| PART ONE LPS/CpG ODN AGGRAVATED AIRWAY INFLAMMATION AND AIRWAY HYPERRESPONSIVENESS IN THE LATE STAGE OF RSV INFECTED MICEObjective: Respiratory syncytial virus(RSV)is the main viral pathogen of lower respiratory tract infections(LRTIs)in children under 5 years old worldwide.Severe RSV infections were associated with recurrent wheezing and asthma.Bacterial colonization is usually accompanied in children with virus-induced repeated wheezing,and viral infection accompanied with bacterial colonization may have impact on disease severity.However,its mechanisim is remain unclear.The bacterial cell wall component Lipopolysaccharide(LPS)and bacterial DNA CpG oligonucleotide(CpG ODN)are widely distributed following the path of pathogen,they can last for a long time after inactivation of bacteria,which can promote inflammation by recongnizing Toll like receptos(TLRs).The virus has been cleared in the late stage of RSV infection,however,weather restimulated with LPS or CpG ODN can affect airway inflammation in the late stage of RSV infection is unclear.Therefore,we established a mice model in this part to imitate bacterial components LPS/CpG ODN stimulation in the late stage of RSV infection,to observe its effect on airway inflammation and AHR.Methods: Balb/c female mice aged 6-8 weeks were randomly divided into control group,CpG group,LPS group,RSV group,RSV+LPS group,RSV+CpG group,each group containing 8-10 mice;On the 0th day post RSV infection,control group,CpG group,LPS group were administrated by intranasal with 100?l PBS;RSV group ? RSV+LPS group and RSV+CpG group were administrated by intranasal with 100?l RSV-A2;On the 35th-41 st days post RSV infection,control group and RSV group were administrated by intranasal with 50?l PBS every 2 days;CpG group,RSV+CpG group were administrated by intranasal with 50?l CpG(0.1?g/?l)every 2 days;LPS group,RSV+LPS group were administrated by intranasal with 50?l LPS(0.2?g/?l)every 2 days;On the 42 nd days post RSV infection,airway hyperreactiveness were measured,mice were sacrificed for bronchoalveolar lavage fluid(BALF)collecting and inflammatory cells counting,lung tissues for pathological assessment.Results: 1,RSV+LPS group mice underwent a significant weight loss when 36 th days(P<0.05),42 nd days(P<0.05)compared to control group;RSV+CpG group mice underwent a significant weight loss when 41 st days(P<0.0001),42 nd days(P<0.0001)compared to control group;There was no significant difference in weight change of LPS group and CpG group compared to control group;2,Macropathological pictures showed that only RSV+CpG group mice with inflammatory nodules formed in lungs(66.7%);3,RSV+CpG group and RSV+LPS group had obvious lung pathological changes compared to RSV group,and RSV+CpG group was more severe for inflammatory nodules formation;RSV+LPS group has higher scores of lung inflammatory pathology compared to the RSV group by pulmonary bronchioles inflammation(P<0.01),pulmonary vessels inflammation(P<0.01),pulmonary interstitial inflammation(P<0.01)and total pulmonary inflammation(P<0.01);RSV+CpG group has higher scores of lung inflammatory pathology than control group,CpG group and RSV group,by pulmonary bronchioles inflammation(P<0.0001),pulmonary vessels inflammation(P<0.0001),pulmonary interstitial inflammation(P<0.0001)and total pulmonary tissue inflammation(P<0.0001);4,LPS group(P<0.05),RSV group(P<0.01),RSV+LPS group(P<0.0001)have higher total inflammatory cells in BALF compared to the control group;RSV+LPS group was further elevated compared to the LPS group(P<0.01),and elevation of neutrophils in RSV+LPS group was most significant,higher than the control group(P<0.05)and RSV group(P<0.01);5,RSV group(P<0.01),RSV+LPS group(P<0.0001)and RSV+CpG group(P<0.01)had significantly higher Penh values than the control group;RSV+LPS group(P<0.001)and the RSV+CpG group(P<0.01)has higher Penh values compared to RSV group;RSV+CpG group has higher Penh values than RSV+LPS group(P<0.01),meanwhile stimulated in a lower concentration of acetylcholine(3.125 mg/ml).Conclusion: The bacterial components LPS/CpG ODN can both