Interleukin-9 Facilitates Osteoclastogenesis in Rheumatoid Arthritis.

In rheumatoid arthritis (RA), inflammatory cytokines play a pivotal role in triggering abnormal osteoclastogenesis leading to articular destruction. Recent studies have demonstrated enhanced levels of interleukin-9 (IL-9) in the serum and synovial fluid of patients with RA. In RA, strong correlation has been observed between tissue inflammation and IL-9 expression in synovial tissue. Therefore, we investigated whether IL-9 influences osteoclastogenesis in patients with RA. We conducted the study in active RA patients. For inducing osteoclast differentiation, mononuclear cells were stimulated with soluble receptor activator of NF-kB ligand (sRANKL) and macrophage-colony-stimulating factor (M-CSF) in the presence or absence of recombinant (r) IL-9. IL-9 stimulation significantly enhanced M-CSF/sRANKL-mediated osteoclast formation and function. Transcriptome analysis revealed differential gene expression induced with IL-9 stimulation in the process of osteoclast differentiation. IL-9 mainly modulates the expression of genes, which are involved in the metabolic pathway. Moreover, we observed that IL-9 modulates the expression of matrix metalloproteinases (MMPs), which are critical players in bone degradation. Our results indicate that IL-9 has the potential to influence the structural damage in the RA by promoting osteoclastogenesis and modulating the expression of MMPs. Thus, blocking IL-9 pathways might be an attractive immunotherapeutic target for preventing bone degradation in RA.

Interleukin-35 Suppresses Interleukin-9-Secreting CD4+ T Cell Activity in Patients With Hepatitis B-Related Hepatocellular Carcinoma.

Chronic hepatitis B virus (HBV) infection induces dysfunction of immune response and chronic liver damage. However, the mechanisms that account for HBV-related hepatocellular carcinoma (HCC) are poorly understood. The aim of present study was to investigate the modulatory role of interleukin (IL)-35, an immunosuppressive cytokine, to IL-9-secreting T cells in hepatitis B-related HCC. Twenty-two HBV-related HCC patients, twenty-seven chronic hepatitis B (CHB) patients, and eleven controls were enrolled. Serum IL-35 and IL-9 concentration was measured by ELISA. Peripheral and liver-infiltrating non-specific and HBV-specific Th9 and Tc9 cells were assessed by flow cytometry. The regulatory activity of IL-35 to peripheral and liver-infiltrating Th9 cells was assessed in co-culture system between CD8+ T cells and HepG2.2.15 cells. Serum IL-35 was up-regulated, while IL-9 was down-regulated in HBV-related HCC patients compared with in CHB patients and controls. Peripheral non-specific and HBV-specific Th9 cells, but not Tc9 cells, were decreased in HBV-related HCC patients. Liver-infiltrating non-specific and HBV-specific Th9 cells were also reduced in HCC tumor sites. CD8+ T cells from CHB and HBV-related HCC patients revealed decreased cytotoxicity compared with those from controls. Autologous Th9 cells mediated the elevation of CD8+ T cell cytotoxicity, and this process was depending on IL-9 secretion. Recombinant IL-35 stimulation inhibited IL-9 secretion and PU.1 mRNA expression in non-specific and HBV-specific Th9 cells, leading to the suppression of Th9-mediated CD8+ T cell cytotoxicity in CHB and HBV-related HCC patients. Our current data indicated that IL-35 might dampen non-specific and HBV-specific Th9 cells activity in HBV-related HCC patients.

Interleukin-9 produced by helper T cells stimulates interleukin-8 expression in endometriosis.

