A Role For BATF3 in Tu9 Differentiation And T-cell-driven L Pathologies MucOsal (Part 2)

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DISCUSSION

T#9 cells are important for the development of inflammatory and allergic diseases. However, the transcriptional program and inflammatory triggers that drive the differentiation of TH9 cells remain incompletely understood. In this study, we identified the pro-inflammatory cytokine TL1A as a potent inducer of murine and human T-9 differentiation and identified BATF3 as an important transcription factor involved in the differentiation of TH9 cells. The transcriptional network that was induced by TL1A included the transcription factors BATF and BATF3 and several BATF-regulated genes (Supplementary Figure 7). In vivo, in adoptive transfer experiments， Tμ9-TL1A cells were highly pro-inflammatory leading to intestinal and lung inflammation. The pro-inflammatory propensity of T-9-TL1A cells was dependent on IL-9 production. Transfers of Batf3-/T and 9-TL1A cells resulted in reduced mucosal inflammation and cytokine expression in vivo.

Although Tμ9 cells have been identified as a distinct T-helper subset, a transcription factor that acts as a master regulator or solely identifies the TH9 phenotype has not been identified. Instead, several transcription factors act in concert to regulate the differentiation of T-9 cells.BATF has been shown to functionally cooperate with IRF4 and to bind to composite elements within the promoter regions of responsive genes.BATF along with IRF4 has been recently proposed as "pioneer factors" in the differentiation of Tp17 cells by contributing to the initial chromatin accessibility and facilitating the access of other transcription factors.36 We observed that TL1A has a direct effect on BATF expression and it synergizes with TGF-β1 and IL-4 to maximally induce BATF expression particularly early in TH9 differentiation. In support of this notion, stimulation with TL1A alone leads to the binding of BATF to the II9 promoter while it has no direct effect on the binding of other transcription factors (IRF4 and BATF3). However, TH9-TL1A conditions synergistically enhance the binding of BATF3 and IRF4, as well as acetylation of histone H3, a permissive chromatin modification that correlates with ll9 promoter activity.14 In line with previously published data, BATF is required for the expression of IL-9 and IL-10 under T,9 conditions.15 TL1A is at least partially able to overcome the requirement for BATF in the induction of IL-9 and IL-13 most likely through enhancement of other transcription factors that might be able to compensate the loss of BATF. One candidate for functional compensation is BATF3 which is transcriptionally induced by TL1A. It has been proposed that BATF and BATF3 are functionally interchangeable for Tu17 and Tp2 development and overexpression of BATF3 in BATF-'- T cells can restore IL-17 production in TH17 cells and IL-4 and IL-10 production in T#2 cells.18323738 However, the role of BATF3 in the development of Tu cells under physiological conditions and potential interaction between BATF and BATF3 during TH9 differentiation has not been defined. We demonstrate here that TL1A facilitates the binding of BATF3 to the ll9 promoter. As a functional consequence, BATF3 contributes to IL-9 production under T-9 conditions, particularly in the context of TL1A stimulation. Surprisingly, BATF3 is dispensable for the early differentiation of Tu9 cells but might be required to stabilize or maintain the TA9 phenotype and IL-9 secretion at later time points. Our findings of the role of BATF3 in TH9 differentiation are in contrast with previous reports of BATF3 being dispensable for Tu1, TA2, and Tu17 differentiation.2'3'In contrast to BATF, BATF3 only contributes to IL-9 secretion and not to other TH9 cytokines such as IL-10 and IL-13, suggesting that BATF3 specifically binds to the ll9 promoter and induces IL-9 secretion. Further studies are required to determine additional BATF3 target genes during TH9 differentiation and its role in other Tu subsets.

