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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

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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 of many diseases, including , , 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 eosinophil numbers at baseline. Exogenous IL-33 administration increased EoMs in both the 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 specific to eosinophil develop- also reside in several tissues, including the intestine, ment (4–6) and mechanistically acts to drive expansion and survival E , and (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 (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 . In its initial description by Schmitz et al. (8), rIL-33 by guest on September 28, 2021 modulate inflammation through the release of 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 – production. Furthermore, ST2, the IL-33R, is expressed on many progenitors (GMPs) give rise to eosinophil lineage– types involved in type 2 effector responses, including Th2 committed progenitors (EoPs), which then develop into fully cells (9), mast cells, , eosinophils (10), and type 2 innate granulated mature eosinophils (EoMs) (3). Although IL-3, GM-CSF, lymphoid cells () (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 and allergic diseases. Studies in asthma (12–14), food (15), and hook- worm models (16) have reported the presence of reduced - *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 development. Macrophage activation has also Institutes of Health Grant 1S10OD011996-01. been implicated in driving IL-33–induced (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 are reduced in ST2-deficient mice described [20]) were provided to Dr. Sergejs Berdnikovs by Dr. James Lee (Mayo Clinic, Phoenix, AZ) and crossed with ST2 KO mice. Depending on Because recently it had been shown that ST2 was expressed on the experimental requirements, both male and female mice (aged 6–36 wk) hematopoietic stem cells (23), we initially asked whether ST2 was were used. Animals were housed under specific pathogen-free conditions necessary for competent hematopoiesis. Naive ST2 KO mice had at Northwestern University. All experiments were approved by the North- lower weight/body weight ratios and total cell numbers in Downloaded from western University Animal Care and Use Committee. bone marrow than WT mice (Fig. 1A, 1B), suggesting a defect in Intraperitoneal injections the hematopoietic compartment. Upon further cytologic examina- tion of hematopoietic-derived cell populations in peripheral blood, Mice were given IL-33 (eBioscience, San Diego, CA) daily by i.p. injection at 0.4 mg/d for a total of seven injections. For some experiments, mice were ST2 KO mice had fewer peripheral blood eosinophils than WT also given either anti–IL-5 (TRFK5; eBioscience) or control (rat mice as determined by with Discombe’s fluid (Fig. 1C).

IgG1 k; eBioscience) at a dose of 25 mg per mouse by i.p. injection on Because IL-33 had been proposed to promote a macrophage-like http://www.jimmunol.org/ days 21, 2, and 5. One day after the last injection, cells and serum were phenotype in vitro (18) and neutrophilic inflammation in vivo collected from blood for eosin staining, flow cytometry, and cytokine (24), the frequency of , , eosinophils, protein analysis. In addition, bone marrow was collected for RNA, cyto- logic, and flow cytometric analysis. and in the blood of ST2 KO and WT mice was also assessed using flow cytometry (Fig. 1D, 1E). In addition to Bone marrow analysis eosinophils, ST2 KO mice had fewer neutrophils than WT mice Real-time RT-PCR was used to determine IL-5 expression, as previously (Fig. 1E). Notably, there were no differences in total monocytes, described. Myeloid-to-erythroid ratios in bone marrow were assessed by CD115+Ly6C+ monocyte progenitors, or CD115+Ly6C2 mature histologic inspection of nuclear and staining profiles on cytologic prepa- monocytes. Similar to ST2 KO mice, IL-33 KO mice also showed rations (Shandon Cytospin; Thermo Fisher Scientific, Waltham, MA) by guest on September 28, 2021 stained with Kwik-Diff stain (Thermo Scientific) using established ap- lower numbers and frequency of both eosinophils and neutrophils in proaches (21); the relative proportions