Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 8 (1), 301

JunB Promotes Th17 Cell Identity and Restrains Alternative CD4 + T-cell Programs During Inflammation

Affiliations

JunB Promotes Th17 Cell Identity and Restrains Alternative CD4 + T-cell Programs During Inflammation

Tiffany M Carr et al. Nat Commun.

Abstract

T helper 17 (Th17) cell plasticity contributes to both immunity and autoimmunity; however, the factors that control lineage flexibility are mostly unknown. Here we show the activator protein-1 (AP-1) factor JunB is an essential regulator of Th17 cell identity. JunB activates expression of Th17 lineage-specifying genes and coordinately represses genes controlling Th1 and regulatory T-cell fate. JunB supports Th17 cell identity by regulating key AP-1 complex constituents. In particular, JunB limits the expression of the subset repressor IRF8, and impedes access of JunD to regulatory regions of alternative effector loci. Although dispensable for homeostatic Th17 cell development, JunB is required for induction and maintenance of Th17 effector responses in the inflammatory contexts of both acute infection and chronic autoimmunity in mice. Through regulatory network analysis, we show that JunB is a core regulator of global transcriptional programs that promote Th17 cell identity and restrict alternative CD4+ T-cell potential.AP-1 family transcription factors regulate CD4+ T helper cell differentiation. Here the authors show that the AP-1 member JunB is a nonredundant regulator of transcriptional programs that support Th17 cell identity and restrain alternative Th1 and Treg cell fates in inflammatory contexts of acute fungal infection and chronic autoimmunity.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
JunB promotes Th17 cell identity and represses Th1 and iTreg cell programs. a Flow cytometry of JunB expression in sort-purified Junb +/+CD4cre (WTCD4cre) naive CD4+ T cells (CD4+CD25CD44loCD62L+) or WTCD4cre naive CD4+ T cells cultured under Th0, Th17, iTreg, Th1, or Th2 conditions, for the indicated times. Junb fl/flCD4cre (KOCD4cre) naive CD4+ T cells were cultured and analyzed in concert as a negative control for staining. Error bars represent SEM of two independent experiments. Histograms depict levels of JunB in Th17 (blue), iTreg (red), and Th1 (green) cells, 48 h post polarization. b Flow cytometry of splenocytes from WTCD4cre and KOCD4cre mice, stained for TCRβ, CD4, and CD8α. Total splenocyte counts, frequencies of CD4+TCRβ+ and CD8+TCRβ+ splenocytes, and total numbers of TCRβ+ cells are shown. c Flow cytometry of WTCD4cre and KOCD4cre sort-purified naive CD4+ T cells cultured under Th0, Th17, iTreg, Th1, or Th2 conditions for 72 h followed by restimulation. Data are representative of ten independent experiments which are compiled into bar graphs. Error bars depict SEM. d Flow cytometric analysis of CFSE dilution in restimulated 72 h Th17 cell cultures relative to IL-17A expression (left), and specifically in IL-17A-expressing cells (right). The frequency of cells in each generation is indicated in the line graph. Error bars represent SEM of two independent experiments with at least five mice per group. ****p<0.0001 (unpaired two-tailed Student’s t-test)
Fig. 2
Fig. 2
Continuous expression of JunB is required to maintain the Th17 cell phenotype. a Flow cytometry of JunB expression in Il17a ZsGreen+ fate-mapped (Il17a ZsGreen-FM+) cells and Il17a ZsGreen-FM- cells from Junb +/+ Il17a cre+/ Rosa26 ZSGreen+/ (WTIl17aCre R26 ZS) and Junb fl/fl Il17a cre+/ Rosa26 ZSGreen+/ (KOIl17aCre R26 ZS) Th17 cell cultures, day 4 post-polarization of naive CD4+ T cells. b Flow cytometry of Il17a ZsGreen-FM+ cells sort purified from day 4 WTIl17aCre R26 ZS and KOIl17aCre R26 ZS Th17 cell cultures re-seeded in Th17 cell polarizing conditions for 4 days followed by restimulation. c Flow cytometry of intracellular IL-17A in Il17a ZsGreen-FM+ cells, plated as in b, following stimulation with PMA and ionomycin. Data are representative of three independent experiments
Fig. 3
Fig. 3