significantly aggravate the airway inflammation and AHR in the late stage of RSV infection.However,CpG ODN induced a more severe aggravation of RSV associated lung inflammation,AHR,with formation of pulmonary inflammatory nodules.PART TWO LPS AGGRAVATED AIRWAY INFLAMMATION AND AIRWAY HYPERRESPONSIVENESS THROUGH ERK/MMP-12 IN THE LATE STAGE OF RSV INFECTED MICEObjective: The results of the first part showed that bacterial components LPS could significantly aggravate airway inflammation and AHR on the 42 nd days post RSV infection.Our preliminary results have demonstrated that matrix metalloproteinases(MMPs),especially MMP-12 are important in RSV pathogenesis.It was reported that MAPK family member ERK participate in the regulation of MMP-12 production.Therefore,specific inhibitor was used in this part to observe the effects of ERK on MMP-12 production,airway inflammation and AHR in mice restimulated with LPS in the late stage of RSV infection.Methods: Balb/c female mice aged 6-8 weeks were randomly divided into control group,LPS group,RSV group,RSV+LPS group,RSV+LPS+SB203580 group,RSV+LPS+SP600125 group and RSV+LPS+PD98059 group,each group containing 8-10 mice;On the 0th day post RSV infection,control group,LPS group were administrated by intranasal with 100?l PBS;RSV group ?RSV+LPS group,RSV+LPS+SB203580 group,RSV+LPS+SP600125 group and RSV+LPS+PD98059 group mice were administrated by intranasal with 100?l RSV-A2;On the 35th-41 st days post RSV infection,RSV+LPS+SB203580 group mice were administrated with p38 inhibitor SB203580,RSV+LPS+SP600125 group mice were administrated with JNK inhibitor SP600125,RSV+LPS+PD98059 group mice were administrated with ERK inhibitor PD98059(every day,bid on 35 th dpi.,8 times in total);Meanwhile,on the 35th-41 st days post RSV infection,control group and RSV group were administrated by intranasal with 50?l PBS per two days;LPS group,RSV+LPS group,RSV+LPS+SB203580 group,RSV+LPS+SP600125 group and RSV+LPS+PD98059 group mice were administrated by intranasal with 50?l LPS(0.2?g/?l)per 2 days;On the 42 nd days post RSV infection,airway hyperreactiveness were measured,mice were sacrificed for bronchoalveolar lavage fluid(BALF)and lung tissues collecting;the inflammatory cells in BALF were counted,lung pathological changes were assessment;MMP-12 expression in BALF were detected by ELISA;p-p38/p38,p-JNK/JNK,p ERK/ERK expression in lung tissues were detected by WB.Results: 1,The p-ERK expression in RSV+LPS group was significantly higher than that in RSV group(P<0.001);p-ERK expression was significantly decreased after treatment with ERK inhibitor PD98059(P<0.0001);2,The expression of MMP-12 in BALF was significantly increased in RSV+LPS group than that in RSV group and LPS group(P<0.01);MMP-12 expression in BALF was significantly decreased after treatment with ERK inhibitor PD98059(P<0.05);MMP-12 expression in BALF did not change in RSV+LPS+SP600125 group and RSV+LPS+SB203580 group,compared to that in RSV+LPS group;3,RSV+LPS group mice showed obvious pathological injury of lung tissues: with higher inflammatory scores in pulmonary bronchioles inflammation(P<0.05),pulmonary interstitial inflammation(P<0.01)and total pulmonary inflammation(P<0.05)compared to RSV group;After treatment with ERK inhibitor PD98059,the inflammatory scores was decresed compared to RSV+LPS group;4,RSV+LPS group mice showed significantly higher inflammatory cells in BALF by total inflammatory cells(P<0.05),monocytes/macrophages(P<0.05),neutrophils(P<0.01)compared to RSV group;After treatment with ERK inhibitor PD98059,inflammatory cells in BALF was significantly decresed compared to RSV+LPS group;5,The airway hyperresponsiveness was much higher in the RSV+LPS group compared to control group(P< 0.001),RSV group(P<0.001),LPS group(P<0.001);After treatment with ERK inhibitor PD98059,airway hyperresponsiveness was significantly decresed.Conclusion: LPS can induce the production of MMP-12 by activate ERK,participate in the airway inflammation,AHR exacerbation in the late stage of RSV infection.PART THREE CpG ODN INDUCE PULMONARY INFLAMMATORY NODULES THROUGH