Mechanism Research Of Innate Immune Response Mediated By NOD1/NF-k?P65 On Fusarium Keratitis

Fungal keratitis is the result of the combined action of the pathogen and the host.In the early stage,Pathogen invasion,virulence,multiple enzymes and adhesion mechanisms are primary pathological factors.Further injury after infection is caused by excess immune reactions of host rather than the pathogen.Conventional anti-fungal drugs can not control corneal tissue damage caused by local excessive inflam-matory reaction.Therefore,It's most important to find a way to control local inflam matory reaction in addition to antifungal therapy.NOD1/NF-k?P65 signaling pathway is highly involved in the innate immunity induced by bacteria,viruses and fungal infections.The incidence of fusarium keratitis is the highest in all filamentous fungal keratitis in China.To date,the mechanism of Innate Immune Response Mediated by NOD1/NF-k?P65 in fusarium keratitis has not been reported.The present study consists of part 1 and 2(in vitro experiments)and part 3 and 4(in vivo experiments),aiming at the regulation of innate immune response by NOD1/NF-k?P65 signaling pathway when the cornea is challenged by Fusarium.Part1:The expression of NOD1?RIP2?NF-k?P65 in human corneal epithelial cells after activatied by Fusarium spore under different conditions[Objectives]To detect the mRNA level of NOD1?RIP2?NF-k?P65 in human corneal epithelial cells line(pRSV-T)when the cells were activatied by Fusarium spore under different Inactivation temperature,different density,different stimulation time.The aim is to obtain The optimal conditions for activating the NOD1/NF-k?P65 signaling pathway.[Methods]Fusarium solani was inoculated into Sabouraud medium,and cultured at 26 ? for 7 days to collect fungal suspension.Spores were extracted by gauze filtering.Quantitative real time polymerase chain reaction(q-PCR)were used to detect the level of NOD1 mRNA?RIP2mRNA?NF-k?P65mRNA in human corneal epithelial cells line after activatied by Fusarium spore with different inactivation temperature(55 ?,65 ?,75 ?,85 ?,95 ?),different density(1×103CFU/ml?1×107CFU/ml),and different stimulation time(2h,4h,8h,12h,24h).[Results]1.Different inactivation temperature:Relative expressions of NOD1mRNA in Control,55?,65?,75?,85?and 95?groups were(1.00±0.00),(1.59±0.15),(2.21 ±0.2),(1.88±0.33),(1.51±0.25)and(1.12±0.13);Relative expressions of RIP2mRNA were(1.00±0.00),(1.07±0.14),(1.91 ±0.22),(1.45±0.23),(1.23±0.12)and(1.02±0.11);Relative expressions of NF-k?P65mRNA were(1.00±0.00),(1.27±0.14),(2.12±0.19),(2.21±0.25),(1.51±0.14),(1.09±0.18).respectively.Compared to Control group,relative expression of NOD1mRNA were all increased in pRSV-T cells in 55 ?,65 ?,75 ?,85 ? group(P<0.05);relative expression of RIP2mRNA were both increased in pRSV-T cells in 65?and 75?group(P<0.05);relative expression of NOD1mRNA and RIP2mRNA were the highest in the 65 ?group.relative expression of NF-k?P65mRNA were all increased in pRSV-T cells in 65 ?,75 ?,85 ? group(P<0.05).relative expression of NF-k?P65mRNA was the highest in the 75 ? group.2.Different density:Relative expressions of NOD1mRNA in Control,1×103CFU/ml,1×104CFU/ml,1×105CFU/ml,1×106CFU/ml and 1×107CFU/ml groups were(1.00±0.00),(1.08±0.13),(1.57±0.2),(2.06±0.15),(1.45±0.06)and(1.04±0.09);Relative expressions of RIP2mRNA were(1.00±0.00),(1.13±0.23),(1.64±0.2),(2.13±0.35),(1.69±0.22)and(1.23±0.26);Relative expressions of NF-k?P65mRNA were(1.00±0.00),(1.67±0.37),(2.27±0.27),(2.8±0.36),(1.77±0.17)and(1.15±0.14).respectively.Compared to Control group,relative expression of NOD1mRNA and RIP2mRNA were all increased in pRSV-T cells