Cutting Edge: Core Binding Factor β Is Required for Group 2 Innate Lymphoid Cell Activation

This information is current as Xiaofei Shen, Mingwei Liang, Xiangyu Chen, Muhammad of September 24, 2021. Asghar Pasha, Shanti S. D'Souza, Kelsi Hidde, Jennifer Howard, Dil Afroz Sultana, Ivan Ting Hin Fung, Longyun Ye, Jiexue Pan, Gang Liu, James R. Drake, Lisa A. Drake, Jinfang Zhu, Avinash Bhandoola and Qi Yang

J Immunol 2019; 202:1669-1673; Prepublished online 6 Downloaded from February 2019; doi: 10.4049/jimmunol.1800852 http://www.jimmunol.org/content/202/6/1669 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/02/05/jimmunol.180085 Material 2.DCSupplemental References This article cites 20 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/202/6/1669.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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: Core Binding Factor b Is Required for Group 2 Innate Lymphoid Cell Activation Xiaofei Shen,*,1 Mingwei Liang,*,1 Xiangyu Chen,*,1 Muhammad Asghar Pasha,† Shanti S. D’Souza,* Kelsi Hidde,* Jennifer Howard,* Dil Afroz Sultana,* Ivan Ting Hin Fung,* Longyun Ye,* Jiexue Pan,* Gangx Liu,* James R. Drake,* Lisa A. Drake,* Jinfang Zhu,‡ Avinash Bhandoola, and Qi Yang* Group 2 innate lymphoid cells (ILC2) are tissue-resident, IL-5 and IL-13 as well as other effector molecules such as long-lived innate effector cells implicated in allergy and VEGFA (1, 2). Activated ILC2 are potent inducers of airway asthma. Upon activation, mature ILC2 rapidly secrete hyperresponsiveness (AHR) and are implicated in allergic asthma and asthma exacerbation. The molecular pathways large amounts of type-2 cytokines and other effector mol- Downloaded from ecules. The molecular pathways that drive ILC2 activa- that drive ILC2 activation, however, are not well understood. b b tion are not well understood. In this study, we report Core binding factor (CBF ) is a non-DNA–binding sub- that the transcriptional controller core binding factor unit that binds with the RUNX proteins to form different CBF transcriptional complexes. CBFs are critical for early lymphocyte b (CBFb) is required for ILC2 activation. Deletion or development. CBFb is required for the generation of thymic inhibition of CBFb did not impair the maintenance of

T cell progenitors, bone marrow B cell precursors, and early http://www.jimmunol.org/ ILC2 at homeostasis but abolished ILC2 activation innate lymphoid progenitors (3–7). CBFs are dispensable for during allergic airway inflammation. Treatment with b the maintenance of mature T cells, but they control several CBF inhibitors prevented ILC2-mediated airway aspects of T cell differentiation. CBFs are known to repress hyperresponsiveness in a mouse model of acute Alternaria CD4+ Th2 differentiation (8–10). Deletion of CBFb or allergen inhalation. CBFb promoted expression of key RUNX proteins in T cells derepressed GATA3 and IL-4 ex- ILC2 genes at both transcriptional and translational pression in CD4+ T cells, resulting in elevated IgE and air- levels. CBF transcriptional complex directly bound way eosinophil inflammation in mice (8–10). Whether CBFs to Il13 and Vegfa promoters and enhancers, and con- play a similar role in repressing the function of innate type-2 by guest on September 24, 2021 trolled gene transcription. CBFb further promoted ri- cytokine–producing cells, such as ILC2, remains unknown. bosome biogenesis and enhanced gene translation in The current study was undertaken to examine the role of b activated ILC2. Together, these data establish an essen- CBF in the activation and function of mature ILC2. To b our surprise, contrary to its suppressive role in Th2 cell dif- tial role for CBF in ILC2 activation. The Journal of b Immunology, 2019, 202: 1669–1673. ferentiation, our data indicate that CBF promotes ILC2 activation. Deletion or inhibition of CBFb abrogated ILC2 activation during allergic airway inflammation and prevented roup 2 innate lymphoid cells (ILC2) are long-lived ILC2-mediated AHR. The requirement of CBFb in ILC2 innate effector cells residing at mucosal barriers such function was cell intrinsic and involved both transcriptional G as lung and gut. Mature ILC2 functionally and and posttranscriptional gene regulatory mechanisms. These transcriptionally mirror Th2 cells, but ILC2 lack clonal Ag data establish a critical role for CBFb in ILC2 activation and receptors and can rapidly respond to the epithelial alarmin function. The positive regulation of ILC2 function by CBFb IL-33 in the absence of Ag stimulus (1). Upon activation, is in contrast with the suppressive role of CBFb in CD4+ Th2 mature ILC2 quickly secrete large amounts of type-2 cytokines cell differentiation (8, 9). Our data thus indicate that adaptive

