IL-33 Precedes IL-5 in Regulating Eosinophil Commitment and Is Required for Eosinophil Homeostasis This information is current as Laura K. Johnston, Chia-Lin Hsu, Rebecca A. Krier-Burris, of September 28, 2021. Krishan D. Chhiba, Karen B. Chien, Andrew McKenzie, Sergejs Berdnikovs and Paul J. Bryce J Immunol 2016; 197:3445-3453; Prepublished online 28 September 2016; doi: 10.4049/jimmunol.1600611 Downloaded from http://www.jimmunol.org/content/197/9/3445 Supplementary http://www.jimmunol.org/content/suppl/2016/09/28/jimmunol.160061 Material 1.DCSupplemental http://www.jimmunol.org/ References This article cites 44 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/197/9/3445.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IL-33 Precedes IL-5 in Regulating Eosinophil Commitment and Is Required for Eosinophil Homeostasis Laura K. Johnston,* Chia-Lin Hsu,* Rebecca A. Krier-Burris,* Krishan D. Chhiba,* Karen B. Chien,* Andrew McKenzie,† Sergejs Berdnikovs,* and Paul J. Bryce* Eosinophils are important in the pathogenesis of many diseases, including asthma, eosinophilic esophagitis, and eczema. Whereas IL-5 is crucial for supporting mature eosinophils (EoMs), the signals that support earlier eosinophil lineage events are less defined. The IL-33R, ST2, is expressed on several inflammatory cells, including eosinophils, and is best characterized for its role during the initiation of allergic responses in peripheral tissues. Recently, ST2 expression was described on hematopoietic progenitor subsets, where its function remains controversial. Our findings demonstrate that IL-33 is required for basal eosinophil homeostasis, because both IL-33– and ST2-deficient mice exhibited diminished peripheral blood eosinophil numbers at baseline. Exogenous IL-33 administration increased EoMs in both the bone marrow and the periphery in wild-type and IL-33–deficient, but not ST2- Downloaded from deficient, mice. Systemic IL-5 was also increased under this treatment, and blocking IL-5 with a neutralizing Ab ablated the IL-33–induced EoM expansion. The homeostatic hypereosinophilia seen in IL-5–transgenic mice was significantly lower with ST2 deficiency despite similar elevations in systemic IL-5. Finally, in vitro treatment of bone marrow cells with IL-33, but not IL-5, led to specific early expansion of IL-5Ra–expressing precursor cells. In summary, our findings establish a basal defect in eosinophilopoiesis in IL-33– and ST2-deficient mice and a mechanism whereby IL-33 supports EoMs by driving both systemic IL-5 production and the expansion of IL-5Ra–expressing precursor cells. The Journal of Immunology, 2016, 197: 3445–3453. http://www.jimmunol.org/ osinophils are immune cells that circulate in the blood and appears to be the critical cytokine specific to eosinophil develop- also reside in several tissues, including the intestine, ment (4–6) and mechanistically acts to drive expansion and survival E thymus, and adipose tissue (1). In addition to their roles in of EoMs within the bone marrow (7). In contrast, the factors in- homeostatic processes, eosinophils contribute to the pathology of volved in driving the initial commitment of GMP into the eosinophil many type 2–mediated diseases, such as asthma, eosinophilic lineage are less clear. esophagitis, and atopic dermatitis (2). Many studies have established IL-33 is the most recently discovered member of the IL-1 family the important effector functions of eosinophils and their ability to of cytokines. In its initial description by Schmitz et al. (8), rIL-33 by guest on September 28, 2021 modulate inflammation through the release of granule contents and was shown to promote several type 2–associated responses, in- cytokines. However, the development of eosinophils in the bone cluding type 2 cytokine expression (IL-4, IL-5, and IL-13) and IgE marrow is less understood. It has been established that granulocyte– production. Furthermore, ST2, the IL-33R, is expressed on many macrophage progenitors (GMPs) give rise to eosinophil lineage– cell types involved in type 2 effector responses, including Th2 committed progenitors (EoPs), which then develop into fully cells (9), mast cells, basophils, eosinophils (10), and type 2 innate granulated mature eosinophils (EoMs) (3). Although IL-3, GM-CSF, lymphoid cells (ILC2s) (11). Subsequently, IL-33 has been ex- and IL-5 can drive this eosinophilopoiesis process in vitro (1), IL-5 tensively studied in the setting of helminth infections and allergic diseases. Studies in asthma (12–14), food allergy (15), and hook- worm models (16) have reported the presence of reduced