DUSP8 induces TGF-β–stimulated IL-9 transcription and Th9-mediated allergic inflammation by promoting nuclear export of Pur-α

Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear. Using T cell–specific Dusp8 conditional KO (T-Dusp8 cKO) mice, mass spectrometry analysis, ChIP-Seq, and immune analysis, we found that DUSP8 interacted with Pur-α, stimulated interleukin-9 (IL-9) gene expression, and promoted Th9 differentiation. Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-β signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation. Furthermore, Il-9 mRNA levels were induced in Pur-α–deficient T cells. In addition, T-Dusp8–cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in the allergic asthma model. Reduction of Il-9 mRNA levels in T cells and allergic responses of T-Dusp8–cKO mice was reversed by Pur-α knockout. Remarkably, DUSP8 protein levels and the DUSP8–Pur-α interaction were indeed increased in the cytoplasm of T cells from people with asthma and patients with atopic dermatitis. Collectively, DUSP8 induces TGF-β–stimulated IL-9 transcription and Th9-induced allergic responses by inhibiting the nuclear translocation of the transcriptional repressor Pur-α. DUSP8 may be a T-cell biomarker and therapeutic target for asthma and atopic dermatitis.


Introduction
Dual-specificity phosphatase 8, or DUSP8 -also named M3/6 and hVH5 -is a member of the dual-specificity phosphatase (DUSP) family phosphatases (1)(2)(3).The subcellular localization of DUSP8 is mainly in the cytoplasm and nucleus (4).DUSP8 plays an important role in cardiac ventricular remodeling (5).DUSP8 dephosphorylates and inactivates the kinase JNK (JNK1 and JNK2 isoforms), specifically, but not p38 and ERK, in Jurkat T cells (6) and 3T3-L1 adipocytes (7).Dusp8-deficient mice show hyperactivation of JNK signaling in the hypothalamus and display glucose intolerance (8).TNF-α induces the expression of DUSP8, which dephosphorylates JNK and subsequently inhibits the production of proinflammatory cytokines MCP-1 and IL-6 in 3T3-L1 adipocytes (7).To date, the in vivo role of DUSP8 in T cell functions and immune responses has not been investigated.

T cell development is normal in T cell-specific Dusp8-cKO mice.
To study the in vivo role of DUSP8 in T cell functions, we generated T cell-specific Dusp8 conditional KO (T-Dusp8 cKO [Dusp8 fl/fl ;Cd4-Cre]) mice by crossing floxed Dusp8 mice and Cd4-Cre transgenic mice (Supplemental Figure 1, A and B; supplemental material available online with this article; https:// doi.org/10.1172/JCI166269DS1).DUSP8 ablation in peripheral Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK.DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear.Using T cell-specific Dusp8 conditional KO (T-Dusp8 cKO) mice, mass spectrometry analysis, ChIP-Seq, and immune analysis, we found that DUSP8 interacted with Pur-α, stimulated interleukin-9 (IL-9) gene expression, and promoted Th9 differentiation.Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-β signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation.Furthermore, Il-9 mRNA levels were induced in Pur-α-deficient T cells.In addition, T-Dusp8-cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in the allergic asthma model.Reduction of Il-9 mRNA levels in T cells and allergic responses of T-Dusp8-cKO mice was reversed by Pur-α knockout.Remarkably, DUSP8 protein levels and the DUSP8-Pur-α interaction were indeed increased in the cytoplasm of T cells from people with asthma and patients with atopic dermatitis.Collectively, DUSP8 induces TGF-β-stimulated IL-9 transcription and Th9-induced allergic responses by inhibiting the nuclear translocation of the transcriptional repressor Pur-α.DUSP8 may be a T-cell biomarker and therapeutic target for asthma and atopic dermatitis.
DUSP8 induces TGF-β-stimulated IL-9 transcription and Th9-mediated allergic inflammation by promoting nuclear export of Pur-α however, the reduction was not statistically significant.These results suggest that DUSP8 does not play an important role in the regulation of TCR signaling.
