Study On Theactivation Of Bone Marrow-derived Mast Cells Pulsed With Wbo A- B.suis In Vitro

Objective:To investigate the recognition mechanism and immune response of bone marrow-derived mast cells(BMMC) to B.suis and the induction of immune response by Wbo A gene, thus provide a reliable experiment basis and a new way for the prevention of Brucellosis and Brucella vaccine which aim Wbo A- S2 strains as target antigen.Methods:1. BMMC were prepared by IL-3 and SCF to induce in vitro; BMMC were identified according to the culturable state and toluidine blue staining;2. BMMC were pulsed by B.suis Wbo A- S2, B.suis S2 according to MOI 100:1 and the normal BMMC acted as control, each group were stained with toluidine blue and Wright-Giemsa. The morphological change of BMMC were observed at different time points after pulsed so as to observe the activation of Brucella and Wbo A gene products in mast cells;3. The intake status of BMMC on B.suis Wbo A- S2 and B.suis S2 was observated by Laser confocal microscopy after BMMC pulsed with B.suis Wbo A- S2 and B.suis S2 which marked by FITC;4. BMMC were pulsed by B.suis Wbo A- S2, B.suis S2 according to MOI 100:1 and the normal BMMC acted as control. The levels of TNF-α, IFN-γ, histamine, IL-1,IL-6 and IL-12 in serum at different time points after pulsed were detected by ELISA so as to observe the biological active medium released by mast cells after pulsed with B.suis Wbo A- S2 and B.suis S2;5. The m RNA expression of TLR2, 4, 8 and 9 of mast cells were observated by RT-PCR;6. BMMC were pulsed by B.suis WboAS2, B.suis S2 according to MOI 100:1 and the normal BMMC acted as control. The cells were collected at 12 h and 24 h, and the m RNA expressions of TLR2, 4, 8 and 9 at different time points after pulsedwere detected by RT-PCR;7. The TLR4 and TLR8 protein expressions of BMMC after pulsed with B.suis Wbo A- S2 and B.suis S2 were detected by flow cytometry at 12 h.Results:1. The morphological change of BMMC after pulsed with Wbo A- S2 and S2(1) BMMC were observed by optical microscope after toluidine blue staining: there were a lot of partical in normal BMMC; the two pulsed groups had a significant degranulation after pulsed 15min; the particles in BMMC gradually reduced over time, and there was no particles in cytoplasm at 60 min, besides, Wbo A- S2-pulsed group degranulation was more obvious than S2-pulsed group;(2) BMMC were observed by optical microscope after Wright-Giemsa staining: the volume of BMMC in both of the two pulsed groups began to increase after pulsed15min; cytomembrane invagination and vacuoles began to appear in the cytoplasm after pulsed 30 min, but the cytomembrane was intact in the two groups;the vacuoles in cytoplasm were significantly increased and enlarged after pulsed45 min, besides, the cytomembrane began to bubble; the vacuoles filled almost all of the cytoplasm after pulsed 60 min, the cytomembrane of the Wbo A- S2-pulsed group was more complete than S2-pulsed group;(3) The results observed by Laser confocal microscopy: both of the Wbo A- S2 and S2 were located on the cytomembrane of BMMC after pulsed 1h, this indicated that Brucella had not been ingested into the mast cell, but adhere on the cell surface.2. The activation of Wbo A- S2 and S2 in BMMC(1) The levels of TNF-α in both of the two pulsed groups were significantly higher than the normal control group at different time points(P<0.05), but the levels of TNF-α in S2-pulsed group had no significant difference between groups at different time points, however, the content of TNF-α in Wbo A- S2-pulsed group gradually increased over time until 12 h, began to decrease at 24 h, and The level of TNF-αin the serum of the Wbo A- S2-pulsed group were significantly higher than the S2-pulsed group at different time points;(2) The levels of IL-6 in the S2-pulsed group had no significant difference compared with normal group(P>0.05), but the Wbo A- S2-pulsed group was significantly higher than the S2-pulsed group and the normal group at the same time points(P<0.01), and with time, the levels of IL-6 had an increasing trend until 12 h reached the peak, began to decrease at 24h;(3) The levels of histamine in both of the two pulsed groups were significantly higher than the normal control group at different time points(P<0.05), and with time, the content of histamine in the S2-pulsed group showed an increasing trend, but the Wbo A- S2-pulsed group had no significant change. Moreover, S2-pulsed group was significantly higher than the Wbo A- S2-pulsed group after pulsed 24h;(4) IFN-γ,IL-1,IL-12 were not detected at all pulsed times both in Wbo A- S2-pulsed group and S2-pulsed group.3. The recognition mechanism of BMMC to Wbo A- S2 and S2(1) The m RNA expressions of TLR2, 4 and 8 were detected in BMMC, but there was no TLR9;(2) The m RNA expressions of TLR4 in both of the two pulsed groups had a significant change at 12 h and 24 h, and it was significantly higher than the normal control group, in addition, the expression of TLR4 in Wbo A- S2-pulsed group was significantly higher than the S2-pulsed group, and it obviously weakened at 24 h in both of the two pulsed groups;(3) The m RNA expression of TLR8 in the S2-pulsed group had an increase trend at12 h, and compared with the normal group had no significant difference, however,the expression of TLR8 in Wbo A- S2-pulsed group was significantly higher than the normal group, and it obviously weakened at 24 h in both of the two pulsed groups;(4) The m RNA expressions of TLR2 in both of the two pulsed groups had no significant change at 12 h and 24h;(5) The protein expression of TLR4 and TLR8 were detected in the normal BMMC;(6) The protein expression of TLR4 in both of the two pulsed groups were significantly higher than the normal group; compared with the normal group, theprotein expression of TLR8 in the S2-pulsed group had no statistical significance,but Wbo A- S2-pulsed group was significantly higher than the normal group.Conclusion:1. Both of B.suis WboA- S2 and B.suis S2 can activate BMMC;2. B.suis WboAS2 and B.suis S2 are bound to mast cell surface and not ingested into the mast cell;3. Both of B.suis WboA- S2 and B.suis S2 can rapidly induce BMMC to secrete large number of cytokine, and B.suis Wbo A- S2 is much more powerful to activate BMMC than B.suis S2;4. TLR4 play an important role in the recognition mechanism of BMMC to Brucella.