Role Of The TLR4/NF-?B Pathway In B Lymphocytes Developmental Disorders Caused By CD38 Gene Deficiency

Objective:The spleen is an important peripheral immune organ in the body,and it is a place to trigger the immune response.The aggregated immune cells are lymphocytes,macrophages and so on,B lymphocytes accounted for about 60%percent of the total number of splenic lymphocyte.In the body,only B lymphocytes can produce antibodies,and it can also act as an antigen presenting cell,participate in humoral immunity and cellular immunity and play an important role.The development of B lymphocytes has muti-steps and undergone complicated signal regulation,and its developmental disorders can cause autoimmune diseases,tumors and so on.Our preliminary study found that in the physiological state,CD38 gene deficiency mice shew splenic B lymphocyte developmental disorder,and more significantly when stimulated with LPS.TLR4^mutut mice shew bone marrow B cell development disorder;These results suggest that CD38 and TLR4 may play an important role in the developmental disorder of B lymphocytes.This study aims at:1.Causes of the developmental disorder of B lymphocytes in CD38 gene deficiency,2.The role of TLR4 in the developmental disorder of B lymphocytes induced by CD38 gene deficiency in physiological state and its'key points.These explorations will provide a new theoretical basis for the study of the factors influencing the B cell developmental disorder,which will help to provide new ideas for the treatment of clinical B-cell developmental disorders.Methods:1.The splenic B lymphocytes were isolated and purified by immunomagnetic beads?MACS?in 6 weeks old female WT?C57BL/6?mice and stained with CD45R PerCP-Cyanine5.5,CD3e FITC,Fluorescence Activated Cell Sorting was used to identify the purity of B lymphocytes after purification.2.The splenic B lymphocytes were isolated and purified,and the mRNA expression levels of CD38 and TLR4 in splenic B lymphocytes were detected by real-time fluorescence quantitative PCR.3.The splenic cells and splenic B lymphocytes were isolated and counted under microscope.The total number of spleen cells and B cells in WT,CD38^-/-C3H/HeN and CD38^-/-C3H/HeJ mice were compared.4.The purified B lymphocytes of spleen of mice were stained with the corresponding antibody,and the B lymphocytes maturation,cell apoptosis and cell cycle were detected by flow cytometry.5.LPS stimulated splenic B lymphocytes of mice were isolated and cultured in vitro,and the proliferation of B lymphocytes in WT,CD38^-/-C3H/HeN and CD38^-/-C3H/HeJ mice was detected by Cell Counting Kit-8 method.6.The expression levels of TLR4,Bcl-2,caspase-8,NF-kappaB1,Phospho-NF-kappaB1,p65,Phospho-p65,and Sirt1 proteins in splenic B lymphocytes of WT,CD38^-/-C3H/HeN and CD38^-/-C3H/HeJ mice were detected by Western blotting.7.The mRNA expression of P53,TNF-?,IL-1?,IL-4,BAFF,BAFFR,RelB in B cells of WT,CD38^-/-C3H/HeN and CD38^-/-C3H/HeJ mice was detected by real-time fluorescent quantitative PCR.8.The protein expression level of TNF-alpha and IL-1?in serum of WT,CD38^-/-C3H/HeN and CD38^-/-C3H/HeJ mice was determined by ELISA.9.Histological differences in spleen were observed by hematoxylin-eosin staining in the spleen of WT,CD38^-/-C3H/HeN and CD38^-/-C3H/HeJ mice.Results:1.The splenic B lymphocytes were isolated and purified by MACS,and the purity of CD3e^-CD45R⁺cells was 96.8%by FACS.2.Compared with WT mice,CD38 gene expression was significantly decreased and the expression of TLR4 was obviously higher in spleen B lymphocytes of CD38^-/-C3H/HeN mice.Compared with CD38^-/-C3H/HeN mice,there was no significant difference in the expression of CD38 gene and the expression of TLR4gene was significantly decreased in spleen B lymphocytes of CD38^-/-C3H/HeJ mice.3.Compared with WT mice,the