The anti-inflammatory regulation of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis

AIM:To analyze the impact of calcitonin gene-related peptide(CGRP)in mouse keratitis after Aspergillus fumigatus(A.fumigatus)infection.METHODS:C57 BL/6 mice were treated subconjunctivally with different concentrations of exogenous CGRP,and BALB/c mice were treated with CGRP8-37(a CGRP antagonist)before corneas were infected with A.fumigatus.The cornea was assessed under the slit-lamp and the clinical score was recorded.The mRNA levels of IL-1β,TNF-α,IL-6,and MIP-2 were detected by quantitative real-time polymerase chain reaction(PCR),while the protein level of IL-1βwas determined by Western blotting.In vitro,RAW264.7 cells were used to investigate NLRP3 and IL-1βexpression induced by A.fumigatus after the pretreatment of exogenous CGRP or CGRP8-37.Cytokines expression in RAW264.7 cells was evaluated by real-time PCR and Western blotting.RESULTS:Using exogenous CGRP resulted in downregulated synthesis of IL-1βand MIP-2 stimulated by A.fumigatus in C57 BL/6 mice keratitis,and the synthesis of IL-1β,MIP-2 and IL-6 was up-regulated in BALB/c mice corneas after the pretreatment with CGRP8-37.Pretreatment with exogenous CGRP and CGRP8-37 did not influence TNF-αmRNA levels either in BALB/c or C57 BL/6 mice keratitis.The levels of NLRP3 and IL-1βwere both reduced in A.fumigatus stimulated-macrophages after treatment with exogenous CGRP.And CGRP8-37 pretreatment would increase NLRP3 and IL-1βlevels.CONCLUSION:CGRP may alleviate the inflammatory reaction in mice keratitis after infection with A.fumigatus.The anti-inflammatory effect may be related to the inhibition of NLRP3 expression by CGRP.

High-mobility group box1 as an amplifier of immune response and target for treatment in Aspergillus fumigatus keratitis

AIM:To determine the roles of high-mobility group box1(HMGB1)in pro-inflammation,host immune response and its potential target for treatment in Aspergillus fumigatus(A.fumigatus)keratitis.METHODS:Expression of HMGB1 was tested in C57 BL/6 normal and infected corneas.Dual immunostaining tested coexpression of HMGB1 with TLR4 or LOX-1.C57 BL/6 mice were pretreated with Box A or PBS and then infected.Clinical scores,polymerase chain reaction,ELISA,and MPO assay were used to assess the disease response.Flow cytometry were used to test the effect of Box A on reactive oxygen species(ROS)expression after A.fumigatus stimulation in polymorphonuclear neutrophilic leukocytes(PMN).C57 BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation,and MIP-2,IL-1β,TNF-α,HMGB1 and LOX-1 were measured.Macrophages were pretreated with Box B or Box B combined with Poly(I)(an inhibitor of LOX-1)before stimulating with A.fumigatus,and MIP-2,IL-1β,TNF-α,LOX-1,p38-MAPK,p-p38-MAPK were measured.RESULTS:HMGB1 levels were elevated in C57 BL/6 mice after infection.HMGB1 co-expressed with TLR4,and LOX-1 in infiltrated cells.Box A vs PBS treated C57 BL/6 mice had lower clinical scores and down-regulated corneal HMGB1,MIP-2,IL-1βexpression and neutrophil influx.Box B treatment amplified expression of MIP-2,IL-1β,TNF-α,HMGB1 and LOX-1 that induced by A.fumigatus in macrophage.Compared to the treatment of Box B only,the protein expression of IL-1β,TNF-αshowed inhibition of Box B combined with Poly(I),which also reduced the A.fumigatusevoked protein level of LOX-1 and phosphorylation level of p38-MAPK.The production of A.fumigatus-stimulated ROS was significantly declined after Box A pretreatment in PMN.CONCLUSION:Blocking HMGB1 reduces the disease response in C57 BL/6 mice.HMGB1 can amplify the host immune response through p38-MAPK,and is a target for treatment of A.fumigatus keratitis.