The Mechanism Of The CCL20/CCR6 System In IL-17 Mediated Autoimmune Response Of Rat’s Degenerated IVDs

ObjectiveInterleukin-17-producing T helper 17(Th17) cells play an important role in various autoimmune diseases. It is known that a higher level of IL-17-producing Thl7cells is observed in patients with intervertebral disc (IVD) degeneration. However, the mechanism of the accumulation of Th17 cells remains to be elucidated. Our aim is to demonstrate whether the interaction of CC chemokine ligand (CCL) 20 and its specific receptor CCR6 is involved in recruitment of Thl7 cells to degenerated disc tissues in rat models.MethodsIn this study, the well-accepted needle puncture model and autologous nucleus pulposus application model of rats were established first. All animals were randomly divided into 4 groups:sham surgery group, needle puncture group (NP group), sham surgery + autologous nucleus pulposus application group (Sham-ANPA group) and needle puncture + autologous nucleus pulposus application group (NP-ANPA group). All of the animals were sacrificed at special point-in-time (5 weeks after the first operation) by an intraperitoneal pentobarbital injection. HE staining was used to reveal the morphology of the degenerated IVD tissues of rats. Immunohistochemical staining was performed to evaluate the expression level of CCL20, IL-17 and CCR6 in IVD samples. The contents of CCL20, IL-17 andCCR6 protein in the degenerated IVD tissues from each group as determined by a western blot analysis. The CCL20, IL-17 and CCR6 mRNA expression in the degenerated and grafted IVD tissues were detected by real-time PCR. Moreover, the IL-17 concentrations in serum were detected by Enzyme-linked immunosorbent assay (ELISA). Pearson’s correlation analyses were performed to analyze the correlation among the expression of CCL20, CCR6 and IL-17.Results1. The results of IHC showed that CCL20, CCR6 and IL-17 immunoreactivity of IVD tissues were detected in both NP group and NP-ANPA group. Compared to sham surgery group, the immunoreactivity was significantly increased (P<0.05). The expression of CCL20, IL17 and CCR6 of NP-ANPA group were dramatically elevated in comparison with NP group (P<0.05).2. Western blot findings showed that the expression of CCL20, CCR6 and IL-17 in the IVD tissues of NP group and NP-ANPA group were significantly increased compared to sham surgery group (P<0.05). The expression of CCL20, IL17 and CCR6 of NP-ANPA group were dramatically elevated in comparison with NP group (P<0.05).3. The results of real-time PCR showed that when we compared the expression levels of the target genes, statistically significant increases in the gene expressions of CCL20, IL-17 and CCR6 were obtained in both the NP and NP-ANPA groups compared with the sham surgery group (P<0.05). The expression of CCL20, IL17 and CCR6 mRNA of NP-ANPA group were dramatically elevated in comparison with NP group (P＜0.05).4. The circulating IL-17 in serum of NP group and NP-ANPA group were apparently elevated compared to the sham surgery group (P<0.05).Similarly, the secretion level of IL-17 in the serum in the NP-ANPA group was elevated compared to the NP group CP<0.05).5.The Pearson correlation analysis demonstrated that the expression level of IL-17 in the degenerated disc tissues was positively correlated with the expressions of CCL20andCCR6 (CCL20vs.IL-17, r=0.916,P< 0.001; CCR6vs.IL-17, r=0.868, P<0.001)ConclusionIL-17 plays an important role in the process of autoimmune response of rat’s degenerated IVD tissues. The degenerated NP cells could secrete elevated levels of CCL20, which recruit Th17 cells to inflammatory sites in the degenerated or herniated intervertebral discs via CCL20/CCR6 system. Inflammatory factors produced by Th17 cells are activated the autoimmune response in the degenerated IVD tissues, furthermore, the local inflammatory environment stimulate degenerated NP cells to secreted more higher level of CCL20. Then, high level ofIL-17 released by the Th17 cells would induce more severe auto-immune responses and inflammatory reaction in the degenerated IVD tissues, IL-17 migrates to the inflammatory area by the positive feedback loop of the CCL20-CCR6-IL-17.We suggest that IL-17-producing cells might traffic to the degenerated disc tissues via an interaction with the CC20/CCR6 system in vivo.