*Department of Immunology and Microbial Diseases, Albany Medical College, Albany, Programs of the National Cancer Institute, the Center for Cancer Research (to A.B.), NY 12208; †Division of Allergy, Asthma and Immunology, Department of Medicine, and the National Institute of Allergy and Infectious Disease (to J.Z.) Albany Medical Center, Albany, NY 12203; ‡Molecular and Cellular Immunoregulation The microarray data presented in this article have been submitted to the Gene Expression Unit, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, x Omnibus (https://www.ncbi.nlm.nih.gov/geo) under accession number GSE116062. National Institutes of Health, Bethesda, MD 20892; and Laboratory of Genome In- tegrity, Center for Cancer Research, National Cancer Institute, National Institutes of Address correspondence and reprint requests to Dr. Qi Yang or Dr. Avinash Bhandoola, Health, Bethesda, MD 20892 Department of Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, MC-151, Albany, NY 12208 (Q.Y.) or Center for Cancer Research, 1X.S., M.L., and X.C. contributed equally to this work. National Cancer Institute, Building 37, Room 1108, Bethesda, MD 20892 (A.B.). ORCIDs: 0000-0002-5206-1353 (M.L.); 0000-0002-3560-9284 (X.C.); 0000-0001- E-mail addresses: [email protected] (Q.Y.) or [email protected] (A.B.) 5542-4511 (S.S.D.); 0000-0001-6773-0743 (I.T.H.F.); 0000-0001-7125-9608 (J.P.); The online version of this article contains supplemental material. 0000-0002-4888-1439 (G.L.); 0000-0002-8365-0986 (Q.Y.). Abbreviations used in this article: AHR, airway hyperresponsiveness; CBFb, core bind- Received for publication June 20, 2018. Accepted for publication January 20, 2019. ing factor b; CHIP, chromatin immunoprecipitation; gRNA, guide RNA; ILC2, group This work was supported by National Institutes of Health (NIH) Grants 2 innate lymphoid cell; 4-OHT, 4-hydroxytamoxifen; qPCR, quantitative PCR. R01HL137813, R01AG057782, and K22AI116728 (to Q.Y.), the Alexandrine and Alexander L. Sinsheimer Scholar Award (to Q.Y.), and NIH Intramural Research Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800852 1670 CUTTING EDGE: A CRITICAL ROLE OF CBFb IN ILC2 ACTIVATION and innate type-2 immune responses are controlled by divergent (Sigma-Aldrich) was added to primary lung ILC2 culture at day 6. Cells were molecular mechanisms. Such divergence in molecular control cultured for nine more days in the presence of 4-OHT. Cytokine production of YFP+ cells was examined by intracellular staining and flow cytometry analysis. might help explain the extreme heterogeneity and complexity In some experiments, YFP+ cells were sorted after 5 d of culture with 4-OHT of human diseases and necessitates consideration of personalized and cultured for another 4 d in the presence of 4-OHT. Growth rate of YFP+ medicine based on the specific immune responses involved. cells were measured as the inverse of doubling time. In vitro Th2 differen- tiation was performed as described (12).