total number of splenic cells and splenic B lymphocytes in CD38^-/-C3H/HeN mice were significantly decreased.Compared with CD38^-/-C3H/HeN mice,there was no significant difference in the number of both spleen cells and B lymphocytes in CD38^-/-C3H/HeJ mice.4.Compared with WT mice,there was significant difference?the proportion of T1 B cell subsets increased from 17.5%to 28.9%?in T1-T2 B cell subsets in spleen of CD38^-/-C3H/HeN mice,and Mature B cells were significantly reduced.Compared with CD38^-/-C3H/HeN mice,the transition of T1-T2 cell subsets was increased slightly in CD38^-/-C3H/HeJ mice?the proportion of T1 B cell subsets increased from28.9%to 33.1%?,but there was no significant difference.And mature B cells decreased slightly with no significant difference.5.Compared with WT mice,the proliferation of B lymphocytes has no significant change in CD38^-/-C3H/HeN mice.But compared with CD38^-/-C3H/HeN mice,the proliferation of splenic B lymphocytes was significantly reduced in CD38^-/-C3H/HeJ mice.6.Compared with WT mice,the apoptosis of splenic B lymphocytes in CD38^-/-C3H/HeN mice was significantly increased.Compared with CD38^-/-C3H/HeN mice,the apoptosis of splenic B lymphocytes in CD38^-/-C3H/HeJ mice was significantly decreased.7.Compared with WT mice,the percentage of S phase cells increased in CD38^-/-C3H/HeN mice?average percentage from 1.32%to 1.89%?.Compared with CD38^-/-C3H/HeN mice,the percentage of S phase decreased in CD38^-/-C3H/HeJ mice?average percentage from 1.89%to 1.03%?.All had significant difference.8.Compared with WT mice,the expression of TLR4,Phospho-NF-kappa B1/NF-kappa B1,Phospho-p65/p65 and Sirt1 protein in splenic B lymphocytes of CD38^-/-C3H/HeN mice were increased significantly.Compared with CD38^-/-C3H/HeN mice,the expression of TLR4,Phospho-NF-?B1/NF-?B1,Phospho-p65/p65 and Sirt1protein decreased significantly.There was no significant difference between Bcl-2and caspase-8.9.Compared with WT mice,the mRNA expression levels of TNF-?,IL-1?,IL-4,BAFF,BAFFR and p53 in splenic B lymphocytes of CD38^-/-C3H/HeN mice decreased significantly.Compared with CD38^-/-C3H/HeN mice,the mRNA expression levels of BAFFR,TNF-?and IL-1?were significantly increased,while RelB,BAFF,IL-4 and p53 had no significant changes in the expression of mRNA.10.Compared with WT mice,the expression level of TNF-?and IL-1?in serum ofCD38^-/-C3H/HeNmicewassignificantlydecreased.Comparedwith CD38^-/-C3H/HeN mice,there was no significant difference in TNF-?and IL-1?with CD38^-/-C3H/HeJ mice.11.Compared with WT mice,the number of inflammatory cells decreased in CD38^-/-C3H/HeN mice.Compared with CD38^-/-C3H/HeN mice,the number of inflammatory cells increased in spleen of CD38^-/-C3H/HeJ mice.Conclusions:1.The genotype of mice was stable,the purification of mouse spleen B lymphocytes separated by magnetic beads is satisfiled with the requirements of experiment.The reliability of the experimental results was ensured.2.In the physiological condition,the development disorders of B lymphocytes in spleen of CD38 gene deficient mice due mainly to obstacled maturation,increased apoptosis and cell cycle arrest,but there was no significant difference in cell proliferation.TLR4 plays an important role in the increasing of apoptosis and arresting of cell cycle of B lymphocytes induced by CD38 gene deficiency in mice.3.The deletion of CD38 gene can activate the TLR4/NF-?B pathway,but the activated NF-?B was deacetylated and inactivated by Sirt1 in the nucleus,the expression of downstream inflammatory factor was inhibited,the apoptosis of splenic B lymphocytes was increased and the infiltration of inflammatory cell was decreased.4.The changes in the species and relative number of inflammatory cells in the spleen of mice were consistent with those of inflammatory factors.