Blimp-1 Functions as a Molecular Switch to Prevent Inflammatory Activity in Foxp3+RORγt+ Regulatory T Cells

Abstract

Foxp3⁺ regulatory T cells (Treg) are essential modulators of immune responses, but the molecular mechanisms underlying their function are not fully understood. Here we show that the transcription factor Blimp-1 is a crucial regulator of the Foxp3⁺RORγt⁺ Treg subset. The intrinsic expression of Blimp-1 in these cells is required to prevent production of Th17-associated cytokines. Direct binding of Blimp-1 to the Il17 locus in Treg is associated with inhibitory histone modifications but unaltered binding of RORγt. In the absence of Blimp-1, the Il17 locus is activated, with increased occupancy of the co-activator p300 and abundant binding of the transcriptional regulator IRF4, which is required, along with RORγt, for IL-17 expression in the absence of Blimp-1. We also show that despite their sustained expression of Foxp3, Blimp-1^-/- RORγt⁺IL-17-producing Treg lose suppressor function and can promote intestinal inflammation, indicating that repression of Th17-associated cytokines by Blimp-1 is a crucial requirement for RORγt⁺ Treg function.

Keywords: Blimp-1; Foxp3; IL-17; IRF4; Prdm1; RORγt; Treg; intestinal; transcription.

Figure 1.. Blimp-1 Is Preferentially Expressed in Microbiota-Specific Foxp3⁺RORγt⁺ Treg and Is Required to Repress IL-17A Expression

(A) Blimp-1^YFP expression in RORγt⁺Helios⁻ (purple) and RORγt⁻Helios⁺ (black) Foxp3⁺ Treg (left) and frequency of RORγt⁺ in Foxp3⁺Blimp-1^YFP+ and Foxp3⁺Blimp-1 ^YFP− cells in the large intestine’s lamina propria (LI-LP) and mesenteric lymph nodes (MLNs) of Blimp-1^YFP reporter mice (right). (B) Prdm1 (Blimp-1) mRNA expression in Foxp3⁺RORγt⁺ and Foxp3⁺RORγt⁻ cells sorted from the small intestine of RORγt^GFPFoxp3^RFP mice. (C) IL-17A expression in splenic CD4⁺Foxp3⁺RORγt⁺ cells of Prdm1^+/+Foxp3^CREYFP+ and Prdm1^F/FFoxp3 ^YFP−CRE+ mice. (D) IL-17A expression in CD4⁺Foxp3⁺RORγt⁺IRF4⁺ cells (bar color indicates IL-17 expression intensity), and RORγt and IRF4 expression in Blimp-1^+/+ (blue) or Blimp-1^−/− (red) Foxp3⁺ cells. (E) IL-17A (top) and IL-10 (middle) expression in Blimp-1^+/+ (CRE⁻) and Blimp-1^−/− (CRE⁺) TCRβ⁺CD4⁺Foxp3⁺ cells in MLNs and spleen (SP) of Prdm1^F/FFoxp3^YFP−CRE+ female mice. Bar graphs (bottom) show the frequency of IL17A⁺ (left) and IL10⁺ (right) cells. (F) Expression of Prdm1, Il17a, Rorc, and Foxp3 mRNA in CD4⁺Foxp3⁺ cells sorted from Prdm1^F/F and Prdm1^+/+Foxp3^CREYFP+ mice and re-stimulated in vitro. (G) Expression of IL-17A in TCRβ⁺CD4⁺Foxp3⁺ cells from MLNs of antibiotic-treated with vancomycin, neomycin, ampycilin, and methonidazole (VNAM) or non-treated (NT) Prdm1^F/FCD4^cre+ mice. Data are representative of at least two independent experiments. Error bars show average and SEM (n ≥ 3 per group); each symbol represents one mouse. *p < 0.05 and **p < 0.01, unpaired Student’s t test (A, C, D, F, and G) and paired Student’s t test (E).

Figure 2.. Binding of Blimp-1 to I117a and Il17f Genes in Foxp3⁺ Treg Is Associated with Decreased Locus Accessibility