JunB actively promotes the Th17 cell program. a Differential mRNA expression in WTCD4cre and KOCD4cre 48 h Th17 cell polarization cultures, displayed as a volcano plot of log2 fold change vs. the –log10(p-value) for each gene. Genes considered significant below FDR < 0.05 are highlighted in orange, and select genes involved in T-cell differentiation are labeled and highlighted in blue. Gray trendlines indicate fold change of 1.5 and p-value = 0.01. p-value capped at 10−30. b JunB occupancy in 48 h polarized Th17 cells. ChIP-Seq tracks at the Il23r, Il21, and Il17a loci are displayed using the Integrative Genomics Viewer (IGV; Broad Institute). Lower track marks the boundaries of significant peaks called by MACS2 with FDR<0.0005. c Luciferase reporter assay of enhancer activity for select JunB-bound regions at the Il17a locus, in WTCD4cre and KOCD4cre naive CD4+ T cells cultured under Th17 cell conditions for 48 h. The JunB-bound CNS of interest at the Il17a locus are indicated in b. Error bars represent SEM of two independent experiments. d Luciferase reporter assay of silencer activity, as in b. Error bars represent SEM of two independent experiments. e QPCR analysis of the expression of Irf8 transcript in WTCD4cre and KOCD4cre 48 h Th17 cell polarization cultures, with Irf8 fl/flCD4cre Th17 cells as a negative control. mRNA expression is presented relative to Actb expression. Data are representative of three independent experiments, with error bars representing SEM. f Flow cytometry of IRF8 expression in WTCD4cre (gray), KOCD4cre (black), and Irf8 fl/+KOCD4cre (teal) Th17 cells 72 h post polarization, with Irf8 fl/fl CD4cre (shaded) Th17 cells as a negative control. Data are representative of three independent experiments. g JunB ChIP-Seq and MACS2 analysis tracks at the Irf8 locus, in 48 h polarized Th17 cells. h Flow cytometry of IL-17A production by WTCD4cre, KOCD4cre, and Irf8 fl/+KOCD4cre 72 h Th17 cell polarization cultures. Relative percentages of IL-17A+ cells compared to WTCD4cre cultures are displayed. *p<0.05; **p<0.01; ****p<0.0001 (unpaired two-tailed Student’s t-test)
Fig. 4
Fig. 4
JunB binding defines novel silencer elements in Th17 cells. a ChIP-Seq tracks of JunB occupancy at the Foxp3, Tbx21, and Ifng loci in WT Th17 cells from 48 h polarization cultures. Lower track marks the boundaries of significant peaks called by MACS2 with FDR < 0.0005. b Gene set enrichment analysis (GSEA; Boad Institute) for KOCD4cre relative to WTCD4cre 48 h Th17 cell polarization cultures demonstrating significant enrichment of gene sets associated with Th1, iTreg and Th17 cell programs in the absence of Junb (p < 1 × 10−4). ES, enrichment score c Luciferase reporter assay of silencer activity for select JunB-bound regions at the Ifng, Foxp3, and Irf8 loci, in WTCD4cre and KOCD4cre naive CD4+ T cells cultured under Th17 polarizing conditions for 48 h. JunB-bound CNS of interest at each locus are indicated in a. Error bars represent SEM of three independent experiments. *p<0.05; **p<0.01 (unpaired two-tailed Student’s t-test)
Fig. 5
Fig. 5
Enhanced JunD occupancy in the absence of JunB. a ChIP-Seq tracks for JunB, JunD, IRF4, BATF, and Fosl2 at the Il17a and Il17f loci in 48 h polarized wild-type Th17 cells. b Clustered heat map of pCRM regions of JunB, JunD, BATF, Fosl2, IRF4, p300, and RORγt occupancy in wild-type 48 h Th17 cell polarization cultures. c QPCR analysis for Batf, Irf4, Fosl2, cJun, and Jund transcripts, relative to Actb expression, in WTCD4cre and KOCD4cre CD4+ naive T cells and naive CD4+ T cells cultured under Th17 cell conditions for 24 and 48 h. d ChIP of WTCD4cre and KOCD4cre 48 h polarized Th17 cells, using α-BATF, α-IRF4, α-Fosl2, or α-JunD followed by QPCR. Primers used encompassed the AP-1 consensus site at select JunB-bound regions or a negative control region, as depicted with boxes in the schematics of the relevant loci. ChIP recovery at negative control loci provides local background for the assay. Error bars represent SEM of two independent experiments. e Differential global occupancy of JunD in KOCD4cre vs. WTCD4cre 48 h Th17 cell polarization cultures displayed as a volcano plot of fold change vs. significance. Differential binding of JunD is considered significant below FDR < 0.05; pCRMs with and without JunB co-occupancy displayed in orange and blue, respectively. The percentage of pCRMs for each subtype with fold change≥2 and p-value<0.01 are indicated for the regions bounded by gray trendlines. p-value capped at 10−25. f Clustered heat map of pCRM regions defined by occupancy of JunB and JunD in WT (C57Bl/6), WTCD4cre, or KOCD4cre 48 h Th17 cell polarization cultures, as indicated. *p<0.05; **p<0.01 (unpaired two-tailed Student’s t-test)
Fig. 6
Fig. 6
