| Objective:To explore the effects of schistosome infection with different time points and egg excretory and secretory products(E/S)on OVA-induced allergic airway inflammation(AAI).Methods:The models of Schistosoma japonicum(Sj)infection and OVA-induced allergic airway inflammation were established.All infected mice were contacted abdominally with 15±2 cercaria of Schistosoma japonicum.OVA-induced allergic airway inflammation:The mice were injected intraperitoneally with two shots of alum adjuvanted OVA protein on day 0 and day 14(Lung-stage intervention and E/S treatment experiment)or day 21 and 35(Sj-21d intervention experiment)and challenging with aerosolized OVA(from day 21 to day 24 or from day 42 to 46,respectively).48 h after last challenge,all mice were executed.BALFs and lung,spleen and lung draining lymph nodes were extracted,sera samples were obtained.The study included schistosome Lung-stage intervention experiment,Sj-21d intervention experiment and schistosome egg E/S treatment experiment.Lung-stage intervention experiment was further divided into lung or post-lung stage schistosome infection,OVA specific CD4 transfer,Treg deletion and RNA-seq analysis.1)In therapeutic experiment,the mice were grouped into OVA group,OVA+INF group(post lung-stage infection),OVA+INF(lung-stage infection),OVA+DXM treatment group,INF group and NOR groups and 5 mice each group.The proportions of inflammatory cell,eosinophils,neutrophils,macrophage and lymphocyte were counted,Haematoxylin-eosin(HE)and periodic acid-schiff(PAS)staining were used to observe inflammatory cell infiltration around pulmonary vasculature and bronchus and hyperplasia of bronchial epithelial cells.IgE and OVA specific IgE were detected by ELISA,IL-4?IL-5?IL-13?Eotaxin?IFN-? and IL-10 were assayed by luminex,and the frequency of CD4+CD25+Foxp3+Treg in spleen and lung were determined by flow cytometry.2)Adoptive transfer experiment was grouped into OVA+CD4 and OVA+CD4+INF groups and 8 mice each group.The frequency of CD4+CD25+Foxp3+Treg,CD4+IL-4+T and CD4+IFN-?+T in CD45.1+or CD 45.2+T cells from lung and LDLNs were assayed by flow cytometry.3)Treg deletion experiment was grouped into OVA+INF+?CD25 and OVA+CD4+IgG-and each group included 8 mice.The proportions of inflammatory cell were counted,haematoxylin-eosin(HE)and periodic acid-schiff(PAS)staining were used to observe inflammatory cell infiltration around pulmonary vasculature and bronchus and hyperplasia of bronchial epithelial cells.OVA specific IgE and IgG were detected by ELISA.4)RNA-seq analysis was grouped into OVA group,OVA+INF(lung-stage infection),OVA+DXM treatment group,INF group and NOR groups.Total RNA was extracted from lung tissue of all groups,RNA sequence was performed and KEGG and GO analysis were conducted.Sj-21d intervention experiment was divided into OVA,OVA+INF,INF,and NOR groups and 5 mice each group.E/S treatment experiment were grouped into OVA,OVA+E/S,E/S and NOR groups.The proportions of inflammatory cell,eosinophils,neutrophils,macrophage and lymphocyte were counted,Haematoxylin-eosin(HE)and periodic acid-schiff(PAS)staining were used to observe inflammatory cell infiltration around pulmonary vasculature and bronchus and hyperplasia of bronchial epithelial cells.IgE and OVA specific IgE were detected by ELIS A,IL-4?IL-5?IL-13?Eotaxin?IFN-? and IL-10 were assayed by luminex,and the frequency of CD4+CD25+Foxp3+Treg in spleen and lung were determined by flow cytometry.Schistosome egg E/S treatment experiment was divided into OVA,OVA+E/S,E/S,and NOR groups and 5 mice each group.E/S treatment experiment were grouped into OVA,OVA+E/S,E/S and NOR groups.The proportions of inflammatory cell,eosinophils,neutrophils,macrophage and lymphocyte were counted,haematoxylin-eosin(HE)and periodic acid-schiff(PAS)staining were used to observe inflammatory cell infiltration around pulmonary vasculature and bronchus and hyperplasia of bronchial epithelial cells.IgE and OVA specific IgE were detected by ELISA,IL-4?IL-5?IL-13?Eotaxin?IFN-? and IL-10 were assayed by luminex,and the frequency of CD4+CD25+Foxp3+Treg in spleen and lung were determined by flow cytometry.Results:1 Schistosome lung-stage intervention experiment:1.1 Lung and post-lung schistosome infection experiment:The counts of inflammatory cell showed that total inflammatory cells and eosinophils in OVA group were significantly increased compared to the NOR group(P<0.01),while that in OVA+DXM group was significantly reduced relative to the OVA group(P<0.01),Total inflammatory cells and eosinophils in OVA+INF group(lung-stage infection)was also significant reduced,while that in OVA+INF(post lung-stage infection)didn't show any difference.Pathological results showed that peribronchial and perivascular inflammation and mucus secretion in OVA group were more aggravated than that in NOR group(P<0.01,P<0.05),whereas that in OVA+INF group(lung-stage infection)were alleviated but not in OVA+INF mice(post lung-stage infection)relative to the OVA group.The results of ELISA showed IgE and OVA specific IgE in O VA group were significantincreased compared to the NOR group(both P<0.01),whereas that in OVA+INF(lung-stage infection)group were reduced but not that in OVA+INF(post lung-stage infection)group compared to the OVA group.IL-4,IL-5,and Eotaxin in OVA group by Luminex were significant upregulated relative to the NOR group,whereas IL-5 was significant reduced in OVA+INF(lung-stage infection)but not OVA+INF(post lung-stage infection)compared to the OVA group.The frequency of Treg from lung and spleen in OVA+INF(lung-stage infection)was upregulated compared to the OVA group,whereas that only in spleen in OVA+INF(post lung-stage infection)were a little increased relative to the OVA group.1.2 Specific CD4+T transfer