of granulocytic and erythrocytic peripheral blood than WT mice (Fig. 2B–D and data not shown). cells were calculated after elimination. Thus, IL-33 and ST2 are necessary for and eosinophil homeostasis in the periphery. Blood analysis Blood was collected into EDTA-coated tubes, and absolute eosinophil IL-33 expands eosinophils in vivo numbers were determined after staining with Discombe’s fluid. Next, we wanted to determine whether exogenous IL-33 was In vitro culture of eosinophils sufficient to induce hematopoiesis in vivo and whether such an effect was dependent on ST2. Following the approach used by Bone marrow from the femur and tibia was recovered by brief centrifu- Schmitz et al. (8), we injected WT, IL-33 KO, and ST2 KO mice gation, and the pellet was resuspended in 10 ml of complete media (RPMI 1640 with 2 mM of L-glutamine, 10% FCS, 100 U/ml , 100 mg/ml with 0.4 mg of rIL-33 or PBS for 7 d and analyzed them 18 h after streptomycin, 1% nonessential amino acids, 1 mM of sodium pyruvate, the last injection. As predicted, exogenous IL-33 increased splenic 25 mM of HEPES, 0.05 mM of 2-ME). Cells were counted and seeded at weight and peripheral blood eosinophils in WT and IL-33 KO mice, 6 3 3 10 cells/ml in 1 ml of complete media supplemented with one of but not ST2 KO mice (Fig. 2A, 2B). This increase in eosinophils by the following conditions for 3 d: 100 ng/ml stem cell factor (SCF) and cytology was also confirmed by flow cytometry, with eosinophils 100 ng/ml Flt3 ligand (Flt3L), 10 ng/ml IL-5, or 10 ng/ml IL-33. Alter- + hi neg2lo + + natively, cells were seeded at 0.5 3 106 cells/ml in 6 ml of complete media being defined as CD45 SSC Ly6G CD11b Siglec-F cells supplemented with 100 ng/ml SCF and 100 ng/ml Flt3L with or without (Fig. 2C, 2D). Although the levels of eosinophils observed after 10 ng/ml IL-33; on days 3 and 7, nonadherent cells were collected, IL-33 treatment were lower in IL-33 KO mice than in WT, this was 6 counted, and readjusted to 0.5 3 10 cells/ml with fresh medium con- not significantly different, and the relative increases were similar taining 10 ng/ml IL-5. when the differences in basal numbers were considered. In terms of Neutrophil isolation from bone marrow the effect of exogenous IL-33 on other cell populations in the blood, IL-33 did not increase the frequency of neutrophils in the blood Bone marrow neutrophils were isolated as previously described (22). The purity of neutrophils (.95%) was confirmed using flow cytometry against (Supplemental Fig. 1) despite ST2 KO mice displaying impaired Ly6C+Gr-1+Siglec-F2 cells. neutrophil numbers in the basal state (Fig. 1E); it did, however, increase the numbers of ILC2s in agreement with previous literature Flow cytometric analysis (11). Because we observed differences in total bone marrow cell Bone marrow cells or whole blood was lysed with RBC lysis buffer numbers (Fig. 1B) and eosinophil numbers in the peripheral blood (eBioscience) following the manufacturer’s protocol. Bone marrow cells between ST2 KO and WT mice (Fig. 1C), we queried whether the were counted, and 5 3 106 cells were used for staining. Cells were washed with PBS and stained with 0.25 ml of LIVE/DEAD Fixable Aqua Dead Cell reduced numbers in ST2 KO mice might be caused by an effect on Stain (Thermo Fisher Scientific) in 500 ml of PBS for 20 min at room the bone marrow itself (and perhaps eosinophilopoiesis) rather than temperature in the dark. In some experiments, cells were washed with FACS just the known effect on the periphery. Indeed, in addition to the The Journal of Immunology 3447

FIGURE 1. ST2 is necessary for basal granulocyte homeostasis. WT and ST2 KO mice were analyzed for (A) spleen weight/body weight, (B) total cells in bone marrow, and (C) eosinophil numbers by staining with Discombe’s fluid. (D and E)Usingthe flow cytometry gating strategy shown in (D), we analyzed blood leuko- cytes (E). Data represent mean 6 SEM (n = 8–18 from three independent ex- periments). *p # 0.05, **p # 0.01, ****p # 0.0001 compared with WT by two-tailed Student t test. Downloaded from changes seen in the blood, the bones of the IL-33–treated WT mice results were observed when CD11b was used instead of the full were noticeably lighter in color than in PBS-treated controls, and lineage mixture (Supplemental Fig. 4A, 4B). Because IL-33 was this color change was ST2 dependent (Fig. 2E). Upon further ex- previously shown to induce IL-5 (8), we hypothesized that the ex- amination, IL-33 treatment increased the myeloid-to-erythroid ratio panded EoM pool might be because of the influence of IL-5. Indeed, in the bone marrow, indicating an expansion in the myeloid com- IL-33 treatment significantly increased IL-5 mRNA levels in bone