PROBLEM: Inflammation and immune responses play crucial roles in the development of endometriosis. Although interleukin-9 (IL-9) has a pro-inflammatory function in chronic inflammatory diseases, its function in endometriosis remains unknown. Here, we aimed to investigate the significance of IL-9 and IL-9-producing lymphocytes in endometriosis. METHOD OF STUDY: Specimens were obtained from patients with and without endometriosis. Peritoneal fluid (PF), peripheral blood (PB), and ovarian endometrioma (OE) tissues were analyzed for the proportion of CD4+ IL-9+ lymphocytes and IL-9 concentration using flow cytometry and enzyme-linked immunosorbent assay. OE, endometrium with endometriosis (EE), and normal endometrium (NE) were analyzed for IL-9 receptor (IL-9R) expression using immunohistochemical staining. IL-9-dependent changes in Interleukin-8 (IL-8) expression in endometrial stromal cells from OE (OESCs) were evaluated using real-time PCR. RESULTS: The proportion of CD4+ IL-9+ lymphocytes was higher in the PF, but not the PB, of patients with endometriosis than individuals without endometriosis (p < .05). However, IL-9 levels in the PF did not differ between those with and without endometriosis. We detected CD4+ IL-9+ lymphocytes in OE tissues and IL-9R in OE tissues and OESCs. In OESC culture, IL-9 significantly elevated IL-8 expression in a dose-dependent manner (p < .05), which was nullified by the addition of the anti-IL-9 receptor antibody. Furthermore, IL-9 additively stimulated IL-8 expression in the presence of TNF-α (p < .05). CONCLUSION: Our findings show that IL-9 produced by helper T cells induces IL-8 expression, suggesting that IL-9 plays an important role in the development of endometriosis by stimulating IL-8 expression.

Interleukin-9 Aggravates Isoproterenol-Induced Heart Failure by Activating Signal Transducer and Activator of Transcription 3 Signalling.

BACKGROUND: Previous studies have demonstrated that inflammation is closely related to the occurrence and development of heart failure (HF). As an inflammation-related cytokine, interleukin (IL)-9 has been reported to be involved in the development of cardiovascular diseases. However, the role of IL-9 in HF in response to isoproterenol (ISO) stimulation has barely been explored. Thus, this study aimed to investigate whether IL-9 participates in HF and the possible associated mechanisms. METHODS: Chronic ISO infusion was used to establish an HF model, and the IL-9 levels in mice and isolated cardiomyocytes were measured. In addition, ISO-treated mice received an injection of recombinant mouse IL-9 (rIL-9) or an antimouse IL-9 neutralizing monoclonal antibody (mAb) to investigate the effects of IL-9 on cardiac function, hypertrophy, and fibrosis. RESULTS: IL-9 levels were significantly increased in mice and isolated cardiomyocytes after ISO treatment. Treatment with rIL-9 resulted in aggravated cardiac dysfunction and amplified cardiac hypertrophy and fibrosis, whereas treatment with the anti-IL-9 neutralizing mAb ameliorated cardiac dysfunction and reduced cardiac hypertrophy and fibrosis in ISO-treated mice. In addition, ISO infusion-induced cardiac inflammation and cardiomyocyte apoptosis was aggravated by rIL-9 but prevented by the anti-IL-9 mAb. IL-9 did not activate signal transducer and activator of transcription (STAT)1 or STAT5 but induced STAT3 phosphorylation in ISO-induced HF. Moreover, S31-201, a specific STAT3 inhibitor, nearly abolished rIL-9-induced increases in cardiac dysfunction, hypertrophy, and fibrosis in response to ISO stimulation. CONCLUSIONS: IL-9 aggravated cardiac dysfunction and amplified cardiac hypertrophy and fibrosis in the ISO-induced HF model by activating STAT3 signalling. These data indicate that blocking IL-9 may be an attractive pharmacotherapeutic strategy for the treatment of cardiac hypertrophy and fibrosis induced by chronic ß-adrenergic receptor activation to limit the progression of HF.

Inhibition of Histone H3K27 Acetylation Orchestrates Interleukin-9-Mediated and Plays an Anti-Inflammatory Role in Cisplatin-Induced Acute Kidney Injury.

Nephrotoxicity is a major side effect of cisplatin (CP)- and platinum-related chemotherapy, and inflammation contributes to disease pathogenesis. Interleukin-9 (IL-9) is a pleiotropic cytokine associated with inflammation. Here, we investigated the key role of IL-9 as a regulator of protective mechanisms in CP-induced acute kidney injury (AKI). We observed that IL-9 was decreased not only in a CP-induced AKI mouse model but also in THP-1 and RAW264.7 cell lines. Seventy-two hours post-CP injection, renal dysfunction and tubule injury were significantly attenuated in IL-9 overexpression adeno-associated virus 9 (AAV9)-treated mice. The levels of serum urea, serum creatinine, kidney injury molecule-1 (KIM-1), and histological damage were partially diminished following treatment with IL-9. The renoprotective effects of IL-9 may be attributed to the regulation of cytokines, and we found that IL-9 acted on macrophages in a regulatory manner, promoting an anti-inflammatory phenotype. Furthermore, IL-9 enhanced the suppression of macrophage-driven renal inflammation. Inhibition of H3K27 acetylation orchestrated IL-9-mediated renoprotection in CP-induced AKI. Thus, our findings indicate novel and potent anti-inflammatory properties of IL-9 that confer preservation of kidney function and structure in CP-induced AKI, which may counteract kidney disease procession.

