| Haemophilus parasuis(H.parasuis,HPS)is a commensal organism of the upper respiratory tract of pigs.This pathogen characterized by fmultiple serositis,arthritis,and meningitis.H.parasuis outer membrane protein P2(Omp P2)is the most abundant protein in the outer membrane,which can induce the host's inflammatory response.The sequence analysis revealed that Omp P2 protein is composed of eight or nine surface-exposed loops.we speculated that loops involved in the Omp P2-induced inflammatory response.In this study,we used the H.parasuis virulent SC096 strains and surface-exposed loops L-L8 of Omp P2 as the research objects,stimulate porcine alveolar macrophages(PAMs)in vitro and detected the types and expression levels of cytokine of loop-induced to study the role of loop in the Omp P2-induced inflammatory response;by analyzing the expression of key protein molecules in the TLR/NF-?B and MAPK pathways,we explored the molecular mechanism of loop in the Omp P2-induced inflammatory response.The results obtained are as follows:1.Loops L7 and L8 are involved in H.parasuis Omp P2-induced cytokine expressionTo research whether Omp P2 and Omp P2 loops L1-L8 can induce the expression of cytokine,we stimulated PAMs by extracting H.parasuis Omp P2 and synthetic Omp P2 loops L1-L8,then collecting samples for Real-time PCR after stimulation 6 h and 12 h,respectively.The resulted showed that the IL-1?,IL-1?,IL-6,IL-8,IL-17,IL-23,CCL-4and CCL-5 m RNA levels were significantly upregulated after PAMs were stimulated with H.parasuis Omp P2 at the concentration of 5 and 10 ?g/m L(P<0.05 or P<0.01).The m RNA levels of cytokines were upregulated after stimulated with loops L1-L8 at the concentration of 130 nmol/m L and 260 nmol/m L,among them,both loops L7 and L8 significantly upregulated the m RNA expression of the cytokine,in a time-and dose-dependent manner(P<0.05 or P<0.01).When the cells were stimulated by loop L7 at260 nmol/m L for 12 h,the transcription level of and IL-1? and IL-6 m RNA were up-regulated,which increased 8.8 times and 7.5 times,respectively.When the cells were stimulated by loop L8 at 260 nmol/m L for 6 h,the transcription level of IL-17 m RNA was increased 8.5 times.When stimulated for 12 h,the transcription level of IL-8 m RNA was increased 7.2 times,and the transcription level of IL-23 m RNA increased 7.4 times.The above results indicate that among the eight loops,loops L7 and L8 are peptides mainly involved in H.parasuis Omp P2-induced cytokine expression.To verification that loops L7 and L8 induce IL-6 and IL-8 protein expression,cells culture supernatants were collected after stimulation with 10 ?g/m L of H.parasuis Omp P2,260 nmol/m L of loops L7 and L8 for 12 h,and the expression of IL-6 and IL-8 cytokine protein was detected by ELISA detection kit.The results showed that the protein expression levels of IL-6 and IL-8 were significantly upregulated after loops L7 and L8 stimulation(P<0.05 or P<0.01).After loop L7 stimulation,the protein expression levels of IL-6 and IL-8 increased 5 times and 3.4 times,respectively;when loop L7 stimulated,the protein expression levels of IL-6 and IL-8 increased 5.60 times and 5.8 times,respectively.The results indicated that loops L7 and L8 are involved in the expression of cytokine proteins induced by H.parasuis Omp P2.In order to further determine that loops L7 and L8 are involved in H.parasuis Omp P2-induced cytokine expression,we constructed both omp P2?Loop7 and omp P2?Loop8 mutants strains,and extracted the mutants strains Omp P2 to stimulate PAMs,then collecting samples for Real-time PCR after stimulation 6 h and 12 h,respectively.Compared with the Omp P2 stimulation,the cytokine m RNA expression induced by the omp P2?Loop7-Omp P2 and omp P2?Loop8-Omp P2 was noticeably decreased,in a concentration-and time-dependent manner(P<0.01).When these mutant Omp P2 s were used at 5 ?g/m L or 10 ?g/m L for 6 h or 12 h,IL-1?,IL-1?,IL-6,IL-8,IL-17,IL-23,CCL-4 and CCL-5 m RNA transcription levels were only 10%~50% of Omp P2 stimulation.This result further illustrates that loops L7 and L8 play an important role in the expression of cytokine induced by H.parasuis Omp P2.By alignments the amino acid sequence of Omp P2 loops L7 and L8 of 15 serovars of H.parasuis reference strains.The results showed that loops L7 and L8 are relatively conserved in Omp P2 protein among the most virulent reference H.parasuis,indicating that both loops L7 and L8 belong to relatively conserved peptides of Omp P2.Therefore,loops L7 and L8,as the most conserved and active peptides in H.parasuis Omp P2,can induce the expression of cytokine in PAMs and play an important role in the inflammatory response caused by H.parasuis Omp P2 infection.2.Loops L7 and L8 are participate in H.parasuis Omp P2-induced activation of TLR/NF-?B and MAPK signaling pathwayIn order to verification that the NF-?B and MAPK signaling pathways are activated by loops L7 and L8 in the H.parasuis Omp P2-induced inflammatory response,we co-transfecting PAMs with luciferase reporter plasmid p NF-?b-Luc or p AP-1-Luc,respectively,with