Materials and Methods Flow cytometry analysis Mice Abs were purchased from eBioscience, BioLegend, or MD Biosciences. Flow f/f tm2.1Ddg CreERt2 tm1.1(cre/ERT2)Blh/ZhuJ CBFb (Cbfb /J, line 028550), Id2 (B6.129S(Cg)-Id2 cytometric analysis was performed on FACSCanto (BD Biosciences). Flow yfp tm1(EYFP)Cos line 016222), and Rosa26R (B6.129 3 1-Gt(ROSA)26Sor /J, line cytometry cell sorting was performed on a FACSAria II (BD Biosciences). 006148) mice were obtained from The Jackson Laboratory (11). CBFbf/f Id2CreERt2 Rosa26Ryfp and control Id2CreERt2 Rosa26Ryfp mice were bred at the Albany Medical Examination of gene transcription and translation College Animal Research Facility. BALB/c mice were purchased from Taconic Biosciences. mRNA was extracted by Qiagen RNeasy kits. Gene expression was exam- For deletion of CBFb in ILC2, five doses of 5 mg of tamoxifen were ined by quantitative PCR (qPCR). Microarray analysis was performed at administrated i.p. every other day. Twenty-eight days after the first tamoxifen Boston University Microarray and Sequencing Resource (accession number treatment, mice were administrated with a single dose of IL-33 (400 ng; GSE116062, https://www.ncbi.nlm.nih.gov/geo). Gene enrichment analyses BioLegend) i.p. BALB/c mice received a single dose of intranasal adminis- were provided by Boston University Clinical and Translational Science tration of Alternaria extracts (100 mg; Greer) as described (2). CBFb inhibitor Institute translational bioinformatics consultation service (CTSA Grant Ro5-3335 (50 mg/kg; EMD) was administered i.p. 24 h before Alternaria U54-TR001012). Downloaded from challenge. AHR was measured 12 h after Alternaria challenge by a flexiVent Sucrose gradients were performed to examine gene translation. Specifically, system (SCIREQ). All mice experiments were approved by the Albany cells were treated with 100 mg/ml cycloheximide (Sigma-Aldrich) for 10 min Medical College Institutional Animal Care and Use Committee. and lysed on ice. Cell lysates were layered onto 10–50% sucrose gradients and centrifuged in an SW 41 Ti rotor at 35,000 rpm for 2 h. Sixty fractions ILC2 culture, in vitro Th2 differentiation, and were carefully collected. OD260 absorption for each fraction was recorded. 4-hydroxytamoxifen treatment Unassociated RNA (pooled from fractions 1–9) and polysome-associated RNA (pooled from fractions 41–59) were extracted and examined by

Sorted lung ILC2 or ILC2 line were cultured with 10 ng/ml IL-2, IL-7, qPCR. Translation was determined by the ratio of polysome-associated RNA http://www.jimmunol.org/ and IL-33 for 5 d. One hundred nanomolar 4-hydroxytamoxifen (4-OHT) versus -unassociated RNA and normalized to Gapdh. by guest on September 24, 2021