The expression of Th9 and Th22 cells in rats with cerebral palsy after hUC-MSC transplantation.

BACKGROUND: This study aimed to investigate the expression of Th9 and Th22 cells in rats with cerebral palsy (CP) after human umbilical cord-derived mesenchymal stem cell (hUC-MSC) transplantation. METHODS: First, hUC-MSCs were isolated from fresh umbilical cords and identified. Rats were divided into the normal group, CP group, and hUC-MSC transplantation group. The Morris water maze and balance beam tests were performed to evaluate the neurobehavioral ability of the rats. The levels of TNF-α, IL-6, IL-9, and IL-22 in rat brain tissues were detected by ELISA. Th9 and Th22 proportions in brain tissues were detected by flow cytometric analysis. The mRNA levels of IL-9, IL-22, PU.1, and AHR in brain tissues were determined by qRT-PCR. RESULTS: hUC-MSC transplantation enhanced the neurobehavioral ability of CP rats. Furthermore, Th9 and Th22 proportions were decreased in brain tissues from CP rats after hUC-MSC transplantation. The levels of proinflammatory cytokines (TNF-α and IL-6), Th9-related IL-9 and PU.1, and Th22-related IL-22 and AHR were markedly higher in brain tissues from CP rats than in brain tissues from control rats, but their levels were significantly decreased after hUC-MSC transplantation. CONCLUSION: Our data indicate that Th9 and Th22 proportions are decreased in CP rats after hUC-MSC transplantation.

Inhibitory effect of activin A on IL-9 production by mouse NK cells through Smad3 signaling.

Interleukin-9 (IL-9) is a cytokine secreted by T-helper (Th)9 cells, and activin A can enhance Th9 cell differentiation. However, whether activin A affects IL-9 production by natural killer (NK) cells remains unclear. Herein, we found that not only Th cells, but also CD3-CD49b+NKp46+ NK cells of Balb/c mice produced IL-9. Although activin A promoted IL-9 expression in CD4+ Th cells, it inhibited IL-9 production by CD49b+NKp46+ NK cells in mice. Furthermore, the enzyme-linked immunosorbent assay (ELISA) results showed that mouse NK cells could secrete mature IL-9 protein, and activin A inhibited IL-9 release by NK cells. Additionally, activin A inhibited interferon (IFN)-γ production in splenic NK cells in mice, but promoted IL-2 production, and did not alter the production of IL-10. Western blotting results showed that levels of activin type IIA receptor (ActRIIA), Smad3 and phosphorylated-Smad3 (p-SMAD3) protein increased in activin A-treated splenic NK cells, compared with that in control NK cells. The inhibitory effects of activin A on IL-9 production by NK cells were attenuated in the presence of activin antagonist follistatin (FST) or Smad3 knockdown to NK cells. These data suggest that although activin A up-regulates IL-9 expression in Th cells, it inhibits IL-9 production in NK cells through Smad3 signaling.

IL-9-producing CD8+ T cells represent a distinctive subset with different transcriptional characteristics from conventional CD8+ T cells, and partially infiltrate breast tumors.