p RL-TK,which served as an internal control.At 24 h after transfection,PAMs were treated with 5 ?g/m L and 10 ?g/m L H.parasuis Omp P2,130 nmol/m L and 260nmol/m L loops L7 and L8 or a scrambled peptide for 12 h,then analysis by dual Luciferase reporting system.Compared with the control group,the fluorescein values of NF-?B and AP-1 significantly increased in a dose-dependent manner in Omp P2 and both loop L7 and L8-treated PAMs(P<0.01).When H.parasuis Omp P2 used at 10 ?g/m L to stimulated the cells,the expressions of NF-?B and AP-1 fluorescence upregulated 22 times and 13 times,respectively(P<0.01).When 260 nmol/m L loops L7 and L8 stimulated,the expressions of NF-?B fluorescence upregulated 25 times and 23.5 times,respectively(P<0.01);and the expressions of AP-1 fluorescence upregulated 15 times and 12 times,respectively(P<0.01).The above results indicate that loops L7 and L8 are involved in activating NF-?B and MAPK signaling pathways during the H.parasuis Omp P2-induced inflammatory response.It is known that the pattern recognition receptors of H.parasuis are mainly TLR1,TLR2,TLR4,TLR5 and TLR6,to further study which TLR receptors are involved in recognizing loops L7 and L8 activation of NF-?B and MAPK signaling pathways,we co-transfecting PAMs with interference molecule and luciferase reporter plasmid,with p RL-TK,which served as an internal control.At 24 h after transfection,PAMs were treated with 5 ?g/m L H.parasuis Omp P2,130 nmol/m L loops L7 and L8 for 12 h,then analysis of dual Luciferase reporting system after stimulation.The result show that,the fluorescence levels of NF-?B and AP-1 were significantly decreased after cell transfected by psi TLR1, psi TLR2,psi TLR4 and psi TLR6(P<0.01).When transfected with psi TLR4,loop L7 induced the fluorescence values of NF-?B and AP-1 were significantly lowest(P<0.01),which were down-regulated 4.3 times and 3 times,respectively.loop L8 induced the fluorescence values of AP-1 were significantly lowest(P<0.01),which were down-regulated 1.9 times.The above results indicate that loops L7 and L8 through the TLR1,TLR2,TLR4 and TLR6 pathways involved in the activation of NF-?B and MAPK signaling pathways during the H.parasuis Omp P2-induced inflammatory response.In order to further understand which signaling pathway-related proteins are involved in loops L7 and L8 activation of NF-?B and MAPK signaling pathways during the H.parasuis Omp P2-induced inflammatory response,we used Western blot to detect the expression of ERK,JNK,p38 and p65 proteins.The results showed that,compared with the control group,after 10 ?g/m L H.parasuis Omp P2 stimulated PAMs,the phospho-ERK and phospho-p65 phosphorylation levels was the highest at 0.5 h,which increased by 1.5 times and 2.7 times(P<0.01).The phosphorylation level of phospho-JNK was the highest at 3 h,increased 1.5 times(P<0.01).The phosphorylation level of phospho-p38 was the highest at1 h,increased 3 times(P<0.01).After 260 nmol/m L loop L7 stimulated PAMs for 0.5 h,the phosphorylation level of phospho-p65 was significantly up-regulated(P<0.05)and increased 1.4 times.The phosphorylation level of phospho-JNK was the highest at 3 h,increased 1.3 times(P<0.05).When 260 nmol/m L loops L7 and L8 stimulated PAMs for 3h,the phosphorylation level of phospho-p38 was the highest(P<0.05),increased 2.3 times and 2.4 times,respectively(P<0.01).The above results indicate that during the inflammatory response induced by H.parasuis Omp P2,loop L7 invovled in activation of NF-?B and MAPK signaling pathways through the p65,p38,and JNK pathways,loop L8 participates in the activation of NF-?B and MAPK signaling pathways through the p38 pathway.When we used p65 and JNK inhibitors to applied PAMs,we used Western blot to detect the expression of JNK and p65 proteins.Compared with the control group,after H.parasuis Omp P2 and loop L7 stimulated PAMs,the phospho-ERK and phospho-p65 phosphorylation levels was significantly decreased(P<0.05 or P<0.01).When 10 ?g/m L of Omp P2 stimulated the cells for 1 h,phospho-ERK phosphorylation levels was down-regulated the most,was 25.8 times.When 260 nmol/m L of Omp P2 stimulated the cells for 0.5 h,phospho-ERK phosphorylation levels was down-regulated the most,was16.7 times.Next,we used Real-time PCR to detect the changes of cytokines transcription levels induced by loop L7.It was found that the transcription level of cytokine m RNA was significantly down-regulated after the inhibitors applied PAMs(P<0.05 or P<0.01).After the ERK inhibitors to applied PAMs,when loop L7 stimulated cells of 260 nmol/m L for 1 h,the cytokine m RNA transcription level was less than 30% of that induced by the control group.After the p65 inhibitors to applied PAMs,when loop L7 stimulated cells of 260nmol/m L for 1 h,the cytokine m RNA transcription level was down-regulated by at least50%.The results indicate that loop L7 activates NF-?B and MAPK signaling pathways through the p65 and JNK pathways,thereby inducing the transcription of cytokines IL-1?,IL-8,IL-17 and CCL-4. |
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