Investigation On Toll-like Receptors Of Bovine Mammary Epithelial Cells Infected By M.bovis

Bovine mastitis is a high incidence disease and large proportion disease of dairy cattle. In the dairy industry, mastitis caused a huge loss annually. Mycoplasma bovis(M. bovis) is an important pathogens of mastitis. M. bovis mastitis has the features of high infection rates, long duration, and often accompanied with arthritis or other diseases. As we all know, M. bovis lack the cell wall structure, which limited the efficient application of most broad-spectrum antibiotics, and currently experimental M. bovis vaccines often have poor immune protection. There is an almost ―helpless‖ circumstance in the prevention of M. bovis mastitis, so efficient prevention of M. bovis mastitis need new ideas and new strategies.There is a complete set of defense system for denfensing pathogens in the mammary gland of cows. Mammary innate immune system starts immediately when infected by pathogens. After the pathogens infect gland cistern through teat canal tissue, mammary epithelial cells as internal solid walls begin to play the role of sentinel. Pattern recognition receptors –TLRs are rich in mammary epithelial cells. Pathogen-associated molecular patterns(PAMPs) could quickly recognized by TLRs, then activating host innate immune cells, and initiating natural or specific immune to destroy the invaders. Investigating the interaction of M. bovis and mammary epithelial cells, and what kind of TLRs participation and how to play the role, this investigation would contributed to a better understanding of the pathological mechanism in bovine M. bovis mastitis, and the theoretical foundation in bovine M. bovis mastitis’ s efficient prevention and treatment.In this study, we are intended to establish a reliable M. bovis infection model of bovine mammary epithelial cells(BMECs) in vitro, through the research on transcript and protein levels of TLRs signaling pathway related molecules in M. bovis infection, determining the components involved in the regulation of innate immunity, providing a theoretical basis for M. bovis mastitis mechanism, and also laying the theoretical foundation of exploring new drug target for intervention bovine mammary inflammatory.The main contents and results were described as follows: 1 Extraction and identification of primary bovine mammary epithelial cells.Cells were extracted from healthy cow mammary tissue, through keratin 18 immunofluorescence and PCR amplification of specificity MUC-1 gene to confirm the extracted cells were primary BMECs. The extracted BMECs grew well in vitro, and had no external mycoplasma contamination. 2 Establishment in vitro model of M. bovis infected BMECs.M. bovis infected BMECs can promote inflammatory cytokines IL-6, TNF-α and chemokines IL-8. According to the release of these cytokines and combine BMECs’ viability under different infection conditions to determine the best conditions for infection.Using 39 YC which originated from clinical M. bovis mastitis, mastitis standard strain PG45 and Mycoplasma pneumonia clinical isolates HB0801 served as two parallel groups, blank control group was not infected with any pathogens. M. bovis infected BMECs with 500 MOI and 1000 MOI respectively, then determined BMECs’ viability by trypan blue staining and MTT assay, finally relative expression of inflammatory cytokines IL-6, TNF-α and chemokines IL-8 were detected by qPCR. The results were taked control group as a standard:(1) BMECs’ viability showed no significant difference under 500 MOI and 1000 MOI;(2) MOI=500, the expression of IL-6 and TNF-α had significant increase(P<0.01) and IL-8 was significantly increased(P<0.001) in 39 YC group from infected 16 h to 24h; The expression of IL-6 in PG45 group was increased after infected 20h(P<0.05), the expression level of IL-8 was significantly increased in 20h(P<0.001), the expression level of TNF-α was increased from 16h(P<0.05), and 24 h was increased significantly(P<0.001); Expression of IL-6, IL-8 and TNF-α in HB0801 group were increased significantly different(P<0.001) in 24h;(3) MOI=1000, the expression of IL-8 and TNF-α in 39 YC group were significantly higher(P<0.001) in 16 h, IL-6 also increased significantly(P<0.01); Expression of IL-6 and IL-8 were significantly higher after