Th9 cell differentiation is attenuated in T cells from T-Dusp8-cKO mice.To investigate whether DUSP8 plays a role in T-cell differentiation, we performed T helper (Th) cell differentiation in vitro.Remarkably, in vitro Th9 differentiation was reduced using splenic CD4 + T cells from T-Dusp8-cKO mice (Figure 2, A and B), whereas differentiation of Th1, Th2, Th17, or Treg was not affected (Figure 2, C-F).Because TGF-β or IL-4 treatment is critical for Th9 differentiation, we examined the IL-9 production in T cells of T-Dusp8-cKO mice under TGF-β or IL-4 stimulation.Strikingly, real-time PCR data showed that Il-9 mRNA induction in murine primary T cells stimulated by TGF-β alone or IL-4 plus TGF-β treatment, but not IL-4 alone, was abolished by Dusp8 cKO (Figure 2G).TGF-β-induced Il-9 mRNA levels in human Jurkat T cells were decreased by DUSP8 shRNA knockdown (Supplemental Figure 4A); conversely, the levels were enhanced by DUSP8 overexpression (Supplemental Figure 4B).Consistently, IL-9 mRNA levels were decreased by DUSP8 shRNA knockdown (Figure 2H) and induced by DUSP8 overexpression (Figure 2I) in human primary T cells under Th9 polarizing conditions (Supplemental Figure 4C).Moreover, DUSP8 phosphatase activity was induced by TGF-β signaling (Figure 2J).These results suggest that DUSP8 plays an important role in controlling IL-9 gene expression in TGF-β-stimulated T cells.
DUSP8 induces IL-9 production in T cells by inhibiting Pur-α function.Next, we tested whether DUSP8 regulates IL-9 production through transcriptional activation of the IL-9 promoter.IL-9 promoter activity in Jurkat T cells was enhanced by DUSP8 overexpression but not by the DUSP8 (C246S) phosphatasedead mutant (Figure 3A).To investigate the mechanism of DUSP8-induced IL-9 transcription in T cells, we characterized DUSP8-interacting proteins by performing mass spectrometrybased proteomics using anti-myc (DUPS8)-immunocomplexes.Among DUSP8-interacting proteins, we identified a DNA-binding protein, Pur-α (Figure 3B), which acts as either a transcriptional repressor (18) or activator (19) for different target genes.Pur-α interacts with the coactivator Smad in myofibroblasts (18).TGF-β-stimulated Smad2 and Smad4 are required for IL-9 transcription (20); however, it is unknown whether Pur-α regulates IL-9 transcription.Next, we examined whether Pur-α regulates IL-9 transcription using luciferase reporter assays of the IL-9 promoter.TGF-β-stimulated IL-9 promoter activity was inhibited by Pur-α overexpression in Jurkat T cells (Figure 3C).Notably, Pur-α-inhibited IL-9 transcription was reversed by DUSP8 overexpression (Figure 3C).To further confirm the suppressive function of Pur-α in the regulation of IL-9 transcription, we generated Pur-α KO mice and characterized Pur-α-KO T cells (Supplemental Figure 5, A and B).Pur-α homozygous-KO mice died prematurely at or before the age of 3 weeks.Due to the lack of mature Pur-α-KO mice, it was not feasible to perform in vitro Th9 differentiation assays or in vivo immunization studies on these mice.Nevertheless, we managed to perform immunoblotting and real-time PCR analyses using the scarce splenocytes from immature Pur-α-KO mice.Prior to premature death, the few available 3-week-old Pur-α homozygous-KO mice displayed blood T cells of T-Dusp8-cKO mice was confirmed by real-time PCR and immunoblotting analysis (Supplemental Figure 1, C and D).Four-week-old T-Dusp8-cKO mice displayed normal T cell development in the thymus, as well as normal peripherally derived natural Treg (nTreg) populations in the spleen and lymph nodes (Supplemental Figure 2, A-F).
T cell profiles and T cell receptor-induced cytokines of T-Dusp8-cKO mice are similar to those of WT mice.To investigate T cell profiles and potential immune phenotypes of T-Dusp8-cKO mice, unstimulated T cells from the spleen and lymph nodes of 6-month-old mice were subjected to single cell-RNA-Seq (scRNA-seq) analysis.Dimensionality reduction and clustering analyses showed that frequencies of individual T cell clusters of mice are comparable between WT and T-Dusp8-cKO mice (Figure 1, A and B).Notably, expression levels of 28 genes were decreased, while 6 other genes were increased, in T cells of T-Dusp8-cKO mice (Figure 1C).However, the fold changes on the mRNA levels of these 34 dysregulated genes were very modest (Figure 1C).Protein levels of CD4 and CD8 detected by the AbSeq antibody-oligonucleotide conjugates were decreased in T-Dusp8-cKO mice (Figure 1C); nevertheless, the frequencies of CD4 + T cells and CD8 + T cells were unchanged in T-Dusp8-cKO mice (Supplemental Figure 2B).Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the 28 downregulated genes in T-Dusp8-cKO T cells belong to protein folding signaling, stress-stimulated cellular responses, cell death/autophagy pathways, and metabolic pathways (Figure 1D).Among these DUSP8-regulated pathways, none of the pathways are associated with T cell-mediated immune responses.These results suggest that basal T cell subsets and immune phenotypes in the absence of stimulation are not affected by DUSP8 ablation.