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Fig.7 Batf3 deficiency leads to reduced mucosal inflammation driven by TH9-TL1A cells. Naive WTor Batf3-/cells were differentiated into T_9-TL1A cells and injected into Rag1-7mice. a Representative H&E stainings of lungs (top panels) and large intestines (bottom panels).Scale bar:200 mm. b Histology scores for lungs.c Total cell counts(left, middle), frequency of CD4* T cells(right).Data represent means±SD.n=10/group.d Intracellular staining of IL-9, IL-13, and IL-17 from spleen, MLN, and lungs after ex vivo restimulation with PMA+lonomycin.Data represent means±SD.n=5/group. One representative experiment out of three independent experiments is shown.*p<0.05, **p<0.01 as determined by Student's t-test

Although other pro-inflammatory cytokines or mediators such as IL-1β, OX40, and TSLP have been described to enhance TH9 differentiation, TL1A seems to be unique in upregulating BATF, thereby opening chromatin for the recruitment of other transcription factors including BATF3.353940 Our RNA-sequencing data support this conclusion identifying a subset of BATF-dependent genes significantly upregulated by TL1A. Furthermore, our RNA-sequencing data reveal that BATF3 regulates the activity of transcription factors, cell proliferation, and intracellular signal transduction suggesting an important role of BATF3 during T#9 differentiation. In vivo, Batf3-'- TA9-TL1A cells resulted in reduced mucosal inflammation and cytokine expression. We did not observe impaired migration of Batf3-'- TH9-TL1A cells to the intestine, which is in contrast to BATF-'- CD4+ T cells that fail to upregulate intestinal migration markers and do not populate the intestines.4' Our data support the role of BATF3 in the pathophysiological function of TA9-TL1A cells.

IL-9 and TA9 cells have been recently associated with the pathogenesis of IBD.*-10 IL-9+ CD4+ T cells were enriched in patients with UC and high serum levels of IL-9 correlated with severe disease in patients with CD.-10In an experimental model of hapten-induced UC-like disease, IL-9-and PU.1-deficient mice are protected from intestinal inflammation and anti-IL-9 antibodies are protective in a chronic prevention model.°However, we did not observe any effects of TL1A on PU.1 expression under T9 conditions. We have shown that TL1A stimulation results in upregulation of BATF and BATF3 but not of PU.1.Although PU.1 has been described as a master regulator of TH9 differentiation, it is only required for the expression of a subset of TH9 genes and mice suggesting binding of PU.1 is not changed in BATF-complete independency of PU.1 and BATF. 75

We demonstrate that T,9-TL1A cells are highly pathogenic in vivo and induce intestinal and lung inflammation. Intestinal inflammation in TH9-TL1A recipients was mainly confined to the small intestine. Furthermore, we observed an increased number of CCR6* and CD103 cells in TH9-TL1A recipients in vivo. These data consistently demonstrate that the chemokine receptor CCR6 and the integrin CD103 are expressed on TH9 cells and facilitate migration to inflammatory sites and mucosal surfaces.15，42 The increased number of CCR6+ cells in Tμ9-TL1A recipients in vivo is consistent with our observation of mainly small intestinal inflammation since the CCR6/CCL20 axis has been shown to specifically facilitate the migration of T,17 cells to the small intestine and it is plausible that similar mechanisms apply to Tμ9 cells.3 Our findings are also consistent with recent findings of elevated IL-9 serum levels in CD patients, particularly with severe diseases.?

TL1A enhances T,1, TH2, and TA17 differentiation and although we did not see any indication of a global shift from T9 cells into other TH subsets in vitro, it could suggest that TH9 cells differentiated with TL1A might be unstable in vivo and"trans-differentiate" into other subsets. The potential of trans-differentiation of TH9 cells has been controversial and might be dependent on the context, disease model, and immune status of the host. In EAE, trans-differentiation of TH9 to IFN-Y-producing cells occurs, however, the T,9 phenotype seems to be stable in cancer models.339 Surprisingly, in vivo T9-TL1A cells continued to produce IL-9 and downstream cytokines such as IL-17 and IL-13 and did not trans-differentiate. This notion is also supported by our data showing that anti-lL-9 treatment alone is sufficient to block the pathogenic effects of T9-TL1A cells. Anti-Lil-9 antibody treatment alone reduced IL-13 production and attenuated intestinal and lung inflammation back to TH9 levels. The development of allergic lung inflammation requires the corporation of TH2 and TH9 cells. However, our model of chronic, nonallergic lung inflammation seems to be mainly driven by IL-9-producing T.9 cells, although we cannot exclude a contribution of IL-9-producing non-T cells in pathogenesis in our model.