FIGURE 1. CBFb is required for ILC2 activation in vivo. (A) Gating strategy to identify lung ILC2. (B) Representative profiles of YFP+ ILC2 in the lungs of tamoxifen-treated CBFbf/f Id2CreERt2 Rosa26Ryfp (CBFbf/f) and control Id2CreERt2 Rosa26Ryfp (CBFb+/+) mice. (C) Numbers of YFP+ ILC2 in the lungs. (D) YFP+ 2 or YFP ILC2 in the lungs of tamoxifen-treated mice were sorted by flow cytometry cell sorting. mRNA levels of Cbfb were examined using primers that targeted the deleted exons. (E) Mice were administrated with a single dose of IL-33 or PBS. Expression of IL-13 and IL-5 was examined by intracellular staining at 12 h after IL-33 or PBS administration. (F) Percentages of IL-13+ or IL-5+ ILC2 after PBS or IL-33 administration. (G) Histograms comparing the expression of IL-5 and IL-13 in YFP+ ILC2. (H) BALB/c mice received i.p. injection of CBFb inhibitor Ro5-3335 or vehicle (DMSO) followed by intranasal administration of Alternaria extracts or PBS. Numbers of bronchoalveolar lavage cells in mice were shown. (I) AHR was examined by flexiVent analysis. Data are from three independent experiments. *p , 0.05, **p , 0.01. n.s., not significant. The Journal of Immunology 1671

FIGURE 2. Cell-intrinsic CBFb is essential for ILC2 cytokine production. (A) Sort-purified ILC2 from CBFbf/f Id2CreERt2 Rosa26Ryfp (CBFbf/f) and control Id2CreERt2 Rosa26Ryfp (CBFb+/+) mice were cultured with IL-33, IL-2, IL-7, and 4-OHT. YFP+ cells were sorted after 5 d of 4-OHT treatment and cultured for 2 another 4 d. Growth rates were calculated as the inverse of doubling time (day 1). (B) Cultured cells were restimulated with fresh IL-33 (10 ng/ml) for 2.5 h in the presence of monensin. IL-13 and IL-5 expression from YFP+ ILC2 were examined by flow cytometry analysis. (C) Mean fluorescence intensity (MFI) of IL-13 and IL-5 in YFP+ ILC2 restimulated with IL-33. Data are from three mice per group. *p , 0.05.

Chromatin immunoprecipitation and CRISPR-mediated administration results in deletion of Cbfb in ILC2 of CBFbf/f gene knockout mice. The deletion efficiency is indicated by expression of YFP. + bf/f ILC2 cell line was described previously (13). Specifically, an immortalized cell Comparable numbers of YFP ILC2 appeared in CBF and +/+ line was established with small intestinal lamina propria ILC2 by selection of control CBFb mice 4 wk after tamoxifen treatment, indicat- Downloaded from spontaneous mutants. The ILC2 line cells have been maintained with IL-2 ing that the loss of CBFb did not affect the survival and and IL-33, and therefore they phenotypically and functionally resemble activated maintenance of ILC2 at homeostasis (Fig. 1A–C). qPCR primary ILC2. Chromatin immunoprecipitation (CHIP) was performed as + f/f we previously described (13). LentiCRISPRv2GFP was a gift from David verified effective deletion of Cbfb in YFP ILC2 of CBFb Feldser (plasmid no. 82416; Addgene). Guide RNA (gRNA)-encoding se- mice (Fig. 1D). quences were cloned into LentiCRISPRv2GFP vectors to achieve gene knock- To examine whether CBFb regulates ILC2 function, we out effects as described (14). Two gRNA sequences targeting Cbfb achieved effective knockout effects. The gRNA (59-CGATCTCCGAGCGACCGTCG-39) activated ILC2 in vivo by treating mice with IL-33. A single http://www.jimmunol.org/ was used throughout this study, but major findings were verified using the other dose of IL-33 induced production of IL-5 and IL-13 in ma- gRNA (59-ACCGCCTCACCTCGCACTCG-39) with similar results. Nontarget ture lung ILC2 within 12 h (Fig. 1E–G). CBFb-deficient 9 9 control (5 -TGCGAATACGCCCACGCGATGGG-3 ) was used to generate ILC2, however, failed to efficiently produce IL-5 or IL-13 control CRISPR constructs. Lentiviral transduction of ILC2 was performed as b described (15). Gene knockout effects were verified by Western blotting. in vivo (Fig. 1E–G). Thus, CBF is required for ILC2 acti- vation in vivo. Statistical analysis To confirm the relevance of CBFb-mediated ILC2 activation, Data are shown as mean 6 SD. Unpaired t test was used to compare the we used a model of acute Alternaria allergen inhalation. difference of two groups.