To study whether T cell receptor (TCR) signaling-stimulated cytokine production is regulated by DUSP8, supernatants from TCR-stimulated splenic T cells of WT and T-Dusp8-cKO mice were subjected to cytokine array analysis.Among the 111 cytokines examined, several cytokines (including IL-2, IFN-γ, IL-10, and GM-CSF) were increased by TCR stimulation; however, none of these cytokines were markedly affected by Dusp8 cKO in T cells (Supplemental Figure 3A).ELISA data also confirmed that IL-2, IFN-γ, IL-4, and IL-17A levels from TCR-stimulated splenic T cells of T-Dusp8-cKO mice were not changed compared with those of WT T cells (Supplemental Figure 3B).Besides the 111 aforementioned cytokines, the protein levels of IL-9 from TCR-stimulated splenic T cells were undetectable, suggesting that TCR signaling is not sufficient to induce IL-9 production from splenic T cells.Moreover, the data suggest that DUSP8 in CD4 + T cells does not regulate the production of the T-cell cytokine IL-2, the Th1-cytokine IFN-γ, the Th2-cytokine IL-4, or the Th17-cytokine IL-17A.To further study whether DUSP8 regulates T cell-mediated immune responses, T-Dusp8-cKO mice were immunized with keyhole limpet hemocyanin (KLH), a T cell-dependent antigen, using alum as an adjuvant.Serum levels of individual KLH-specific antibodies in the mice after primary and secondary immunization were not markedly changed in T-Dusp8-cKO mice (Supplemental Figure 3C).Only the production of KLH-specific IgG1 antibody (Th2 marker) was slightly reduced (P = 0.216) by Dusp8 cKO (Supplemental Figure 3C); Remarkably, the reduction of Il-9 mRNA levels in peripheral T cells of T-Dusp8-cKO mice was efficiently reversed by Pur-α heterozygous KO (Figure 3H).Collectively, these results suggest that DUSP8 induces IL-9 production in T cells by blocking the binding of Pur-α to the Il-9 promoter.
DUSP8 dephosphorylates Pur-α at Ser-127 and induces its nuclear export.We next studied the mechanism by which DUSP8 regulates Pur-α suppressor to induce IL-9 transcription.Pur-α can translocate from the nucleus to the cytoplasm in neurons (24); therefore, we studied whether DUSP8 suppresses Pur-α binding to the Il-9 promoter by regulating the subcellular localization of Pur-α.Confocal imaging showed that TGF-β treatment indeed induced Pur-α nuclear export in WT T cells; in contrast, Pur-α localized to the nucleus in T-Dusp8-cKO T cells (Figure 4A).Notably, the nuclear localizations of the Th9-related transcription factors PU.1, BATF, and IRF-4 in the in vitro differentiated Th9 cells were not significantly reduced by DUSP8 cKO (Supplemental Figure 8, A and B).In addition, the mRNA levels of the Th9 suppressor Foxp3 were not induced in the in vitro differentiated Th9 cells of T-Dusp8-cKO mice (Supplemental Figure 8C).These results suggest that DUSP8 may regulate Th9 differentiation mainly via inhibiting Pur-α.Our confocal imaging data support that DUSP8 interacts with Pur-α and induces Pur-α nuclear export.The interaction between DUSP8 and Pur-α was confirmed by co-IP analysis (Figure 4B) and fluorescence resonance energy transfer (FRET) analysis, which showed a direct interaction (< 10 nm) (Figure 4C).In situ proximity ligation assay (PLA) signals also showed a direct interaction (< 40 nm) between DUSP8 and Pur-α; these PLA signals were further enhanced by TGF-β stimulation in murine primary T cells (Supplemental Figure 9).Because DUSP8 phosphatase activity was required for Il-9 transcriptional activation (Figure 3A), we studied whether DUSP8 directly dephosphorylates and subsequently induces Pur-α nuclear export.In vitro phosphatase assays using purified proteins showed that DUSP8 dephosphorylated Pur-α at a serine residue(s) but not threonine residue(s) (Figure 4D).To identify the DUSP8-targeted dephosphorylation residue on Pur-α, Pur-α proteins and in vitro DUSP8-dephosphorylated Pur-α proteins were isolated and subjected to mass spectrometry analyses.Ser127 on the Pur-α protein was identified as a DUSP8-dephosphorylated site (Figure 4E).Similar to DUSP8-dephosphorylated Pur-α protein, phospho-deficient Pur-α (S127A) mutant failed to inhibit Il-9 promoter activity (Figure 4F) and localized to the cytoplasm (Figure 4G).Moreover, TGF-β-induced IL-9 mRNA levels in human primary Th9 cells or human Jurkat T cells were inhibited by Pur-α overexpression but not by phospho-deficient Pur-α (S127A) mutation (Figure 4H and Supplemental Figure 10).Collectively, DUSP8 blocks Pur-α function by dephosphorylating Pur-α and promoting Pur-α nuclear export, thus derepressing IL-9 transcription in T cells.
