IL-6 Mediated Transcriptional Programming of Naïve CD4+ T Cells in Early Rheumatoid Arthritis Drives Dysregulated Effector Function

Abstract

Objective: We have previously shown that increased circulating interleukin-6 (IL-6) results in enhanced CD4+ T cell signaling via signal transduction and activator of transcription-3 (STAT3) in early rheumatoid arthritis (RA). We tested the hypothesis that transcriptional "imprinting" of T-cells by this mechanism skews downstream effector responses, reinforcing immune dysregulation at a critical, but targetable, disease phase. Methods: We modeled naïve CD4+ T cell exposure to pathophysiological concentrations of IL-6 in vitro, assessing the dynamic transcriptional and functional consequences for downstream effector cells utilizing microarray and flow cytometry. Fresh blood from treatment-naïve early arthritis patients was phenotyped in parallel for comparison. Results: T cell sensitivity to IL-6 was most marked in the naïve subset, and related to gp130 rather than IL-6R expression. Exposure of healthy naïve CD4+ T cells to IL-6 induced the same STAT3 target genes as previously seen to discriminate RA patients from disease controls. After TCR stimulation IL-6 pre-exposed cells exhibited enhanced proliferative capacity, activation, and a propensity toward Th1 differentiation, compared to non-exposed cells. An entirely analogous phenotype was observed in early RA compared to control CD4+ T cells. Conclusions: Sustained IL-6 exposure at a critical point in the natural history of RA "primes" the adaptive immune system to respond aberrantly to TCR stimulation, potentiating disease induction with implications for the optimal timing of targeted therapy.

Keywords: CD4+ T cell; early rheumatoid arthritis; interleukin-6; pathogenesis; transcriptional programming.

Figure 1

(A) pSTAT-3 expression was determined in true naïve (TN; CD62L+ CD45RA+), central memory (CM; CD62L+ CD45RA-), and effector memory (EM; CD62L- CD45RA-) CD4+ T-cells in peripheral blood of a previously described early arthritis patient cohort (3) using flow cytometry. (B) In the same cohort, the relationship between pSTAT-3 and paired circulating interleukin (IL)-6, measured by MSD immunoassay, was assessed by Spearman correlation coefficients (rho); gradients of best-fit lines differ significantly (analyses of covariance p < 0.001). Amongst newly-enrolled early arthritis patients (cohort A; see Table 1), surface IL-6R expression (C) and surface gp130 expression (D) was determined in TN, CM and EM CD4+ T-cells in the peripheral blood using flow cytometry [left panels; n = 26; ^**p < 0.001, ^***p < 0.0001, non-parametric analysis of variance (Friedman's) with Dunn's post hoc pairwise analyses; associated p-value are depicted]. Exemplar FACS plots are also shown (right panels).

Figure 2

Naïve or memory CD4+ T cells were isolated from 3 healthy donors and cultured with 0.5 ng/ml IL-6 and equimolar sIL-6R for 72 h prior to washing and stimulation with 0.2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 for 4 h. RNA was extracted at baseline (t0), after 6 or 72 h exposure to IL-6 (t1, t2) and 4 h post-TCR stimulation (t3) as depicted by the red arrows.

Figure 3

(A) Venn diagrams showing the number of differentially expressed genes between untreated and IL-6 pre-exposed naïve and memory CD4+ T cells isolated from 3 healthy donors at t1–t3 using a moderated paired t-test with fold change of 1.5 and Benjamini-Hochberg adjusted p-value of <0.05. (B) Dynamic profiles of genes significantly dysregulated at indicated time-points amongst naïve CD4+ T-cells. Fold- expression in IL-6 exposed cells is depicted relative to that in non-exposed cells at each time-point (FC > 1.5, corrected p < 0.05 for inclusion). Genes previously observed to be differentially expressed (DE) in early RA (eRA) are indicated (heavy lines), and specific STAT-3 targets labeled (solid lines), FC, fold-change in expression, see text.

Figure 4

(A) Naïve CD4+ T-cells isolated from 6 healthy donors display increased division index after 6 days anti-CD3/CD28 stimulation following prior exposure to increasing concentrations of IL-6. (B) CD4+ T cells isolated from healthy donors display increased Ki67 expression after 3 days anti-CD3/CD28 stimulation following prior exposure to 0.5ng/ml IL-6. Expression of CD25 (C) and CD40L (D) is increased in naïve CD4+ T-cells after 6 days anti-CD3/CD28 stimulation following prior exposure to 0.5 ng/ml compared to naïve CD4+ T-cells with no prior exposure to IL-6. (E,F) Increased level of IFN-γ producing cells from naïve CD4+ T cells differentiated toward Th1 following exposure to 0.5 ng/ml IL-6 and equimolar sIL-6R prior to differentiation. (G,H) Decreased level of IL-17 producing cells from memory CD4+ T cells differentiated toward Th17 following exposure to 0.5 ng/ml IL-6 and equimolar sIL-6R prior to differentiation. N = 6; *p < 0.05, non-parametric analysis of variance (Friedman's) with Dunn's post hoc pairwise analyses (A) and Wilcoxon matched-pairs signed rank test (B–D,F,H); associated p-values are depicted.

Figure 5

(A) PBMC were isolated form peripheral blood of healthy controls (HC) and early drug-naïve RA patients (eRA). Expression of Ki67 in CD4+ T cells was assessed in a previously described cohort (16) by flow cytometry. (B) Expression of CD25 in CD4+ T cells from 16 healthy controls and 20 eRA patients in cohort B was assessed by flow cytometry. (C,D) CD4+ T cells were isolated from 9 healthy controls and 14 eRA patients in cohort C, and stimulated for 6 days with 0.5 μg/ml anti-CD3 and 1 μg/ml anti-CD28; IFN-γ (C) or IL-17 (D) were assessed by flow cytometry. **p < 0.001, ***p < 0.0001; Mann Whitney test.