Global regulatory trends for targets of JunB, BATF, and Fosl2. a Comparison of differential expression (DE) targets of JunB in combination with BATF or Fosl2 from RNA-Seq of 48 h Th17 cell polarization cultures with individual TF KO. Genes highlighted in red show DE (FDR<0.05) in both datasets, and represent the subset of genes used for subsequent analyses. Numbers in corners indicate the percentage of all significant genes that fall within that quadrant. Genes with opposing AP-1 mediated regulation fall in quadrants 1 and 3, and select genes in these categories are labeled. b Network representation of shared targets of JunB and BATF or Fosl2. Networks were constructed by combining RNA-Seq and ChIP-seq data to determine direct regulatory targets. Green edges indicate activation, red edges indicate repression based on DE measured by RNA-Seq. Nodes are colored based on log2FC of DE in Th17 vs. Th0 RNA-Seq (only significantly DE nodes are included; FDR < 0.05), and are grouped based on combinatorial interactions between JunB and BATF or Fosl2 (i.e., activation:activation, activation:repression, etc.). c Comparison of the net effect of JunB:BATF and JunB:Fosl2 interactions on Th17-specific gene expression. Common targets were classified based on positive or negative regulatory action (ACT or REP) for each TF, then grouped based on 4 possible modes of cooperative regulation. The log2FC for DE genes (FDR<0.05) from Th17 vs. Th0 RNA-Seq is shown as a boxplot for each group. d Comparison of the Th17 vs. Th1 preference of activation and repression targets of Th17 TFs. Target genes were given regulatory classification as in c. For each category, the log2FC for genes with DE in Th17 vs. Th1 (FDR<0.05) is plotted. e Network representation of Th17 TF targets with functional categorization. Networks were constructed as in b, except with node coloring indicating log2FC of DE in Th17 vs. Th1 (FDR<0.05). Genes were classified and grouped based on molecular function. Only targets with JunB-connected edges are shown. Dashed lines indicated direct targets validated using ChIP-QPCR and luciferase assay that fall outside the 5 kb threshold used for global target classification
Fig. 7
Fig. 7
JunB is not required for homeostatic CD4+ Th cell differentiation. a Flow cytometry of intracellular IL-17A vs. IFNγ in CD4+TCRβ+ cells from the spleen, mesenteric lymph node (mLN), and small intestine lamina propria (SILP), of WTCD4cre and KOCD4cre mice. The frequencies of total IL-17A and IFNγ producing CD4+TCRβ+ cells from each tissue are shown. b Flow cytometry of RORγt and IL-17A expression in CD4+TCRβ+ cells from the SILP of WTCD4cre and KOCD4cre mice. c Frequencies of RORγt+ and T-bet+ CD4+TCRβ+ cells from the SILP of WTCD4cre and KOCD4cre mice are shown. d Flow cytometry of Foxp3 expression by CD4+TCRβ+ cells from the thymus, spleen, and mLN of WTCD4cre and KOCD4cre mice. Frequencies of Foxp3+CD4+TCRβ+ cells from each tissue are shown. **p<0.01; ****p<0.0001 (unpaired two-tailed Student’s t-test)
Fig. 8
Fig. 8
JunB promotes inflammatory Th17 cells and limits alternative CD4+ Th cell programs in vivo. a Flow cytometry of JunB expression by Il17a ZsGreen-FM+CD4+TCRβ+ cells producing IL-17A, IFNγ, or IL-17A-IFNγ in the draining lymph node of WTIl17aCre R26 ZS mice day 6 post induction of EAE, with KOCD4cre as a negative staining control (left). Flow cytometry of JunB expression in CD4+TCRβ+ IL-17A or IFNγ producing cells from the inguinal lymph node of WTCD4cre mice, with KOCD4cre cells as a negative staining control (right). Data are representative of two independent experiments. b WTCD4cre and KOCD4cre mice were injected with an emulsification of Complete Freund’s Adjuvant and MOG35-55 peptide and monitored for disease severity over time. Error bars represent SEM of six mice in each cohort, from three independent experiments. c Flow cytometry of IL-17A, IFNγ, and Foxp3 expression in CD4+CD45+ cells isolated from the cerebellum at day 18 post induction of EAE and restimulated ex vivo. Plots are representative of all mice analyzed in a. Total cell numbers of CD4+CD45+ cells in the cerebellum and spinal column of WTCD4cre and KOCD4cre mice, as well as naive control, when WTCD4cre mice were at max score are shown. d Flow cytometry of cytokine production in WTCD4cre and KOCD4cre CD4+TCRβ+ cells from the draining lymph node of mice on day 6 post induction of EAE and restimulated ex vivo. Total cell numbers of CD4+TCRβ+ cells producing IL-17A, IFNγ, or GM-CSF are shown. ns, not significant. e Relative frequency of Foxp3+CD4+TCRβ+ cells in the draining lymph node of WTCD4cre and KOCD4cre mice at day 6 post-induction of EAE relative to naive mice of the same genotype. f C. albicans. cutaneous infection model in WTCD4cre and KOCD4cre mice. Flow cytometry of cytokine production by ex vivo restimulated CD4+CD45+ cells harvested from the skin 5 days post infection, as well as naive control. Data are compiled from three independent experiments. g Relative frequencies of Foxp3+CD4+CD45+ by ex vivo restimulated WTCD4cre and KO CD4cre cells harvested from the skin 5 days post infection, compared to their respective naive controls. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (unpaired two tailed Student’s t-test)