Accumulating evidence suggests that IL-9 and IL-9-producing cells exert various roles in antitumor immunity. Our study examined the IL-9 production in CD8+ T cells from breast cancer patients as compared to healthy controls. IL-9 secretion was undetectable in CD8+ T cells ex vivo, but could be readily detected following anti-TCR or PMA + ionomycin stimulation, and was higher in breast cancer patients than in healthy controls. The capacity to express IL-9 was not universal to all CD8+ T cells, but was favored in IL-9Rhigh CD8+ T cells, which were also present in breast cancer patients at significantly higher frequency than in healthy controls. Interestingly, exogenous IL-9 could significantly increase the expression of both IL-9 and IL-9R in IL-9Rhigh, but not IL-9Rlow, CD8+ T cells. IL-9Rhigh CD8+ T cells ex vivo presented lower expression of KLRG-1, PD-1, and Tim-3 than IL-9Rlow CD8+ T cells. Additionally, IL-9Rhigh CD8+ T cells following anti-TCR and PMA + ionomycin stimulation presented higher IL-2 and IL-17 expression, and lower IFN-γ expression, than IL-9Rlow CD8+ T cells. IL-9-expressing CD8+ T cells could be found in some, but not all, resected breast tumors. IL-9R expression, on the other hand, was readily present in CD8+ T cells, but with high variability from patient to patient. Patients with high intratumoral IL-9 expression also tended to present high IL-9R expression. Together, these data demonstrate that a transcriptionally distinctive IL-9-producing CD8+ T cell subset was elevated in breast cancer patients and could be found inside the tumor, with higher capacity to produce IL-2 and IL-17 and lower expression of inhibitory receptors.

The Il9 CNS-25 Regulatory Element Controls Mast Cell and Basophil IL-9 Production.

IL-9 is an important mediator of allergic disease that is critical for mast cell-driven diseases. IL-9 is produced by many cell types, including T cells, basophils, and mast cells. Yet, how IL-9 is regulated in mast cells or basophils is not well characterized. In this report, we tested the effects of deficiency of a mouse Il9 gene regulatory element (Il9 CNS-25) in these cells in vivo and in vitro. In mast cells stimulated with IL-3 and IL-33, the Il9 CNS-25 enhancer is a potent regulator of mast cell Il9 gene transcription and epigenetic modification at the Il9 locus. Our data show preferential binding of STAT5 and GATA1 to CNS-25 over the Il9 promoter in mast cells and that T cells and mast cells have differing requirements for the induction of IL-9 production. Il9 CNS-25 is required for IL-9 production from T cells, basophils, and mast cells in a food allergy model, and deficiency in IL-9 expression results in decreased mast cell expansion. In a Nippostrongylus brasiliensis infection model, we observed a similar decrease in mast cell accumulation. Although decreased mast cells correlated with higher parasite egg burden and delayed clearance in vivo, T cell deficiency in IL-9 also likely contributes to the phenotype. Thus, our data demonstrate IL-9 production in mast cells and basophils in vivo requires Il9 CNS-25, and that Il9 CNS-25-dependent IL-9 production is required for mast cell expansion during allergic intestinal inflammation.

ATP Triggers Human Th9 Cell Differentiation via Nitric Oxide-Mediated mTOR-HIF1α Pathway.

Interleukin 9 (IL-9)-producing helper T (Th9) cells have a crucial effector function in inducing allergic inflammation, autoimmunity, immunity to extracellular pathogens and anti-tumor immune responses. Although the cytokines that lead to the differentiation of human Th9 cells have been identified, other factors that support the differentiation of Th9 cells have not been identified yet. Here we show that the extracellular ATP (eATP) induces the differentiation of Th9 cells. We further show that eATP induces the production of nitric oxide (NO), which create a feed forward loop in the differentiation of human Th9 cells, as inhibition of purinergic receptor signaling suppressed the generation of human Th9 cells while exogenous NO could rescue generation of Th9 cells even upon inhibition of purinergic receptor signaling. Moreover, we show that ATP promotes mTOR and HIF1α dependent generation of Th9 cells. Our findings thus identify that ATP induced nitric oxide potentiate HIF1α-mediated metabolic pathway that leads to IL-9 induction in Th9 cells. Here we identified that the ATP-NO-mTOR-HIF1α axis is essential for the generation of human Th9 cells and modulation of this axis may lead to therapeutic intervention of Th9-associated disease conditions.

Cholesterol negatively regulates IL-9-producing CD8+ T cell differentiation and antitumor activity.

CD8+ T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8+ or CD4+ T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function.

Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation.