DUSP8 plays a critical role in T cell-mediated allergic responses.Th9 cells contribute to allergic responses (9); therefore, we performed allergic asthma mouse models using T-Dusp8-cKO mice.Ovalbumin-stimulated (OVA-stimulated) inflammation in the lung tissues was induced in WT mice, whereas the lung inflammation was attenuated by T cell-specific DUSP8 cKO (Figure 5A).To study whether DUSP8 mediates allergic responses through normal T cell development in the thymus (Supplemental Figure 5, C and D).Splenic T cells of 3-week-old Pur-α homozygous-KO mice showed the anticipated deletion of Pur-α (Figure 3D) and a drastic induction of Il-9 mRNA levels without any stimulation (Figure 3E).Notably, splenic T cells of Pur-α heterozygous-KO mice also showed an efficient reduction of Pur-α protein levels (Figure 3D) and enhancement of Il-9 mRNA levels (Figure 3E).These results support that Pur-α acts as a repressor for Il-9 transcription.Collectively, the above results suggest that Pur-α constitutively binds to the Il-9 promoter and inhibits Il-9 promoter activity in the absence of DUSP8.
To characterize the binding regions of Pur-α within the IL-9 gene locus, we performed ChIP-Seq using Pur-α immunocomplexes isolated from the lysates of TGF-β-stimulated T cells.The data showed the enhanced bindings of Pur-α to multiple genes in T cells of T-Dusp8-cKO mice, suggesting that Pur-α may be involved in multiple T cell signaling pathways (Supplemental Figure 6).Moreover, ChIP data showed enhanced Pur-α bindings to 2 distinct regions (-555 to -360 and -1,186 to -856) of the Il-9 promoter in T-Dusp8-cKO mice (Figure 3F and Supplemental Figure 7A).After close examination of the Il-9 promoter sequence for Pur-α-binding repeats, (GGN)n (19,21,22) or (CAG)n ( 23), we found 3 putative Pur-α-binding repeats coinciding with the 2 regions that showed the most enhanced Pur-α bindings to the Il-9 promoter in T-Dusp8-cKO T cells detected by ChIP assays (Figure 3F and Supplemental Figure 7B).Furthermore, the TGF-β-stimulated Il-9 reporter activity was suppressed by Pur-α overexpression, whereas the Pur-α-mediated suppression was reversed by individual mutations of the 3 putative Pur-α-binding repeats (-451 to -426, -1,043 to -1,031, -1,066 to -1,059) (Figure 3G).To verify that DUSP8 induces Il-9 transcription by blocking the suppressive function of Pur-α, T-Dusp8-cKO mice were crossed with Pur-α heterozygous-KO mice.Due to the premature death of Pur-α homozygous-KO mice, we thus characterized T cells isolated from T-Dusp8 cKO/Pur-α heterozygous-KO mice.Pur-α, we also performed OVA model using T-Dusp8 cKO/Pur-α heterozygous-KO mice, as Pur-α homozygous mice showed premature death.The attenuated lung inflammation in OVA-challenged T-Dusp8-cKO mice were indeed restored by Pur-α heterozygous KO (Figure 5A).Consistently, BALF IL-9 levels were significantly decreased in OVA-challenged T-Dusp8-cKO mice compared with those of WT mice (Figure 5B), while the reduction of BALF IL-9 levels in OVA-challenged T-Dusp8-cKO mice was reversed by Pur-α heterozygous KO (Figure 5B).Similarly, serum IgE levels were significantly decreased in OVA-challenged T-Dusp8-cKO mice, whereas IgE levels were restored in T-Dusp8 cKO/Pur-α heterozygous-KO mice (Figure 5C).In contrast, the levels of Th2-cytokines IL-4, IL-5, IL-13, and IL-33 were only slightly decreased in OVA-challenged T-Dusp8-cKO mice (Figure 5B); the slight reduction of Th2 cytokines in T-Dusp8-cKO mice may be due to the secondary effect of Th9 reduction.In contrast, the BALF levels of the Th1 cytokine IFN-γ were unchanged in OVA-challenged T-Dusp8-cKO mice (Figure 5B).For the lunginfiltrating immune cells, the IL-9-producing T cells (Figure 5D and Supplemental Figure 11A), eosinophils (Supplemental Figure 11, B and D), and mast cells (Supplemental Figure 11, C and D) were decreased in the lung tissues of OVA-challenged T-Dusp8-cKO mice.Notably, the IL-9-producing T cells were mainly CD4 + T cells but not CD8 + T cells (Supplemental Figure 11A).In contrast, the reduction of Th9 cells, eosinophils, and mast cells in the lung tissues of T-Dusp8-cKO mice was restored by Pur-α heterozygous KO (Figure 5D and Supplemental Figure 11, A-D).Moreover, Pur-α localized to the