partment within the bone marrow compartment and that IL-33 marrow (Fig. 3G) and IL-5 protein levels in serum (Fig. 3H). Taken http://www.jimmunol.org/ might exert direct function at this important eosinophil develop- together, these data define that IL-33 supports both the expansion of ment site (Fig. 2F). Taken together, these data suggest that, within the EoM pool and the elevation of IL-5 in the bone marrow and granulocyte populations, IL-33 can expand EoMs in the blood and periphery. alter myeloid cells in the bone marrow. IL-33–driven eosinophilopoiesis is IL-5 dependent IL-33 expands EoMs in the bone marrow To address the contribution of this elevated IL-5 in IL-33–driven The increased myeloid-to-erythroid ratio led us to further examine eosinophil expansion, we treated mice with i.p. injection of 0.4 mg eosinophil development specifically in the bone marrow using flow of IL-33 for 7 d in the presence of a neutralizing anti–IL-5 Ab or + cytometry. Because we observed an increase in the pool of Siglec-F its isotype control (Fig. 4A). In this particular experiment, we by guest on September 28, 2021 eosinophils in peripheral blood after IL-33 treatment, we used noticed that anti–IL-5 treatment dramatically altered IL-5R ex- this Siglec-F marker to initially examine the cells in the bone pression levels on cells from those treated mice, most likely be- marrow. Indeed, the total Siglec-F+ cell population in the bone cause of feedback from IL-5R being internalized upon binding to marrow was similarly increased in WT and IL-33 KO, but not IL-5 (26). We therefore felt it was inappropriate to define the ST2 KO, mice after IL-33 treatment (Fig. 3A, 3B). Interestingly, EoPre and EoM populations in this study, because proper identi- strict gating based on the fluorescence minus one controls in- fication of these populations relied on IL-5R staining as a defining corporated a range of Siglec-F–expressing cells and higher marker. Instead, we focused on the significant increase in Siglec-F+ overall percentages than have been previously reported for EoMs cells, as shown in Fig. 3B, and the significant decrease in GMP-like (25). To further examine eosinophil-related populations within cells, as shown in Fig. 3F. In mice that received PBS, anti–IL-5 this Siglec-F+ pool, we defined a population of GMP-like cells treatment did not have any significant effect on the frequency of 2 2 + 2 lo hi + + (Lin Sca1 Siglec-F IL-5Ra SSC ckit CD34 ST2 ), EoPre cells Siglec-F+ cells (Fig. 4B, 4C) or GMP-like cells (Fig. 4D) in the bone hi 2 lo + lo 2 2 2 (Lin Sca1 Siglec-F IL-5Ra SSC ckit CD34 ST2 ), and EoM marrow; moreover, despite a trend toward being lower, no signifi- lo 2 hi + hi 2 2 lo cells (Lin Sca1 Siglec-F IL-5Ra SSC ckit CD34 ST2 ) (char- cant difference was observed in the number of eosinophils in the acterization and fluorescence minus one control staining are shown peripheral blood after anti–IL-5 treatment (Fig. 4E). As before, the in Supplemental Fig. 2). Because these GMP-like and EoPre cells addition of IL-33 significantly increased the Siglec-F+ population, expressed slightly different markers than the classically defined and this was prevented by anti–IL-5 treatment (Fig. 4C), suggesting GMP and EoP cells, we also used traditional staining regimens to that this response was regulated by the elevated IL-5 levels upon 2 2 hi + hi examine the GMP (Lin Sca1 ckit CD34 CD16/CD32 ), EoP IL-33 treatment. Anti–IL-5 treatment was also sufficient to com- 2 2 + lo + (Lin Sca1 IL-5Ra ckit CD34 ), and common myeloid progeni- pletely block the increases in peripheral EoMs (Fig. 4E). In contrast, 2 2 hi + hi tor (Lin Sca1 ckit CD34 CD16/CD32 ), which precedes the GMP the significant decrease in GMP-like cells upon IL-33 treatment was (Supplemental Fig. 3) (3). Importantly, EoM frequency in the bone unaffected by blockade of IL-5. Collectively, these data suggest that marrow was significantly