CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9–producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

This article is featured in Top Reads, p. 517

Interleukin-9 is a pleiotropic cytokine associated with type 2 immunity, supporting the clearance of helminth infections and tumor cells (1–5). Although appropriate production of IL-9 is important for human health, inappropriate or overproduction of IL-9 results in a myriad of diseases ranging from allergic to autoimmune disorders (6–8). Allergic asthma is a respiratory disease initiated by sensitization to inhaled aeroallergens and has been strongly correlated with IL-9 production (9). In chronic allergic airway disease (AAD), IL-9–producing CD4 TH9 cells lead to accumulation of eosinophils and mast cells in the airway, increased type 2 cytokines, stimulated type 2 innate lymphoid cell proliferation, and induced mucus production from airway epithelium (2, 10–12). In response to Ag receptor activation in the presence of IL-4 and TGF-β, naive CD4 T cells differentiate into TH9 cells by activating a complex network of transcription factors that can regulate the production of IL-9 (6, 13). Transcription factors including basic leucine zipper ATF-like transcription factor (BATF) and IFN regulatory factor (IRF)4 are induced by the IL-4–mediated STAT6 signaling pathway (14–17). TGF-β signal activates SMAD proteins that lead to the induction of PU.1 and E26 transformation-specific (Ets) translocation variant 5 (ETV5) (18, 19). Although several transcription factors have been shown to affect IL-9 production in TH9 cells, the transcriptional network that governs the development of TH9 cells and their function during allergic responses is not entirely understood.

The Ets family of transcription factors plays important roles in cellular proliferation, differentiation, angiogenesis, and apoptosis, and it is defined by the presence of a conserved ∼85-aa-long Ets domain, which facilitates DNA binding at the consensus binding site GGAA/T (20, 21). In humans, the Ets family of transcription factors is comprised of 28 family members; there are 27 members in mice. The existence of a large family of closely related transcription factors suggests that individual Ets family members evolved with specific roles in the regulation of target genes. The Ets family member PU.1 is expressed in larger amounts in TH9 cells compared with TH2 cells and inhibits the production of TH2 cytokines (22). Interestingly, ectopic expression of PU.1 enhanced IL-9 production in TH2 cells. Moreover, deficiency in PU.1 expression impaired IL-9 production in vitro as well as TH9 function in AAD models (18). Additional studies also indicate that PU.1 binds at the Il9 promoter and facilitates recruitment of GCN5, a histone acetyltransferase (HAT) that induces IL-9 production in TH9 cells (23). PU.1-dependent TH9 function is associated with both allergic airway inflammation as well as inflammatory bowel disease (23).

ETV5, another member of the Ets family of transcription factors, functions in parallel with PU.1 to modulate IL-9 secretion in TH9 cells. IL-9 production was decreased in the absence of ETV5, whereas overexpression of ETV5 in TH9 cultures led to increased IL-9 production. Chromatin immunoprecipitation studies revealed that, although both PU.1 and ETV5 can regulate Il9 gene expression in a parallel manner, they bind to distinct conserved noncoding regions at the Il9 locus. In the absence of ETV5, lower binding of HAT p300 was observed at the Il9 locus, whereas GCN5 binding remained unaffected (19). Besides the similar roles of ETV5 and PU.1 in regulation of TH9 function, they had distinct effects on the overall development of allergic inflammation (19). In addition to PU.1 and ETV5, Koh et al. (19) showed additional members of the Ets family of transcription factors that were expressed in the TH9 subset, including ELK3 and ETV6 (19). However, ectopic expression of either of these transcription factors did not alter IL-9 production.

In a microarray analysis, Jabeen et al. (17) provided strong evidence that the TH9 subset has a unique transcription signature from other CD4 TH cell subsets. In the analysis, another member of the Ets family of transcription factors, Erg, was preferentially expressed in TH9 cells compared with TH2 and the T regulatory (Treg) cell subset, and expression was dependent on STAT6 and BATF. The function of ERG in TH9 cells has not been examined. ERG is primarily expressed in hematopoietic and vascular endothelial cells (24). Translocations associated with ERG are involved in the generation of oncogenic fusion proteins, including TMPRSS2-ERG in prostate cancer and EWS-ERG in Ewing’s sarcoma (25, 26). Furthermore, aberrant expression of ERG has been strongly associated with poor prognosis of acute lymphoblastic leukemia (ALL) (27). Chromatin immunoprecipitation (ChIP) sequencing analysis revealed that ERG modulates gene expression patterns in prostate cancer by promoting chromatin rearrangements (28–31). Additionally, overexpression of ERG facilitates recruitment of transcription factors and coactivator proteins through the pointed (PNT) domain that can alter transcriptional activity of target genes (21). Some of the known Ets transcription factor binding partners of ERG include PU.1, FLI-1, ETS-2, ER81, and ERG itself (32). In the immune system, ERG controls B cell development and is induced during the initial stages of T cell lineage commitment and repressed once T cells are committed (20, 33). However, the functions of ERG in mature T cells, Th cell subset differentiation, and the development of effector function have not been studied. In this study, we demonstrate the function of ERG in the regulation of IL-9 and TH9 function in allergic airway inflammation.

C57BL/6, dLck-Cre, CD4-Cre, and inducible CD4 (iCD4)-Cre mice (on a C57BL/6 background) were purchased from The Jackson Laboratory. Ergfl/fl mice were obtained from Dr. Anna Randi (24) and crossed with dLck-Cre, iCD4-Cre, or CD4-Cre mice respectively. Spifl/flEtv5fl/flErgfl/fl CD4-Cre mice were generated by crossing Spifl/flEtv5fl/fl CD4-Cre mice (19, 34–37) with Ergfl/fl CD4-Cre. Ergfl/fl Mx1-cre mice were maintained by Dr. Bo T. Porse and treated with polyinosinic-polycytidylic acid 2 wk before harvesting for in vitro assays (33, 38). Erg exon 3-mutant mice were generated by Taconic as outlined in Supplemental Fig. 1. Mutation was confirmed by sequencing. In this study, 6- to 8-wk-old male or female mice were used. All mice were used with the approval of the Indiana University Institutional Animal Care and Use Committee.