in1×104CFU/ml,1×105CFU/ml and 1×106CFU/ml group(P<0.05);relative expression of NF-k?P65mRNA were all increased in pRSV-T cells in 1±103CFU/ml,1×104CFU/ml,1×115CFU/ml and 1×x106CFU/ml group(P<0.05).The relative expression of NOD 1 mRNA?RIP2mRNA?NF-k?P65mRNA were the highest in 1×105CFU/ml group.3.Different stimulation time:Relative expressions of NOD1mRNA in 0h,2h,4h,8h,12h and 24h groups were(1.00±0.00),(3.01±0.47),(1.97±0.33),(4.29±1.07),(3.32±0.51)and(2.87±0.27);Relative expressions of RIP2mRNA were(1.00±0.00),(2.68±0.26),(2.28±0.44),(5.27±1.16),(2.64±0.37)and(1.05±0.14);Relative expressions of NF-k?P65mRNA were(1.00±0.00),(2.81±0.29),(1.38±0.17),(3.27±0.51),(1.77±0.22)and(1.47±10.23).respectively.Compared to Oh group,relative expression of NOD1mRNA were all increased in pRSV-T cells in 2h,8h,12h and 24h group(P<0.05);relative expression of RIP2mRNA were all increased in pRSV-T cells in 2h,4h,8h and 12h group(P<0.05);The relative expression of NF-k?P65mRNA were all increased in pRSV-T cells in 2h,8h and 12h group(P<0.05).relative expression of NOD1mRNA?RIP2mRNA?NF-k?P65mRNA were highest in 8h group.[Conclutions]The expression of NOD1mRNA?RIP2mRNA?NF-k?P65 mRNA in pRSV-T cells were all increased after activatied by Fusarium spore under different conditions.Relative expression of NOD1mRNA and RIP2mRNA were the highest when Fusarium spore inactivation temperature was 65 ?,the density was 1×105CFU/ml,and stimulation time was 8h;the relative expression of NF-k?P65mRNA was the highest when Fusarium spore inactivation temperature was 75 ?.and when other comditions were the same.Part 2:Mechanism research of Proinflammatory reaction in Innate Immune Response Mediated by NOD1/NF-k?P65 in pRSV-T cells activiated by Fusarium spore[Objectives]To verify whether NODI regulates the releasing of its downstream inflammatory factors(IL-6,IL-8,TNF-alpha)through NF-k?P65 in the Innate Immune Response in pRSV-T cells activiated by Fusarium spore.[Methods]1.To activate NODI/NF-k?P65 signaling pathway:we use 3 groups:Experi-mental group(inactivated Fusarium spore),control group(NOD1 specific ligand iE-DAP),and blank control group(cell culture medium).In all the three groups cells and supernatants were collected after pRSV-T cells were stimulated for 8h.Quantitative real time polymerase chain reaction(q-PCR)and western blot were used to detect the expression of NOD1,RIP2 and NF-k?P65;Immunofluorescence was used to detect the expression of NF-k?P65;Flow cytometry was used to detect the concentration of IL-6,IL-8 and TNF-a in Culture supernatant.2.To detect effective concentration of NOD1 inhibitor(ML130):pRSV-T cells were treated with different concentration of ML 130 for 24h,then stimulated by inactivated Fusarium spore for 8h,cells were collected and Western blot were used to detect the expression of NOD1,RIP2 and NF-k?P65.3.Inhibition of NOD1/NF-k?P65 signal pathway:cells were divided into 3 groups:Experimental group(inactivated Fusarium spore),control group(NOD1 inhibitor ML 130+ inactivated Fusarium spore),and blank control group(cell culture medium).pRSV-T cells in Experimental group were stimulated for 8h.Cells in control group was treated with ML 130 for 24h before stimulated by inactivated Fusarium spore for 8h.Quantitative real time polymerase chain reaction(q-PCR)and western blot were used to detect the expression of NODI,RIP2 and NF-k?P65;Immunofluorescence was used to detect the expression of NF-k?P65;Flow cytometry was used to detect the concentration of IL-6,IL-8 and TNF-a of Culture supernatant.