Study On The Signaling Pathways Of Inflammation Activated By Haemophilus Parasuis Infection

Gl?sser’s disease caused by Haemophilus parasuis(HPS) has become one of the most important bacterial infectious diseases in swine herd, leading to great economic losses in the swine industry worldwide. The most distinct characteristic after HPS infection is causing severve systemic inflammation, including polyserositis, arthritis and meningitis, etc. To date, the molecular mechanisms of inflammation caused by HPS remain unclear. Our study focus on related signaling pathways of inflammation, growth and differentiation, analyze the molecular mechanism of TLR/NF-κB, TLR/MAPK and Wnt/β-catenin signaling pathways and discuss mechanisms of adheren junctions and biological effects among cells, which provide a basis for studying further infectious mechanism and pathogenesis. The main studies were as following: 1. HPS infection activates TLR/NF-κB signaling pathway in PK-15 cells(1) HPS infection activates NF-κB signaling pathway in PK-15 cellsReal-time RT-PCR was performed to detect the expression of IL-8, CCL4 and CCL5 regulated by NF-κB were significantly upregulated after HPS infection. HPS-infected cells treated with NF-κB inhibitor BAY11-7082 exhibited a reduced ability to upregulation of IL-8 and CCL4. Furthermore, Western blot was performed to detect the decrease of IκBα and the increase of p-p65. Laser scanning confocal microscope was used to observe NF-κB nuclear translocation. Dual-luciferase reporter system was used to detect that HPS infection activated NF-κB in a time- and dose-dependent manner.(2) The role of TLRs and adaptor molecules in NF-κB activation by HPS infection in PK-15 cellsThe transfection of specific siRNA for TLRs and adaptor molecules significantly inhibited the activation of NF-κB after HPS infection. Western blot found that TLR1, 2, 4, 6 and TRAF6 were upregulated in a time-dependent manner. These results showed that HPS infection induced NF-κB activation by TLR signaling pathway. 2. HPS infection activates TLR/MAPK signaling pathway in PK-15 cells(1) HPS infection activates p38 and JNK MAPK signaling pathway in PK-15 cellsReal-time RT-PCR was performed to detect treatment with SB202190(p38 inhibitor) and SP600125(JNK inhibitor) exhibited a reduced ability to upregulation of IL-8 and CCL4. Western blot detected that HPS infection upregulated the expression of p-p38 and p-JNK but not p-ERK, which showed that HPS infection activated p38 and JNK MAPK signaling pathway, but not ERK.(2) TLRs were involved in p38 and JNK MAPK signaling pathway in PK-15 cells afterHPS infectionThe siRNA experiment showed that psiTLR1, psi TLR2, psiTLR4 and psiTLR6 inhibited the phosphorylation of p38 and JNK but not ERK. These results showed that HPS infection activated p38 and JNK MAPK pathways by TLR1, TLR2, TLR4 and TLR6, but ERK MAPK was not involved in it. 3. The mechanism whereby HPS infection regulated RANTES transcription inPK-15 cells(1) HPS infection activates RANTES through NF-κB pathway in PK-15 cellsPromoter analysis by site-directed mutagenesis indicated that NF-κB, CRE and ISRE sites play essential roles in RANTES transcription, among which NF-κB is the most important. Through treated with the dominant-negative mutant of IκBα and NF-κB inhibitor BAY11-7082, HPS infection significantly inhibited the induction of RANTES. These results showed that HPS infection activates RANTES through NF-κB pathway.(2) HPS infection activates RANTES through MAPK pathway in PK-15 cellsThe inhibitor experiment showed that treatment with SP600125(JNK inhibitor) exhibited a reduced ability to upregulation of RANTES, but SB202190(p38 inhibitor) and U0126(ERK inhibitor) have no effect. PK-15 cells were transfected with the dominant-negative mutant of JNK and ERK, followed by infection with HPS. Dual-luciferase reporter system was performed to detect the dominant-negative mutant of JNK but not ERK significantly inhibited RANTES production. These results showed that HPS infection activated RANTES through JNK MAPK pathway.(3) HPS infection activates RANTES through TLRs pathway in PK-15 cellsTransfected with siRNA and dominant-negative mutants of TLRs and adaptor molecules significantly inhibited the induction of RANTES after HPS infection. These results showed that HPS infection activates RANTES through TLR1/TLR2/TLR4/TLR6-MyD88/TRIF-NF-κB and JNK MAPK pathway. 4. HPS infection activates Wnt/β-catenin signaling pathway in PK-15 and NPTRcells(1) HPS infection activates Wnt/β-catenin siganaling pathway in PK-15 and NPTR cellsDual-luciferase reporter system showed that HPS infection activates Wnt/β-catenin signaling pathway in PK-15 and NPTR cells. Western blot analysis showed that HPS infection decresed p-β-catenin and increased p-GSK3β. Laser scanning confocal microscope experiment showed that HPS infection induced β-catenin to translocate to the nucleus to regulate downstream target genes MMP7, COX2 and PAI-1, which enhanced cell mobility and weaken cell adhesion.(2) HPS infection disrupts cell adheren junctions in PK-15 and NPTR cellsCell adheren junctions comprise β-catenin and E-cadherin. The previous study has shown that HPS infection induced β-catenin to tanslocate from cell membrane to cell nucleus. Western blot and laser scanning confocal microscope experiment showed that E-cadherin expression was sharply reduced after HPS infection. CO-IP and laser scanning confocal microscope experiment showed that HPS infection broke the interaction between β-catenin and E-cadherin, indicating cell adheren junction was disrupted. These results showed that HPS infection weakened the interaction between β-catenin and E-cadherin to disrupt cell adheren junctions, causing bacteria easily to invade cells and systemic inflammation.In all, our study firstly reports that the signaling pathways of inflammation activated by HPS infection, NF-κB, TLR, MAPK and Wnt/β-catenin signaling pathway. The mechanism of chemokines IL-8, CCL4 and RANTES production was elucidated clearly. From a new angle, we demonstrated the pathogenesis and molecular mechanism of HPS infection, which established theoretical basis to develop safe and effective vaccine and medicine.