Enhanced T cell responses to IL-6 in type 1 diabetes are associated with early clinical disease and increased IL-6 receptor expression

Abstract

Interleukin-6 (IL-6) is a key pathogenic cytokine in multiple autoimmune diseases including rheumatoid arthritis and multiple sclerosis, suggesting that dysregulation of the IL-6 pathway may be a common feature of autoimmunity. The role of IL-6 in type 1 diabetes (T1D) is not well understood. We show that signal transducer and activator of transcription 3 (STAT3) and STAT1 responses to IL-6 are significantly enhanced in CD4 and CD8 T cells from individuals with T1D compared to healthy controls. The effect is IL-6-specific because it is not seen with IL-10 or IL-27 stimulation, two cytokines that signal via STAT3. An important determinant of enhanced IL-6 responsiveness in T1D is IL-6 receptor surface expression, which correlated with phospho-STAT3 levels. Further, reduced expression of the IL-6R sheddase ADAM17 in T cells from patients indicated a mechanistic link to enhanced IL-6 responses in T1D. IL-6-induced STAT3 phosphorylation was inversely correlated with time from diagnosis, suggesting that dysregulation of IL-6 signaling may be a marker of early disease. Finally, whole-transcriptome analysis of IL-6-stimulated CD4(+) T cells from patients revealed previously unreported IL-6 targets involved in T cell migration and inflammation, including lymph node homing markers CCR7 and L-selectin. In summary, our study demonstrates enhanced T cell responses to IL-6 in T1D due, in part, to an increase in IL-6R surface expression. Dysregulated IL-6 responsiveness may contribute to diabetes through multiple mechanisms including altered T cell trafficking and indicates that individuals with T1D may benefit from IL-6-targeted therapeutic intervention such as the one that is being currently tested (NCT02293837).

Copyright © 2016, American Association for the Advancement of Science.

Figures

Figure 1. IL-6-induced pSTAT3 and pSTAT1 are increased in T cells from patients with T1D

Thawed and rested PBMCs from healthy controls and subjects with T1D were treated with recombinant cytokine (IL-6, IL-10, or IL-27) followed by staining for CD4, CD8, CD45RA, pSTAT3 (pY705), and pSTAT1 (pY701). (A) CD4 T cell response to IL-6 as determined by frequency of IL-6-induced pSTAT3-positive cells [pSTAT3+ (%)] and fold change in pSTAT3 MFI (FC MFI pSTAT3) compared to unstimulated cells (left and right panels, respectively); n = 24 (Ctrl) and n = 27 (T1D). (B) Dose-response curve showing IL-6-induced pSTAT3 (MFI) in total CD4 T cells from controls and patients; n = 5 (Ctrl) and n = 5 (T1D). (C) CD8 T cell response to IL-6 as determined by frequency of IL-6-induced pSTAT3-positive cells and fold change in pSTAT3 MFI (left and right panels, respectively); n = 24 (Ctrl) and n = 27 (T1D). (D) Linear regression showing positive correlation between IL-6-induced STAT3 activation in CD4 and CD8 T cells; n = 24 (Ctrl) and n = 27 (T1D). (E) pSTAT1 in response to IL-6 in total CD4 and CD8 T cells; n = 22 (Ctrl) and n = 23 (T1D). (F and G) pSTAT3 in total CD4 and CD8 T cells following stimulation with IL-27 (F) [n = 13 (Ctrl) and n = 13 (T1D)] or IL-10 (G) [n = 12 (Ctrl) and n = 14 (T1D)]. Statistical tests: Mann-Whitney U; r = Pearson correlation coefficient.

Figure 2. T cell responses to IL-6 decrease with time from diagnosis

(A) Linear regression showing that IL-6-induced pSTAT3 is independent from age at draw, BMI, age at diagnosis, blood glucose levels, and glycated hemoglobin (HbA1c). (B) Linear regression showing the inverse correlation between time from diagnosis (disease duration) and IL-6/pSTAT3 in CD4 and CD8 T cells from subjects with T1D (left and right panels, respectively); 21≤ n ≤29 (A) and n = 26 (B) ; r = Pearson correlation coefficient.

