Rheumatoid arthritis-associated RBPJ polymorphism alters memory CD4+ T cells

Abstract

Notch signaling has recently emerged as an important regulator of immune responses in autoimmune diseases. The recombination signal-binding protein for immunoglobulin kappa J region (RBPJ) is a transcriptional repressor, but converts into a transcriptional activator upon activation of the canonical Notch pathway. Genome-wide association studies of rheumatoid arthritis (RA) identified a susceptibility locus, rs874040(CC), which implicated the RBPJ gene. Here, chromatin state mapping generated using the chromHMM algorithm reveals strong enhancer regions containing DNase I hypersensitive sites overlapping the rs874040 linkage disequilibrium block in human memory, but not in naïve CD4(+) T cells. The rs874040 overlapping this chromatin state was associated with increased RBPJ expression in stimulated memory CD4(+) T cells from healthy subjects homozygous for the risk allele (CC) compared with memory CD4(+) T cells bearing the protective allele (GG). Transcriptomic analysis of rs874040(CC) memory T cells showed a repression of canonical Notch target genes IL (interleukin)-9, IL-17 and interferon (IFN)γ in the basal state. Interestingly, activation of the Notch pathway using soluble Notch ligand, Jagged2-Fc, induced IL-9 and IL-17A while delta-like 4Fc, another Notch ligand, induced higher IFNγ expression in the rs874040(CC) memory CD4(+) T cells compared with their rs874040(GG) counterparts. In RA, RBPJ expression is elevated in memory T cells from RA patients compared with control subjects, and this was associated with induced inflammatory cytokines IL-9, IL-17A and IFNγ in response to Notch ligation in vitro. These findings demonstrate that the rs874040(CC) allele skews memory T cells toward a pro-inflammatory phenotype involving Notch signaling, thus increasing the susceptibility to develop RA.

Figure 1.

SNAP plots of chromatin state around the rs874040 reveal enhancers and active transcription histone marks in memory CD4⁺ T Cells. (A) Tissue-specific chromatin state mapping were generated using the chromHMM algorithm for naïve CD4⁺CD25⁻CD45RA⁺ and memory CD4⁺CD25⁻CD45RO⁺ T cells as shown in XX and YY. ENCODE ChIP-seq data for various chromatin marks including DNase I hypersensitive sites (enhancers; red), H3K9me3 (heterochromatin; purple), H3K27me3 (polycomb; gray), H3K9ac, H3K4me3 and H3K36me3 (transcription; green) and quiescent regions (other; white) are color-coded. TSS of the three neighboring genes RBPJ, C4orf52 and SEL1L3 are shown (blue). The figure shows all the discovered SNPs from dbGAP137, their P-value of association to RA, the recombination rate as calculated from Broad SNAP online server and genomic information for hg19 version at the bottom of the figure. The LD structure is represented in r-square value calculated from the Broad SNAP server and is assigned in red color. (B) A SNAP plot showing a 100 kb window around the reference SNP rs874040 in both naïve and memory T cells.

Figure 2.

The risk allele rs874040^C is associated with increased RBPJ expression in stimulated memory CD4⁺CD45RO⁺ T cells from healthy genotyped subjects. (A) RBPJ gene expression in naïve CD4⁺ T cells from 213 healthy European-American subjects, (ρ = 0.016, P = 0.8). The expression data have been adjusted for confounding factors in the model (batch, age and gender). (B) Naïve CD4⁺ T cells were isolated from peripheral blood of healthy subjects bearing risk (CC), protective (GG) or heterozygous for the reference rs874040 allele, cells were stimulated with anti-CD3/CD28 for 3 h and RBPJ expression was measured by Taqman PCR. (C and D) Memory CD4⁺CD45RO⁺ T cells were isolated from peripheral blood of genotyped healthy subjects, cells were stimulated with anti-CD3/CD28 for 3 h and total RBPJ expression measured at the gene level by (C) Taqman PCR and at the protein level by (D) ELISA. Groups were compared by the Wilcoxon matched pairs test and adjusted for batch, age and gender. Each dot represents an individual (n = 12–15/group). (E and F) RBPJ gene silencing in CD4⁺ T cells up-regulates inflammatory cytokine expression. Naïve CD4⁺ cells from healthy donors were transfected with RBPJ siRNA or mock scrambled siRNA by electroporation and cells were incubated in the transfection medium for 4 h. Cells were then harvested, washed and incubated in serum-free medium (X-vivo) with anti-CD3/CD28 for 24 h. (E) Cytokine expression was analyzed by Taqman qPCR normalized to β2-microglobulin. (F) RBPJ mRNA expression in CD4⁺ T cells transfected with RBPJ siRNA and scrambled siRNA. Data were compared using the unpaired Student t-test.

Figure 3.

The risk allele rs874040^C is associated with repressed inflammatory genes in memory CD4⁺CD45RO⁺ T cells of healthy genotyped subjects. CD4⁺CD45RO⁺ memory T cells were isolated from peripheral blood of healthy subjects bearing risk (CC), protective (GG) or heterozygous for the rs874040 allele. Cells were stimulated with anti-CD3/CD28 and the expression of (A) Il9, Il17a and Ifng as well as (B) Irf4, Rorc and Stat1 was measured at the gene level by the Fluidigm BioMark technology. Data are shown at 3 h after stimulation. Groups were compared by one-way ANOVA test. Each dot represents an individual (n = 12–15/group).

Figure 4.

Notch stimulation enhanced inflammatory molecules in the rs874040^CC memory T cells of healthy subjects. Memory CD4⁺CD45RO⁺ T cells were isolated from peripheral blood of healthy subjects bearing risk (CC), protective (GG) or heterozygous for the rs874040 allele and cells were stimulated for 7 days with anti-CD3/CD28 in the presence of immobilized Delta-like 4 (DLL4-Fc), Jagged2-Fc or control IgG fusion proteins. IL-9, IL-17A and IFNγ production in the culture supernatants was measured by Luminex bead-based assays. Statistical analyses of log10-transformed values were performed by Spearman rank correlations. Each dot represents an individual (n = 8/group).

Figure 5.

Elevated RBPJ expression and Notch signaling in memory T cells of RA patients. Memory CD4⁺CD45RO⁺ T cells were isolated from frozen PBMCs of RA patients and control subjects affected with a non-inflammatory musculoskeletal condition. (A) RBPJ mRNA was measured by Taqman PCR 3 h following memory T cells stimulation with anti-CD3/CD28 (n = 20/group). (B) Cells were stimulated for 7 days with anti-CD3/CD28 in the presence of immobilized DLL4-Fc, Jagged2-Fc or control IgG. Culture supernatants were collected and the expression of IL-9, IL-17A and IFNγ was measured by Luminex bead-based assays. Statistical analyses of log10-transformed values were performed by the Student t-test. Each dot represents an individual (n = 8–10/group).

Figure 6.

The summary of the involvement of rs874040 and RBPJ in the regulation of memory CD4⁺ T-cell phenotype. The rs874040 lays over an enhancer region upstream of the TSS of RBPJ, a transcriptional repressor that is involved in the differentiation of T cells. We detected a higher expression of RBPJ in memory, but not in naive T cells exposed to TCR signaling (1). Induction of canonical Notch signaling in memory CD4⁺ T cells carrying the reference rs874040 risk allele (2) using two soluble forms of Notch ligands, Jagged2 (3) or DLL4 (4), differentiated memory T cells into IL-9/IL-17A or IFNγ producers, respectively.