(A) IL-17A expression in Blimp-1^−/− Foxp3^GFP+ Treg and in wild-type Th17 cells transduced with MIG-R1 or Blimp-1-expressing MIG-R1 retrovirus (MIG-R1Blimp1). (B) Top: representation of the Il17 locus (arrows, Il17a and Il17f genes; black ovals, promoter; gray ovals, CNS regions) indicating Blimp-1 consensus binding sites and a non-related (NR) site; bottom: ChIP of Blimp-1 in wild-type CD4⁺Foxp3^GPP+Treg and pTh17 cells (NG, negative control: non-related gene lacking Blimp-1 binding sites). (C) ChIP of H3K4me3 (black bars) and H3K27me3 (gray bars) at different regions of the Il17 locus in Blimp-1^+/+ (Ctrl, control) or Blimp-1^−/− (CKO) CD4⁺Foxp3^GFP+ Treg (empty bars, Ctrl antibody immunoprecipitation [IP]). (D) ChIP of p300 at the Il17a promoter and Il17 CNS7 regions in CD4⁺Foxp3^GPP+ Treg from Ctrl and CKO mice and in wild-type pTh17 cells. (E) Representation of promoter constructs (top) and luciferase activity assay (bottom). Striped bars, control plasmid; black bars, full-length Blimp-1; gray bars, truncated Blimp-1 lacking the zinc finger-containing region. Data shown are from two (A) or three (B–E) independent experiments. Error bars show SEM (n ≥ 3 per group). *p < 0.05 and **p < 0.01, one-way ANOVA.

Figure 3.. Increased Binding of IRF4 at the Il17 Locus and IRF4 and RORγt Requirement for IL-17A Production in Blimp-1 ^−/−Foxp3⁺ Treg

(A) Representation of the Il17 locus showing RORγt and IRF4 consensus binding sites (top) and RORgt ChIP (bottom) in Blimp-1^+/+ and Blimp-1 ^−/−CD4⁺ Foxp3^GFP+ Treg and wild-type pTh17 cells. (B) IL-17A expression in TCRβ⁺CD4⁺Foxp3⁺ Treg (MLNs) from Prdm1^+/+Rorc^+/+Foxp3^CREYFP+ (Ctrl), Prdm1^F/FRorc^+/+Foxp^3CREYFP+ (knockout [KO]), and Prdm1 ^F/FRorc^F/FFoxp3^CREYFP+ (double knockout [DKO]) mice and expression of Foxp3, Prdm1, Rorc, and Il17a mRNA (bottom graphs) in sorted and in vitro-stimulated (phorbol myristate acetate [PMA] and ionomycin for 4 hr) CD4⁺Foxp3^CREYFP+ cells. (C) IRF4 ChIP at the Il17 CNS7 and Il10 Intron I regions in Blimp1^+/+ and Blimp1 ^−/−CD4⁺Foxp3^GFP+ Treg and wild-type pTh17 cells. (D) Irf4, Il17a, and Il10 mRNA expression following Irf4 siRNA knockdown (SC, scrambled control). Data shown are representative of at least two independent experiments. Error bars show SEM (n ≥ 3 per group). *p < 0.05 and **p < 0.01, one-way ANOVA.

Figure 4.. IL-17A-Producing Blimp1 ^−/− Foxp3⁺ Treg Have Impaired Suppressive Function and Can Cause Intestinal Inflammation

(A) Body weight of RAG^−/− mice injected with naive CD45.1⁺CD4⁺T cells alone or in combination with (CD45.2⁺) Blimp-1 ^−/−IL-17A-producing (IL17A^GFP+ Treg) or non-producing (IL-17A^GFP− Treg) Foxp3⁺ Treg from the same donor mice. (B and C) Colon length (B) and H&E-stained histological sections (C) (10x magnification; scale bar, 100 μm) in mice shown in (A) 8 weeks after transfer. (D) Frequency (fluorescence-activated cell sorting [FACS] plots) and absolute numbers (bar graphs) of IL-17A^GFP+ cells from LI-LP of mice shown in (A)-(C). (E) Numbers of CD45.1 ⁺ cells in the LI-LP, MLNs, and SP from mice shown in (A)–(D). (F) IL-17A expression (ELISA) in LI-LP cells of mice shown in (A)ߝ(E). (G) Body weight of RAG^−/− mice adoptively transferred with IL-17⁺ Th17 cells (Th17), Blimp1 ^−/− IL17A^GFP+, or IL17A^GFP−Foxp3⁺ Treg. (H) Histology scores (colon, rectum, and cecum; left), and pictures of colon histological sections (H&E stained; 10x magnification; scale bar, 100 μm) of mice shown in (G). (I) IL-17A expression in TCRβ⁺ T cells from the LI-LP of mice show in (G). Foxp3⁺, cells that maintained Foxp3; Foxp3⁻, cells that had lost Foxp3 expression after transfer. (J) Expression of IL-17A and IL-17F in TCRβ⁺Foxp3⁺ and Foxp3⁻ cells in SP of mice shown in (G)–(I). Data shown are from three different experiments. Error bars show SEM (n = 3–7 mice per group). *p < 0.05 and **p < 0.01, one-way ANOVA (A, B, D, and F–I) and unpaired Student’s t test (E and I).