[Results]1.Activated NOD1/NF-k?P65 signal pathway1.1 The changes in transcriptional and translation levels of NOD1?RIP2?NF-k?P65 were as follows:NOD1mRNA:The Relative expressions of NOD1mRNA in control group,iE-DAP group and F.solani group were(1.00±0.00),(3.65±0.44),(2.95±0.36).respectively.Compared to Control group,relative expression of NOD1mRNA in pRSV-T cells were both increased in iE-DAP group and F.solani group(P<0.05);There was no statistical difference between iE-DAP group and F.solani group(P>0.05).The NODI Protein expression trend was consistent with the transcriptional level.RIP2mRNA:The Relative expressions of RIP2mRNA in control group,iE-DAP group and F.solani group were(1.00±0.00),(4.11 ±0.49),(3.47±0.34).respectively.Compared to Control group,relative expression of RIP2mRNA in pRSV-T cells were both increased in iE-DAP group and F.solani group(P<0.05);There was no statistical difference between iE-DAP group and F.solani group(P>0.05).The RIP2Protein expression trend was consistent with the transcriptional level.NF-k?P65mRNA:The Relative expressions of NF-k?P65mRNA in control group,iE-DAP group and F.solani group were(1.00±0.00),(4.60±0.26),(4.14±0.29).respectively.Compared to Control group,relative expression of NF-k?P65mRNA in pRSV-T cells were both increased in iE-DAP group and F.solani group(P<0.05);There was no statistical difference between iE-DAP group and F.solani group(P>0.05).The RIP2Protein expression trend was consistent with the transcriptional level.1.2 Immunofluorescence results:NF-k?P65 protein was expressed in cytoplasm,Compared to Control group,NF-k?P65 protein expression were both increased in pRSV-T cells in iE-DAP group and F.solani group;Activated p-NF-k?P65 protein was expressed in the nucleus,Compared to Control group,p-NF-k?P65 protein expression were both increased in pRSV-T cells in iE-DAP group and F.solani group.1.3 Inflammatory factor concentration:The concentration changes of IL-6,IL-8 and TNF-a in Culture supernatant of pRSV-T cells were as follows:IL-6 concentration:The concentration of IL-6 in control group,iE-DAP group and F.solani group were(57.50±5.21)pg/ml,(331.94±42.78)pg/ml and(240.94±30.15)pg/ml.respectively.Compared to Control group,The concentration of IL-6 were both increased in Culture supernatant of pRSV-T cells in iE-DAP group and F.solani group(P<0.05);Compared to iE-DAP group,The IL-6 concentration in F.solani group was decreased(P<0.05).IL-8 concentration:The concentration of IL-8 in control group,iE-DAP group and F.solani group were(3.58±0.42)pg/ml,(16.09±2.62)pg/ml and(13.03±1.54)pg/ml.respectively.Compared to Control group,The concentration of IL-8 were both increased in Culture supernatant of pRSV-T cells in iE-DAP group and F.solani group(P<0.05);Compared to iE-DAP group,The IL-8 concentration in F.solani group was decreased(P>0.05).TNF-? concentration:The concentration of TNF-a in control group,iE-DAP group and F.solani group were(13.87±2.16)pg/ml,(74.42±6.52)pg/ml and(80.41±11.33)pg/ml.respectively.Compared to Control group,The concentration of TNF-a were both increased in Culture supernatant of pRSV-T cells in iE-DAP group and F.solani group(P<0.05);Compared to iE-DAP group,The TNF-? concentration in F.solani group was decreased(P>0.05).2.The results of the effective concentration of NOD1 inhibitor(ML130):different concentration(0.1uM/ml,0.5 uM/ml,1 uM/ml,5 uM/ml,10 uM/ml,20 uM/ml,30 uM/ml)of ML130 were influenced the protein expression of RIP2 and NF-k(3P65 in pRSV-T cells,respectively.Compared to F.solani group,The protein expression of RIP2 were both decreased in 0.1uM/ml group,5uM/ml group and 20uM/ml group(P<0.05);Compared to F.solani group,The protein expression of NF-k?P65 were both decreased in 0.1uM/ml group and 20uM/ml