Naive CD4 T cells were isolated from mouse spleens using a naive CD4 T cell isolation kit provided by the supplier (Miltenyi Biotec). Cells were cultured in complete RPMI 1640 media on anti-CD3 (2 μg/ml)–coated plates and soluble anti-CD28 (0.5 μg/ml) under TH cell subset polarizing conditions, including TH9 (TGF-β1): TGF-β1 (2 ng/ml), IL-4 (20 ng/ml), human (h)IL-2 (50 U/ml), and anti–IFN-γ (10 μg/ml); TH9 (activin A): TGF-β1 (2 ng/ml), IL-4 (20 ng/ml), hIL-2 (50 U/ml), and anti–IFN-γ (10 μg/ml); TH2 polarizing condition: IL-4 (20 ng/ml), hIL-2 (50 U/ml), and anti–IFN-γ (10 μg/ml); TH1 polarizing condition: IL-12 (20 ng/ml), hIL-2 (50 U/ml), and anti–IL-4 (10 μg/ml); TH17 polarizing condition: IL-6 (100 ng/ml), TGF-β1 (2 ng/ml), IL-1β (10 ng/ml), IL-23 (10 ng/ml), anti–IFN-γ (10 μg/ml), anti–IL-4 (10 μg/ml), and anti–IL-2 (10 μg/ml); Treg cell polarizing condition: TGF-β1 (2 ng/ml), hIL-2 (50 U/ml), anti–IFN-γ (10 μg/ml), and anti–IL-4 (μg/ml). On day 3, cells were expanded into fresh media containing the original concentrations of cytokines in the absence of costimulatory signals for an additional 2 d. On day 5, mature TH9 or TH2 cells were harvested for further analysis.

PBMCs were isolated from deidentified buffy coat blood packs from healthy anonymous donors (Indiana Blood Center, Indianapolis, IN) by density gradient centrifugation using Ficoll-Paque (GE Healthcare). Human naive CD4+ T cells were isolated from human PBMCs using magnetic separation (Miltenyi Biotec). Isolated naive CD4 T cells were activated with an equal ratio of receptor crosslinking beads, that is, Dynabeads human T-activator CD3/CD28 (Thermo Fisher Scientific), in complete RPMI 1640 to generate TH9 cells in the TH9 subset polarizing condition, that is, hIL-4 (20 ng/ml), hTGF-β1 (2 ng/ml), and anti–IFN-γ (10 μg/ml), and cultured at 37°C under 5% CO2. Cells were harvested on day 5 for analysis.

Total RNA was isolated from cells using TRIzol (Life Technologies). RNA was reverse transcribed according to the manufacturer’s directions (Quantabio, Beverly, MA). Quantitative RT-PCR was performed with commercially available primers (Life Technologies) with a 7500-Fast PCR machine (Life Technologies). Gene expression was normalized to housekeeping gene expression (β2-microglobulin). In the case of quantitative PCR (qPCR) for a ChIP assay, SYBR Green master mix (Applied Biosystems) was used for gene expression analysis (39).

Platinum E cells were grown in 10 ml of DMEM 1640 with 10% FBS and 1% antibiotics in a 100-mm tissue culture dish. When confluency reached 80–90%, cells were transfected with control vector or retroviral vector expressing PU.1-internal ribosome entry site (IRES)-hCD4 (18), ETV5-IRES-Thy1.1 (19, 34), ERG-IRES-enhanced GFP (EGFP) (ERG3 cDNA was cloned into the Mieg3-GFP plasmid) (40), or Cre-IRES-EGFP (41) using Lipofectamine 3000 (Thermo Fisher Scientific). For transfection, 18 μg of vector, 6 μg of pCL-Eco, and 25 μl of P3000 were mixed in 500 μl of Opti-MEM I reduced-serum medium (Thermo Fisher Scientific), and 25 μl of Lipofectamine 3000 was mixed in another 500 μl of Opti-MEM I. After combining, this mixture was incubated for 10-15 mins at room temperature (RT). The mixture was gently pipetted into a culture dish. After 16 h, the medium containing retrovirus was collected and changed with new fresh medium. After 24 h, the medium was collected and centrifuged at 1500 rpm for 5 min to remove cell debris. Supernatant-containing retrovirus was used for retroviral transduction or stored at −80°C for subsequent use.

Activated mouse CD4+ T cells were infected on day 1 with retrovirus containing control or expressing the interested gene by centrifugation at 2300 rpm at 32°C for 90 min in the presence of 8 μg/ml Polybrene (Sigma-Aldrich). After spin infection, the supernatant was replaced with the fresh TH subset cell condition medium. Cells were expanded on day 3 and analyzed on day 4 or 5.

Mouse or human naive CD4 T cells were cultured in TH9 polarizing condition as described under In vitro human T cell isolation and differentiation. On day 1 of TH9 differentiation, the TH9 cell culture medium was replaced with small interfering RNA (siRNA) delivery medium containing 1 μM SMARTpool Accell siRNA against mouse or human Erg (provided by Dharmacon) along with TH9 subset polarizing cytokines and growth factors for 48 h. For control, nontargeting siRNA (provided by Dharmacon) was used at the same concentration as the targeting siRNA. On day 3, cells were expanded into fresh medium containing the original concentrations of cytokines in the absence of costimulatory signals for an additional 2 d. On day 5, mature TH9 cells were harvested for further analysis.

Jurkat cells were transfected with an Il9 promoter Renilla luciferase reporter construct in combination with the control or Erg-overexpressing plasmid using FuGENE 6 reagent (Roche). After 24 h of transfection, cells were stimulated with PMA and ionomycin for 6 h and luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Firefly values were normalized to Renilla values.

