The Effects Of TLR4 Gene In The Obstacled B Cell Development In CD38^-/- Mice

Objective:TLR4 is an important molecule related to innate immune, it play an important role in the body’s inflammatory response by recognizing pathogen associatedmolecular patterns. At the meantime, TLR4 is also involved in the development and progression of cancer, atherosclerosis and autoimmune diseases. Studies showed that CD38 gene knockout mice have the obstacled B cell development,especially obstacled at immature B cells phase in which the apoptosis of T1 cells is decreasing and the development of T2 cells is obstacled. Our previously study found that the expression of TLR4 gene is specifically expressed in mature B cells in bone marrow, and it’s expression is increased significantly with the migration of mature B cells from bone marrow to spleen. The B cell development of TLR4 mut mice is also obstacled. In addition, the expression of TLR4 in spleen is also decreased in CD38-/- mice. These results hint that the influence of TLR4 and CD38 on B cell development may have some inner relations.Our study aims at, to further explore the role of CD38 and TLR4 in B cell development and its mechanism, to clarify the interaction of CD38 and TLR4 in promoting the B cell development and it’s possible signal pathway, to provide new clues and basis for further research on B cell development.Methods:1. Female mice of both WT and CD38-/-(C57BL/6J, 6w) was injected with LPS intraperitoneally, spleen B cells were separated by magnetic bead separation technique 2 weeks later, and the development, cell cycle and apoptosis of B cells were detected by FACS. 2. CD38-/- and CD38-/- TLR4 mut mice were hybrided by CD38-/- female mice(C57BL/6J) with C3H/He N and C3H/He J male mice respectively. 3. DNA were extracted from the tail of mice, and the expression of CD38 genewere detected by polymerase chain reaction(PCR), which can help to identify the successfully knockout of CD38 gene. Extract the RNA from ears of CD38-/- and CD38-/- TLR4 mut mice respectively, and the expression level of m RNA were detected by RT-PCR. 4. Spleen B cells of both CD38-/- and CD38-/- TLR4 mut mice were purified by magnetic bead separation technique, the m RNA expression level of TLR4 and inflammatory factors(including TNF-α, MCP-1 and IL-1β) were detected by through the Realtime PCR. 5. Extract protein of spleen B cells from CD38-/- and CD38-/- TLR4 mut mice respectively, the expression level of TLR4, Sirt1, NF-κB(p65)and AC- NF-κB(AC-p65) were detected by Western Blot. 6. The histological differences in spleen of CD38-/- and CD38-/- TLR4 mut mice were identified by both HE and IHC-P staining. 7. The development, cell cycle and apoptosis of B cells were detected by Flow Cytometry with the staining of related surface antibody(including CD21 FITC, CD24 PE and CD45 R PE Cy5.5, etc.).Results:1. B cell development was promoted by LPS stimulation significantly. But compared with WT mice, the development and maturation of B cells in CD38-/- mice were decreased significantly, and the cell cycle was retardant in S phase. 2. CD38-/- TLR4 mut mice were prepared and identificationed successfully. 3. Compared with the CD38-/- mice, the m RNA expression level of CD38-/-TLR4 mut mice in spleen B cells were decreased significantly in TLR4 and inflammatory factors including MCP-1 and TNF-α. 4. Compared with the CD38-/- mice, the protein expression level of CD38-/-TLR4 mut mice in spleen B cells were decreased significantly in TLR4 but showed no obvious changing in the expression of Sirt1, NF-κB(p65)and AC- NF-κB. 5. Compared with the CD38-/- mice, the number of macrophages and neutrophils in CD38-/- TLR4 mut mice in spleen were decreased significantly. 6. Compared with CD38-/- mice, the obstacle of spleen B cell development weresevere in CD38-/- TLR4 mut mice, which focus on the obstacle transformation of T1 to T2 cells, but we haven’t seen significant change in both cell cycle and apoptosis.Conclusion:1. TLR4 gene is involved in the obstacled B cell development in CD38-/- mice, and this have no correlation with Sirt1 pathway. 2. CD38 gene knockout can help to inhibit the expression of TLR4 gene, reduce the expression of its downstream inflammatory factors, such as MCP-1 and TNF-a, reduce the infiltration of inflammatory cells such as macrophages and neutrophils. 3. CD38-/- and TLR4 mut inhibit the maturation of B cells synergistically, especially inhibit the transformation of T1 to T2 cells.