group(P<0.05);The protein expression of RIP2 and NF-k?P65 were lowest in the 20 uM/ml group.3.Inhibitoried NOD1/NF-k?P65 signal pathway3.1 The changes in transcriptional and translation levels of NOD1?RIP2?NF-k?P65 were as follows:NOD1mRNA:The Relative expressions of NOD1mRNA in control group,F.solani group and ML130 group were(1.00±0.00),(3.65±0.5),(2.91 ±0.26).respectively.Compared to Control group,relative expression of NOD1mRNA were both increased in pRSV-T cells in F.solani group and ML 130 group(P<0.05);Compared to F.solani group,relative expression of NOD1mRNA was decreased in ML130 group(P>0.05).The protein expression of NOD1 was similar with the mRNA expression.RIP2mRNA:The Relative expressions of RIP2mRNA in control group,F.solani group and ML130 group were(1.00±0.00),(3.86±0.44),(2.13±0.16).respectively.Compared to Control group,relative expression of RIP2mRNA were both increased in pRSV-T cells in F.solani group and ML130 group(P<0.05);Compared to.solani group,relative expression of RIP2mRNA was decreased in ML 130 group(P<0.05).The protein expression of RIP2 was similar with the mRNA expression.NF-k?P65mRNA:The Relative expressions of NF-k?P65 in control group,F.solani group and ML 130 group were(1.00±0.00),(3.11±0.39),(1.67±0.18).respectively.Compared to Control group,relative expression of NF-k?P65 were both increased in pRSV-T cells in F.solani group and ML 130 group(P<0.05);Compared to F.solani group,relative expression of NF-k?P65 was decreased in ML 130 group(P<0.05).The protein expression of NF-k?P65 was similar with the mRNA expression.3.2 Immunofluorescence results:NF-k?P65 protein was expressed in cytoplasm,Compared to Control group,NF-k?P65 protein expression were both increased in pRSV-T cells in F.solani group and ML 130 group;Compared to F.solani group,NF-k?P65 protein expression was decreased in ML 130 group.The activated p-NF-k?P65 protein was expressed in the nucleus,Compared to Control group,p-NF-k?P65 protein expression were both increased in pRSV-T cells in F.solani group and ML 130 group;Compared to F.solani group,NF-k?P65 protein expression was decreased in ML130 group.3.3 Inflammatory factor concentration:The concentration changes of IL-6,IL-8 and TNF-a in Culture supernatant of pRSV-T cells were as follows:IL-6 concentration:The concentration of TL-6 in control group,F.solani group and ML130 group were(45.02±5.5)pg/ml,(314.28±33.56)pg/ml and(192.72±23.04)pg/ml.respectively.Compared to Control group,The concentration of IL-6 were both increased in Culture supernatant of pRSV-T cells in F.solani group and ML 130 group(P<0.05);Compared to F.solani group,The IL-6 concentration in ML130 group was decreased(P<0.05).IL-8 concentration:The concentration of IL-8 in control group,F.solani group and ML130 group were(4.48±0.38)pg/ml,(18.59±2.64)pg/ml and(8.81±1.50)pg/ml.respectively.Compared to Control group,The concentration of IL-8 were both increased in Culture supernatant of pRSV-T cells in F.solani group and ML 130 group(P<0.05);Compared to F.solani group,The IL-8 concentration in ML130 group was decreased(P<0.05).TNF-? concentration:The concentration of TNF-a in control group,F.solani group and ML130 group were(11.55±2.27)pg/ml,(65.91±9.6)pg/ml and(36.85±4.89)pg/ml.respectively.Compared to Control group,The concentration of TNF-a were both increased in Culture supernatant of pRSV-T cells in F.solani group and ML130 group(P<0.05);Compared to F.solani group,The TNF-a concentration in ML130 group was decreased(P<0.05).