experiment:The results of adoptive transfer showed that CD45.1+Treg in lung in O VA+CD4+INF group was significantly up-regulated compared to that in OVA+CD4 group(P<0.001),total Treg(P<0.05)but not CD45.2+Treg(P>0.05)was also upregulated compared to the OVA+CD4 group.However,CD45.2+Treg and total Treg(both P<0.05)but not CD45.1+Treg(P>0.05)in lung draining lymph nodes(LDLNs)in OVAH-CD4+INF group were upregulated compared to OVA+CD4 group.The ratio of OVA specific CD4+IL-4+ T and CD4+IFN-y+T in OVA+CD4+INF group was reduced compared to O VA+CD4 group(P<0.05).1.3 Treg deletion experiment:The results showed that inflammatory cell in BALFs in OVA+INF+?CD25 were increased,mucus secretion was more aggravated and OVA specific IgE was also upregulated relative to the OVA+INF+IgG group.1.4 RNA-seq analysis:The results showed that differential expressed genes(DEGs)between OVA+INF(lung-stage infection)and OVA group has upregulated 203 and downregulated 279 genes(Padj<0.05).GO analysis of DEGs showed that the significant enriched top 3 pathway includes the T cell activation,lymphocyte proliferation and the regulation of lymphocyte proliferation.Panther analysis showed that 482 DEGs were divided into 8 biological process,including immune system process(84).biological regulation and adhesion(86),cell and multicellular process(78),development(11),localization(14),metabolic process(29),response to stimulus(21)and not annotated(159).70 among 84 genes of immune system were downregulated after lung-stage schistosome infection.Further analysis showed that genes Clec7a?CCR6?Spi-B?ADA?Ptgir?Ctss?Ctsk?Epor?ABCG1?CD46 and Klra17(P<0.05)were predicated to be involved in Treg response.DOCK2,IRF4.Rac2,Lgals3,H2-Oa,Pdcdllg2,Sash3,and Mzb1 were involved in the differentiation and function of B cell or plasm cell.FOXF1,ANO9,TRIM6,MMP27,Epor,Gatal and Serpina were contributed to lung development and Villin and CRB1 were related to cell integrity.2 Sj-21d intervention experiment:ELISA showed that the A450 values of IgE in the INF group on day 35,42 and 49 were all significantly increased compared to the healthy control(all P<0.01).TheA4500 values of IgE in the OVA group on days 42 and 49 significantly increased compared to the healthy control(P<0.01),that on day 35.The A450 values of IgE in the INF+OVA group on those days were all significantly increased compared to the OVA group(P<0.01).On day 35,42 and 49 after infection,the A450 values of OVA-specific IgE in OVA group were all significantly increased compared to the control group(P<0.01,0.05,0.05).However,those in the OVA+INF group were all significantly lower than those in the OVA group(P<0.01,0.01,0.05).Pathological examinations on day 49 after infection showed that the lung inflammation scores around blood vessels and bronchi in the healthy control group,INF group,OVA group and INF+OVA group were,respectively.The inflammation score in the INF and OVA groups were both significant increased compared to the healthy control group(P<0.01),while that in the INF+OVA group was significantly reduced(P<0.05).The mucus score in the OVA group was significantly increased compared to the healthy control group(P<0.05),whereas it did not show any difference between INF+OVA group and OVA group(P>0.05).The results of flow cytometry on day 49 after infection showed that the proportions of Tregs in spleen and lung tissues in the INF group were significantly up-regulated compared to the healthy control group(both P<0.05),while there was no difference between the OVA and NOR groups(P>0.05).The splenic Treg proportion showed a significant increase in the INF+OVA group compared to the OVA group(P<0.01),while the lung Treg proportion showed no significant difference(P>0.05).3 Schistosome egg-E/S treatment experiment:Inflammatory cell counts showed that total inflammatory cells and eosinophils in OVA group were significantly increased compared to the NOR group(P<0.01),whereas total inflammatory cells and eosinophils in OVA+E/S group were significant reduced compared to the OVA group(both P<0.01).Pathological results showed that peribronchial and perivascular inflammation and mucus secretion in OVA group were significant aggravated relative to the control group,whereas that in OVA+E/S group were alleviated compared to the OVA group.ELISA showed that A450 value of total IgE on day 20 and 26 and OVA specific IgE in OVA group on day 20 were significant increased relative to control group,whereas that in OVA+E/S group was reduced compared to the OVA group(P<0.001,P<0.001,P<0.05).The frequency of Treg in spleen and lung in OVA group and infection group didn't show any difference compared to the normal control(P>0.05),whereas that in OVA+E/S group was upregulated compared to the OVA group(P<0.01).Conclusion:1).Lung-stage schistosome infection could relieve OVA induced asthma in a mouse model.The therapeutic effect was mediated by the upregulated OVA specific Treg which suppressed IgE production and Th2 cytokine secretion.2).S.japonicum infection(21 days after infection)showed significant regulations on OVA-induced allergic airway inflammation by reducing the OVA-specific IgE levels in serum at the sensitization and challenge phases of allergic airway inflammation,relieving peribronchi and perivascular inflammatory cell infiltration induced by OVA,and up-regulating Treg proportions in the spleen.3).Schistosome egg E/S treatment showed significant regulations on OVA-induced allergic airway inflammation by reducing the OVA-specific IgE levels in serum at the sensitization and challenge phases of allergic airway inflammation,relieving peribronchi and perivascular inflammatory cell infiltration induced by OVA,and up-regulating Treg proportions in the lung. |
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