cytoplasm of the infiltrating CD3 + T cells within the lung tissues of WT mice, whereas Pur-α mainly localized to the nucleus of the lung-infiltrating T cells of OVAchallenged T-Dusp8-cKO mice (Figure 5E).Interestingly, the signals of nuclear Pur-α were greatly decreased by Pur-α heterozygous KO (Figure 5E).Furthermore, in vitro OVA-restimulated Il-9 induction in lymph node T cells was blocked by DUSP8 cKO; in contrast, the Il-9 reduction in DUSP8 conditional-KO T cells was restored by Pur-α heterozygous KO (Figure 5F).Unlike Il-9 induction, OVA-restimulated Il-4 induction was modestly decreased by DUSP8 cKO, and Il-4 levels were not restored by Pur-α heterozygous KO (Figure 5F).These results suggest that DUSP8 intrinsically induces IL-9 production, but not IL-4 production, through Pur-α in OVA-immunized T cells.Nevertheless, we cannot rule the possibility that the DUSP8-Pur-α axis may also regulate other allergy-associated cytokines directly or indirectly.Collectively, DUSP8 blocks the suppressive function of Pur-α in T cells, contributing to T cell-mediated allergic responses.
DUSP8 production and DUSP8-Pur-α interaction are induced in the cytoplasm of Th9 cells from people with asthma and patients with atopic dermatitis.To study whether our findings are translatable to human allergic diseases, we enrolled people with allergic asthma and characterized their peripheral blood T cells (Supplemental Tables 1 and 2).To understand whether protein levels of DUSP8 or other DUSP family members are increased in asthma patients, protein lysates of freshly isolated peripheral blood T cells were digested by trypsin and subjected to mass spectrometry-based proteomics analysis.DUSP4, DUSP6, DUSP7, DUSP8, DUSP10, DUSP16, DUSP19, and DUSP21 were detectable in T cells from either individuals in the control group or patients with asthma (Table 1).Remarkably, DUSP8 was detected in 5 of 6 patients with asthma, whereas DUSP8 was detected in only 1 of 6 people in the control group (Table 1).Furthermore, the protein scores of DUSP8 were significantly increased in people with asthma compared with those of people in the control group (Table 1).Nevertheless, we could not rule out the possibility that additional DUSPs may be detected by proteomics analysis using peptidases other than the proteolytic enzyme trypsin.Consistent with the above mentioned results of animal studies, the flow cytometry analysis showed that the frequencies of DUSP8-positive T cells were increased in people with asthma compared with those of healthy controls (Figure 6A).The DUSP8 induction was concomitant with IL-9 production in patient T cells (Figure 6B).The induction of DUSP8 and IL-9 in the same T cells of people with asthma were also detected by immunofluorescence staining (Figure 6C and Supplemental Figure 12).As expected, DUSP8 proteins were colocalized with Pur-α in the cytoplasm of T cells from people with asthma (Figure 6C and Supplemental Figure 12).Moreover, in situ PLA showed that the interaction between DUSP8 and Pur-α was induced in the cytoplasm of the T cells from people with asthma of 2 independent cohorts (Figure 6D); the frequencies of the PLA-positive T cells were drastically increased in people with asthma compared with those of healthy controls (Figure 6E).To study whether the DUSP8-Pur-α-IL-9 axis contributes to other human allergic diseases, we further enrolled people with atopic dermatitis (AD).Interestingly, the induction of DUSP8 in the peripheral blood T cells of people with AD were also detected by immunofluorescence staining (Supplemental Figure 12).The colocalization of DUSP8 and Pur-α was detected predominantly in the cytoplasm of peripheral T cells from people with AD by immunofluorescence staining (Supplemental Figure 12).Furthermore, the DUSP8 and Pur-α interactions in the cytoplasm of the dephosphorylated Pur-α and subsequent induction of IL-9 transcription.The complex of Pur-α and the Purα-binding protein (PurBP) translocates from the nucleus to the cytoplasm (24).Pur-α also regulates the subcellular localization of Rac1 and RhoA in developing neurons (25).To date, the mechanism of Pur-α nuclear export is unclear.In this report, our findings indicate that Ser127 phosphorylation controls Pur-α nuclear translocation, which is inhibited by DUSP8.Il-9 mRNA levels in T cells were decreased in T-Dusp8-cKO mice, whereas the reduction of Il-9 mRNA levels in T-Dusp8-cKO T cells was reversed by Pur-α heterozygous KO.Consistently, we examined detected by PLA assays were also significantly induced in T cells of people with AD (Figure 6, D and F).These results suggest that DUSP8 interacts with the transcription repressor Pur-α and induces Pur-α nuclear export, resulting in IL-9 induction in the T cells of people with asthma or AD (Figure 7).