Similar articles

See all similar articles

Cited by 17 PubMed Central articles

See all "Cited by" articles

References

    1. Nakayamada S, Takahashi H, Kanno Y, O’Shea JJ. Helper T cell diversity and plasticity. Curr. Opin. Immunol. 2012;24:297–302. doi: 10.1016/j.coi.2012.01.014. - DOI - PMC - PubMed
    1. Lee YK, et al. Late developmental plasticity in the T helper 17 lineage. Immunity. 2009;30:92–107. doi: 10.1016/j.immuni.2008.11.005. - DOI - PMC - PubMed
    1. Mukasa R, et al. Epigenetic instability of cytokine and transcription factor gene loci underlies plasticity of the T helper 17 cell lineage. Immunity. 2010;32:616–627. doi: 10.1016/j.immuni.2010.04.016. - DOI - PMC - PubMed
    1. Guglani L, Khader SA. Th17 cytokines in mucosal immunity and inflammation. Curr Opin HIV AIDS. 2010;5:120–127. doi: 10.1097/COH.0b013e328335c2f6. - DOI - PMC - PubMed
    1. Zielinski CE, et al. Pathogen-induced human TH17 cells produce IFN-gamma or IL-10 and are regulated by IL-1beta. Nature. 2012;484:514–518. doi: 10.1038/nature10957. - DOI - PubMed

Publication types

MeSH terms

Feedback