lower in PBS-treated ST2 KO and IL-33 the IL-33–driven expansion of EoMs we observed is dependent on KO mice than in WT mice (Fig. 3C, 3D), which is in agreement with IL-5. In contrast, the decrease in the GMP-like population is IL-5 our analysis of EoMs in the peripheral blood (Fig. 1C, 1E). Similar independent and demonstrates that IL-33 exerts its influence on less frequencies of EoPre, GMP-like, and GMP cells were observed at mature populations that are separable from the influences of ele- baseline for all genotypes (Fig. 3C, 3E, 3F, Supplemental Fig. 3). vated systemic IL-5. ST2 KO mice had significantly lower EoPs at baseline (Supplemental Fig. 3). After treatment with IL-33 for 7 d, WT and IL-33 KO mice IL-5–driven eosinophilopoiesis is ST2 dependent had dramatically increased frequency of EoMs (Fig. 3C, 3D), lower We next sought to better define whether the requirement for IL-33 GMP-like cells (Fig. 3F), and similar EoPre (Fig. 3C, 3E). Similar on basal eosinophil homeostasis lay upstream or downstream of IL-5. 3448 IL-33 SUPPORTS EOSINOPHIL PRECURSOR EXPANSION

was assessed during these later stages of culture. Because our data suggest that IL-33 is upstream of IL-5, we sought to examine the effects of IL-33 during the initial progenitor expansion phase. We cultured freshly isolated bone marrow cells with SCF and Flt3L, IL-5, or IL-33 for 3 d and assessed for changes in EoPre and EoM populations by flow cytometry (Fig. 5A, 5B). As shown in Fig. 5A and quantified in Fig. 5B, both populations were maintained after 3 d when cultured in SCF and Flt3L or IL-5. In contrast, IL-33 drastically increased the EoPre population in WT and IL-33 KO cultures, but not in ST2 KO cultures. Assessment of cytology after cell sorting of the EoPre and EoM populations showed clear multilobed nuclear morphology consistent with eosinophils but poor granule staining in the EoPre, whereas the EoM population possessed clear eosin-stained granules (Fig. 5C). Moreover, bone marrow cultured with IL-33, but not SCF and Flt3L or IL-5, led to a substantial increase in IL-5Ra mean fluorescence intensity on the EoPre (Fig. 5D), further indicating their commit- ment to an eosinophil lineage. When CD11b was specifically used

instead of the full lineage mixture, a similar decline in EoMs and Downloaded from expansion of EoPre was observed, as well as an upregulation of IL-5Ra on the EoPre population (Supplemental Fig. 4C–F). Although the difference in granularity we see by cytology is consistent with the differences in side scatter we observe in our flow cytometry analysis, it contrasts with the previously described EoPre,

which have been reported to possess granule proteins (29). More- http://www.jimmunol.org/ over, IL-5Ra can also be expressed on neutrophils under certain FIGURE 2. IL-33 is sufficient to drive eosinophil expansion in vivo. conditions (30). Therefore, to further confirm that this population WT, ST2 KO, and IL-33 KO mice were given 0.4 mg of IL-33 or PBS i.p. we considered to be EoPre cells was truly within the eosinophil for 7 d and analyzed 18 h after the last injection. (A) Spleen weight was lineage, we interrogated the gene expression profiles of these EoPre measured, and blood eosinophils were counted by (B) Discombe’s fluid cells to assess whether they expressed eosinophil-associated genes. and (C and D) flow cytometry. (E) Representative photograph of color In this experiment, we used the EoM population as a positive change of bone marrow. (F) Quantification of the myeloid-to-erythroid control and bone marrow neutrophils as a negative control lying still 6 ratio in bone marrow. Data represent mean SEM (n = 4–12 from three within the granulocyte lineage. Whereas bone marrow neutrophils, independent experiments). *p # 0.05, ***p # 0.001, ****p # 0.0001 by EoPre, and EoMs had similar expression of granulocyte-associated by guest on September 28, 2021 two-way ANOVA. genes Csf2ra (GM-CSFRa)andSpi1 (PU.1), EoPre and EoMs had significantly lower expression of the neutrophil-associated We used the NJ.1638 strain of mice, which possess transgenic gene Csf3r (G-CSFR) than neutrophils (Fig. 5E). Instead, the overexpression of IL-5 and develop a profound, age-dependent EoM and EoPre populations expressed significantly higher levels hypereosinophilia in the blood (27), and