Th9 cells are prominently featured in allergic lung inflammation, but the mechanism that regulates IL-9 induction in T helper cells remains poorly defined. Here we demonstrate that formation of super-enhancers (SEs) is critical in robust induction of IL-9 and that assembly of the Il9 SEs in Th cells requires OX40-triggered chromatin acetylation. Mechanistically, we found that OX40 costimulation induces RelB expression, which recruits the histone acetyltransferase p300 to the Il9 locus to catalyze H3K27 acetylation. This allows binding of the SE factor Brd4 to organize assembly of the SE complex, which in turn drives robust IL-9 expression and Th9 cell induction. Thus, Th9 cells are strongly induced upon OX40 stimulation, and disruption of SEs abolished Th9 cell induction in vitro and inhibited Th9 cell-mediated allergic airway inflammation in vivo. Together, our data suggest that formation of SEs is essential in IL-9 expression and Th9 cell induction. These findings may have important clinical implications.

Microbe-Dependent Induction of IL-9 by CLA+ T Cells in Psoriasis and Relationship with IL-17A.

IL-9 is present in psoriatic lesions and is produced by lymphocytes. However, it is not known whether this cytokine is induced by relevant pathogenic triggers of psoriasis, such as Streptococcus pyogenes. Here we addressed the production of IL-9 in response to various pathogens in a psoriatic ex vivo model. Extracts of S. pyogenes and Candida albicans triggered the production of IL-9 and also IL-17A and IFN-γ. This induction was dependent on the interaction between CLA+ T cells and epidermal cells. Neutralization of IL-9 reduced S. pyogenes-induced IL-17A production by CLA+ T cells but had no effect on IFN-γ production. Also, IL-9 increased the survival of circulating psoriatic CLA+ T cells. Co-cultures from patients with guttate or plaque psoriasis with S. pyogenes produced similar amounts of IL-9. High cytokine responses in streptococcal-driven guttate patients paralleled peaks in Psoriasis Area Severity Index and anti-streptolysin O levels. Our results confirm that IL-9 promotes inflammation in psoriasis by up-regulating IL-17A production and support the clinical association of the immune response by streptococcal-sensitized CLA+ T cells with this cytokine, especially in guttate psoriasis.

IL-9 and Th9 cells in health and diseases-From tolerance to immunopathology.

CD4+ T cells have the capacity to differentiate into various T helper (Th) cell subsets after activation, and by acquiring distinct cytokine profiles and effector functions, they regulate the nature as well as the outcomes of immune responses. Th9 cells are a relatively new member in the Th cell family. The signature cytokine for Th9 cells is IL-9, a cytokine in the IL-2Rγc-chain family. Over the past few years, there has been an explosion of knowledge on the roles of Th9 cells in immunity and immunopathology, but the exact mechanisms in the control of Th9 cells remain poorly defined. This apparent paradox presents both challenges and opportunities. Here we review recent advances in our understanding of the fundamental biology of IL-9 and Th9 cells, highlighting the challenges and unanswered questions in the field. We also discuss potential opportunities in targeting Th9 cells for therapeutic purposes in the clinic.

Paracrine IL-2 Is Required for Optimal Type 2 Effector Cytokine Production.

IL-2 is a pleiotropic cytokine that promotes the differentiation of Th cell subsets, including Th1, Th2, and Th9 cells, but it impairs the development of Th17 and T follicular helper cells. Although IL-2 is produced by all polarized Th subsets to some level, how it impacts cytokine production when effector T cells are restimulated is unknown. We show in this article that Golgi transport inhibitors (GTIs) blocked IL-9 production. Mechanistically, GTIs blocked secretion of IL-2 that normally feeds back in a paracrine manner to promote STAT5 activation and IL-9 production. IL-2 feedback had no effect on Th1- or Th17-signature cytokine production, but it promoted Th2- and Th9-associated cytokine expression. These data suggest that the use of GTIs results in an underestimation of the presence of type 2 cytokine-secreting cells and highlight IL-2 as a critical component in optimal cytokine production by Th2 and Th9 cells in vitro and in vivo.

IL-9-producing cells in the development of IgE-mediated food allergy.

