The Mechanism Research Of The CCL20/CCR6System In Th17Mediated Discogenic Pain

ObjectiveLumbar disc herniation (LDH) is a widespread and debilitating disorder. According to the present statistical materials, the number of LDH patients accounts for15percent of lumbocrural pain patients. Annually,5.7million individuals develop an intervertebral disc (IVD) related disease in the U.S. alone and as many as40percent of these cases are the results of IVD pathologies. As people life rhythm speeding up, the incidence of LDH is in a rising trend year by year and the pain caused by LDH is becoming an important factor influcencing the patients’quality of life. Therefore, fully understanding of the pathological mechanism of lumbocrural pain caused by LDH and trying to find the etiological factor are important to explore the most effective and appropriate treatment strategy and to improve the clinical effect.MethodsPatient Enrollment took place between April2012and July2012at Qilu Hospital, Shandong University, China. For immunohistochemistry analyses,50disc tissues were collected during surgery from50patients who have received diagnoses of disc degeneration and3disc tissue samples from3patients diagnosed with spinal burst fracture. According to the surgical and imaging findings, we divided the specimens into three groups:the protrusion group, in which the annulus fibrosus was intact (Group P, n=20), the extrusion group, in which the annulus fibrosus was ruptured (Group E, n=30) and the normal control group (Normal Control, n=3). We used immunohistochemistry and double immunofluorescence staining to detect the expression of CCL20, TNF-a and Thl7cells (CD4+IL-17A+�'�CD4+CCR6+) in the degenerated intervetebral discs and normal controlled discs. For nucleus pulposus culture,8disc samples were collected from8patients diagnosed with LDH. The NP cells were cultured either alone or with one of the following cytokines:IL-17Aand TNF-a. At the appointed time intervals after receiving cytokine stimulation, the supernatants and NP cells were harvested for CCL20protein detection and mRNA quantification, respectively. Then we collected the peripheral blood of other20LDH patients pre-and post-operation and15normal people to detect the Th17frequency by flow cytometry and IL-17concentration by enzyme-linked immuno sorbent assay (ELISA). To investigate whether the pain sensation is related to the proportion of Th17cells and the IL-17concentration, we analysed the correlations between the VAS pain scores, the Th17cell frequency, and the IL-17concentration, including the pre-and post-operation levels and the changes induced by the surgery.Results1. The results of IHC showed that CCL20and TNF-a immunoreactivity were detected in both Group P and Group E. Compared to the normal control, the immunoreactivity was significantly increased (p<0.001). The results of double immunofluorescence staining showed that Th17cells were detected in the IVDs of Group E. In contrast, there were few or no positive cells in the Group P and normal control samples.2. Th17cell associated cytokines, IL-17A and TNF-a,can significantly enhance CCL20secretion in the NP cells from degenerated IVD tissues in a dose-dependent manner.3. Compared to the healthy controls, the percentage of peripheral Th17cells significantly increased in the patients with degenerated IVD (1.039±0.156%vs.2.973±0.689%, P<0.0001). The rate of surface CCR6expression on the Th17cells was profoundly increased in the patients with degenerated IVD when compared to the healthy controls (28.75±2.09%vs.59.69±4.48%, P<0.0001). 4. The correlative analysis results showed that there was a positive correlation between the Th17cell frequency, the IL-17concentration, and the VAS pain scores in patients with degenerated IVD, including the pre-and post-operation levels and the changes induced by the surgery.(Pre-operation:Th17vs. IL-17, r=0.723, p<0.01; Th17vs.VAS, r=0.805, p<0.01; IL-17vs.VAS, r=0.675, p<0.01; Post-operation: Th17vs. IL-17, r=0.551, p<0.01; Thl7vs.VAS, r=0.536, p<0.01; IL-17vs.VAS, r=0.507, p<0.01; During operation:Th17vs. IL-17, r=0.729, p<0.01; Th17vs.VAS, r=0.732, p<0.01; IL-17vs.VAS, r=0.536, p<0.01)ConclusionsOur results provide a potential explanation for involvement of the CCL20-CCR6system in the trafficking of IL-17-producing cells to degenerated IVD tissues. Additionally, our results explain the contribution of Th17associated cytokines to the development of degenerated discs via the up-regulation of CCL20secretion from NP cells, which forms a positive chemotactic feedback loop. After Th17cells were transferred via chemotaxis to the lesion areas, they could secrete many inflammatory cytokines, leading to the appearance of a cytokine milieu with the cytokines produced by macrophages or other inflammatory cells. This cytokine milieu promotes the inflammatory process and stimulates the production of CCL20, including the trafficking of more Th17cells to the inflammatory lesion. As a result, we have a potential explanation for how the CCR6-positive Th17cells maintain a continuous presence in the degenerated discs through a positive chemotactic feedback loop.