examined the effects of of the eosinophil-associated genes Epx (), ST2 deficiency on this response. Evaluating the homeostatic pe- Prg2 (), Cebpa (C/EBPa), Gata1 (GATA-1), ripheral blood eosinophil numbers over time, we observed that the and Gata2 (GATA-2) than neutrophils (Fig. 5E). Furthermore, substantial elevations in eosinophils seen in the NJ.1638 mice were we noted that the EoPre population expressed intermediate levels significantly diminished in the absence of IL-33 signaling in the of these eosinophil-associated genes, suggesting that they pos- NJ.1638/ST2KO mice (Fig. 4F). Although we observed a trend sessed an immature eosinophil phenotype. Together, these find- toward higher serum IL-5 at early time points in the NJ.1638/ST2KO ings suggest that IL-33 may function as a for the mice versus NJ.1638, this was not statistically significant and there early commitment toward the EoPre population and/or drive their were no significant differences as we tracked these animals during expansion. aging (Fig. 4G). Interestingly, NJ.1638/ST2KO mice still had more To test this idea further, we cultured bone marrow cells in SCF eosinophils than WT and ST2 KO mice, implying that ST2 is not an and Flt3L with or without IL-33 for 3 d before reculturing them in essential checkpoint for eosinophilopoiesis and that ST2-independent IL-5 for another 7 d; cell populations were assessed by flow mechanisms exist to support IL-5–responsive eosinophil devel- cytometry at various stages of the culture. Although no difference opment. Regardless, these studies do establish that ST2 clearly was seen in the GMP populations (data not shown), WT cultures regulates IL-5–driven eosinophil homeostasis and that IL-33 sig- grown with IL-33 exhibited high numbers of EoPre populations naling lies upstream of the effect of IL-5 on eosinophils. at early stages of culture, as well as a rapid increase in total EoMs that was sustained throughout the entire culture period IL-33 expands EoPre and upregulates IL-5R in vitro (Fig. 5F). In contrast, the WT culture without early IL-33 treat- With our in vivo data showing that IL-33 and IL-5 both play a role ment showed little change in EoPre cells, and increases in EoM in increasing eosinophils, we next turned to in vitro culture systems appeared only after day 10 of culture. ST2 KO cells showed no to assess the mechanisms by which IL-33 and IL-5 cooperate to expanded EoPre on IL-33 treatment and generated even fewer promote development of EoMs. Previously, Dyer et al. (18, 28) EoMs over the course of culture with IL-5. Taken together, these used a protocol for generating eosinophils in vitro in which bone data suggest that IL-33 precedes the need for IL-5 and functions marrow cells were cultured in SCF and Flt3L for the first 4 d to mainly to promote not only the expansion of EoPre cells but also expand the progenitor cells, followed by a switch to IL-5 in the IL-5Ra upregulation, which then sustains the EoM pool if IL-5 culture to promote eosinophil development; the effect of IL-33 is provided. The Journal of Immunology 3449

FIGURE 3. In vivo IL-33 expands EoMs. WT, ST2 KO, and IL-33 KO mice were given 0.4 mg of IL-33 i.p. for 7 d and ana- lyzed 18 h after the last injection. (A) Rep- resentative flow plots of the expansion of the Siglec-F+ population in bone marrow. (B) Frequency of the Siglec-F+ population. (C) Representative flow plots of eosinophil Downloaded from populations in bone marrow. (D) Frequency of EoMs (Siglec-F+LinloSSChi). (E)Fre- quency of EoPre (Siglec-F+LinhiSSClo). (F) Frequency of GMP-like (Siglec-F+Lin2ckithi). (G) IL-5 mRNA expression in bone marrow. (H) Serum IL-5 concentration. All frequen- +

cies are shown as the percent of CD45 cells. http://www.jimmunol.org/ Data represent mean 6 SEM [n = 3–9 from three (A–D)ortwo(E and F) independent experiments]. **p # 0.01, ***p # 0.001, ****p # 0.0001 by two-way ANOVA. by guest on September 28, 2021