In vitro–differentiated TH cells were activated with anti-CD3 for 3 h and were crosslinked for 15 min with 1% formaldehyde at RT with rotation. The reaction was quenched by adding 0.125 M glycine and incubated at RT for 5 min. Fixed cells were lysed with cell lysis buffer, followed by nuclear lysis buffer. Nuclei were degraded and chromosomal DNA was fragmented to a size range of 200–500 bp through an ultrasonic processor (Vibra-Cell). After sonication, the supernatant was diluted 10-fold with ChIP dilution buffer. After preclearing, the supernatant was incubated with the ChIP Abs against ERG (EPR3864(2), rabbit mAb, Abcam), PU.1 (9G7, rabbit mAb, Cell Signaling Technology), H3K27ac (rabbit polyclonal, Abcam), p300 (N-15, Rabbit mAb, Santa Cruz Biotechnology), and IgG (rabbit polyclonal, Abcam) at 4°C overnight with rotation. The following day, immunocomplexes were precipitated with protein agarose A or G beads at 4°C for 2–4 h. Immunocomplexes were washed with low salt, high salt, LiCl, and twice with TE (Tris-EDTA) buffer. After elution followed by reverse crosslinks, DNA was purified and analyzed by qPCR. After normalization to the input DNA, the amount of output DNA of each target protein was calculated by subtracting that of the IgG control. Quantification of the ChIP assay by qPCR was performed using primers as previously described (39).

In the chronic allergic airway model, mice were intranasally challenged with A. fumigatus protein extract (Greer Laboratories, Lenoir, NC) in PBS (25 μg/25 μl PBS) three times per week for either 3 or 6 wk to develop a chronic response. Bronchoalveolar lavage (BAL) fluid and blood were collected prior to harvesting lung tissue samples. The mesenteric lymph node and spleen were collected and manually dissociated to generate single-cell suspensions. The lung vascular bed was perfused through the injection of 10 ml of cold PBS via the right ventricle of the heart. Lungs were digested with 1 mg/ml collagenase A (Roche) for 1 h at 37°C followed by pressing digested lungs through a wire mesh sieve (Bellco Glass) to generate single-cell suspensions. RBCs were lysed using ACK (ammonium-chloride-potassium) lysis buffer (Lonza). CD4 T cells were isolated via magnetic bead positive selection (clone L3T4, Miltenyi Biotec).

For cytokine staining, CD4+ T cells were stimulated with PMA (50 ng/ml, Sigma-Aldrich) and ionomycin (1 μg/ml, Sigma-Aldrich) for 3 h followed by monensin (2 μM, BioLegend) for 6 h at 37°C. After restimulation, cells were washed with FACS buffer (PBS with 0.5% BSA). CD4+ T cells were then stained with a fixable viability dye (eFluor 780, eBioscience) and surface markers (CD4, GK1.5, BioLegend) for 30 min at 4°C followed by washing and fixation with 4% formaldehyde for 10 min at RT. For cytokine staining, cells were then permeabilized with permeabilization buffer (eBioscience) for 30 min at 4°C and stained for cytokines, including IL-9 (RM9A4, BioLegend), IL-4 (11B11, BioLegend), IL-5 (TRFK5, BioLegend), IL-13 (eBio13A, Invitrogen), IL-10 (JES6-5H4, BioLegend), and IFN-γ (XMG.1, BioLegend), for 30 min at 4°C. To analyze cytokine production from CD4 T cells in retroviral transduction assays, cytokine expression was detected from viable CD4+ T cells that expressed respective reporters, including human-CD4 (PU.1 overexpression), Thy1.1 (ETV5 overexpression), and EGFP (ERG or Cre recombinase overexpression). For detection of cellular populations in the lung, single-cell suspensions of lung cells were stained for different panels, including a lymphocyte panel (CD4 T cells, CD8 T cells, and B cells), a granulocyte panel (eosinophils, neutrophils, and macrophages), and Treg cells. CD4 T cells were identified by staining for CD4 and TCRβ (H57-597, BioLegend). CD8 T cells were identified as CD4, CD8+ (53-6.7, BioLegend), and TCRβ+. B cells were identified as B220+ (RA3-6B2, BioLegend) and CD19+ (6D5, BioLegend) cells. Among the granulocyte populations, eosinophils were identified as CD11b+ (M1/70, BD Biosciences), CD11c (N418, BioLegend), Siglec F+ (E50-2440, BD Biosciences), F4/80+ (BM8, BioLegend), CD45+ (104, BioLegend), and Ly6G (IA8, BioLegend), and neutrophils were detected as CD11b+, Siglec F, F4/80, CD45+, Ly6G+ cells. Alveolar macrophages were detected as CD11c+, CD11b, Siglec F+, F4/80+, CD64+ (X54-5/7.1, BioLegend), and Mertk+ (2B10C42, BioLegend) cells, and interstitial macrophages were detected as CD11b+, CD11c, Siglec F, F4/80+, CD64+, Mertk+ cells. For transcription factor staining for the detection of Treg cells, a Foxp3 staining kit provided by eBioscience was used. After surface staining, cells were fixed using Fix/Perm buffer provided in the kit overnight at 4°C in the dark. Samples were washed and incubated in the 1× permeabilization buffer along with the Foxp3 Ab (FJK-16s, Invitrogen) for 1 h at 4°C. Cells were washed and resuspended in FACS buffer for further analysis.

CD4+ T cells were sorted from the lungs of A. fumigatus–challenged mice by magnetic CD4+ positive selection (Miltenyi Biotec). The 50,000 purified CD4+ T cells were cocultured with an equal number of CD11c+ APCs isolated from splenocytes from naive C57BL/6 mice in the presence of either BSA or A. fumigatus (100 mg/ml) for 72 h in RPMI 1640 complete medium in a 96-well U-bottom plate. In some experiments, additional controls and innate cytokines such as IL-33 (50 ng/ml) were added to the coculture. IL-9 was measured by ELISA MAX deluxe set mouse IL-9 (BioLegend).

