Monocytes from patients with type 1 diabetes spontaneously secrete proinflammatory cytokines inducing Th17 cells

Abstract

Autoimmune diseases including type 1 diabetes (T1D) are thought to have a Th1/Th17 bias. The underlying mechanisms driving the activation and differentiation of these proinflammatory T cells are unknown. We examined the monocytes isolated directly from the blood of T1D patients and found they spontaneously secreted the proinflammatory cytokines IL-1beta and IL-6, which are known to induce and expand Th17 cells. Moreover, these in vivo-activated monocytes from T1D subjects induced more IL-17-secreting cells from memory T cells compared with monocytes from healthy control subjects. The induction of IL-17-secreting T cells by monocytes from T1D subjects was reduced in vitro with a combination of an IL-6-blocking Ab and IL-1R antagonist. In this study, we report a significant although modest increase in the frequency of IL-17-secreting cells in lymphocytes from long-term patients with T1D compared with healthy controls. These data suggest that the innate immune system in T1D may drive the adaptive immune system by expanding the Th17 population of effector T cells.

Figure 1. Type 1 diabetic subjects have a higher number of ex vivo IL-6 and IL-1β secreting PBMCs compared to control subjects

Unstimulated PBMCs were incubated in HL-1 media with 1% human serum for 18 hours and then analyzed by ELISpot. Each circle represents the number of positive PBMCs derived from a single subject. 250,000 PBMCs were added per well. Horizontal bars indicate the mean. Significant differences in cytokine positive cells (p<0.05) between groups are shown in the figure. (A) The number of ex vivo IL-6 secreting cells from PBMCs from recent-onset T1D subjects was significantly greater than from healthy control subjects or T2D subjects. Long-term T1D subjects also had a greater number of IL-6 secreting cells compared to healthy control subjects. (B) The number of ex vivo IL-1β secreting cells from recent-onset and long-term T1D subjects was significantly greater than that from healthy controls. Recent-onset T1D subjects had a greater number of IL-1β secreting cells compared with T2D subjects.

Figure 2. Type 1 diabetic subjects have a higher number of ex vivo IL-6 and IL-1β secreting monocytes compared to control subjects

Monocytes from T1D and healthy control subjects were negatively selected, and then both the monocytes and the monocyte-depleted PBMCs were analyzed by ELISpot. Significant differences in cytokine positive cells (p<0.05) between groups are shown in the figure. (A) The number of ex vivo IL-6 secreting cells from monocytes and monocyte-depleted PBMCs was significantly greater in the T1D subjects than from healthy control subjects, N=7. (B) The number of ex vivo IL-1β secreting cells from monocytes, but not monocyte-depleted PBMCs was significantly greater than that from healthy controls, N=8.

Figure 3. Negatively-isolated monocytes and CD11c+ cells from type 1 diabetic subjects have high gene expression for IL-6 and IL-1β

Gene expression of IL-6 and IL-1β from negatively isolated monocytes, and FACS sorted CD11c+, CD19+ and CD3+ cells from five T1D subjects were analyzed by quantitative RT-PCR. The CD16- monocytes were removed by negative isolation, and the monocyte-depleted cells were FACS sorted into CD3⁺, CD19⁺, and CD3⁻CD19⁻CD11c⁺ populations. Significant differences are shown in the figure. Relative gene expression of (A) IL-6, and (B) IL-1β are increased in the classical monocyte and CD11c populations compared to the CD19 and CD3 populations. All gene expression is relative to β2-microglobulin.

Figure 4. Unstimulated CD14⁺CD16⁻ and CD14⁺CD16⁺ monocytes from T1D subjects, but not CD14^dimCD16⁺ monocytes have increased IL-6 gene expression compared to healthy control subjects

Gene expression of IL-6 from CD14⁺CD16⁻, CD14⁺CD16⁺ and CD14^dimCD16⁺ FACS sorted monocytes from five T1D subjects and five healthy control subjects were analyzed by quantitative RT-PCR. Significant differences are shown in the figure. All gene expression is relative to b2-microglobulin.

Figure 5. Ex vivo monocytes from type 1 diabetic subjects have increased cytokine gene expression in comparison to healthy control subjects

Monocytes from recent onset (<1 years) T1D subjects were negatively selected, RNA was immediately isolated, and the relative gene expression was measured using quantitative RT-PCR. Relative (A) IL-6 (B) IL-1β and (C) TNFα gene expression for T1D and healthy control subjects is shown. All gene expression is relative to β2-microglobulin. Each circle represents a subject; 13 recent-onset T1D subjects were compared to 8 healthy control subjects. Horizontal bars indicate the mean. Correlation of gene expression between (D) IL-6 and IL-1β and (E) IL-6 and TNFα for monocytes from recent-onset T1D is shown. The monocytes derived from T1D subjects are in a more activated state as shown by increased expression of (F) PDL1 and (G) CD80 as compared to those from healthy controls. Significant differences are shown in the figure.

Figure 6. IL-1R antagonist reduced the number of IL-17 secreting memory T cells in monocyte-T cell co-cultures

Healthy control memory T cells were cultured with monocytes from T1D subjects for 5 days in the presence of plate-bound anti-CD3. T cells were then expanded for 7 days with the addition of IL-2. The cells were stimulated with PMA and ionomycin and analyzed by intracellular staining for IL-17/IFNγ expression. The number of (A) IL-17⁺, (B) IL-17⁺/IFNγ⁺ and (C) IL-17⁺/IFNγ⁻ secreting cells, but not (D) IL-17⁻/IFNγ⁺ secreting cells, (N=8) could be reduced by IL-1R antagonist and anti-IL-6 or IL-1R antagonist alone, but were slightly increased by anti-TNFα. ***, p<0.0005; **, p<0.005; *, p<0.05.

Figure 7. IL-17 secreting cells from anti-CD3 stimulated PBMCs from long-term T1D subjects was greater as compared to those from healthy control subjects

Anti-CD3 stimulated PBMCs were incubated in HL-1 media with 1% human serum for 18 hours and then analyzed by ELISpot. Each circle represents the number of positive PBMCs derived from a single subject. 250,000 PBMCs were added per well. Horizontal bars indicate the mean. Significant differences in cytokine positive cells between groups are shown in the figure.