Discussion using eosinophil numbers as a key response readout in previous IL-33 has emerged as an important cytokine in allergic diseases, studies may be warranted. largely because of its potential to activate cells that are hallmarks of Although our data clearly show a role for IL-33 in the bone allergy, including eosinophils, mast cells, and basophils (31). marrow, other studies examining ST2 expression on hematopoietic Outside of allergy, IL-33 has also been proposed to be involved in stem and progenitor cells in the bone marrow have shown conflicting bacterial and viral infections, tumorigenesis, , fibrosis findings. Initial reports claimed that ST2 was present on multiple 2 (32), and more recently, hematopoiesis (23, 33). In this article, we subsets of Lin ckit+ progenitor cells, including GMP cells (23). In define a previously unappreciated mechanism for IL-33 in regu- contrast, recent work using a spontaneous mutant mouse model of lating eosinophil commitment. myeloproliferative neoplastic tumorigenesis failed to detect ST2 Our data demonstrate that IL-33 directs the eosinophil com- expression on a variety of and progenitor partment by expanding the EoPre frequency and upregulating IL-5Ra lineages (35). However, this work did demonstrate a functional role to license the responsiveness of these precursors to IL-5 within for IL-33 in regulating myeloid cell fate in this model. From our the bone marrow. Importantly, the defects in basal eosinophil pop- own characterization of the Siglec-F–expressing compartment ulations we identified in the IL-33 KO and ST2 KO mice strongly (Supplemental Fig. 2) in which we define the stages of eosinophil implicate a homeostatic contribution of this cytokine that functions development based on surface markers as well as size and granu- outside of a disease pathogenesis setting. Indeed, the previously larity, ST2 does appear to be expressed on GMP-like cells and defined function of IL-33 as an alarmin released upon tissue damage EoMs, but its expression is lost at the EoPre stage. One possibility or injury (34) seems unlikely to explain such homeostatic regulation for this diminished ST2 expression on the EoPre population is that in healthy animals. Thus, the underlying alteration in eosinophil IL-33 binding leads to receptor internalization, as occurs when IL-5 homeostasis in IL-33 KO and ST2 KO mice we describe in this binds to IL-5R (26). ST2 can also be shed from the surface of cells article may impact the numerous interpretations others have made after activation (36). Alternatively, differences in gating strategy in from studying the IL-33 KO and ST2 KO mice in disease models; terms of how to define GMP cells has influenced our conclusions; consequently, reconsideration of some conclusions made from data certainly, the expression of low Siglec-F levels on a progenitor cell, 3450 IL-33 SUPPORTS EOSINOPHIL PRECURSOR EXPANSION

suggested for eosinophil maintenance (39), or only under disease conditions remains to be determined. In agreement with others, we show that administration of IL-33 is capable of inducing significant elevation of IL-5, both system- ically and within the bone marrow itself (Fig. 3G, 3H). However, the source of this IL-5 remains to be determined. Because the mice in this study were not primed toward a Th2 adaptive re- sponse, it seems reasonable to predict that innate cells, such as ILC2s, might be a significant source. ST2-expressing ILC2 pop- ulations that produce IL-5 were previously shown to regulate eosinophil homeostasis in the intestine and lung, but this pro- duction was spontaneous; moreover, these cells were shown to be largely absent from the bone marrow compartment (39). Inter- estingly, a Sca-1+ precursor population within the bone marrow that expresses ST2 and produces IL-5 in response to IL-33 has been reported (40), suggesting that both the expansion of the IL-5Ra+ EoPre population and the elevation of IL-5 itself could occur locally within the bone marrow and be driven by IL-33. In

our in vitro cultures, IL-5 was detected in the media after 3 d of Downloaded from culture with SCF, Flt3L, and IL-33 (54.6 6 6.7 pg/ml) but was undetectable (,31.25 pg/ml) with SCF and Flt3L alone. This might explain the rapid transition from precursors toward EoMs that we observed. Importantly, through the use of the IL-5–transgenic mouse, our studies clearly define a role for IL-33 signaling in

