Lymphatic Endothelial Cells Regulate B Cell Lymph Node Homing Through NIK And The Related Research Of SAPHO Syndrome Peripheral Blood ??T Cells

Objective:After developing and maturing in central lymphoid organs,lymphocytes settle in peripheral lymphoid organs by blood flow and exert various biological functions in the body,organs,tissues and inflammation sites.Lymphocyte homing is a special form of lymphocyte migration.Lymphocytes from different populations or subgroups are relatively selective during migration,that is,a specific lymphocyte population or subgroup is homed to the corresponding tissue or organs.The molecular basis of lymphocyte homing is the interaction of lymphocytes with the adhesion molecules of vascular endothelial cells in various tissues and organs.B cells home to the lymph nodes(LNs)via high endothelial venules(HEVs)under the guidance of chemokines,particularly CXCL13.However,since CXCL13 is not directly made in HEVs,the molecular mechanism mediating B cell homing to LNs has remained unclear.NF-?B signaling pathway is involved in various biological processes such as immune response,inflammatory reaction,apoptosis and tumorigenesis.NIK as a key molecule in the non-canonical NF-?B pathway also maintains intact immune system function,especially in maintaining intact lymph node and B cell populations.However,its role in lymphatic endothelial cells(LECs)is still unclear.The purpose of this study is to explore the effect of NIK molecules in lymphatic endothelial cells on lymphatic functions and its molecular mechanism.Methods:To study the function of NIK in lymphatic vessels,we generated conditional KO mice in which NIK was specifically deleted in LECs(NIKLEC-KO,NIKfl/flLyvel+/Cre)and littermate controls(NIK+/+Lyvel+/Cre)by crossing NIK-flox mice with Lyvel EGFP-hCre mice.Flow cytometry analyzed the phenotype of mouse immune cells.The lymph flow functional assay in the ear and fluorescein isothiocyanate painting assay were used to evaluate the lymphatic drainage functions of NIKLEC-KO mice.The homing of major cell subsets of NIKLEC-KO mice was detected by in vivo homing experiment.RT-qPCR was used to analyze the expression of related important chemokines.Finally,the immunized mice were tested for their antibody secretory capacity by enzyme-linked immunosorbent assay to evaluate their humoral immune function.Results:The data show here that NIK,a kinase mediating activation of the non-canonical NF-?B pathway,functions in lymphatic endothelial cells to regulate B cell homing to LNs.LEC-conditional deletion of NIK in mice did not affect the integrity or global function of lymphatic vessels but caused a severe reduction in the frequency of B cells in LNs.The LEC-specific NIK deficiency did not affect the survival of B cells or the frequency of B cells in the spleen.B cell adoptive transfer studies revealed that the LEC-specific NIK deletion impairs the ability of LNs to recruit B cells.We further show that NIK mediates expression of the chemokines CXCL13 and CCL19 in LECs.Conclusions:These results suggest that NIK regulate naive B cell homing to LNs via mediating production of the B cell homing chemokine CXCL13 in LECs.Objective:SAPHO syndrome,which is distinguished by a variable combination of synovitis,acne,pustulosis,hyperostosis and osteitis,is a rare and often unrecognized disease.The pathogenesis of SAPHO is still unclear although TH17 cells have been found associated with SAPHO.Since y8T cells are also an important source of IL-17 then probably involved in the disease progression ofSAPHO syndrome.In this study,the phenotype of peripheral ??T cells in Chinese patients with SAPHO syndrome were analyzed to assess whether any changes of ??T cells would be observed in SAPHO patients.Then,try to find evidence for the involvement of ?? T cells in SAPHO syndrome based on the characteristics of y8T cell receptor(TCR??)complementary determinant region 3(CDR3)repertoire.Bisphosphonate treatment clinical trail may affect the change of peripheral blood y8 T cells.This study attempts to explore whether ?? T cells are involved in the progression of SAPHO syndrome in these three ways.Methods:42 patients with SAPHO syndrome and 16 healthy controls were involved in this study.In accordance with marks of VAS(Visual Analogue Scale)pain and BASDAI(Bath Ankylosing Spondylitis Activity Index),SAPHO patients were divided into active group(n=18)and stable group(n=24).By means of flow cytometry,the proportions of total and different subsets of y8T cells in PBMCs(peripheral blood mononuclear cells)of different groups of SAPHO syndrome and healthy controls were studied.Next,4 active SAPHO patients and 4 stable group SAPHO patients and 4 healthy volunteers(age and gender matched)were enrolled in the experiment.Specific PCR and high-throughput sequencing technique were used to analyzed and compared the characteristics of TCR??CDR3 repertoire in peripheral blood mononuclear cells from above 3 groups.In the third part,we collected 30 SAPHO patients and registered borine-pamidronate treatment clinical trail.The peripheral blood before and after borine-pamidronate treatment of SAPHO patients were collected and analyzed the related phenotypic changes of peripheral blood y8T cells by flow cytometry.Finally,the CCT5 concentrations in plasma of SAPHO patients were detected by ELISA.Results:The proportion of y8T cells in peripheral blood of SAPHO patients was significantly lower than that in healthy controls,especially in the active group;V?1 T cells were significantly decreased in patients while no difference in V?2 T cells;The ratio of CD27+/CD27-??T cells was significantly lower in SAPHO active group than healthy controls and patients with stable stage.There was no significant correlation between the proportion of y8T cells and VAS or BASDAI in all SAPHO patients.However,the two ratio(V?2/V?1 y8T cells and CD27+/CD27-?8T cells)showed a significant negative correlation with the disease progression of SAPHO.The diversity of CDR3? in all SAPHO patients and stable stage patients was significantly lower than that in healthy controls.The clone size>10%CDR3 number in SAPHO active patients were significantly lower.The clone size>1%CDR3 number in SAPHO stable patients were significantly higher than healthy controls in the top 50 CDR35 sequences.Compared with the control,the patient tends to use a shorter CDR3?,which is not caused by random insertion fragment length.In the germ line gene usage of TCRy8 V/J fragment,the differences between SAPHO patients and healthy controls were mainly focused on the CDR3y,including TRGV3 reduction and TRGV5,TRGJ1 increases.In the third part,after bisphosphonate treatment,the proportion of V82 ??T cells in the peripheral blood of SAPHO patients decreased,the proportion of V81 ??T cells did not change,the proportion of memory-type ?? T cells decreased,and the differentiation of V82 ??T cells to effector cells increased.Finally,plasma CCT5 concentrations in active group SAPHO patients were lower than that in healthy controls and stable group patients.The bisphosphonate treatment increased plasma CCT5 levels,and plasma CCT5 levels were significantly negatively correlated with SAPHO disease activity scores VAS and BASDAI.Conclusions:??T cells,in particular,CD27-??T cell subsets may play a role in the pathogenesis of SAPHO syndrome.y8T cells in SAPHO patients had a number of changes in the TCR?? CDR3 repertoire,providing evidence for the role of y8T cells in SAPHO syndrome.V82??T cells may play a role in the treatment of bisphosphonates in SAPHO syndrome.This study provides the evidences of y5T cells enrolled the progress of SAPHO syndrome.