Activated IL-1RI Signaling Pathway Induces Th17 Cell Differentiation via Interferon Regulatory Factor 4 Signaling in Patients with Relapsing-Remitting Multiple Sclerosis

Abstract

IL-1β plays a crucial role in the differentiation of human Th17 cells. We report here that IL-1RI expression is significantly increased in both naive and memory CD4⁺ T cells derived from relapsing-remitting multiple sclerosis (RR MS) patients in comparison to healthy controls. Interleukin 1 receptor (IL-1R)I expression is upregulated in the in vitro-differentiated Th17 cells from RR MS patients in comparison to the Th1 and Th2 cell subsets, indicating the role of IL-1R signaling in the Th17 cell differentiation in RR MS. When IL-1RI gene expression was silenced using siRNA, human naive CD4⁺ T cells cultured in the presence of Th17-polarizing cytokines had a significantly decreased expression of interleukin regulatory factor 4 (IRF4), RORc, IL-17A, IL-17F, IL-21, IL-22, and IL-23R genes, confirming that IL-1RI signaling induces Th17 cell differentiation. Since IL-1R gene expression silencing inhibited IRF4 expression and Th17 differentiation, and IRF4 gene expression silencing inhibited Th17 cell differentiation, our results indicate that IL-1RI induces human Th17 cell differentiation in an IRF4-dependant manner. Our study has identified that IL-1RI-mediated signaling pathway is constitutively activated, leading to an increased Th17 cell differentiation in IRF4-dependent manner in patients with RR MS.

Keywords: IL-1; IL-1R; IRF4; Th17 cells; multiple sclerosis.

Figure 1

IL-1RI gene expression is increased in CD4⁺, CD4⁺CD45RA⁺, and CD4⁺CD45RO⁺ cells from RR MS patients in comparison to HCs. CD4⁺, CD4⁺CD45RA⁺ naive, and CD4⁺CD45RO⁺ memory T cells derived from six RR MS patients and six HCs were separated using magnetic beads. The total RNA was harvested and the gene expression of IL-1RI and 18S was measured using qRT-PCR. The results are presented as the relative gene expression normalized against 18S mRNA. Statistical analysis was performed using unpaired t-test.

Figure 2

IL-1R1 expression is higher in the in vitro-differentiated Th17 cells in comparison to Th1 and Th2 cultures. CD4⁺CD45RA⁺ cells were derived from three HCs and stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb in a serum-free medium in the absence or presence of Th1, Th2, or Th17-polarizing cytokines. After 72 h, total RNA was extracted and gene expression of IL-1R1 and 18S was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using ANOVA. ***p < 0.001.

Figure 3

IL-1R1 signaling induces Th17 cell differentiation. CD4⁺CD45RA⁺ cells were derived from three HCs (A) and seven RR MS patients (B) and transfected with a control siRNA A or siRNA IL-1R1, stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb and cultured in serum-free medium in the absence or presence of Th17-polarizing cytokines. The total RNA was extracted at 72 h, and the expression of the indicated genes was measured using RT-PCR. The results are expressed as relative gene expression normalized against 18S mRNA expression. Statistical analysis was performed using repeated measures ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

IL-1R1 signaling induces secretion of Th17 cytokines. CD4⁺CD45RA⁺ cells were derived from three HCs (A) and three RR MS patients (B), transfected with a control siRNA A or siRNA IL-1R1, stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb and cultured in serum-free medium in the absence or presence of Th17-polarizing cytokines. After 72 h, the supernatants were collected and the cytokine production measured by ELISA. Statistical analysis was performed using a repeated measures ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

IL-1R1 and IRF4 are required for human Th17 cell differentiation. (A) IRF4 gene expression in the CD4⁺ T cell from RR MS patients is significantly increased in comparison to HCs. CD4⁺ T cells derived from six RR MS patients and six HCs were separated using magnetic beads, and the total RNA was extracted. The gene expression of IRF4 and 18S was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using t-tests. *p < 0.05. (B) IL-1R1 gene silencing downregulates IRF4 and RORc protein expression in CD4⁺CD45RA⁺ cells from RR MS patients and inhibits Th17 cell differentiation.

Figure 6

IL-1R1 positively regulates human Th17 cell differentiation in an IRF4-dependent manner. CD4⁺CD45RA⁺ cells from six RR MS patients transfected with a control siRNA A or siRNA IRF4 were stimulated with plate-immobilized anti-CD3 and anti-CD28 mAb and cultured in serum-free medium in the absence or presence of Th17-polarizing cytokines. After 72 h, the total RNA was extracted. The gene expression was measured by RT-PCR. The results are expressed as relative gene expression normalized for 18S mRNA expression. Statistical analysis was performed using ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.