In summary, TL1A is a strong inducer of murine and human T9 differentiation and we elucidated the signaling pathways involved. Given the pro-inflammatory properties of TH9-TL1A cell in vivo, targeting the TL1A-BATF3-IL-9 axis may be a promising therapeutic approach in T19-driven pathologies such as IBD or allergic lung disease.

METHODS

Mice C57BL/6, C57BL/6 Rag1-/, B6.SJL-Ptprc" Pepc/BoyJ(CD45.1),(B6.Cg-Nfkb1*m130l/J), Stat6-/-(B6.129S2(C)-Stat6"miGr"/J), p50-/-B6.Cg-Tg(TcraTcrb)425Cbn/ (OT-I),Batrf/(B6.129s-Batm1.1kmm/), and Bat3-/(B6.129 S(C)-Batf3m1km"/) mice were purchased from the Jackson Laboratory. Dr3-/- n mice have been described elsewhere.4 Mice were maintained under SPF conditions. All animal studies were approved by the Cedars-Sinai Medical Center Animal Care and Use Committee. T-cell isolation and differentiation Naive T cells (CD62LhichcD44'o"CD25~; clones MEL-14, IM7, PC61.5; eBioscience)were isolated from spleens and MLN using the EasySeptM Mouse CD4+ Isolation Kit (Stem Cell Technologies)followed by cell sorting (MoFlo, Beckman Coulter). Cells were cultured in RPMI-1640 medium(10% FBS) with anti-CD3s (145-2C11;BD Biosciences), anti-CD28(37.51; eBioscience)(TH0 conditions), and murine TL1A(100 ng/ml; R&D Systems)under T-9 conditions (human TGF-β1 [3 ng/ml], Biolegend, IL-4 [20ng/ml, PeproTech).After 2 days, IL-2(20 U/ml) was added. To evaluate cell proliferation, sorted cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen), differentiated for 5 days, and CFSE dilution was assessed by flow cytometry. To evaluate antigen-specific T,9 differentiation, naive CD4+ T cells from OT-Il mice were stimulated for 3 days with 1 μg/ml OVA;23-339 peptide in the presence of syngeneic APCs(ratio 1:3). APCs were prepared by depletion of CD90.2T cells from splenocytes(Stem Cell Technologies)followed by mitomycin C treatment. isolation of human CD4+ T cells and differentiation Blood was obtained from healthy volunteers after informed consent in accordance with procedures established by the Cedars-Sinai Institutional Review Board (IRB # 3358, 2673). Naive CD4+ T cells(CD4+CD25-CD45RA+CD45RO7)were isolated from PBMC using the EasySepl Human CD4 T cell Enrichment Kit (Stem Cell Technology) followed by cell sorting. Cells were cultured with anti-CD3（5 ug/ml） and anti-CD28（2 μg/ml） with human TL1A(100ng/ml; Fitzgerald) under TH9 conditions (human TGF-β1 [5 ng/ml], human IL-4 [10 ng/ml]). ELISA Cytokine concentration in culture supernatants were assayed by ELISA for murine or human IL-9, IL-10, and IL-13(eBioscience). Intracellular staining Cells were restimulated with 50 ng/ml PMA(phorbol 12-myristate 13-acetate), 500ng/ml ionomycin, and monensin (eBioscience) for 4 h,stained with anti-CD4 (RM4-5,eBioscience),fixed and permeabilized using the FoxP3 staining buffer set (eBioscience), and stained with antibodies against murine IL-9 (RM9A4, BioLegend),IL-10(JES5-16E3),IL-13(13A),IL-17A (eBio17B7), IL-17F (eBio18F10),IFN-y(XMG1.2),IL-4 (BVD6-24G2),IL-22 (1H8PWSR),Ki67(SolA15),BATF(MBM7C7), IRF4 (3E4),human IL-9(MH9A4, all eBioscience),and BATF3(841792, R&D Systems). Samples were analyzed using a CyAnADP flow cytometer(Dako Cytomation) and FlowJo software (TreeStar Inc.).