Discussion
A key finding of this study was the identification of what we believe to be 2 novel IL-9 upstream regulators, DUSP8 and Pur-α.DUSP8 directly dephosphorylates the transcriptional repressor Pur-α at the Ser127 residue, resulting in nuclear export An exciting finding in this report is that DUSP8 plays a critical role in Th9-mediated allergic diseases.Our data showed that the induction of asthma phenotypes in the mouse model was attenuated by T-cell-specific DUSP8 cKO.Consistently, among 25 phosphatases of the DUSP phosphatase family, only DUSP8 proteins in T cells of people with asthma were induced, detected by mass spectrometry-based proteomics.Furthermore, DUSP8 induction in T cells also occurred in T cells of people with atopic dermatitis.The concomitant induction of DUSP8 and IL-9 in peripheral blood T cells of people with asthma and AD supports that DUSP8 induces IL-9 production.Moreover, the nuclear export of Pur-α and the cytoplasmic interaction between DUSP8 and Pur-α also occurred in the peripheral blood T cells of people with asthma and atopic dermatitis.These results suggest that DUSP8 interacts with Pur-α and induces Pur-α nuclear export in T cells, leading to the inhibition of Pur-α suppressive function and the induction of IL-9 transcription (Figure 7).Interestingly, the DUSP8 SNP table in GeneCards (Human Gene Database) website shows that a SNP (rs61867538) within the DUSP8 gene is associated with IL-9 levels (www.genecards.org/cgi-bin/carddisp.pl?gene=DUSP8).Moreover, 2 other SNPs (rs17885785 and rs1004446), mapped to the putative enhancers of the DUSP8 gene by GeneHancer database of the GeneCards website, are associated with several autoimmune diseases, including systemic lupus erythematosus, chronic childhood arthritis, psoriasis, ulcerative colitis, and type I diabetes mellitus.Our findings and the database information support that DUSP8 is a key activator of IL-9 production and may be involved in human allergic the GEO Data set (GDS5343) (26) and found that Pur-α mRNA levels were inversely correlated with Il-9 mRNA levels in differentiated murine Th9 cells under additional TCR stimulation, supporting the inhibitory role of Pur-α in controlling Il-9 transcription.To our knowledge, this is the first report that Pur-α is a transcriptional repressor of IL-9 transcription.
Pur-α is also a transcriptional repressor for other genes such as CD43 (27); in contrast, Pur-α is a transcriptional activator for c-Myc and PCK2 (28).The dual roles of Pur-α in transcriptional activation or suppression may be due to its interaction with different proteins, its different posttranslational modifications, or different contexts of the Pur-α-binding sites within different promoters.Pur-α ChIP-Seq data derived from T-Dusp8-cKO T cells showed that, besides the Il-9 promoter, Pur-α may also bind to other regions of the Il-9 locus, leading to the inhibition of Il-9 transcription.In addition, Pur-α also bound to multiple genes belonging to the pathways of mitophagy, cholesterol metabolism, TNF signaling, ubiquitin-mediated proteolysis, and protein processing (Supplemental Figure 6).Similarly, scRNA-Seq data of T-Dusp8-cKO T cells showed that DUSP8 is involved in the pathways of autophagy reactome, cellular metabolic process, cellular responses to stress, and protein folding (Figure 1D).Notably, ChIP-Seq data using T-Dusp8-cKO T cells showed that Pur-α targeted signaling molecules of mTOR signaling and HIF-1α signaling (Supplemental Figure 6) which also regulate Th9 generation (29) and T-cell mediated immune responses (30).Collectively, DUSP8 induces IL-9 production and may also regulate other genes/pathways by inhibiting Pur-α function.