eosinophil frequency that is independent of the IL-33–induced http://www.jimmunol.org/ IL-5 response and establishes that the contribution of IL-33 lies upstream of IL-5–driven eosinophilopoiesis. Similarly, it has pre- viously been shown that IL-33 cannot elicit eosinophilia in IL-5 KO mice, helping to define that IL-5 lies downstream of IL-33–driven responses (18). Furthermore, blocking IL-5 in vivo prevented the increased mature blood eosinophil expansion that occurs in re- sponse to IL-33 but failed to prevent the observed decreases in the GMP-like cells in the bone marrow. Currently, we postulate that the GMP-like cells, shown as expressing ST2 in Supplemental Fig. 2, by guest on September 28, 2021 FIGURE 4. Eosinophilopoiesis is both IL-5 dependent and ST2 de- are being driven toward the EoPre phenotype by IL-33, and that the pendent. (A) Injection scheme for (B)–(E). (B) Representative flow plots of the expansion of the Siglec-F+ population in bone marrow. (C) Frequency high expression of IL-5R on these cells combined with the elevated of the Siglec-F+ population shown in (B). (D) Frequency of GMP-like IL-5 (from currently unknown cells) results in rapid increases in (Siglec-F+Sca-12Lin2ckithi). (E) Blood eosinophil number determined EoMs. This model is supported by our studies in which we neu- by staining with Discombe’s fluid. (F) Blood eosinophil numbers over tralized IL-5 and affected only the IL-33–induced changes in EoMs, time as determined by staining with Discombe’s fluid. (G)SerumIL-5 but this concept requires further studies to define fully. concentration. Data represent mean 6 SEM [n = 5–14 from one (B–E) Previous studies using in vitro culture approaches to eosino- or four (F and G) independent experiments]. *p # 0.05, **p # 0.01, philopoiesis have led to confusing findings. Whereas Stolarski et al. A D F G ****p # 0.0001 by one-way ( – ) and two-way ANOVA ( and ). (17) claimed that culture with IL-33 alone for 5 or 8 d was suf- n.s., not significant. ficient to induce eosinophil differentiation, Dyer et al. (18) con- cluded that IL-33 did not support eosinophilopoiesis and, instead, which we are choosing to term GMP-like because of the other antagonized the effects of IL-5 and promoted monocyte differ- marker profiles (Lin2Sca12IL-5Ra2c-kit+CD34+), has not been entiation. Data from our initial in vitro studies were in agreement previously described to the best of our knowledge. Interestingly, with Dyer et al. (18) in that IL-33 failed to sustain the cell cultures publicly available gene expression microarray data sets do support to day 5 or beyond, with cells dying through (data not the potential for expression of Siglec-F on GMP cells, as well as on shown). We also observed that the addition of IL-33 to SCF and other progenitor subsets (http://biogps.org/gene/233186). Flt3L during the first 3 d of culture led to significant expansion of Recently, significant increases in EoMs, CD34+ hematopoietic total cell numbers at day 3 compared with cultures with SCF and progenitor cells, and committed eosinophil progenitors were Flt3L alone and that this was not seen with ST2KO cells. As shown reported within the of asthmatic patients; this report further in Fig. 5, these cells, although clearly not EoMs, possessed hallmark showed that IL-33 treatment primed the CD34+ population for characteristics indicative of EoPre. Interestingly, they expressed migration toward stromal cell–derived factor 1a (CXCL12) (37). mRNA for eosinophil granule proteins and eosinophil-associated It has also been shown that CD34+ cells circulating in the blood transcription factors, but lacked eosin-positive granules. Because express ST2, and activation of these cells with IL-33 leads to of this and the clearly defined nuclear morphology of an eosinophil, significant production of Th2-type cytokines, including IL-5 (38). we postulate that these cells represent an alternative precursor state Although our data define the responses to IL-33 that occur within than those defined from long-term IL-5 culture approaches (29). the bone marrow compartment, the recruitment of progenitor Initially, when these cultures were switched into IL-5, many of these populations after IL-33 exposure might support similar eosinophil expanded cells (.50%) were dying by apoptosis at day 7, even when developmental processes that occur within peripheral tissues; a 10-fold higher concentration of IL-5 was used (data not shown). however, whether this might occur under homeostasis, as has been We subsequently found that readjusting the cell density with each The Journal of Immunology 3451 