Quantitative RT-PCR

Total RNA was isolated using RNeasy kits and reverse transcribed into cDNA with Omniscript RT kit (both Qiagen).qPCR was performed using the Mastercycler" ep realplex² System （Eppendorf). Platinum Quantitative PCR SuperMix-UDG(Invitrogen) and TaqMan probes and primers were used for Actb, I9, I10, 13, and BATF （IDT）（Supplementary Table 7）. RT² SYBR"Green qPCR Mastermix(Qiagen) and primer sets were used for Actb and Batf3 (IDT). mRNA expression of target genes was normalized to the expression of Actb. The relative gene expression was calculated by the method.

the 2-AACt Chromatin immunoprecipitation

Chloe was performed using EZ ChIP chromatin immunoprecipitation kit(Millipore) followed by qPCR analysis. Totally,1×10°cells were stimulated, fixed, sonicated, and immunoprecipitated using 2ug of ChIP-grade antibodies: anti-BATF(sc-100974),anti-BATF3(sc-162246),normal mouse lgG(sc-2025), anti-IRF4(sc-6059), normal goat lgG(sc-2028)(all Santa Cruz Biotechnology), anti-acetyl-histone H3(17-615), and normal rabbit lgG (Milli-pore). Immunoprecipitated DNA was reverse cross-linked, purified using spin columns, and analyzed by qPCR(primer sequences: Supplementary Table 7). To quantify immunoprecipitated DNA, we generated a standard curve from serial dilutions of input DNA. Data are presented as the percentage of input DNA based on normalization against the amount of input DNA.

RNA sequencing (GEO accession numbers: GSE60362 and GSE106926)

Low-input RNA-Seg (TH9 vs.TH9-TL1A): Clontech's SMARTer Ultra"Low Input RNA Kit for Sequencing v3 was employed to generate double-stranded cDNA libraries from 1.5 to 2.5 ng of total RNA from each sample as per the manufacturer's recommendations. Double-stranded cDNA libraries were individually fragmented and ligated with lon Torrent lon Xpress" Barcode Adapters using lon Xpress" Plus Fragment Library Kit with limited modifications including size-selected final libraries with a double-bead cleanup and V volume of ion adapters. RNA-Seq libraries were assessed for concentration and length using Invitrogen's QubitdsDNA HS Assay Kit and Agilent High Sensitivity DNA Kit, respectively. Samples were multiplexed to obtain >10 million reads each for sequencing. The pooled libraries were amplified onto lon Sphere" and sequenced using the lon PIM Sequencing 200 Kit v2,lon Torrent".

mRNA-Seg (WT vs.Baft3-7T,9-TL1A): Illumina TruSeq Stranded mRNA library preparation kit was employed for RNA-Seq. Of total RNA, 1 ug per sample was used for poly-A mRNA selection using streptavidin-coated magnetic beads. cDNA was synthesized from enriched and fragmented RNA using reverse transcriptase (Super-Script ll, Invitrogen) and random primers. The cDNA was converted into double-stranded DNA, and enriched with PCR for library preparation. The PCR-amplified library was purified using Agencourt AMPure XP beads(Beckman Coulter). The concentration of the amplified library was measured with a NanoDrop spectrophotometer and on an Agilent 2100 Bioanalyzer. Samples were multiplexed to obtain >20 million reads/sample and sequenced on a NextSeq 500 platform(Illumina) using 75-bp single-end sequencing.