Figure 3. Surface IL-6R levels are increased in T1D T cells and correlate with IL-6-induced pSTAT3

Unstimulated PBMC from controls and patients obtained from the same blood draw as shown in Fig. 1 were stained for CD4, CD8, CD45RA, gp130 and IL-6R. IL-6R surface expression (mbIL-6R) was calculated as the antigen-specific MFI minus the MFI of a matched isotype control (see also fig. S5). (A) mbIL-6R expression in CD4 and CD8 T cells (left and right panels, respectively); n = 24 (Ctrl) and n = 27 (T1D). (B) gp130 expression (MFI) in CD4 T cells; n = 24 (Ctrl) and n = 27 (T1D). (C) Linear regression showing the positive relationship between mbIL-6R expression and IL-6-induced pSTAT3 in total CD4 T cells from subjects with T1D and controls (left and right panel, respectively); n = 24 (Ctrl) and n = 27 (T1D). (D) Effect of IL-6R rs2228145 A/C polymorphism on IL-6R surface levels and IL-6-induced pSTAT3 in CD4 T cells from subjects with T1D; n = 24. (E) Real-time PCR analysis for baseline expression of IL-6 signaling components in CD4+CD25− T cells; n = 12 (Ctrl) and n = 12 (T1D). Statistical tests: Mann-Whitney U; r = Pearson correlation coefficient.

Figure 4. Reduced expression of IL-6R sheddase ADAM17 in T cells from patients with T1D

(A) Real-time PCR for ADAM17 and ADAM10 transcript in unstimulated CD4+CD25−_ cells form controls and patients with T1D; n = 12 (Ctrl) and n = 12 (T1D). (B) (Left) Western blot for ADAM17 in CD3 T cell lysates from controls and patients; TFIIB protein levels were determined as loading control. (Right) Densitometric analysis of the ratio between mature and pro-form of ADAM17; n = 3 (Ctrl) and n = 3 (T1D). (C) Flow cytometry showing ADAM17 surface expression on resting T cells from controls and patients; n = 13 (Ctrl) and n = 12 (T1D)] (D to F) IL-6R shedding assay demonstrating the role of ADAM17 in constitutive and TCR-activation induced shedding of mbIL-6R. CD3 T cells from patients with T1D were incubated for 4 hours in the presence or absence of anti-CD3/CD28 beads (anti-TCR) and the ADAM17 inhibitor TAPI. sIL-6R levels in the supernatant were determined by (D) ELISA and (E) surface expression of mbIL-6R and ADAM17 by flow cytometry. (F) Linear regression showing the inverse correlation between mbIL-6R and ADAM17 expression (left panel) and mbIL-6R and sIL-6R concentrations (right panel). Triangles, nonactivated cells at 0 hours; circles, nonactivated cells at 4 hours; squares, anti-CD3/CD28 bead-activated cells at 4 hours; n = 8 (Ctrl) and n = 8 (T1D). Statistical tests: Mann-Whitney U (A to C); Wilcoxon matched pairs (E and F); r = Pearson correlation coefficient.

Figure 5. Transcriptome analysis of IL-6 treated CD4+CD25− T cells from patients with T1D

(A) Network of enriched KEGG pathway cytokine- cytokine receptor interaction illustrates cluster of IL-6-induced genes involved in T cell trafficking. Chemokine receptors are shown in color. (B) Fold change up-regulation, expression level (logCPM) and P value of the chemokine receptors identified in (A). (C) GO analysis of IL-6-induced genes shows enrichment of terms associated with cell migration and regulation of inflammatory responses. (D) Heat map of immune-relevant genes demonstrating consistent up-regulation by IL-6 across subjects. (Left) Unstimulated cells. (Right) IL-6-stimulated cells from the same subjects. (E) Linear regression showing positive correlation between IL-6/pSTAT3 and expression level of CCR7 and SELL in CD3+ T cells; n = 6 (Ctrl) and n = 6 (T1D). r = Pearson correlation coefficient.

Figure 6. IL-6 upregulates inflammatory homing makers CXCR6 and CCR5 on TEM cells

Purified CD3 T cells were cultured in the absence or presence of IL-6 (10 ng/ml) for 48 hours, followed by surface staining and flow cytometric analysis. (A and B) Effect of IL-6 on frequency of CXCR6+ (A) and CCR5+ (B) CD4 and CD8 TEM cells. (C and D) Effect of IL-6 on CD62L (C) and CCR7 (D) expression in naïve (CD45RA+) and memory (CD45RA−) CD4 T cells. (E) Linear regression analysis demonstrating the relationship between IL-6/pSTAT3 and baseline CD62L (left panel) and IL-6-induced CD62L (right panel) in CD4 naïve T cells; n=7 (Ctrl) and n=11 (T1D) in all panels. Statistical tests: Wilcoxon matched pairs, Mann Whitney U; r = Pearson correlation coefficient.