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. IL-33 expands IL-5Ra+ progenitors. (A) Representative flow cytometry, (B) quantification of (A), and (C) images of sorted EoPre and EoMs of bone marrow cells cultured with SCF and Flt3L, IL-5, or IL-33 for 3 d. Scale bar, 100 mm. (D) IL-5Ra mean fluorescence intensity of EoPre after 3 d in culture. (E) Gene expression of EoPre and EoMs sorted from bone marrow cultured in SCF, Flt3L, and IL-33 for 3 d. Gene expression is normalized to the levels seen in bone marrow neutrophils. (F) Total number of EoPre and EoMs over the course of 10 d as determined by flow cytometry. Data represent the mean percent gated (A)andmean6 SEM (B, D,andE). n = 3–5 from 4 (A–D)or3(E and F) independent experiments. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001 by two-way ANOVA (A, B, D,andF)ortwo-tailedStudentt test (E). media change helped facilitate survival of these cells, and these Our findings showing that NJ.1638/ST2KO mice have dimin- cultures were used as the source of the data shown in Fig. 5F. Po- ished eosinophil numbers (Fig. 4F) also provide in vivo evidence tentially, because the IL-33–treated cultures have significantly more suggesting that IL-33 signaling positively regulates eosinophil IL-5Ra+ EoPre cells, consumption of the available IL-5 and out- differentiation rather than antagonizing it. Furthermore, we did not growth of other cell types might explain the elevated monocyte find basal differences in blood monocyte subsets between IL-33 numbers seen by Dyer et al. (18). An interesting aspect of our data in KO and ST2 KO mice or WT mice (Fig. 1E). Instead, we did Fig. 5F is that we observed a rapid expansion of both the EoPre and observe a diminished frequency of blood neutrophils that, unlike EoM populations within the first few days, but that the overall the eosinophil response, was not altered by exogenous IL-33 numbers of EoM plateaued, rather than continued to increase after treatment (Fig. 1E, Supplemental Fig. 1). Sustaining eosinophil removal of IL-33. Although further work is required to understand and neutrophil populations requires a competent GMP population, this fully, one possibility is that the pool of precursors became a but we did not see any significant effect on the homeostatic fre- limiting factor and that the balance between development of mature quency of GMP or GMP-like cells in the bone marrow of either EoMs and the apoptotic death we observed in the expanded IL-33 ST2 KO or IL-33 KO (Fig. 3F, Supplemental Fig. 3B), although cultures simply maintained the EoM populations during the IL-5 exogenous IL-33 treatment did diminish the GMP-like pool (Figs. treatment. Nonetheless, the overall numbers of EoMs generated by 3F, 4D). Intriguingly, ST2KO bone marrow did seem to generate IL-33 treatment during the early days of bone marrow cultures sig- fewer EoM cells by day 10 of culture. Although we saw no ob- nificantly outpaced those cultures without IL-33 and supports the vious difference in the frequency of either EoPre (Fig. 5B) or concept that early expansion of precursors supports a more rapid basal IL-5R levels (Fig. 5D) between ST2KO and WT that did not establishment of eosinophil populations. receive IL-33 treatment, subtle differences in the basal numbers of 3452 IL-33 SUPPORTS EOSINOPHIL PRECURSOR EXPANSION

GMP-like cells or their basal levels of IL-5R expression could 1996. IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal and cytotoxic responses. Im- explain this. Alternatively, WT bone marrow might contain a cell munity 4: 15–24. capable of generating endogenous IL-33 that supports eosinophil 6. Dent, L. A., M. Strath, A. L. Mellor, and C. J. Sanderson. 1990. Eosinophilia in development during the course of culture. These ideas require transgenic mice expressing 5. J. Exp. Med. 172: 1425–1431. 7. Bystro¨m, J., T. A. Wynn, J. B. Domachowske, and H. F. Rosenberg. 2004. Gene further investigation to define properly. Previous reports suggested microarray analysis reveals interleukin-5-dependent transcriptional targets in that IL-33 might directly influence neutrophils during inflamma- mouse bone marrow. Blood 103: 868–877. tion (41), and mice overexpressing IL-33 under the CMV pro- 8. Schmitz, J., A. Owyang, E. Oldham, Y. Song, E. 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