymphocytes in the PBMC populations applied to the upper chamber. FIG 1 ILC2 FACS: Lymphocytes were identified from whole PBMCs (left panel), and lineage-negative cells gated (middle panel). Lineage-negative lymphocytes were further assessed for the expression of CD127 and CRTH2 (right panel). ILC2 were identified as lineage-negative ... In allergic subjects we detected a dose-dependent increase in ILC2 migration in the presence of 5 to 25 nMof PGD2 (Fig 2, A). Higher concentrations of PGD2 did not increase ILC2 chemotaxis (data not shown). PGD2 also induced chemotaxis of ILC2 in normal healthy controls, but to a lesser degree than in allergic donors (Fig 2, B). ILC2 in allergic individuals may therefore be primed for chemotaxis. Preincubation of PBMCs containing ILC2 with ramatroban, a pharmacologic CRTH2 antagonist,9,10 blocked ILC2 chemotaxis in response to PGD2 (see Fig E1 in this article’s Online Repository at www.jacionline.org). In experiments in which PGD2 was added to both the upper and lower wells, the predominant effect of 25 nM of PGD2 was on ILC2 chemotaxis (P < .01) (see Fig E2 in this article’s Online Repository at www.jacionline.org). Previous studies have demonstrated that the effect of PGD2 on eosinophils and basophils is predominantly chemotactic11 but also chemokinetic especially at low 1 to 10 nM concentrations of PGD2.12 Because we studied impure preparations of ILC2, we were not able to determine whether PGD2 acts directly on ILC2 or indirectly through other cells present in the PBMCs studied. FIG 2 ILC2 chemotaxis to PGD2: PBMCs (Fig 2, A: n = 4 allergic subjects; Fig 2, B: n = 6 nonallergic controls) were incubated in duplicate in the upper chamber of a 5-µm pore-size plate. Varying concentrations of PGD2 were placed in the lower chamber. ... Thus, PGD2 induces chemotaxis of human peripheral blood ILC2 that express the PGD2 receptor CRTH2. PGD2 plays a role in allergic disease and has previously been shown to induce chemotaxis of TH2 cells, eosinophils, and basophils via CRTH2.11 Our novel findings suggest that human ILC2 chemotaxis is also induced by PGD2. Human ILC2 have been detected in blood, lung, BAL, and nasal polyps, and potently produce TH2 cytokines.5,6,8 The mechanism by which human (or mouse) ILC2 migrate from the bone marrow through the circulation into the tissues has not been reported. Our studies suggest that PGD2 may contribute to one step of the tissue recruitment of ILC2, namely, the chemotaxis of ILC2 to tissue sites in which PGD2 is released such as the upper and lower airway in asthma and allergic rhinitis. CRTH2 antagonists currently undergoing clinical trials in asthma and allergic rhinitis for their inhibitory effects on TH2 cell recruitment may also inhibit ILC2 recruitment into tissues.

Increased ILC2s in the eosinophilic nasal polyp endotype are associated with corticosteroid responsiveness

Group 2 innate lymphoid cells (ILC2s) have recently been identified in human nasal polyps, but whether numbers of ILC2s differ by polyp endotype or are influenced by corticosteroid use is unknown. Here, we show that eosinophilic nasal polyps contained double the number of ILC2s vs. non-eosinophilic polyps. Polyp ILC2s were also reduced by 50% in patients treated with systemic corticosteroids. Further, using a fungal allergen challenge mouse model, we detected greatly reduced Th2 cytokine-producing and Ki-67+ proliferating lung ILC2s in mice receiving dexamethasone. Finally, ILC2 Annexin V staining revealed extensive apoptosis after corticosteroid treatment in vivo and in vitro. Thus, ILC2s are elevated in the eosinophilic nasal polyp endotype and systemic corticosteroid treatment correlated with reduced polyp ILC2s. Finally, allergen-challenged mice showed reduced ILC2s and increased ILC2 apoptosis after corticosteroid treatment suggesting that ILC2 may be responsive to corticosteroids in eosinophilic respiratory disease.

Alternaria Induces STAT6-Dependent Acute Airway Eosinophilia and Epithelial FIZZ1 Expression That Promotes Airway Fibrosis and Epithelial Thickness

The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when analyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ≥5 d and was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1 demonstrated that CD45+CD11c+ cells (macrophages and dendritic cells), as well as collagen-1–producing CD45− cells (fibroblasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6–dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma.

ORMDL3 transgenic mice have increased airway remodeling and airway responsiveness characteristic of asthma

Orosomucoid-like (ORMDL)3 has been strongly linked with asthma in genetic association studies. Because allergen challenge induces lung ORMDL3 expression in wild-type mice, we have generated human ORMDL3 zona pellucida 3 Cre (hORMDL3zp3-Cre) mice that overexpress human ORMDL3 universally to investigate the role of ORMDL3 in regulating airway inflammation and remodeling. These hORMDL3zp3-Cre mice have significantly increased levels of airway remodeling, including increased airway smooth muscle, subepithelial fibrosis, and mucus. hORMDL3zp3-Cre mice had spontaneously increased airway responsiveness to methacholine compared to wild-type mice. This increased airway remodeling was associated with selective activation of the unfolded protein response pathway transcription factor ATF6 (but not Ire1 or PERK). The ATF6 target gene SERCA2b, implicated in airway remodeling in asthma, was strongly induced in the lungs of hORMDL3zp3-Cre mice. Additionally, increased levels of expression of genes associated with airway remodeling (TGF-β1, ADAM8) were detected in airway epithelium of these mice. Increased levels of airway remodeling preceded increased levels of airway inflammation in hORMDL3zp3-Cre mice. hORMDL3zp3-Cre mice had increased levels of IgE, with no change in levels of IgG, IgM, and IgA. These studies provide evidence that ORMDL3 plays an important role in vivo in airway remodeling potentially through ATF6 target genes such as SERCA2b and/or through ATF6-independent genes (TGF-β1, ADAM8).