Food allergy is a harmful immune reaction driven by uncontrolled type 2 immune responses. Considerable evidence demonstrates the key roles of mast cells, IgE, and TH2 cytokines in mediating food allergy. However, this evidence provides limited insight into why only some, rather than all, food allergic individuals are prone to develop life-threatening anaphylaxis. Clinical observations suggest that patients sensitized to food through the skin early in life may later develop severe food allergies. Aberrant epidermal thymic stromal lymphopoietin and interleukin (IL) 33 production and genetic predisposition can initiate an allergic immune response mediated by dendritic cells and CD4+TH2 cells in inflamed skin. After allergic sensitization, intestinal IL-25 and food ingestion enhance concerted interactions between type 2 innate lymphoid cells (ILC2s) and CD4+TH2 cells, which perpetuate allergic reactions from the skin to the gut. IL-4 and cross-linking of antigen/IgE/FcεR complexes induce emigrated mast cell progenitors to develop into the multi-functional IL-9-producing mucosal mast cells, which produce prodigious amounts of IL-9 and mast cell mediators to drive intestinal mastocytosis in an autocrine loop. ILC2s and TH9 cells may also serve as alternative cellular sources of IL-9 to augment the amplification of intestinal mastocytosis, which is the key cellular checkpoint in developing systemic anaphylaxis. These findings provide a plausible view of how food allergy develops and progresses in a stepwise manner and that atopic signals, dietary allergen ingestion, and inflammatory cues are fundamental in promoting life-threatening anaphylaxis. This information will aid in improving diagnosis and developing more effective therapies for food allergy-triggered anaphylaxis.

T-bet inhibits innate lymphoid cell-mediated eosinophilic airway inflammation by suppressing IL-9 production.

BACKGROUND: Innate lymphoid cells (ILCs) are emerging subsets of immune cells that produce large amounts of cytokines upon cytokine and/or alarmin stimulation. Recent studies have shown that T-bet plays pivotal roles in the development of ILC3s and type 1 ILCs; however, the roles of T-bet in lung type 2 innate lymphoid cells (ILC2s) remain unknown. OBJECTIVE: We sought to determine the role of T-bet in ILC2-mediated airway inflammation. METHODS: The expression of T-bet in lung ILCs (defined as Thy1.2+ Lin- cells) was examined. The roles of T-bet in the development of lung ILC2s and airway inflammation induced by IL-33 administration were examined by using T-bet-deficient (T-bet-/-) mice. Gene expression profiles of T-bet-/- lung ILCs were analyzed by RNA sequencing. RESULTS: T-bet was expressed in lung ILC2s (defined as Thy1.2+ Lin- cells expressing ST2 or CD25) and IFN-γ enhanced its expression. Although the development of lung ILC2s at steady-state conditions was normal in T-bet-/- mice, IL-33-induced accumulation of lung ILC2s and eosinophilic airway inflammation were exacerbated in T-bet-/- mice. The exacerbated accumulation of ILC2s and eosinophilic airway inflammation by the absence of T-bet were evident even in a RAG2-/- background, suggesting that T-bet expressed in non-T/non-B population is involved in the suppression of IL-33-induced eosinophilic airway inflammation. Transcriptome analysis revealed that IL-9 expression in IL-33-stimulated lung ILCs was upregulated in T-bet-/- mice compared with that in wild-type mice. Importantly, neutralization of IL-9 markedly attenuated IL-33-induced accumulation of lung ILC2s and eosinophilic inflammation in T-bet-/- mice. CONCLUSIONS: T-bet suppresses IL-9 production from lung ILC2s and thereby inhibits IL-33-induced eosinophilic airway inflammation.

The ETS Family Transcription Factors Etv5 and PU.1 Function in Parallel To Promote Th9 Cell Development.

The IL-9-secreting Th9 subset of CD4 Th cells develop in response to an environment containing IL-4 and TGF-ß, promoting allergic disease, autoimmunity, and resistance to pathogens. We previously identified a requirement for the ETS family transcription factor PU.1 in Th9 development. In this report, we demonstrate that the ETS transcription factor ETS variant 5 (ETV5) promotes IL-9 production in Th9 cells by binding and recruiting histone acetyltransferases to the Il9 locus at sites distinct from PU.1. In cells that are deficient in both PU.1 and ETV5 there is lower IL-9 production than in cells lacking either factor alone. In vivo loss of PU.1 and ETV5 in T cells results in distinct effects on allergic inflammation in the lung, suggesting that these factors function in parallel. Together, these data define a role for ETV5 in Th9 development and extend the paradigm of related transcription factors having complementary functions during differentiation.