infection 16h(P<0.001) in PG45 group, but the expression of TNF-α was significant increased(P<0.01) after infection 4h, until 16 h there was a very significant increased(P<0.001); HB0801 group’s IL-6, IL-8 and TNF-α were increased significantly under 24h(P<0.001), and in 20 h began to significantly elevated;(4) MOI=1000, the expression of IL-6, IL-8 and TNF-α had a higher degree than MOI=500 after M. bovis infected BMECs.The BMECs’ survival rate did not change significantly after M. bovis infected, three M. bovis strains were induced the expression of inflammatory / chemokine factors such as IL-6, IL-8 and TNF-α, and the infection time and dose dependent changes in the production of inflammatory / chemokine factors were also presented, which indicated that BMECs initiated the inflammatory response to resist the invasion of M. bovis, the model of M. bovis infected BMECs was successfully established in vitro. 3 Screening TLRs in BMECs and TLRs cross-talk after M. bovis infected.BMECs were infected by the three different M. bovis strains within 24 h. At the same time, the control group was not infected, cells were collected in different time point, qPCR and Western blot were used to detect the TLRs species after infection in mRNA and protein levels respectively.qPCR results showed that the normal BMECs expressed TLRs(TLR1-10) except TLR8; After infected by M. bovis, TLR1, TLR2, TLR3, TLR6 and TLR9 involved in the regulation of M. bovis infection. Results were also compared with control group:(1) After infection 10 h, 39 YC group’s TLR2, TLR3 and PG45 group’s TLR9 mRNA expression were significantly increased(P < 0.001), the expression of TLR6 increased significantly in 39 YC group(P < 0.05);(2) After infected 16 h, 39 YC group’s TLR2, PG45 group’s TLR6, HB0801 group’s TLR2 and TLR6 mRNA expression were increased significantly(P <0.001);(3) After infection 20 h, the mRNA expression of TLR2 in 39 YC group and PG45 group’s TLR9 had a significant increase too(P<0.001), the expression of TLR1 increased significantly in 39 YC group(P<0.05);(4) After infection 24 h, TLR2 expression in 39 YC group, HB0801 group’s TLR9 expression and PG45 group’s TLR2, TLR9 mRNA expression were all increased significantly(P <0.001); Western-blot results showed that the expression of TLR1/2/3/6/9 protein were up-regulated in all infection groups compared with the control group.After BMECs were infected by M. bovis, cells were collected every 2 hrs, and the relative mRNA expression of TLR1, TLR2 and TLR6 were determined by qPCR, respectively. Results: After infection, the expression of these three kinds of TLRs increased, and the expression of TLR1 and TLR2 issued a certain similarity trend, the same as expression of TLR2 and TLR6. In order to further verify the synergy between TLR1, 2 and 6, cells were collected from M. bovis infection in 16 h and 20 h, respectively. Using Co-immunoprecipitation assay to detect interactions between TLRs, which showed that these M. bovis strains could cause TLR1/TLR2 interaction and TLR2/TLR6 interaction. 4 Activation of TLRs signaling pathway downstream NF-κB and MAPK signal pathwayActivation of downstream signaling pathways required signal transduction in adaptor proteins. BMECs were infected by three M. bovis strains, blank group served as control group. The expression levels of MyD88 and TRIF increased in different degrees. The increased expression of P-P65 and the reduced expression of P-IκBα indicated the activation of NF-κB signaling pathway, and the increased of P-c-Jun expression marked the activation of MAPK signaling pathway. P-P65, P-IκBα and P-c-Jun protein expression was measured by Western Blot after M. bovis infected BMECs. The expression of P-P65 and P-c-Jun in three M. bovis groups were higher than those in the control group, the expression of P-IκBα was lower than that of control group, and the gray value is almost zero.PDTC is an inhibitor of the NF-κB signal pathway, and could block the activation of NF-κB signaling pathway. Pretreated BMECs with 50μM PDTC for 1h, then M. bovis infected BMECs for 24 h, the release of inflammatory factors IL-6 and TNF-α were detected by qPCR. Expression of IL-6 and TNF-α did not increased during infection, and it proved that M. bovis infected BMECs could activate the NF-κB signaling pathway.