Role And Mechanism Of NADPH Oxidase 2 (NOX2) In Experimental Aspergillus Fumigates Keratitis In Mice

Purpose: In the process of fungal keratitis,both the direct injury of pathogenic fungi and the inflammatory response result in damage to the cornea that leads to loss of visual acuity.Neutrophils play a critical role in the defense against invasive fungal infections.They can directly kill fungi through a variety of mechanisms,express a variety of pattern recognition receptors(PRRs)to start the immune response,and participate in mediating various signal transduction pathways and the release of the downstream inflammatory factors.The first response of neutrophils to foreign microbial infection is to produce Reactive oxygen species(ROS),and the NADPH oxidase(NOX)family is the main origin of ROS in the phagocytes and non-phagocytes.NOXs can transport electrons between the intracellular and extracellular environment,leading to the reduction of oxygen to superoxide.NOXs play diverse roles in different organisms through redox signaling.Among this family,the key member--NOX2 subtype is very critical to host anti-microbial responses.Once the activity of NOX2 defective,production of ROS is inhibited,and both human and experimental animals are hypersensitive to life-threatening bacterial and fungal infections due to their inability to kill invasive pathogens and control the degree of inflammations,which indicates that NOX2 plays a key role in anti-infection immunity and regulation of inflammatory responses.NOX2 has a specific effect on the resistance to Aspergillus fumigatus infection.A.fumigatus is very susceptible to ROS,and phagocytes kill A.fumigatus by NOX-dependent mechanisms.After blocking NOX2,the ability to kill A.fumigatus hyphae is significantly reduced.Studies have confirmed that there was expression of NOX2 in corneal tissue.However,whether NOX2 is expressed in fungal keratitis in mice and its role in this disease have not yet been investigated.This study was to explore expression of NOX2 and detect its role in the immune response of A.fumigatus keratitis in C57BL/6 mice.Methods: 1.Neutrophils in the abdominal cavity of C57BL/6 mice were collected by 9% Casein injected i.p.and purified using 64% and 80% Percoll solutions.Then the inactivated A.fumigatus hyphae suspensions were added and NOX2 expression was detected by real-time PCR and Western blot.The experimental fungal keratitis models of mice were established and NOX2 expression was detected by real-time PCR,Western blot and immunofluorescent staining.2.The inhibition of NOX2 in vivo was carried out by subconjunctivally injecting the experimental eyes with diphenyleneiodonium chloride(DPI),and in vitro DPI was added in the culture of neutrophils with different concentrations.After testing the inhibitory effect of DPI on NOX2,mice were divided to three groups: A.fumigatus infection group,DPI pretreated fungal infection group and solvent DMSO control fungal infection group.The clinical performance in 1d,3d and 5d postinfection(p.i.)between three groups were recorded and evaluated by clinical score.The generations of ROS were detected by flow cytometry in vivo in fungi infected corneas and immunofluorescent staining in vitro in fungi stimulated neutrophils with or without treatment of DPI.To detect the form of neutrophil extracellular traps(NETs),the levels of H3 were tested by ELISA in vivo in fungi infected corneas and in vitro in fungi stimulated neutrophils with or without treatment of DPI.The numbers of CFUs of fungal infected corneas were taken to explore the impact of NOX2 on the clearance of invasive fungi.The MPO levels and immunofluorescent staining were done to detect the impact of NOX2 on neutrophils infiltration.The release of cytokines: Nrf2,NF-κB,IL-17 A,IL-6,IL-10,TGF-β,and TNF-α were checked by real-time PCR,ELISA and Western blot.3.Neutrophils were collected and isolated,and Dectin-1,SYK and PKC-δ were blocked by pretreatment into the culture of their inhibitior-laminarin,PRT062608 and rottlerin.Then cells were stimulated by A.fumigatus and collected to detect the expression of NOX2,dectin-1,SYK and PKC-δ by real-timePCR and Western blot.Results: 1.Expression of NOX2 in Experimental A.fumigatus keratitis in mice In neutrophils isolated from the abdominal cavity,NOX2 mRNA levels were significantly increased 12 h,16 h,20 h and 24 h p.i.compared to the levels measured without infection(p<0.01,p<0.01,p<0.01,p<0.01,respectively),and NOX2 protein levels were significantly increased 24 h p.i.The relative NOX2 mRNA levels were significantly higher in the A.fumigatus-infected corneas than those in the normal and control groups at 1 d,3 d and 5 d p.i.(p<0.05,p<0.01,p<0.01,respectively).The protein levels also increased at 1 d,3 d and 5 d p.i.shown by Western blot.At 3d p.i.,there was stronger staining for NOX2 in the corneal epithelial cells and stroma than those in normal corneas by immunofluorescent staining.2.Role of NOX2 in Experimental A.fumigatus keratitis in mice(1)Pretreatment with 1 μM DPI solution inhibited the expression of NOX2 in neutrophils(p<0.01),and DPI inhibited NOX2 protein expression 24 h p.i.compared to that measured without DPI treatment.