| Natural killer T (NKT) cells possess the immune properties of both T cells and NK cells. CD1d-restricted invariant natural killer T (iNKT) cells are potent regulators of diverse immune responses and play an important role in regulating immune system balance, tumorigenesis and autoimmune diseases, including skin diseaes. However, the detailed mechanisms involved in the iNKT cell development are still poorly understood.MicroRNAs (miRNAs) are a class of short (~22nt) noncoding RNAs that regulate gene expression through either translation repression or mRNA degradation. More and more evidence are emerging that miRNAs are key regulators in the control of wide range of biological functions in variety of mammalian cell types; including cells of the immune system. Using bone marrow Dicer deletion mouse model, our previous studies indicate that lack of mature, functional miRNAs by Dicer deletion dramatically interrupts the development and maturation of iNKT cells in the thymus and decreases the number of iNKT cells in peripheral immune organs. In our group study, we detected the miRNA expression patterns in different development stages of iNKT cells. Interestingly, we found microRNA155 and microRNA223 specifically highly expressed in the immature stages of iNKT cells (stage 1 CD44 lowNK1.1- and stage 2 CD44highNK1.1-). These findings indicate that microRNA155 and microRNA223 may have some regulatory roles in NKT cell development and functions.MethodsIn this study, we have used two distinct but complementary approaches (conventional miR155 deficient mouse model and hematopoitic stem cell specific miR155 over expression mouse model) and conventional miR223 deficient mouse model to better understand the role of miR155 and miR223 in the development and function of iNKT cells. We usedα-GalCer loaded CD1d tetramers, which is specifice for NKT cells, based on flow cytometer technology detected NKT cells number and distribution in different immune organs. We also detected NKT cell development markers and NKT cells'functionsResults1.NKTcells'development and function in miR223KO miceComparied with WT mice, conventional T cells subsets in miR223 deficent mice was not changed, CD4+ T cells in miR223 -/Y KO mice and WT mice are 10.56% vs.11.35%,CD8+ T cells are 5.02% vs. 5.71%,CD4+CD8+ T cells are 81.60% vs. 80.65%,CD4-CD8- T cells are 2.83% vs. 2.30%。With conventional miR223 deficient mouse model, we did not find remarkable defects of NKT cell number. NTK cells ratio in miR223 -/Y KO mice and WT mice are 0.15±0.02% vs. 0.15±0.01%, P=0.805 in thymus, 0.80±0.14% vs. 0.73±0.13%, P=0.715 in spleen, 14.67±3.51% vs. 13.37±2.83%, P=0.764 in liver, 0.12±0.05% vs. 0.14±0.03%, P=0.557 in lymph nodes and 0.99±0.44% vs. 1.71±0.58%,P=0.376 in bone marrow. NKT cell in miR223 deficient mouse thymus showed same development as wild type controls. NTK cells ratio in WT mice and miR223 -/Y KO mice are 5..59±0.92% vs.6.57±2.32%, P=0.781 in stage 1 CD44lowNK1.1-, 15.59±4.44% vs. 13.08±2.39%, P=0.593in stage 2 CD44highNK1.1- and 77.83±4.69% vs. 78.71±3.85%, P=0.894 in stage 3. All NKT cell's development markers did not find any different. Also, miR223 deficient mouse NKT cell number was not change in periphery immune organs.2.NKTcells'development in miR155KO miceWith conventional miR155 deficient mouse model, we did not find remarkable changes of NKT cell number. NTK cells ratio in miR155 -/- KO mice and WT mice are 0.32±0.02% vs. 0.30±0.04%, P=0.745 in thymus, 0.83±0.12% vs. 0.83±0.13%, P=0.987 in spleen, 17.19±2.35% vs. 11.63±1.26%, P=0.694 in liver. NKT cell in miR155 deficient mouse thymus showed same development as wild type controls. All NKT cell's development markers did not find any different. NTK cells ratio in WT mice and miR155 -/- KO mice are 12.94±2.90% vs.12.73±3.49%, P=0.965 in stage 1 CD44lowNK1.1-, 17.36±2.12% vs. 14.88±2.23%, P=0.439 in stage 2 CD44highNK1.1- and 63.53±6.54% vs. 64.94±6.75%, P=0.894 in stage 3. Also, miR155 deficient mouse NKT cell number was not change in periphery immune organs.3.NKTcells'development and function in miR155KO miceConversely, using hematopoitic stem cell specific miR155 over expression mouse model, we found that miR155 over expression results in developmental defect and impaired developmental progression of NKT cells to stage 3, NTK cells ratio in WT mice and miR155KI mice are 6.56±1.29% vs.13.01±2.91%, P=0.077 in stage 1 CD44lowNK1.1-, 21.43±6.65% vs. 63.82±5.10%, P<0.001 in stage 2 CD44highNK1.1- and 69.03±9.37% vs. 15.93±2.59%, P<0.001 in stage 3.As well as fundamental functional defects even though the NKT cell number was not dramatically changed. NTK cells ratio in WT mice and miR155KI mice are 0.17±0.04% vs. 0.20±0.04%, P=0.616 in thymus, 0.85±0.14% vs. 0.66±0.10%, P=0.314 in spleen, 18.70±4.53% vs. 15.31±2.08%, P=0.522 in liver and 0.16±0.03% vs. 0.19±0.02%, P=0.464 in lymphy node.We also fund the consistend increased both in NKT cell proliferation and NKT cell apoptosis. Afterα-GalCer injection, miR155 KI NKT cells showed a markable decreased production of cytokine IL-4 and IFN-γ. Stimulated by PMA and ionomycin, comparable and decreased proportions of iNKT cells from miR155 KI mice stained positive for IL-4 (55% vs. 60%) and IFN-γ(39% vs. 70%) compared to those of WT controls, in agreement with the less mature phenotype of miR155 KI NKT cells that corresponds to a bias toward IL-4 production vs the predominant IFN-γproduction observed from WT NKT cells. These data indicating that the majority of the defect in cytokine production is due to a defect in proximal TCR signaling.Based on our present data, we made a conclusion that the defect of miR155 and miR223 was not affect the NKT cell development and number. But this does not mean the miR155 and miR223 don't has any regulator roles in NKT cell development. The sharply decreased of miR155 and miR223 in the mature stage of NKT cells may contributing NKT cell maturation. If continue high expression of miR155 as our miR155 over expression mouse model, NKT cell development and function also impressing disrupted. In the future, generate miR223 over expression mouse model in necessary for figure out whether miR223 has regulatory roles in NKT cell development and functions. |
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