All statistics were done using Prism software version 9 (GraphPad Software). An unpaired Student t test was used for the comparison of two samples. ANOVA with Tukey’s multiple comparison test was used for the comparison of three or more groups unless otherwise stated. Flow cytometry data were collected using a Nxt Attune flow cytometer (Life Technologies) and were analyzed using FlowJo version 10.8.0.

To investigate the function of ERG in the development of TH9 cells, we first assessed the expression of Erg in TH cell subset populations differentiated from naive CD4 T cells. Among the subsets, Erg was preferentially expressed in the TH9 subset (Fig. 1A). This observation was consistent with previous findings reported by Jabeen et al. (17), where Erg expression was enriched in TH9 cells compared with TH2 and Treg cell populations in microarray and by qPCR analysis. To determine whether ERG contributes to the regulation of IL-9 production in TH9 cells, we performed in vitro siRNA-mediated knockdown of Erg in TH9 cells. Erg knockdown in TH9 cells led to a decrease in IL-9 secretion by TH9 cells (Fig. 1B, 1C). We then performed a retroviral transduction assay to examine whether ectopic expression of Erg increased production of IL-9 by CD4 TH subsets. Ectopic expression of Erg enhanced IL-9 production not only in TH9 cells but also in the TH2 subset (Fig. 1D–G). Ectopic expression of Erg in cells cultured with activin A, a member of the TGF-β superfamily that can also promote IL-9 production in combination with IL-4 (2), similarly enhanced IL-9 (Fig. 1H). Ectopic expression of Erg in TH1, TH17, and Treg cells failed to induce IL-9 production (Fig. 1H). We examined whether ERG was required for IL-9 production in TH9 cells derived from human PBMCs (h-TH9 cells). Knockdown of ERG in h-TH9 cells resulted in reduced expression of IL9 transcript (Fig. 1I, 1J). To investigate whether ERG regulated activity of the Il9 promoter, we performed a Dual-Luciferase reporter assay. In comparison with the control plasmid, transfection of Erg-expressing plasmid induced an ∼5-fold increase in Il9 promoter activity in Jurkat cells cultured in vitro (Fig. 1K). Overall, these findings indicate that Erg promotes IL-9 production in CD4 TH cells.

FIGURE 1.

ERG promotes IL-9 production in murine and human CD4 T cells in vitro. (A) Expression of Erg transcript in TH cell subsets. (B and C) siRNA knockdown of Erg in TH9 cell cultures in vitro showing Erg expression and IL-9 production, respectively. (D) IL-9 production in TH9 subsets after retroviral transductions of ERG overexpressing plasmid on day 1 of T cell differentiation in vitro. (E) Quantification of IL-9 protein secreted by TH9 cells overexpressing ERG. (F) IL-9 production in TH2 subsets after retroviral transductions of ERG overexpressing plasmid on day 1 of T cell differentiation in vitro. (G) Quantification of IL-9 protein secreted by TH2 cells overexpressing ERG. (H) IL-9 production in other TH cell subsets transduced with ERG overexpressing plasmid on day 1 of T cell differentiation in vitro. (I and J) ERG and IL9 transcript expression after siRNA-mediated knockdown of ERG in TH9 cells cultured in vitro from human PBMCs collected from healthy donors. (K) Il9 reporter activity measured as the ratio of Renilla/luciferase with or without overexpression of Erg in Jurkat cells cultured in vitro. (L) ChIP-qPCR assay indicating ERG interactions at the Il9 regulatory regions in TH9 cells during differentiation through day 0, day 3 (expansion), and day 5. (M) ChIP-qPCR assay showing binding of ERG at Il9 regulatory regions on day 5 after restimulation with anti-CD3 Ab for 3 h in TH9 cells compared with TH0 and TH2 cells cultured in vitro. Data are means of three to four mice or human PBMC donors per experiment and representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 1.

ERG promotes IL-9 production in murine and human CD4 T cells in vitro. (A) Expression of Erg transcript in TH cell subsets. (B and C) siRNA knockdown of Erg in TH9 cell cultures in vitro showing Erg expression and IL-9 production, respectively. (D) IL-9 production in TH9 subsets after retroviral transductions of ERG overexpressing plasmid on day 1 of T cell differentiation in vitro. (E) Quantification of IL-9 protein secreted by TH9 cells overexpressing ERG. (F) IL-9 production in TH2 subsets after retroviral transductions of ERG overexpressing plasmid on day 1 of T cell differentiation in vitro. (G) Quantification of IL-9 protein secreted by TH2 cells overexpressing ERG. (H) IL-9 production in other TH cell subsets transduced with ERG overexpressing plasmid on day 1 of T cell differentiation in vitro. (I and J) ERG and IL9 transcript expression after siRNA-mediated knockdown of ERG in TH9 cells cultured in vitro from human PBMCs collected from healthy donors. (K) Il9 reporter activity measured as the ratio of Renilla/luciferase with or without overexpression of Erg in Jurkat cells cultured in vitro. (L) ChIP-qPCR assay indicating ERG interactions at the Il9 regulatory regions in TH9 cells during differentiation through day 0, day 3 (expansion), and day 5. (M) ChIP-qPCR assay showing binding of ERG at Il9 regulatory regions on day 5 after restimulation with anti-CD3 Ab for 3 h in TH9 cells compared with TH0 and TH2 cells cultured in vitro. Data are means of three to four mice or human PBMC donors per experiment and representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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To begin to define the function of ERG, we investigated whether ERG binds the Il9 gene using ChIP from cells isolated during TH9 differentiation. We assessed the interaction of ERG at conserved noncoding sequence (CNS) regions across the Il9 locus including CNS1 (Il9 promoter region), CNS2 (located 2 kb downstream of the Il9 promoter), CNS-6 (located 6 kb upstream of the Il9 promoter), CNS-25 (located 25 kb upstream of the Il9 promoter), and an adjacent site as a negative control (located 35 kb upstream of the Il9 promoter) (39). ERG interaction at the CNS-25 region, recently identified as the Il9 enhancer region, was observed from day 0 through day 5 of TH9 polarization (Fig. 1L). On day 5, ERG interaction was modestly enriched at the Il9 promoter region (CNS1) and CNS-6 region. ERG interaction at CNS-1 was higher in TH9 cells compared with TH0 and TH2 subsets (Fig. 1M). ERG binding at the Il9 enhancer region (CNS-25) also trended higher in TH9 cells compared with TH2 cells (Fig. 1M). These observations indicate that ERG interacts with the Il9 regulatory regions in TH9 cells.