(2)Different concentrations of DPI solutions were used to inhibit the expression of NOX2 in mice.Corneal NOX2 mRNA levels were significantly reduced following treatment with 1 μM DPI(p<0.01).Western blotting also verified the inhibitory effect of DPI on NOX2 protein expression in corneas.(3)With DPI treatment,clinical performances were worse after A.fumigatus infection and marked by a larger ulcer area,more severe edema and more neovascularization than those observed with DMSO control and A.fumigatus infection group.The analysis of the clinical scores were higher significantly(p<0.01,p<0.05,p<0.01 at 1 d,3 d,and 5 d p.i.,respectively).The inflammation degree of A.fumigatus infection without DPI treatment in corneas was most severe at 3 d and began to recover at 5 d,but with DPI treatment,the inflammation degree at 5 d was worse than that at 3 d and the lesion showed a gradual aggravation trend.(4)The corneas were collected 24 h p.i.to detect ROS expression in vivo by flow cytometry.Compared with the A.fumigatus-infected group(43.98% ROS-positive)and the DMSO control group(47.04% ROS-positive),there were fewer ROS-positive cells(17.34%)in the DPI treatment group,which showed that the inhibition of NOX2 by DPI suppressed ROS expression in mouse fungal keratitis.Then,immunofluorescent staining of ROS in neutrophils showed that normal neutrophils with little ROS-positive staining.After fungi infection,there was an increase in FITC staining.DPI treatment showed less staining than that with DMSO control group.(5)Twenty four hours after the corneas were infection with fungi,the concentration of H3 was increased,while after treatment with DPI,the concentration of H3 was decreased(p< 0.01,p<0.01,respectively).In neutrophils 4 h p.i.,the concentration of H3 in the culture medium increased significantly compared to that observed for normal cells,while following treatment with DPI,the H3 concentration decreased(p < 0.01,p< 0.05,respectively).(6)Fungi infection with DPI treatment increased the number of CFUs counted compared with without DPI treatment in corneas(p< 0.05).(7)In vivo treatment with DPI increased the infiltration and intensity of neutrophil labeling with NIMP-R14 after A.fumigatus infection.The expression of MPO in the corneas increased significantly,and with DPI treatment,its expression was enhanced(p<0.01,p<0.05,respectively).(8)Compared with their levels in normal corneas,the relative mRNA levels of Nrf2,NF-κB,TNF-α,IL-6,IL-10 and TGF-β increased following fungal infection at 3 d p.i.(p<0.05,p<0.05,p<0.01,p<0.01,p<0.05,p<0.05,respectively).Treatment with DPI increased the mRNA levels of NF-κB,TNF-α and IL-6(p<0.05,p<0.05,p<0.05,respectively)and decreased the mRNA levels of Nrf2,IL-10 and TGF-β(p< 0.05,p<0.01,p<0.05,respectively)compared to those observed in control corneas.The protein level of TNF-α in the corneas 3 d p.i.increased following fungal infection(p<0.01)and then,with DPI treatment,it increased further(p< 0.05).(9)Compared with their levels in normal corneas,the relative mRNA levels of IL-17 A increased following fungal infection at 5 d p.i.(p<0.05).Treatment with DPI increased its level(p<0.05)compared to those observed in control groups.Western blot indicated that the protein level of IL-17 A increased in corneas at 5 d p.i.after A.fumigatus infection,and treatment with DPI further increased its level.3.Pathway of NOX2 in Experimental A.fumigatus keratitis in mice Treatment with an inhibitor of dectin-1(laminarin),an inhibitor of SYK(PRT062608)and an inhibitor of PKC-δ(rottlerin)decreased the mRNA level of NOX2 expression(p<0.01,p<0.01,p<0.05,respectively)in fungi infected neutrophils.The Western blot shows the reduction of NOX2 protein expression by treatment with laminarin,PRT062608 and rottlerin.The level of dectin-1 increased upon infection and could be blocked by laminarin but not PRT062608 and rottlerin.p-SYK is the active form of SYK,and its levels increased upon fungal infection.Treatment with laminarin and PRT062608 reduced the expression of p-SYK but not rottlerin.The level of PKC-δ increased during infection,and treatment with laminarin,PRT062608 and rottlerin reduced PKC-δ expression.Conclusions: In summary,the data presented herein indicated that increased NOX2 levels in mouse corneas and neutrophils infected by A.fumigatus and they could be inhibited by DPI treatment.The inhibition of NOX2 resulted in an excessive inflammatory response with a decreased fungal clearance.Fewer ROS were produced,fewer NETs were formed.There was increased neutrophils infiltration than that observed in control cells.With DPI treatment,the expression of proinflammatory cytokines increased while the expression of anti-inflammatory cytokines decreased.The inhibition of dectin-1,SYK and PKC-δ all could decrease the expression of NOX2.All of the data indicated that NOX2 plays an important role against A.fumigatus in the mouse cornea that can be modified by the dectin-1,SYK and PKC-δ pathways.