MACROPHAGES AND NEUTROPHILS IN THE LATE STAGE OF RSV INFECTIONObjective: It was found in the first part that bacterial DNA CpG ODN stimulation could significantly aggravate airway inflammation and AHR in the late stage of RSV infection,with pulmonary inflammatory nodules formation.There are studies shown that intestinal microbial product CpG ODN transport by the system plays an important role in liver inflammation and injury exacerbating in chronic HBV infection.In addition to mimic the effects of bacterial components on tissues,local inhalation CpG ODN can alleviate asthma by inhibiting Th2 immune responses.However,it is not clear whether the CpG ODN transported by circulation can also aggravate pulmonary inflammation in the late stage of RSV infection and induce pulmonary inflammatory nodules.Our previous study showed that there was a kind of Th2 responses in lung tissues in the late stage of RSV infection,which come into a similar immune microenvironment in lung tissue as asthma,and played an important role in persistent airway inflammation and AHR.However,whether the systemic sources of CpG ODN can aggravate pulmonary inflammation in the late stage of RSV infection and induce pulmonary nodules formation is unclear.Therefore,RSV+CpG ip.group mice models were established in this part,to observe its effect on airway inflammation and AHR in the late stage of RSV infection.Histopathological images showed that there were foam-like cells distributed around inflammatory cells in the center of inflammatory nodules.Foamy cells are formed by lipids phagocytosis of macrophages,and AT?s regulate lipid metabolism homeostasis by participating in the production of pulmonary surfactant.But the main cellular composition and forming process of the inflammatory nodules remain unclear.Therefore,we try to determine the cellular composition and possible formation process of the inflammatory nodules in this part.Methods: Balb/c female mice aged 6-8 weeks were randomly divided into control group,CpG in.group,CpG ip.group,RSV group,RSV+CpG in.group and RSV+CpG ip.group,each group containing 8-10 mice.On the 0th day post RSV infection,control group,CpG in.group,CpG ip.group were administrated by intranasal with 100?l PBS;RSV group,RSV+CpG ip.group and RSV+CpG in.group were administrated by intranasal with 100?l RSV-A2;On the 35th-41 st days post RSV infection,control group and RSV group were administrated by intranasal/intraperitoneal with 50?l PBS every 2 days;CpG in.group,RSV+CpG in.group were administrated by intranasal with 50?l CpG(0.1?g/?l)every 2 days;CpG ip.group,RSV+CpG ip.group were administrated by intraperitoneal with 50?l CpG(0.1?g/?l)every 2 days,on the 42 nd days post RSV infection,airway hyperreactiveness were measured;mice were sacrificed for bronchoalveolar lavage fluid(BALF)collecting and inflammatory cells counting;lung tissues for pathological assessment,PAS and Oil Red O staining;The foam-like cells and inflammatory cells were observed under high magnificated pictures of HE staining;The immunofluorescence staining was used to identify macrophage(marker,CD68)and AT?s(marker,SPC);The fluorescence intensity and average fluorescence intensity were analysed by the software;The whole lung tissue of mice was taken for flow cytometry;Immunocytes were labeled as T lymphocytes(CD45+ CD3+),B lymphocytes(CD45+ CD19+),NK cells(CD45+ CD49+),neutrophils(CD45+ CD11b+ Ly6G+ Ly6C-),macrophages(CD45+ CD11b+ Ly6G-Ly6C+),interstitial macrophages(CD45+ F4/80+ CD11c+);ATIIs were labeled as SPC+;The upper pole of left lobi pulmonis of mice was fixed with 4% glutaraldehyde for transmission electron microscopy.Results: 1,The kinetics of mice weight in RSV+CpG ip.group were consistent with that in RSV group;There was no significant change in weight change of CpG in.group and CpG ip.group compared to control group;Macropathological picture shows no inflammatory nodules formation in RSV+CpG ip.group;inflammatory nodules formation was only found in RSV+CpG in.group(83.3%);2,RSV+CpG in.group and RSV+CpG ip.group were observed with obvious lung pathological changes compared to