Effects Of Granulocyte-macrophage Colony-stimulating Factor On Pathological Changes And Mechanism Study In Fungal Keraitis

ObjectiveTo study effects of granulocyte-macrophage colony-stimulating factor(GM-CSF) on pathological changes in fungal keratitis and its mechanism.MethodsAnimal models were built with Fusarium solani and Aspergillus fumigatus isolated from the eyes of fungal keratitis patients in BALB/c mice;two models were respectively divided into 2 groups,one of which was injected with GM-CSF while the other not; Control groups included pure GM-CSF injection and normal corneas.Pathological lesions were examined with slit-lamp microscope,Hematoxylin-eosin(H&E) staining,and periodic acid-Schiff(PAS) staining.Fungal burdens were examined in the infected corneas.Expression of TLR-2,TLR-4,TLR-9,and MMP-9 mRNA were detected with reverse transcription-polymerase chain reaction;Protein expression of TLR-2,TLR-4, TLR-9,MMP-9,tumor ncerosis factor a(TNF-a),and interleukin 10(IL-10) were examined with immunohistochmistry;Proteins quantity of MMP-9,TNF-a and IL-10 were detected with gelatin zymography and enzyme linked immunosorbent assay (ELISA),respectively.ResultsIn the corneas infected with Fusarium solani and Aspergillus fumigatus,the pathological lesions were more serious in the groups without GM-CSF injection than those with GM-CSF injection.The hyphae of Fusarium solani grew horizontally,and Aspergillus fumigatus grew vertically in the corneal stroma.On day 1,3,5,and 8,fungal colonies were cultured positive in the corneas infected with Fusarium solani and Aspergillus fumigatus,which were much less in the groups with GM-CSF injection than those without GM-CSF injection.Expression of TLR2,TLR4,TLR9,and MMP-9 mRNA were detected in all groups; and the expression quantity in the normal corneas and pure GM-CSF injection groups were similar and much less than those in the fungal infection groups.On day 1 after infection,expression of TLR2,TLR4,TLR9,and MMP-9 mRNA were much more in the groups with GM-CSF injection than those without GM-CSF injection,which reduced gradully later,especialUy obvious in the former;TLR2 and TLR9 were not detected with western-blot or immunohistochemistry in every groups;TLR4 was negative in the normal corneas and pure GM-CSF injection groups,but positive in the infected corneas,which were consistent with the expression of TLR4 mRNA.Bands of MMP-9 protein were detected with gelatin zymography,whose expression was higher in the groups with GM-CSF injection than in those without GM-CSF on day 1 and 3 after inoculation,while lower in the former than in the latter on day 5 and 8.TNF-a and IL-10 was not detected in the normal corneas and pure GM-CSF injection groups with immunohistochmistry or ELISA,but found positive in the corneas with fungal infection,with the highest expression on day 3.ConclusionsAt the early stage of fungal infection,GM-CSF can enhence the phagocytosis capability of inflammatory cells,which benefit for the clearance of pathogens.GM-CSF may play important roles in antifungal immunity by increasing the expression of TLRs, especially TLR4,and promoting cytokine secretion.Meanwhile,GM-CSF do not enhance the immune injury caused by overexpression of MMP-9.Appropriate timepoint and dosage of GM-CSF administration is key to sucessful intervene in fungal keratitis.