eosin- *Division of Allergy-Immunology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60610; and †Medical Research ophilic inflammation in IL-33– or ST2-deficient mice, suggesting Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom a positive interplay between IL-33 and eosinophils. Indeed, the ORCIDs: 0000-0002-4967-1257 (L.K.J.); 0000-0002-6997-7667 (K.D.C.). initial description of IL-33 demonstrated that in vivo administra- Received for publication April 7, 2016. Accepted for publication August 31, 2016. tion of rIL-33 was sufficient to increase peripheral blood eosino- This work was supported by National Institutes of Health Grants RO1AI105839 and phil numbers (8). Similarly, in vitro IL-33 was proposed to support RO1AI076456 (to P.J.B.) and T32AI007476-16 (to L.K.J.). Imaging work was eosinophil differentiation from bone marrow (17). In sharp con- performed at the Northwestern University Center for Advanced Microscopy, which was supported by National Cancer Institute Grant CCSG P30 CA060553 awarded to trast, Dyer et al. (18) examined the effects of IL-33 on eosinophil the Robert H. Lurie Comprehensive Cancer Center. Flow cytometry cell sorting was development using in vitro differentiation approaches and concluded supported by the Northwestern University Flow Cytometry Core Facility, which was supported by Cancer Center Support Grant NCI CA060553, and was performed on a that IL-33 antagonized IL-5–dependent eosinophilopoiesis and BD FACSAria SORP system that was purchased through the support of National supported monocyte development. Macrophage activation has also Institutes of Health Grant 1S10OD011996-01. been implicated in driving IL-33–induced lung eosinophilia (19). Address correspondence and reprint requests to Dr. Paul J. Bryce, Division of In this study, we sought to reconcile these conflicting results by Allergy-Immunology, Feinberg School of Medicine, Northwestern University, 240 E. Huron Street, Chicago, IL 60610. E-mail address: [email protected] examining the role of IL-33 in eosinophil development in vivo and The online version of this article contains supplemental material. in vitro. We demonstrate that IL-33– and ST2-knockout (KO) mice show homeostatic dysregulation of granulocyte responses in both Abbreviations used in this article: EoM, mature eosinophil; EoP, eosinophil lineage– committed progenitor; EoPre, eosinophil precursor; Flt3L, Flt3 ligand; GMP, granulocyte– the blood and the bone marrow compartments. Furthermore, our macrophage progenitor; ILC2, type 2 innate lymphoid cell; KO, knockout; SCF, stem cell data show not only that IL-33 is a potent stimulus for expansion of factor; WT, wild-type. the Siglec-F+ eosinophil pool, but also that the functional influ- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 ence of IL-33 lies in expansion of an eosinophil precursor (EoPre) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600611 3446 IL-33 SUPPORTS EOSINOPHIL PRECURSOR EXPANSION population, as well as in upregulation of the IL-5Ra on this buffer (1% FBS in PBS) and incubated with allophycocyanin-Cy7–labeled population. As already established, IL-33 also strongly induces anti-CD16/32 for 30 min (Supplemental Table I, panel 5). After washing in FACS buffer, cells were blocked with anti-CD16/CD32 (BD Biosciences, IL-5, which further fuels the development of EoPre cells into an San Jose, CA) for 10 min and then stained in 100 ml of Ab mixture in FACS EoM phenotype. Consequently, we propose that IL-33 and IL-5 buffer (as detailed in Supplemental Table I) for 30 min at 4˚C in the dark. are cooperative cytokines for eosinophilopoiesis and that IL-33 Cells were then washed in FACS buffer and fixed in 4% paraformaldehyde. precedes the need for IL-5 support in the progression toward Samples were run on an LSRII flow cytometer (BD Biosciences) or sorted eosinophil maturity. on a FACSAria SORP system. Data were analyzed on FlowJo 10.7 (Tree Star, Ashland, OR). Compensation on samples collected by the LSRII was performed in FlowJo postcollection. Materials and Methods Mice Statistical analysis Appropriate statistical testing was performed using GraphPad Prism 6 Wild-type (WT) C57BL/6J mice were purchased from Jackson Laboratories software (GraphPad, La Jolla, CA). (Bar Harbor, ME). ST2 KO mice were previously generated by Andrew McKenzie and backcrossed to C57BL/6J for eight generations. IL-33 KO mice on the C57BL/6J background were provided by Dr. Dirk Smith Results (Amgen, Seattle, WA). IL-5–transgenic mice (strain NJ.1638, previously Granulocytes are reduced in ST2-deficient mice described [20]) were provided to Dr.