Erg is expressed in multiple isoforms that include translational start sites in alternatively spliced exons 3 or 4 (42). We assessed expression of the isoforms and observed that preferential expression in TH9 cells was predominantly from exon 3 (Supplemental Fig. 1A). We then generated mice using a CRISPR/Cas9 approach to mutate the initiation codon of exon 3 (Supplemental Fig. 1B). Mutant mice carrying the mutant exon 3 allele were born in normal Mendelian frequencies and did not have any gross abnormalities. Mutation of exon 3 resulted in a decrease of exon 3–containing transcripts without obvious effects on levels of exon 4–containing transcripts and insignificant effects on overall Erg mRNA (Supplemental Fig. 1C–E). Culture of exon 3 mutant CD4 T cells in TH9 culture conditions resulted in IL-9 production that was similar to wild-type cells (Supplemental Fig. 1F–H).

To further assess the role of ERG in the development of TH9 cells, Ergfl/fl mice were crossed with loxP sites flanking exon 6 (24), common to both isoforms, to multiple Cre-transgenic mice to generate Erg conditional knockout mice. Multiple Cre-transgenic were used to test whether deletion at differing developmental time points resulted in distinct phenotypes. Deletion of Erg with CD4-Cre that deletes during thymic development was efficient but had negligible effects on IL-9 production in TH9 cells cultured in vitro (Fig. 2A–C). The same Ergfl/fl mice crossed with dLck-Cre mice allowed restricted deletion of Erg in peripheral T cells and resulted in a modest decrease in IL-9 production by cultured TH9 cells (Supplemental Fig. 2A, 2B). We confirmed these results using Ergfl/fl Mx1-Cre mice with loxP sites located on either side of the exon 11 region of Erg (33, 38). Excision of Erg was achieved by treating Ergfl/fl Mx1-Cre mice with polyinosinic-polycytidylic acid (33). The deletion of Erg in peripheral cells using Mx1-Cre led to a significant though modest reduction (∼5% decrease) in IL-9–producing CD4 T cells cultured in vitro (Fig. 2D, 2E); however, we did not observe significant differences in IL-9 secretion (Fig. 2F). We then tested whether deletion of Erg during in vitro differentiation of TH9 cells would affect IL-9 production. Ectopic expression of Cre recombinase in Ergfl/fl CD4 T cells during in vitro TH9 differentiation led to a greater decrease in IL-9 production in TH9 cells, similar to what was observed in siRNA-treated cells (Fig. 2G–I). These results support the conclusion that ERG contributes to Il9 regulation, but that deletion of Erg early in T cell development or before activation has less of an effect on IL-9 production in TH9 cells. One possibility is that other factors, such as other ETS transcription factors, could compensate for loss of ERG.

FIGURE 2.

ERG regulates IL-9 production by TH9 cells during acute activation. (AF) Detection of IL-9–producing CD4 T cells, Erg gene expression, and secretion of IL-9 in TH9 cells derived from Ergfl/fl CD4-Cre mice (A–C) and Ergfl/fl Mx1-Cre mice (D–F). (G and H) Production of IL-9 and Erg transcript expression in Ergfl/fl TH9 cells with or without ectopic Cre expression on day 3 of TH9 differentiation. (I) Quantification of IL-9 protein secreted. Data are means of three mice donors per experiment and representative of two independent experiments. *p < 0.05.

FIGURE 2.

ERG regulates IL-9 production by TH9 cells during acute activation. (AF) Detection of IL-9–producing CD4 T cells, Erg gene expression, and secretion of IL-9 in TH9 cells derived from Ergfl/fl CD4-Cre mice (A–C) and Ergfl/fl Mx1-Cre mice (D–F). (G and H) Production of IL-9 and Erg transcript expression in Ergfl/fl TH9 cells with or without ectopic Cre expression on day 3 of TH9 differentiation. (I) Quantification of IL-9 protein secreted. Data are means of three mice donors per experiment and representative of two independent experiments. *p < 0.05.

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With increasing evidence for the involvement of multiple Ets transcription factors (PU.1, ETV5, and ERG) in the development of TH9 cells, we profiled transcript expression of TH9-associated Ets transcription factors during TH9 cell differentiation (Fig. 3A–D). Previous studies have reported that PU.1 mediates TH9 differentiation during the early stages of TH9 development. Consistent with these findings, the Spi-1 (PU.1) transcript was observed to peak early during TH9 differentiation and declined during the later stages of differentiation (Fig. 3B). Erg and Etv5 gene expression peaked on days 3 and 4 of TH9 differentiation, along with Il9 expression (Fig. 3A, 3C, 3D). Interestingly, Erg expression preceded Il9 transcription by 12 h and a very similar pattern with a peak after 24 h of transcription induction. These differing patterns of expression suggest distinct temporal but potentially overlapping roles for the factors in regulation of Il9.

FIGURE 3.

TH9-associated Ets transcription factors in differentiating TH9 cells. (AD) Transcript expression of Il9 (A), Spi1 (B), Erg (C), and Etv5 (D) during TH9 polarization in vitro. (E) IL-9 production in TH9 subsets ectopically coexpressing ERG, PU.1, and ETV5. Data are means of three mice per experiment and representative of two independent experiments. **p < 0.01.

FIGURE 3.