Table 1. DUSP protein profiles in peripheral T cells of people with asthma and people in the healthy-control group from Cohort 1
diseases.In addition, 1 SNP (rs11554776), mapped to the putative enhancers of the Pur-α gene by the GeneHancer database, is also associated with psoriasis.It would be valuable to explore whether DUSP8 and Pur-α also play an important role in the pathogenesis of autoimmune diseases.Taken together, our findings suggest that DUSP8 is a T cell biomarker and potential therapeutic target for IL-9-mediated allergic diseases.

Methods
Study participants.This study was conducted in accordance with the Helsinki Declaration.A total of 58 individuals, including 23 people who were healthy, 27 people with asthma, and 8 people with AD were enrolled in this study.For Cohort 1, 18 people in the control group, 16 people with asthma, and 6 people with AD were referred to National Taiwan University Hsin-Chu Hospital in Taiwan (Supplemental Table 1).For Cohort 2, 5 people in the control group, 11 people with asthma, and 2 people with AD were referred to the Division of Immunology and Rheumatology at Taipei Veterans General Hospital or Taichung Veterans General Hospital in Taiwan (Supplemental Table 2).
Mice.All animal experiments were performed in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited (AAALAC-accredited) animal housing facilities according to the protocols and guidelines approved by the IACUC of National Health Research Institutes (NHRI).A C57BL/6J mouse embryonic stem cell clone (ID 25378, clone EPD0754_2_D07) with Dusp8 KO-first allele from the Knockout Mouse Project (KOMP) was injected into C57BL/6J blastocysts to generate chimeric mice by NHRI Transgenic Mouse Core.The KO-first allele is initially a nonexpressing state, but can be converted to a conditional floxed allele via Flp recombination using Actin-Flp transgenic mice (JAX T cell purification and in vitro T cell differentiation using murine primary T cells.Murine T cell purification and in vitro Th1/Th2/ Th17/Treg differentiation assays were performed using the methods described previously (31)(32)(33).For Th9 differentiation, 5 × 10 5 splenic CD4 + T cells were cultured for 5 days in 1 mL Iscove's Modified Dulbecco's Media (IMDM) in 48-well plates coated with anti-CD3 (2.5 μg) and anti-CD28 (2.5 μg) antibodies.The IMDM media contained TGF-β (10 ng/mL), mIL-4 (20 ng/mL), mIL-2 (20 ng/mL), and anti-IFN-γ antibodies (10 μg/mL).Before harvesting cells, murine T cells were stimulated for 4 hours with 0.1% PMA and 0.1% ionomycin in the presence of 0.1% GolgiStop reagent (BD Biosciences).
Flow cytometry using human PBLs.Human PBLs were freshly isolated from 10 mL whole blood by centrifugation, followed by washing twice with 50 mL ACK (ammonium-chloridepotassium) buffer. 1 × 10 6 PBLs were first incubated in MACS buffer containing anti-hCD3-PE-Cy7 (1:100, BD Biosciences) and GolgiStop (1:1,000, BD Biosciences) for 3 hours at room temperature without any other stimulation (32).The PBLs were incubated in 200 μL Cytofix/Cytoperm buffer (BD Biosciences) at 4 °C overnight.The permeabilized cells were washed with Perm/Wash buffer (BD Biosciences) and then incubated in the Perm/Wash buffer containing anti-DUSP8 antibody (1:500) and anti-hIL-9-PerCP-Cy5.5 (1:50) at 4 °C for 2 hours, followed by staining with anti-rabbit IgG-FITC (1:400) for 1 hour.After washing with MACS buffer, the cells were detected by FACSCanto II flow cytometer (BD Biosciences).Notably, when we processed clinical samples using a previously published method (34), the frequency of IL-9-producing T cells in an asthma patient shown in Figure 6B would be decreased from 10.2% to 1.17%.It is likely that the incubation time for GolgiStop and permeabilization is critical.
scRNA-Seq.Murine T cells were isolated by negative selection from the spleen and lymph nodes of T-Dusp8-cKO and WT mice.CD4 + T cells and CD8 + T cells were labelled by the AbSeq anti-CD4 and anti-CD8 antibody-oligonucleotide conjugates (BD Biosciences), respectively.scRNA-Seq was performed using BD Rhapsody Single-Cell Analysis System (BD Biosciences).The scRNA-Seq data were analyzed as described previously (35)(36)(37); scRNA-Seq data are available in a public, open access, NIH repository (SRA no.SRR25734299).
