CXCL12/CXCR4 In OB Cells Of MRL/Lpr Mice Regulate Plasma Cell Homing Through MEK/ERK Signaling Pathway

Objective:To study the chemotaxis and mechanism of MRL/lpr lupus mice osteoblasts on plasma cells through CXCL12/CXCR4 axis and MEK/ERK signaling pathway.To investigate the proportion of Ki67-CD138+ long-lived plasma cells(LLPCs)in bone marrow and peripheral blood of patients with systemic lupus erythematosus(SLE)and their correlation with disease activity markers,in order to explore new markers for predicting disease activity.Methods:1.The expression ratios of CXCR4+CD138+ plasma cells and Ki67-CD138+ LLPCs in peripheral blood,bone marrow,spleen,and kidney of 12-week-old C57BL/6 mice and MRL/lpr lupus mice models were detected by flow cytometry.Immunofluorescence was used to observe the spatial distribution of LLPCs and osteoblasts OB in the spleen and bone marrow microenvironment of MRL/lpr lupus mice and C57BL/6 mice.2.In vitro magnetic beads were used to isolate CD138+ spleen plasma cells,and flow cytometry was used to detect the purity of the selected plasma cells.Using the skulls of MRL/lpr lupus mice and C57BL/6 mice as experimental materials,osteoblasts were isolated and cultured in vitro for 2 weeks.Using Transwell technology,the differences in chemotaxis of spleen plasma cells to the skulls of MRL/lpr mice and C57BL/6 mice were studied,and they were grouped as follows:(1)Plasma cells from MRL/lpr mice were placed in the upper chamber,and osteoblasts from MRL/lpr mice were placed in the lower chamber.(2)Plasma cells from MRL/lpr mice were placed in the upper chamber,and osteoblasts from MRL/lpr mice+CXCL12 factor were placed in the lower chamber.(3)Plasma cells from MRL/lpr mice were placed in the upper chamber,and osteoblasts from MRL/lpr mice+CXCR4 antagonist AMD3100 were placed in the lower chamber.(4)Plasma cells from MRL/lpr mice were placed in the upper chamber,and osteoblasts from MRL/lpr mice+ MEK1/2 antagonist U0126 were placed in the lower chamber.(5)Plasma cells from MRL/lpr mice were placed in the upper chamber,and osteoblasts from MRL/lpr mice+AMD3100+U0126 were placed in the lower chamber.(6)Plasma cells from MRL/lpr mice were placed in the upper chamber,and osteoblasts from MRL/lpr mice+ CXCL12factor+AMD3100+U0126 were placed in the lower chamber.(7)Plasma cells from C57BL/6mice were placed in the upper chamber,and osteoblasts from C57BL/6 mice were placed in the lower chamber.(The number of cells per well in the upper chamber was 1×10^7,and the number of cells per well in the lower chamber was 1×10^4)3.Plasma cells from C57BL/6 mice OB to C57BL/6 mice spleen plasma cells and from different groups of MRL/lpr lupus mice OB to MRL/lpr lupus mice spleen plasma cells after Transwell were collected.Flow cytometry was used to analyze the expression ratio of P-ERK+plasma cells,and the grouping is the same as 2.4.In clinical practice,peripheral blood samples from 36 patients diagnosed with SLE and 30 healthy controls,as well as bone marrow samples from 6 patients diagnosed with SLE and 5healthy controls were collected in our hospital.Flow cytometry was used to detect Ki67-CD138+LLPCs ratio and analyze the difference.Meanwhile,clinical data of SLE patients were collected to analyze the correlation between Ki67-CD138+ LLPCs ratio in peripheral blood and markers of disease activity,so as to speculate the predictive efficacy of SLE.Results:1.Compared with C57BL/6 mice,the proportion of plasma cells expressing CXCR4+CD138+ and LLPCs expressing Ki67-CD138+ in peripheral blood,bone marrow,spleen,and kidney tissues of MRL/lpr lupus mice increased,while the proportion of LLPCs in total plasma cells was lower in peripheral blood,kidney and spleen,but higher in bone marrow.Immunofluorescence staining showed that compared to C57BL/6 mice,MRL/lpr lupus mice had more long-lived plasma cells in the bone marrow and spleen,and they were more prominent in the red pulp region of spleen,bone marrow cavity and around bone trabeculae.2.Compared with C57BL/6 mice,MRL/lpr lupus mice have stronger migration ability of spleen plasma cells and bone marrow OB cells.After the addition of CXCR4 antagonist AMD3100 and MEK1/2 antagonist U0126,the migration ability of spleen plasma cells to bone marrow OB cells in MRL/lpr mice decreased.3.Flow cytometry of Transwell plasma cells showed that compared with C57BL/6 mice,the proportion of P-ERK+ expression in bone marrow OB cells of splenic plasma cells of MRL/lpr lupus mice increased after migration.The expression ratio of P-ERK+ decreased after the addition of MEK1/2 antagonist U0126 or CXCR4 antagonist AMD3100+U0126.4.Compared with the healthy control group,the proportion of LLPCs in the bone marrow and peripheral blood of SLE patients increased.In peripheral blood,the increased expression of LLPCs was positively correlated with SLEDAI score.The percentage of LLPCs in SLE patients with light,moderate and heavy activity was significantly higher than that in healthy donors and SLE patients who were basically inactive.here was a significant negative correlation between the increased expression of LLPCs and C3 and C4 scores.The AUC of the percentage of LLPCs in diagnosing SLE was 0.8377,which can be regarded as a potential biomarker for the early diagnosis of SLE and its disease activity.Conclusion:The proportion of CXCR4+CD138+ plasma cells and Ki67-CD138+ LLPCs increased in peripheral blood,bone marrow,spleen and kidney tissues of MRL/lpr mice.The proportion of LLPCs in total plasma cells was lower in peripheral blood,kidney and spleen,but higher in bone marrow.There were more LLPCs in the red pulp of the spleen,bone marrow cavity and bone trabecula,and they coexisted with osteoblasts expressing CXCL12 around the bone marrow cavity,thus attracting more peripheral plasma cells to the bone marrow homing.The CXCL12-CXCR4 axis of MRL/lpr lupus mice regulates the directed migration of peripheral plasma cells to bone marrow through the MEK/ERK signaling pathway.The proportion of LLPCs in both bone marrow and peripheral blood of SLE patients is increased,and there is a significant positive correlation with SLEDAI scores,and a significant negative correlation with C3 and C4 scores,which can be regarded as a potential biomarker for the early diagnosis of SLE and its disease activity.