TH9-associated Ets transcription factors in differentiating TH9 cells. (AD) Transcript expression of Il9 (A), Spi1 (B), Erg (C), and Etv5 (D) during TH9 polarization in vitro. (E) IL-9 production in TH9 subsets ectopically coexpressing ERG, PU.1, and ETV5. Data are means of three mice per experiment and representative of two independent experiments. **p < 0.01.

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As mentioned previously, overlapping roles of Ets transcription factors in the regulation of Il9 gene expression may explain the minimal effects of Erg deletion on TH9 differentiation in some Cre models (Fig. 2). To begin to test whether there was cooperativity between the Ets transcription factors PU.1, ERG, and ETV5 on the regulation of IL-9 production during later stages of CD4 T cell differentiation, we performed retroviral transductions to ectopically express combinations of Ets transcription factors on day 3 of TH cell differentiation in vitro. Although transduction of ERG resulted in a significant increase of IL-9 (Fig. 1D), paired transduction of ERG with PU.1 or ETV5 did not further increase IL-9 production (Fig. 3E). However, combined ectopic expression of PU.1, ETV5, and ERG led to a further significant increase of IL-9 production in TH9 cells. These data suggested that PU.1, ETV5, and ERG cooperatively regulate IL-9 production in TH9 cells.

To dissect the function of ERG in TH9 development in the absence of other Ets transcription factors including PU.1 and ETV5, we developed double and triple knockout mice that lacked either PU.1 and ETV5 (Spifl/flEtv5fl/fl CD4-Cre mice) or PU.1, ETV5, and ERG (Spifl/flEtv5fl/flErgfl/fl CD4-Cre mice). The deletion of Ets transcription factors in CD4 T cells was confirmed by assessing transcript expression of Erg, Spi1 (PU.1), and Etv5 in TH9 cultures derived from the knockout mice (Fig. 4A–C). Consistent with previous studies (19), deficiency in PU.1 and ETV5 resulted in reduced production of IL-9 in TH9 cells cultured in vitro (Fig. 4D, 4E). Deletion of ERG in the absence of PU.1 and ETV5 led to a further decrease in IL-9 production (Fig. 4D–F).

FIGURE 4.

ERG regulated IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Experiments were performed using transgenic conditional knockout mice, including Spifl/flEtv5fl/flErgfl/fl CD4-Cre, Spifl/flEtv5fl/fl CD4-Cre+, and Spifl/flEtv5fl/flErgfl/fl CD4-Cre+, respectively (n = 3 per group). (AD) Transcript expression of Erg, Spi1, Etv5, and Il9, respectively. (E) IL-9 production in TH9 cells cultured measured via flow cytometry. (F) IL-9 secretion measured by performing ELISA. (GJ) ChIP qPCR assay to assess interactions of PU.1, ERG, p300, and H3K27Ac at the Il9 locus in resting TH9 cells. Data are means of three to four mice per experiment and representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

ERG regulated IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Experiments were performed using transgenic conditional knockout mice, including Spifl/flEtv5fl/flErgfl/fl CD4-Cre, Spifl/flEtv5fl/fl CD4-Cre+, and Spifl/flEtv5fl/flErgfl/fl CD4-Cre+, respectively (n = 3 per group). (AD) Transcript expression of Erg, Spi1, Etv5, and Il9, respectively. (E) IL-9 production in TH9 cells cultured measured via flow cytometry. (F) IL-9 secretion measured by performing ELISA. (GJ) ChIP qPCR assay to assess interactions of PU.1, ERG, p300, and H3K27Ac at the Il9 locus in resting TH9 cells. Data are means of three to four mice per experiment and representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

We further performed ChIP assays to define the binding of Ets factors at Il9 regulatory regions. As expected, there was reduced binding of PU.1 at both Il9 promoter and enhancer regions in cells lacking PU.1 and ETV5 (Fig. 4G). ERG binding was observed at the Il9 enhancer region in PU.1-ETV5 double-deficient TH9 cells, albeit at lower abundance compared with the TH9 cells derived from Cre-negative mice. ERG binding was nearly absent from Spifl/flEtv5fl/flErgfl/fl CD4-Cre mice (Fig. 4H). Binding of HAT p300 was reduced at the Il9 enhancer and CNS-6 region in the absence of PU.1 and ETV5. Deletion of Erg further led to a decrease in p300 binding at both CNS-6 and CNS-25 (enhancer) regions (Fig. 4I). Both double and triple deficiency of Ets proteins led to a decrease in H3K27 histone acetylation modifications across the Il9 locus in TH9 cells (Fig. 4J). The deletion of Ets transcription factors did not affect H3K27 histone acetylation or p300 interactions at the Il9 promoter regions (Fig. 4I, 4J). Taken together, these findings indicate that ERG interacts with the Il9 locus in the absence of PU.1 and ETV5 and may contribute to Il9 gene regulation in the absence of these Ets proteins. Deletion of Erg in the absence of PU.1 and ETV5 led to a decrease in p300 interactions at the Il9 enhancer regions, suggesting that at least one mechanism though which ERG regulates transcription of Il9 is by recruiting p300 to the Il9 regulatory regions.

In the absence of PU.1 and ETV5, ERG facilitates IL-9 production in TH9 cells in vitro. We further wanted to determine whether ERG played a role in TH9 function in vivo in AADs. To addresses this question, we measured TH9 function in A. fumigatus–induced chronic AAD models in conditional knockout mice including Spifl/flEtv5fl/flErgfl/fl CD4 Cre, Ergfl/fl CD4-Cre+, Spifl/flEtv5fl/fl CD4-Cre+, and Spifl/flEtv5fl/flErgfl/fl CD4-Cre+ mice (Fig. 5A). Mice were intranasally challenged with A. fumigatus during a period of 6 wk, and airway inflammation was examined at the end of 6 wk. Deletion of Erg alone led to a modest decrease in total number of cells in the inflamed lungs as well as BAL compared with CD4-Cre mice (Fig. 5B, 5C). The absence of ERG alone failed to significantly reduce the total CD4 T cell population or frequency of IL-9–producing CD4 T cells isolated from the lungs (Fig. 5D, 5J).