Alternaria alternata is a fungal allergen that triggers acute and by guest on September 24, 2021 severe asthma attacks. As described (2, 16), a single dose of Results and Discussion Alternaria inhalation rapidly upregulated IL-5 and IL-13 ex- b CBF is required for ILC2 activation in vivo pression in lung ILC2 within 12 h (Supplemental Fig. 1). CD4+ To examine the role of CBFb in mature ILC2 maintenance Th2 cells were not activated at this time point (2). Treatment and function, we generated CBFbf/f Id2CreERt2 Rosa26Ryfp with CBFb inhibitor (Ro5-3335) reduced production of IL-5 (CBFbf/f) and control Id2CreERt2 Rosa26Ryfp (CBFb+/+) mice. and IL-13 from ILC2 (Supplemental Fig. 1). Alternaria Because mature ILC2 express high amounts of Id2, tamoxifen inhalation rapidly induced airway eosinophil and neutrophil

FIGURE 3. CBFs directly regulate the transcription of Il13 and Vegfa in ILC2. (A) YFP+ ILC2 were sort purified from the lungs of CBFbf/f Id2CreERt2 Rosa26Ryfp (CBFbf/f) and control Id2CreERt2 Rosa26Ryfp (CBFb+/+) mice that were treated with tamoxifen and challenged with IL-33. Gene expression was examined by qPCR analyses. (B) CHIP assay was performed with ILC2 cell line. DNA region lacking CBF binding site was used as a negative control. Data are from triplicate samples representative of three independent experiments. *p , 0.05. n.s., not significant. 1672 CUTTING EDGE: A CRITICAL ROLE OF CBFb IN ILC2 ACTIVATION infiltration that was prevented by Ro5-3335 (Fig. 1H). Acute CBFb controls the expression of key ILC2 product genes at both Alternaria inhalation also rapidly elicited strong AHR that transcriptional and translational levels b was suppressed by Ro5-3335 (Fig. 1I). Together, CBF in- To determine the mechanisms through which CBFb controls hibition repressed ILC2 activation in response to acute ILC2 activation, we examined gene expression of purified Alternaria allergen inhalation and prevented ILC2-mediated YFP+ ILC2 from the lungs of CBFbf/f and CBFb+/+ mice that AHR. weretreatedwithtamoxifenandchallengedwithIL-33. GATA3 is a critical transcriptional controller in ILC2. b Cell-intrinsic CBF is required for ILC2 activation However, deletion of Cbfb did not affect Gata3 mRNA We sought to understand whether CBFb controls ILC2 ac- levels in ILC2 in vivo, indicating that CBFs might control tivation through cell-intrinsic mechanisms. We cultured ILC2 activation through GATA3-independent mechanisms highly purified lung ILC2 from CBFbf/f or control CBFb+/+ (Fig. 3A). Interestingly, mRNA expression for Il13 and Vegfa, mice in the presence of IL-7, IL-2, IL-33, and 4-OHT. In- but not Il5, was reduced in CBFb-deficient ILC2 (Fig. 3A). duction of CRE expression by 4-OHT resulted in the ap- Conserved CBF binding sites were identified at the 290-bp pearance of YFP+ cells in both CBFbf/f and control CBFb+/+ Il13 promoter and the 27.4-Kb Vegfa enhancer regions. ILC2 cultures. However, CBFb-deficient ILC2 (YFP+ CBFbf/f) CHIP indicated that RUNX proteins directly bound to both proliferated at a much lower rate than control ILC2 (YFP+ conserved regulatory regions in ILC2 line cells (Fig. 3B). CBFb+/+)(Fig.2A).CBFb-deficient ILC2 also failed to effi- Thus, CBF transcriptional complex may directly control the ciently produce IL-13 or IL-5 in response to IL-33 (Fig. 2B, transcription of some ILC2 genes. Downloaded from 2C). Thus, cell-intrinsic CBFb is required for ILC2 activation. Nevertheless, the changes in Il5 and Il13 mRNA were rather Similar results have been obtained with CRISPR technique– moderate when compared with the remarkable decrease in mediated knockout of CBFb in an ILC2 cell line, verifying protein expression in CBFb-deficient ILC2 (Fig. 1E). Thus, an essential cell-intrinsic role for CBFb in ILC2 activation additional posttranscriptional mechanisms are likely to be (Supplemental Fig. 2A–D). involved. We further examined the gene expression of purified http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. CBFb promotes the translation of key genes in ILC2. (A)YFP+ ILC2 were sort purified from the lungs of CBFbf/f Id2CreERt2 Rosa26Ryfp (CBFbf/f)orcontrol Id2CreERt2 Rosa26Ryfp (CBFb+/+) mice that were treated with tamoxifen and challenged with IL-33. Gene expression was examined by qPCR analyses with freshly isolated cells. (B) ILC2 cell line was transduced with LentiCRISPRv2GFP lentivirus that targeted CBFb (CBFb CRISPR) or nontarget control (CRISPR). Microarray analyses were performedwithsortedGFP+ cells at day 6 posttransduction. Gene set enrichment analyses were performed. (C) Sort-purified ILC2 from CBFbf/f and CBFb+/+ mice were cultured in the presence of IL-33, IL-2, IL-7, and 4-OHT. YFP+ cells were sorted after 5 d of 4-OHT treatment and recultured with IL-2, IL-7, and IL-33 overnight. Sucrose gradient centrifugation was performed to separate polysome-associated RNA and free RNA. Translational rates of the indicated genes were determinedbytheratioof polysome-associated RNA versus free RNA. Data were normalized to Gapdh.(D) ILC2 cell line was transduced with the indicated LentiCRISPRv2GFP lentivirus. GFP+ ILC2weresortedat10dposttransductionandreculturedwithIL-33andIL-2overnight. Sucrose gradient centrifugation was performed, and 60 fractions were carefully collected from the top to the bottom of the centrifuge tubes. Shown is OD260 absorption of each fraction. Data are from three independent experiments. *p , 0.05. The Journal of Immunology 1673

