The Role Of Netrin-1 In The Mouse Cornea During Aspergillus Fumigatus Infection

Purpose To explore the effects of Netrin-1 on inflammation in A.fumigatus-Infected mouse cornea and proliferation and migration of human corneal epithelial cells(HCECs).Methods 1.Netrin-1 m RNA and protein expression were detected in normal and infected C57BL/6 mouse corneas by PCR,western blot and immunofluorescent staining.A2 BAR m RNA expression was tested in normal and infected mice corneas by PCR.2.Mice were injected subconjunctivally with or without recombinant Netrin-1.The severity of the disease was determined by clinical scores,photography with a slit lamp,myeloperoxidase(MPO)assay and immunofluorescence staining of polymorphonuclear neutrophilic leukocytes(PMNs).PCR,Western blot tested expression of IL-1β,TNF-α and IL-10.3.RAW 264.7 cells were stimulated by A.fumigates.Netrin-1 and A2 BAR m RNA expression was tested by PCR.Netrin-1 protein expression was tested by Western blot.PCR tested expression of IL-1β,TNF-α and IL-10.4.The role of A2 BAR receptor was demonstrated in vivo by detecting the expression of the inflammatory factors IL-1β,TNF-α,and IL-10 in corneas pretreated subconjunctivally with A2 BAR antagonist(PSB1115).In vitro,RAW 264.7 cells were stimulated with A.fumigatus and netrin-1,with or without PSB1115 pretreatment.PCR tested expression of IL-1β,TNF-α and IL-10.5.A Cell Counting Kit-8(CCK-8)assay was used to evaluate HCECs proliferation ability.Cell migration ability was determined by cell scratch experiments with HCECs.Results1.Netrin-1 expression decreased slightly in 1 day post-infection(p.i.)and then rose to a peak in 3 days p.i..Immunofluorescence staining showed that Netrin-1 protein was predominantly expressed in the epithelium of the normal mouse cornea,and expression levels in the corneal epithelium and stroma were both increased 3 days after infection with A.fumigates.A2 BAR expression increased in 1 day p.i.,with a subsequent decline in 3 days p.i.2.Exogenous Netrin-1 attenuated the inflammatory response,PMN infiltration,and expression of pro-inflammatory factors IL-1β and TNF-α compared with PBS group,while anti-inflammatory cytokine IL-10 expression was up-regulated.3.In RAW 264.7 cells,Netrin-1 expression decreased slightly in 4 hours and then rose to a peak in 8 hours.A2 BAR expression was significantly elevated by 75% ethanol-killed A.fumigatus at 2,4,6,8,10 hours and peaked at 4 hours.Recombinant Netrin-1 obviously inhibited m RNA expression of IL-1β and TNF-α and promoted expression of IL-10 m RNA.4.PSB1115 pretreatment resulted in disease aggravation in mice corneas.Levels of IL-1β and TNF-α were increased both in vivo and in vitro,while IL-10 expression was decreased.5.Moreover,exogenous Netrin-1 significantly facilitated the proliferation and migration of HCECs compared with normal control.ConclusionsNetrin-1 attenuated inflammation via the A2 BAR receptor after A.fumigatus infection in C57BL/6 mice.Besides,Netrin-1 can promote proliferation and migration of HCECs.