FIGURE 5.

ERG regulates TH9 function in the absence of PU.1 and ETV5 in chronic AAD model. (A) Schematic of AAD model using transgenic mice of specified genotype. (B) Total number of cells in the lung. (C) Number of cells per milliliter in the BAL fluid. (DI) Number of CD4 T cells (D), B cells (E), neutrophils (F), eosinophils (G), macrophages (H), and CD8 T cell counts (I) in the lung. (JO) Frequency of IL-9–, IL-13–, IL-5–, IL-4–, IL-10–, and IFN-γ–producing CD4 T cells in the lung. (P) IL-9 secretion from lung CD4 T cells cocultured with naive CD11c+ cells in the presence of IL-33 and A. fumigatus (100 mg/ml) for 72 h ex vivo. Data are means of five mice per experiment and representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

ERG regulates TH9 function in the absence of PU.1 and ETV5 in chronic AAD model. (A) Schematic of AAD model using transgenic mice of specified genotype. (B) Total number of cells in the lung. (C) Number of cells per milliliter in the BAL fluid. (DI) Number of CD4 T cells (D), B cells (E), neutrophils (F), eosinophils (G), macrophages (H), and CD8 T cell counts (I) in the lung. (JO) Frequency of IL-9–, IL-13–, IL-5–, IL-4–, IL-10–, and IFN-γ–producing CD4 T cells in the lung. (P) IL-9 secretion from lung CD4 T cells cocultured with naive CD11c+ cells in the presence of IL-33 and A. fumigatus (100 mg/ml) for 72 h ex vivo. Data are means of five mice per experiment and representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

In parallel experiments, we crossed Ergfl/fl mice with tamoxifen inducible CD4-Cre mice to generate Ergfl/fl iCD4-Cre transgenic mice. Mice were sensitized for 6 wk with A. fumigatus, and CD4-specific Cre expression was induced by administering 3 mg of tamoxifen per mouse via oral gavage for 5 d during week 5 and week 6 of the chronic model (Supplemental Fig. 3A). The deletion of Erg with tamoxifen treatment was confirmed by measuring the Erg transcript in CD4 T cells isolated from the lung (Supplemental Fig. 3B). Erg deletion in the last 2 wk of the chronic AAD model led to a modest decrease in cellularity in the lung and BAL (Supplemental Fig. 3C, 3D). ERG-deficient CD4 T cells had a significant decrease in total CD4 T cells and the IL-9–producing CD4 T cell population in the lung (Supplemental Fig. 2E–G). CD4 T cells from Ergfl/fl iCD4-Cre mice demonstrated reduced secretion of IL-9 by CD4 T cells in ex vivo, when cultured with naive CD11c+ APCs for 3 d in the presence of IL-33 and A. fumigatus (Supplemental Fig. 3H). Thus, as with in vitro results (Fig. 2G–I), deletion of ERG in peripheral cells in vivo results in a decrease of IL-9 production, although this was still not sufficient to impact allergic inflammation.

We then determined whether ERG cooperated with other Ets factors in the regulation of allergic airway inflammation. Deficiency in both PU.1 and ETV5 resulted in reduced cellularity in both lung and BAL compared with the CD4-Cre mice (Fig. 5B, 5C). Spifl/flEtv5fl/fl CD4-Cre+ mice also showed a decrease in CD4 T cells and the percentage of IL-9–producing CD4 T cell in the lung (Fig. 5D, 5J). Deletion of Erg in the context of PU.1 and Etv5 double deficiency resulted in a further reduction in the total cellularity in both lung and BAL (Fig. 5B, 5C). Spifl/flEtv5fl/flErgfl/fl CD4-Cre+ mice had the greatest reduction in CD4 T cell populations and the percentage of IL-9–secreting CD4 T cells in the lung (Fig. 5D, 5J). Among the IL-9 responding immune cell populations, B cells and neutrophils showed a significant drop in cellularity in Spifl/flEtv5fl/flErgfl/fl CD4-Cre+ mice (Fig. 5E, 5F). In addition to IL-9+ CD4 T cell populations, we observed decreases in percentages of IL-4–, IL-5–, and IL-10–producing CD4 T cells in the lung with the deletion of Spi1, Etv5, and Erg (Fig. 5L–O). Despite the reduced frequency of IL-5–secreting CD4 T cells in the Spifl/flEtv5fl/flErgfl/fl CD4-Cre+ mice, the eosinophil population remained unaffected (Fig. 5G). The populations of macrophages and CD8 T cells were only affected with the deletion of all three transcription factors, that is, PU.1, ETV5, and ERG (Fig. 5H, 5I). Moreover, deficiency of PU.1, ETV5, and ERG failed to affect IFN-γ or IL-13 production by CD4 T cells, with the latter playing an important function in mucus production by airway epithelial cells (Fig. 5K, 5O). Lastly, we also performed coculture assays to measure IL-9 production by lung CD4 T cells in response to A. fumigatus Ag. The combination of deficiency in PU.1, ETV5, and ERG leads to a significant decrease in IL-9 secretion by lung CD4 T cells specifically in response to A. fumigatus (Fig. 5P). Taken together, these observations indicated that in the absence of PU.1 and ETV5, ERG regulated the development and function of TH9 cells in AADs.

Recent advances in TH9 biology have provided more evidence for the significance of IL-9–secreting CD4 T cells in mediating inflammatory responses by recruiting and activating innate and adaptive immune cells. Most TH cell subsets express a unique transcription factor that orchestrates the development of naive CD4 T cells into specialized effector CD4 TH cells characterized by the production of signature cytokines. In the case of TH9 cells, a master regulator involved in the development of TH9 cells remains to be identified. Many transcription factors including PU.1, ETV5, STAT5, BATF, IRF4, IRF8, and, more recently, PPAR-γ have been reported to regulate IL-9 production in TH9 cells (1, 15–17, 19, 43–45). These findings suggest that TH9 differentiation is governed by a complex transcriptional network that may require recruitment of multiple transcription factors and coactivators to facilitate Il9 gene expression. In this study, we identify the Ets transcription factor ERG that cooperates with PU.1 and ETV5 to enhance IL-9 production in TH9 cells.