CBFb-deficient and control ILC2 from the lungs of (19, 20). Thus, CBFs control the expression of ribosome bio- tamoxifen-treated mice (Fig. 4A). Interestingly, CBFb-deficient genesis genes through cell type–specific mechanisms. Similarly, ILC2 had reduced expression of many ribosomal protein genes CBFs repress the expression of type-2 cytokines in CD4+ (Fig. 4A). They included the ribosome structural genes (rpl3, T cells but promote cytokine production in ILC2. The exis- rpl5, rps2, rps15a), p component molecules (pop4, rpp30,and tence of lineage-specific coactivators and corepressors might rpp38), and other key molecules involved in ribosome bio- help explain the differential and even opposing roles of CBFs genesis and function (riok2, ddx21, rrp1b, rcl1,andnol11). in different cell types. Searching for ILC2-specific functional Microarray analysis with CBFb knockout and control ILC2 partners of CBFb might reveal unique lineage-specific molec- line cells verified that loss of CBFb expression led to extensive ular pathways that control ILC2 activation and function. reduction in mRNA expression for genes related to ribosome biogenesis, RNA processing, and translation (Fig. 4B). Thus, CBFb promotes the expression of ribosomal protein genes in Disclosures activated ILC2. The authors have no financial conflicts of interest. To directly examine whether CBFb promotes gene trans- lation in ILC2, we deleted Cbfb in cultured ILC2 by in vitro 4-OHT treatment and then performed sucrose gradient References 1. Artis, D., and H. Spits. 2015. The biology of innate lymphoid cells. Nature 517: centrifugation to separate polysome-associated RNA from 293–301.

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