| Objective:Recent genomic studies have proved that the common’C allele of single nucleotide polymorphisms (SNPs) rs6897932that encoded IL-7Ra was associated with the susceptibility of Multiple Sclerosis (MS). Effects of IL-7/IL-7Ra pathway on CD4+, CD8+T cells, Regulatory T cells (Treg) have been evident in pathogenesis of MS; however, its impact on natural killer (NK) cells in MS has not yet been reported. The objective of this study was to reveal the biological effects of IL-7/IL-7Ra pathway on NK cell functions in MS patients, which include intracellular cytokines IFN-y secretion, cytolytic effects towards tumor cells, proliferation ability, and potential of anti-apoptosis. We attempted to explore the potential mechanism. In addition, we analyzed the possible correlation between IL-7/IL-7Ra and functional deficiency of NK cells in MS patients.Method:The peripheral blood was collected from MS patients and Healthy Control (HC). Sera were isolated, and the levels of IL-2, IL-7, IL-15and IL-21were tested by ELISA. The peripheral blood mononuclear cell (PBMC) from MS and HC were cultured with or without IL-7. The surface expression of IL-7Ra (CD127) and levels of intracellular cytokine IFN-y were detected and quantified by flow cytometry. NK cells were purified via magnetic sorting. NK cytolytic effects towards tumor cells were evaluated by detecting the release of Lactate dehydrogenase (LDH) from target cells. Bromodeoxyuridine (BrdU) was used to label the proliferating cells, and the percentage of proliferating NK cells were detected by flow cytometry.7-AAD (7-amino-actinomycin D staining) and Annexin V were used to label the apoptotic and dying cells. The percentage of survival cells and intracellular expression of anti-apoptotic factor Bcl-2were detected by flow cytometry.Result:MS patients have significant lower serum IL-7levels compared with HC. CD127is expressed on NK cells, with higher expression levels in MS patients than HC. Upon IL-7stimulation, downregulation of CD127was observed in both groups, with a significantly lower CD127expression on CD56bright and CD56dim populations in MS patients. A significantly higher percentage of IFN-y+CD56bright NK cells and dramatically increased IFN-y intensity in CD56bright NK cells were found in MS patients compared to those of HC after IL-7stimulation. Cytolytic effects of IL-7-stimulated NK cells towards K562tumor cells were significantly enhanced in MS patients at ratio of effector to target at2.5:1,5:1,10:1. The changes in percentage of CD3-CD56brightBrdU+and CD3-CD56dimBrdU+cells were not significantly different between MS patients and HC. The percentage of survival NK cells labeled as Annexin V-7-AAD-was significantly increased in CD3-CD56brightNK subpopulation in MS patients, correlating with the significantly up-regulated Bcl-2expression; however, the changes were not significant in CD3-CD56dimNK subpopulation.Conclusion:1. IL-7/IL-7Ra pathway was aberrantly activated in NK cells of MS patients;2. Compared with HC, IL-7stimulation leads to a higher increase of IFN-y production and enhanced cytolytic effects of NK cells in MS patients. It also promotes NK cells survival by upregulating anti-apoptotic factor Bcl-2. However, IL-7has little effects in NK cell proliferation.3. IL-7/IL-7Rα pathway has important regulatory effects on NK cells especially in CD56brightNK cells. We speculate that aberration of IL-7/IL-7Ra pathway leads to NK functional deficiency, thus cause MS pathogenesis. Targeting the IL-7/IL-7Ra pathway on NK cells might be a compelling potential for therapeutic intervention of MS. |
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