Given the conserved Ets DNA-binding domain and similar DNA target sequence among the members of Ets family of transcription factors, it is not surprising that there are many examples of cooperativity. PU.1 and Spi-B exhibit redundant functions in the myeloid and lymphoid cell development, B cell function, and the progression of leukemia (46, 47). Both ERG and Fli-1 have been implicated in the development of Ewing sarcoma due to the highly homologous Ets-DNA binding region in the C terminus (25, 26, 48). More relevant, PU.1 and ETV5 have also been reported to cooperatively regulate TH9 development in the AAD model (19). That cooperation likely results in the modest decreases in IL-9 when ERG is deleted during T cell development or before activation. In vivo, deficiency of Erg expression alone leads to a modest decrease in IL-9–producing CD4 T cells in the lung and did not affect overall airway inflammation. However, when T cells in vivo are already deficient in PU.1 and ETV5, the additional deficiency in ERG results in greater decreases in IL-9 and allergic airway inflammation. The cooperativity among the Ets factors undoubtedly contributes to the differential effects of ERG deletion in vivo and during differentiation in vitro. The cooperativity, which is further supported by retroviral transduction and ChIP assays, allows for other factors to compensate for ERG deficiency, at least in some circumstances. Importantly, however, note that in the absence of all three Ets factors we could still observe IL-9–producing TH9 cells, albeit at much lower frequency, suggesting that other factors could be facilitating IL-9 production by CD4 T cells.

Erg splice forms add additional complexity to understanding the contribution to IL-9 regulation. Mutation of the TH9-enriched exon 3 resulted in diminished expression of that isoform without changes in the exon 4 transcript or the overall Erg mRNA expression. This suggests that while enriched, exon 3–containing Erg transcripts represent a minor component of total Erg transcripts. It also suggests, in combination with other data presented, that either exon 4–containing Erg transcripts are sufficient to contribute to IL-9 production, or that there are additional cryptic splice forms with translational start sites that are not yet characterized. Importantly, utilizing two conditional mutant mouse strains that targeted distinct exons common to both exon 3– and exon 4–containing transcripts and deleted at the mature T cell stage, we observed modest phenotypes, suggesting that it is redundant function with other ETS proteins and not other splice forms that are responsible for the phenotype.

Mechanistically, ERG promotes Il9 expression by impacting binding of other transcription factors and histone-modifying enzymes. Deletion of ERG results in decreased PU.1 binding, and deletion of ERG in the absence of PU.1 and ETV5 significantly reduced HAT p300 binding at the Il9 enhancer region. HAT p300 is a histone acetyltransferase that has been previously shown to bind at the Il9 locus to catalyze H3K27 acetylation in TH9 cells costimulated with OX40 (49). Studies on angiogenesis in endothelial cells show that vascular endothelial growth factor (VEGF) signaling induces activation of the VEGF/ERK signaling pathway that facilitates ERG-dependent recruitment of p300 to promote transcription of genes associated with VEGF-dependent angiogenesis (50). In prostate cancer, ERG has been reported to interact with BAF chromatin remodeling complexes to promote expression of oncogenes (30). These findings suggest that ERG could enhance Il9 gene expression in TH9 cells by recruiting transcription coactivators and chromatin remodeling proteins to facilitate chromatin vascular endothelial growth factor chromatin interactions among cis-regulatory regions at the Il9 locus.

As noted, there is also evidence of ERG-dependent TH9 function in AADs. Although deletion of Erg alone by either a constitutive or inducible CD4-Cre has modest effects on overall inflammation in the lung, in the absence of PU.1 and Etv5, Erg deficiency results in significant decreases in cellularity in the lung, including decreased CD4 T cells, CD8 T cells, and B cell populations. The combined deficiency of ERG, PU.1, and ETV5 results in a significant decrease in IL-9 production by Ag-specific CD4 T cells in the lung. Erg deletion also leads to a decrease in production of some of the other proinflammatory cytokines, including IL-5, IL-10, and IL-4. Taken together, these findings show that although Ets transcription factors PU.1, ETV5, and ERG each contribute to IL-9 regulation, they can partially compensate for the absence of one another. Our data suggest that Ets proteins function most effectively in concert to regulate IL-9 production and TH9-mediated inflammation in AADs and that the redundancy suggests the importance of maintaining IL-9 expression in the organism.

The authors have no financial conflicts of interest.

The visual abstract was created with BioRender.com.

This work was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant AI057459. B.J.U. was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant T32 AI060519 and National Institutes of Health/National Heart, Lung, and Blood Institute Grant F30 HL147515. M.C. was supported by National Institutes of Health/National Heart, Lung, and Blood Institute Grant T32 HL091816 and National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant F30AI174762. M.M.H. was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant T32 AI060519. Core facility usage was also supported by IU Simon Cancer Center Support Grants P30 CA082709 and U54 DK106846. Support provided by the Herman B. Wells Center was in part from the Riley Children’s Foundation. Work in the Porse laboratory was supported by Novo Nordisk Foundation Center for Stem Cell Biology Grant NNF17CC0027852.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AAD

allergic airway disease

BAL

bronchoalveolar lavage

BATF

basic leucine zipper ATF-like transcription factor

ChIP

chromatin immunoprecipitation

CNS

conserved noncoding sequence

EGFP

enhanced GFP

ERG

Ets-related gene

Ets

E26 transformation-specific

ETV5

Ets translocation variant 5

h

human

HAT

histone acetyltransferase

iCD4

inducible CD4

IRES

internal ribosome entry site

IRF

IFN regulatory factor

qPCR

quantitative PCR

RT

room temperature

siRNA

small interfering RNA

Treg

T regulatory

VEGF

vascular endothelial growth factor

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Supplementary data