RSV group,and RSV+CpG in.group was more severe with inflammatory cell nodules formation;RSV+CpG in.group had significantly higher scores than those in the RSV group and RSV+CpG ip.group,by pulmonary bronchioles inflammation(P<0.001),pulmonary vessels inflammation(P<0.05),pulmonary interstitial inflammation(P<0.0001)and total pulmonary tissue inflammation(P<0.05);RSV group(P<0.01)and RSV+CpG ip.group(P<0.0001)had higher total inflammatory cells in BALF compared to the control group;Inflammatory cells in BALF of RSV+CpG ip.group was further elevated compared to the RSV group(P<0.0001),and macrophages elevation was most significant,higher than RSV group(P<0.001);RSV group(P<0.05),RSV+CpG ip.group(P<0.0001)and RSV+CpG in.group(P<0.01)had significantly higher airway resistance than the control group;RSV+CpG ip.group(P<0.05)and the RSV+CpG in.group(P<0.01)had higher Penh values compared to RSV group;Meanwhile RSV+CpG in.group stimulated in a lower concentration of acetylcholine(3.125 mg/ml);3,HE staining showed that RSV+CpG group mice lungs infiltrated with a large number of granulocyte-like inflammatory cells in inflammatory nodule center;Flow cytometry shows that RSV+CpG group has significantly higher percentage of neutrophils in lung tissues than that in control group(P<0.05),CpG group(P<0.05)and RSV group(P<0.01);There was no significant change in percentage of T lymphocyte in RSV+CpG group lung tissues compared to other groups;Though RSV+CpG group have a higher percentage of B lymphocytes and a lower percentage of lung NK cells in lung tissues compared to control group,there were no significant changes compared to RSV group;A large amount of neutrophils infiltration was observed under transmission electron microscopy;4,There were mild mucus distribution in the pulmonary inflammatory nodules in RSV+CpG group,and a large amount of lipids located in the surroundings of inflammatory nodules;5,HE staining showed RSV+CpG group had a large number of foam-like cells around the inflammatory nodules compared to other groups;Immunofluorescence staining showed an increased macrophages(CD68+),and has same locations with foam-like cells in HE staining;RSV+CpG group has significantly higher CD68 fluorescence intensity than that of control group(P<0.0001),CpG group(P<0.0001)and RSV group(P< 0.0001);RSV+CpG group also has a significantly higher average CD68 fluorescence intensity than that of the control group(P<0.01),CpG group(P<0.0001)and RSV group(P<0.0001);The percentage of macrophages in lung tissues were significantly higher in RSV+CpG group than that in control group(P<0.05),CpG group(P<0.05)and RSV group(P<0.01);However,the percentage of interstitial macrophages did not increase in RSV+CpG group;6,Immunofluorescence staining shows AT?s(SPC+)increased in RSV+CpG group;The SPC fluorescence intensity of RSV+CpG group was significantly higher than that of control group(P<0.001),CpG group(P<0.01)and RSV group(P< 0.01);The average SPC fluorescence intensity in the RSV+CpG group was also significantly higher than that in the control group(P<0.0001),CpG group(P<0.0001)and RSV group(P<0.0001);The percentage of AT?s in lung tissues was significantly higher in RSV+CpG group than that in control group(P<05),CpG group(P<0.01)and RSV group(P< 0.05);7,Significant activation of AT?s lamellar corpuscles were observed in RSV+CpG group compared to RSV group,with a increases in number,volume and distribution density.Conclusion: Both CpG ODN inhalation or systemic administration can aggravate airway inflammation and AHR in the late stage of RSV infection,however,RSV+CpG in.group mice with the most severe airway inflammation and AHR,and pulmonary inflammatory nodule formation only occurred in RSV+CpG in.group mice(? 83.3%).Among inflammatory nodules in the lung tissues of RSV+CpG in.group mice,inflammatory cells in the center were mainly neutrophils,lipid accumulation and foamy cells were found in the periphery;Increasing and dysfuction of AT?s may participated in the formation process of the pulmonary inflammatory nodules. |
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