[Conclutions]The expression of NOD1,RIP2 and NF-k?P65 and the release of pro-inflammatory factors(IL-6,IL-8,TNFa)were increased by stimulated with both iE-DAP and inactivated Fusarium spore;NOD1 inhibitor ML 130 can inhibit the activation of RIP2 and NF-k(3P65 and decreased their expression,Meanwhile,ML130 decreased the release of pro-inflammatory factors(IL-6,IL-8,TNFa).NOD1 regulated the release of proinflammatory factors IL-6,IL-8 and TNFa by monitoring the downstream molecules RIP2 and NF-k(3P65,thus plays an important role in the immune response in pRSV-T cells induced by Fusarium spores.Part three:Establishment of A tree shrew Model of FusariumKeratitis[Objectives]To establish a tree shrew model of Fusariumsolani keractitis by scratching and dropping the Fusariumsolani spores.[Methods]Fusariumsolani were inoculated into Sabouraud culture medium and incubated at 26 ? for 7 days.Fungal suspensions were collected and the density of spores was adjusted to 1 ×109CFU/mL on the blood cell count plate.thirty healthy tree shrews were randomly divided into experimental group(n = 20)and control group(n = 10).3 days before the experiment,experimental group and blank control group were injected 0.1ml dexamethasone(5mg/ml)subconjunctivally in the right eyes.For the experimental group,the corneal epithelium was ripped about 5*5mm,and was scratched by 29G needle,50ul of fungal spore suspension was dropped on the cornea surface,and 50uL saline was dropped on the control group.The animals were evaluated by anterior segment photography,confocal microscopy,histopathological changes,and corneal tissue culture.[Results]The infiltration of fungus,the degree of coneal epithelial cells and endothelial cells edema.and the number of mycelium were positively correlated with time.The infiltration of inflammatory cells reached the peak on the 7th day after modeling,in which the neutrophils were the major protion.The mycelial growth was parallel to the stroma fiber.After the success of the model,the corneal tissue culture showed the growth of Fusariumsolani.The successful rate of modeling was 90%.[Conclusions]The model of Fusariumsolani keratitis was established by Corneal epithelium scratches and drops the Fusariumsolani spores.Part 4:The effect of Proinflammatory reaction Mediated by NOD1 and NF-k?-P65 on Fusarium keractitis[Objectives]To explore whether the expression of NOD1 and NF-k?P65 were related to Inflammatory cells or Pro-inflammatory factor on tree shrew Fusarium keratitis model[Methods]western blot were used to detect the expression of NOD1 and NF-k?P65;Flow cytometry was used to detect the concentration of IL-6,IL-8 and TNF-a.To explore the correlation between the NOD1/NF-k?P65 signaling pathway and the proinflammatory response in vivo.[Results]The expression of NOD1 and NF-k?P65 were higher in experiment group than in control group;Immunofluorescence results:NOD1 protein was expressed in cytoplasm.Compared to Control group,NOD1 protein of experimental group was Strong positive staining;NF-k?P65 protein was expressed in the nucleus of neutrophils in Corneal stromal and corneal endothelial cells.Compared to Control group,NF-k?P65 protein of experimental group was Strong positive staining.The concentration of IL-6?IL-8 and TNF-? in the aqueous humor of experimental group was increased at day 1 and day 3 after establishing mode(P<0.05).[Conclutions]There were High expression of NOD1 and NF-k?P65 protein in the Fasurium keractitis of tree threw,NOD1 protein was expressed on Corneal epithelial cells,NF-k?P65 protein was expressed in the nucleus of neutrophils in Corneal stromal and corbeal endothelial cells.The expression of NOD1 protein had a positive correlation with the number of neutrophils and the concentration of IL-6,IL-8 and TNF-a in aqueous humor.