Rachel Baum

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.

Allergen challenge in allergic rhinitis rapidly induces increased peripheral blood type 2 innate lymphoid cells that express CD84.

Type 2 innate lymphoid cells (ILC2) produce high levels of Th2 cytokines. Our study demonstrates that cat allergen challenge in allergic rhinitis subjects rapidly induces increased peripheral blood ILC2.

Group 2 innate lymphocytes (ILC2) are enriched in active eosinophilic esophagitis

Author(s): Doherty, Taylor A; Baum, Rachel; Newbury, Robert O; Yang, Tom; Dohil, Ranjan; Aquino, Melissa; Doshi, Ashmi; Walford, Hannah H; Kurten, Richard C; Broide, David H; Aceves, Seema

Innate Type 2 Response to Alternaria Extract Enhances Ryegrass-Induced Lung Inflammation

Background: Exposure to the fungal allergen Alternaria alternata as well as ryegrass pollen has been implicated in severe asthma symptoms during thunderstorms. We have previously shown that Alternaria extract induces innate type 2 lung inflammation in mice. We hypothesized that the innate eosinophilic response to Alternaria extract may enhance lung inflammation induced by ryegrass. Methods: Mice were sensitized to ryegrass allergen and administered a single challenge with A. alternata extract before or after final ryegrass challenges. Levels of eosinophils, neutrophils, Th2 cells, innate lymphoid cells (ILC2), interleukin (IL)-5 and IL-13 in bronchoalveolar lavage (BAL) as well as inflammation and mucus were assessed. Results: Mice receiving ryegrass sensitization and challenge developed an eosinophilic lung response. A single challenge with Alternaria extract given 3 days before or 3 days after ryegrass challenges resulted in increased eosinophils, peribronchial inflammation and mucus production in the airways compared with ryegrass-only challenges. Type 2 ILC2 and Th2 cell recruitment to the airways was increased after Alternaria extract exposure in ryegrass-challenged mice. Innate immune challenges with Alternaria extract induced BAL eosinophilia, Th2 cell recruitment as well as ILC2 expansion and proliferation. Conclusions: A single exposure to Alternaria extract in ryegrass-sensitized and -challenged mice enhances the type 2 lung inflammatory response, including airway eosinophilia, peribronchial infiltrate, and mucus production, possibly through Th2 cell recruitment and ILC2 expansion. If translated to humans, exposure to both grass pollen and Alternaria may be a potential cause of thunderstorm-related asthma.

Leukotriene C4 Potentiates IL-33–Induced Group 2 Innate Lymphoid Cell Activation and Lung Inflammation

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R−/− mice, but not CysLT2R−/− mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R−/− mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata. Thus, CysLT1R promotes LTC4- and Alternaria-induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses.

Rigid substrate induces esophageal smooth muscle hypertrophy and eosinophilic esophagitis fibrotic gene expression

Cheng-Chiu Huang, PhD Yu Shin Kim, PhD William P. Olson, BS Fengxian Li, MD Changxiong Guo, BA Wenqin Luo, MD, PhD Andrew J. W. Huang, MD, MPH Qin Liu, PhD From the Department of Anesthesiology and the Center for the Study of Itch, Washington University School of Medicine, St Louis, Mo; the Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Md; the Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pa; the Department of Anesthesiology, Zhujiang Hospital, Southern Medical University, Guangzhou, China; and the Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Mo. E-mail: liuqi@anest. wustl.edu. This work was supported in part by the National Institutes of Health (grant no. R01EY024704 to Q. L.). Disclosure of conflict of interest: Q. Liu received research funding from the National Institutes of Health. The rest of the authors declare that they have no relevant conflicts of interest.

Impaired induction of allergic lung inflammation by Alternaria alternata mutant MAPK homologue Fus3

ABSTRACT The fungal allergen Alternaria alternata is associated with development of asthma, though the mechanisms underlying the allergenicity of Alternaria are largely unknown. The aim of this study was to identify whether the MAP kinase homologue Fus3 of Alternaria contributed to allergic airway responses. Wild-type (WT) and Fus3 deficient Alternaria extracts were given intranasal to mice. Extracts from Fus3 deficient Alternaria that had a functional copy of Fus3 introduced were also administered (CpFus3). Mice were challenged once and levels of BAL eosinophils and innate cytokines IL-33, thymic stromal lymphopoeitin (TSLP), and IL-25 (IL-17E) were assessed. Alternaria extracts or protease-inhibited extract were administered with (OVA) during sensitization prior to ovalbumin only challenges to determine extract adjuvant activity. Levels of BAL inflammatory cells, Th2 cytokines, and OX40-expressing Th2 cells as well as airway infiltration and mucus production were measured. WT Alternaria induced innate airway eosinophilia within 3 days. Mice given Fus3 deficient Alternaria were significantly impaired in developing airway eosinophilia that was largely restored by CpFus3. Further, BAL IL-33, TSLP, and Eotaxin-1 levels were reduced after challenge with Fus3 mutant extract compared with WT and CpFus3 extracts. WT and CpFus3 extracts demonstrated strong adjuvant activity in vivo as levels of BAL eosinophils, Th2 cytokines, and OX40-expressing Th2 cells as well as peribronchial inflammation and mucus production were induced. In contrast, the adjuvant activity of Fus3 extract or protease-inhibited WT extract was largely impaired. Finally, protease activity and Alt a1 levels were reduced in Fus3 mutant extract. Thus, Fus3 contributes to the Th2-sensitizing properties of Alternaria.