Data analysis. Raw reads were filtered and trimmed by the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and aligned using Tophat version 2.0.8 with UCSC GRCm38/mm10 mouse reference genome annotation (http://genome.ucsc.edu). Gene read counts were generated using HTSeq (v0.5.4) and then normalized by the trimmed mean of the M-values normalization method with edgeR (v3.0.8) in Bioconductor in R(v2.15), which uses a weighted trimmed mean of the log expression ratios. For all analyses, only quality signals(threshold: ten counts per million in at least two out of four samples)were used. An unsupervised analysis was performed using the top 100 ranked genes by the interquartile range and then hierarchical clustering was generated using  (v2.11) using two-way Pearson correlations to visualize unbiased pervasive gene expression patterns. Supervised analysis using a modified Fisher's exact test in edgeR and a false discovery rate cutoff of 10% using the Benjamini and Hochberg procedure was used to determine differential expressions between TH9 and T.9-TL1A or WT and Batf3-7T,9-TL1A. Pathway enrichment analysis was performed using DAVID6.7 (http://david.abcc.ncifcrf.gov/) with count thresh-old =5 and ease score threshold=0.05.

T-cell transfer model

CD4+CD62LighcD44"cD25- T cells from CD45.1 mice were differentiated into T9 or T,9-TL1A cells for 3 days. Male Rag1-/recipient mice were injected i.p.with 0.5×10°TH9, or TH9-TL1A cells. Mice were weighed for 6-8 weeks. For neutralization experiments, mice were injected i.p. with anti-IL-9 or lgG2b control antibody (both 50 ug/dose; R&D Systems) three times per week for 4 weeks and twice per week for the remaining time starting at the day of T-cell transfer. Tissues were formalin-fixed, paraffin-embedded, and stained with H&E or AB-PAS. Inflammation was scored by a semiquantitative scoring system by a trained pathologist blinded to the experimental conditions.45,46 LPMC were isolated from the large intestine as previously described.7 Lung tissue was digested for 45 min at 37℃C with 10 ml of HBSS containing 15 ug/ml Liberase"（Roche）and 25μg/ml DNase I （Sigma）and filtering through a 40-μm cell strainer followed by red blood cell lysis. Single-cell suspensions were stained with anti-CD4, anti-CD45.1(A20), anti-CCR6 (29-2L17), and anti-CD103(2E7, all eBioscience), and anti-Ki-67(SolA15, BD PharMingen)antibodies for flow cytometry or restimulated with anti-CD3c/anti-CD28 antibodies for 3 days. Cytokine levels in supernatants were measured by ELISA.

Statistics

Statistical significance was calculated using SPSS software (IBM SPSS Statistics 20, IBM Machines Corp., Armonk, NY, USA). Data were analyzed using one-way analysis of variance(ANOVA)followed by a post hoc unpaired two-tailed Student t-test, or Mann-Whitney U test as indicated. Differences were considered significant at p<0.05.

ACKNOWLEDGEMENTS

This work was supported by the NIH (DK056328 to S.R.T.) and the F. Widjaja Foundation (S.R.T. and K.S.M.). Anita Vibsig Neutzsky-Wulff received postdoctoral fellowships from The Carlsberg Foundation (Denmark) and the Lundbeck Foundation (Denmark). Jordan Nunnelee received a Student Research Award from the Crohn's and Colitis Foundation of America. The Cedars-Sinai MIRIAD IBD Biobank is supported by the F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, NIH/NIDDK grants P01 DK046763, U01 DK062413, and The Leona M and Harry B Helmsley Charitable Trust.

AUTHOR CONTRIBUTIONS

M.T,H.H,LT,M.W,B.S,M.W, B.S,M.W,J.N,JS, A.N.-W,R.B,Y.W,J.T,V.F, and KM. performed experiments analyzed data and critically reviewed the manuscript. A.P, J. T, and Y.W. performed RNA-Seq data analysis. M.T, S.T, and K.M. designed the experiments. M.T. and K.M. wrote the manuscript.

ADDITIONAL INFORMATION

The online version of this article (https://doi.org/10.1038/s41385-018-0122-4)contains supplementary material, which is available to authorized users.

Competing interests: The authors declare no competing interests.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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