Luciferase reporter assays of the Il-9 promoter.Luciferase reporter assays for the mouse Il-9 promoter activity were performed using the Secrete-Pair Dual Luminescence Assay kit (GeneCopoeia) according to the manufacturer's instructions as described previously (38).5 × 10 6 Jurkat T cells were transfected with the 1,123-bp (-1,097 to For OVA-specific T-cell responses, T cells were isolated from draining lymph nodes of individual mice on day 21 after immunization.5 × 10 6 T cells and 5,000 WT B cells were cocultured in RPMI media containing OVA (0, 20, or 50 μg/mL) for 72 hours.

Figure 1 .
Figure 1.Basal T cell profiles of T-Dusp8-cKO mice are similar to those of WT mice.(A and B) Uniform Manifold Approximation and Projection (UMAP) plot of the scRNA-Seq data showed the distribution and classification of unstimulated T cells from T-Dusp8-cKO and WT mice.(C) The volcano plot of the selected differentially expressed genes in the unstimulated T cells of T-Dusp8-cKO and WT mice.Q value was determined using Fisher's exact test.(D) KEGG pathway enrichment analysis of 28 downregulated genes in unstimulated T cells of T-Dusp8-cKO mice compared with WT mice.The different classification pathways are listed on the left of the plot.Varied numbers of genes enriched in individual pathways are presented by different diameter sizes and numbers for individual dots.T-Dusp8 cKO, T cell-specific Dusp8 cKO (Dusp8 fl/fl ;Cd4-Cre); WT (Dusp8 fl/fl ).

Figure 6 .
Figure 6.DUSP8 production and DUSP8-Pur-α interaction are induced in Th9 cells of people with asthma and atopic dermatitis.(A) Flow cytometry analyses of DUSP8-postivie T cells in the PBLs of 1 representative person from the healthy-control group (HC) and person with asthma.Data shown are mean ± SEM, n = 6 (right panel).There were no antibodies in Pacific Blue channel (left panel).FMO, fluorescence minus 1 (isotype control).(B) Flow cytometry analyses of DUSP8-postive and IL-9-producing T cells in the PBLs of a representative person from the healthy-control (HC) group and person with asthma.Data shown are mean ± SEM, n = 4. Isotype denotes the control group with isotype controls for anti-IL-9 and anti-DUSP8 antibodies.(C) Confocal microscopy of DUSP8, Pur-α, DAPI, and IL-9 in the peripheral blood T cells of 1 representative person from the HC group and person with asthma.Mean ± SEM of DUSP8 levels (green intensity) and Pur-α nuclear localization (red over blue, %) from 3 images are shown.Additional 11 people from the HC group, 23 people with asthma, and 7 people with atopic dermatitis (AD) are shown in Supplemental Figure 12.Scale bars: 25 μm.(D) Confocal microscopy of PLA for the DUSP8-Pur-α interaction in peripheral blood T cells, which were freshly isolated from 27 people with asthma and 18 people from the healthy-control group.One representative person from the HC group and person with asthma from Cohort 1 are shown (upper).One representative person from the HC group and person with AD from Cohort 2 are shown (lower).For PLA, red dots represent direct interaction signals.Scale bars: 50 μm.(E) Quantification plots of PLA signals (panel D) in asthma T cells from Cohort 1 (left), Cohort 2 (center), and the combination of 2 cohorts (right).(F) Quantification plots of PLA signals in AD T cells (panel D) from combination of 2 cohorts.*P < 0.05; **P < 0.01; ****P < 0.0001 (2-tailed Student's t test).

Figure 7 .
Figure 7. Schematic diagram for the induction of IL-9 transcription by DUSP8.TGF-β signaling induces DUSP8 phosphatase activity.DUSP8 induces IL-9 transcription by dephosphorylating the transcriptional repressor Pur-α in the nucleus.Dephosphorylated Pur-α is exported from the nucleus to the cytoplasm in T cells, resulting in induction of IL-9 transcription.As a result, DUSP8 promotes Th9-mediated allergic diseases.