Platelets Promote Neutrophil Migration To Fusarium Solani Sites And Its Mechanism In Vitro

Background and Objective:In the current treatment of fungal keratitis（FK）,the treatment effect is often poor due to the lack of effective antifungal drugs.Filamentous fungi,such as fusarium solani and Aspergillus fumigatus,are the most common pathogenic fungi of FK in China.In our previous study,which established a mouse model of Fusarium solani keratitis,we found that neutrophil recruitment to the fungal infection site was reduced after platelet removal;Meanwhile it was also found in the in vitro experiments that fungi induced AMP dependent protein kinase phosphorylation enhancement of corneal epithelial cells,causing proinflammatory factor IL-6 elevation;And platelets can promote neutrophils to phagocytose fungi and inhibit the growth of fungal hyphae.Therefore,this study aimed to examine whether platelets affect neutrophil chemotaxis under the stimulation of Fusarium solani by simulating the chemotaxis of neutrophils to fungal sites in the corneal site in vitro,and to clarify the mechanism of platelet promoting neutrophil migration to Fusarium solani,so as to provide new ideas for better treatment of FK.Methods:Neutrophils and platelets were isolated from human peripheral venous blood.Human immortalized corneal epithelial cells were cultured at 37℃in 5%CO₂environment,after pancreatic enzyme digestion and used later;The spores were extracted from the standard strain of Fusarium solani cultured in potato dextrose agar medium and used up to date.To investigate the effect of platelets on neutrophil chemotaxis under stimulation with Fusarium solani spores in the corneal epithelial environment,a Transwell migration assay was performed to measure the neutrophil migration ability,and the cells were divided into four groups:corneal epithelial cells（CE）group,platelets（P）group,corneal epithelial cells+spores（CE+S）group,corneal epithelial cells+platelets（CE+P）group,corneal epithelial cells+spores+platelets（CE+S+P）group and medium alone（control group）.Human corneal epithelium was seeded into 24 well plates and adherent for 4 hours;Spores and platelets were added into the well plates according to the proportion and grouping;The cells was incubated in a 37℃and 5%CO2 incubator environment for 2 hours;Their broth was collected by centrifugation;The centrifuged supernatant was added to the lower chamber of the Transwell plate;The upper chamber was seeded with equal numbers of neutrophils in each well according to the proportion;Transwell plates were placed in 37℃,5%CO₂incubator for 30 min to allow neutrophils to migrate;Neutrophils that migrated to the lower chamber were photographed using an inverted microscope,six fields were selected per well,and the amount of neutrophil migration was counted by the software Image J.To further define the effects of platelets on the expression of cell adhesion factors and pattern recognition receptors on the surface of neutrophils under inflammatory conditions,the cells were divided into three groups:neutrophils（N）group,neutrophils+spores（N+S）group,and neutrophils+spores+platelets（N+S+P）group and co-cultured at 37℃in a 5%CO₂incubator environment.Neutrophils were examined by immunofluorescence and flow cytometry for the expression of fluorescence intensity of adhesion factors:P-selectin glycoprotein ligand-1（PSGL-1/CD162）,L-selectin（CD62L）,galectin-3（aβ-Galactoside binding protein）and pattern recognition receptors:Dendritic cell-associated C-type lectin-2（Dectin-2）,Macrophage-induced C-type lectin（Mincle）;The mean fluorescence intensities of PSGL-1,CD62L,galectin-3,Dectin-2,Mincle expressed by neutrophils were counted by Image J.Results:The results of neutrophil migration showed that after culturing for 30 min,the amount of neutrophil migration down the chamber in the CE+S+P group was significantly higher than that in the Control,CE,CE+S,CE+P and P group（P<0.05）.There was no significant difference in the amount of neutrophil migration among the Control,CE,CE+S,CE+P and P group（P>0.05）.Platelets promote the expression of adhesion factors on the surface of neutrophils.Immunofluorescence staining showed that after 15 min of co-culture,the fluorescence intensity of PSGL-1and CD62L was low levels in the N group,whereas after the addition of spores and platelets（N+S+P group）,the fluorescence intensity of PSGL-1 and CD62L was significantly higher than that in the N and N+S group（P<0.001）;After 5 min of co-culture,the fluorescence intensity of galectin-3 expression on the surface of neutrophils was lower in the N group,and the addition of spores（N+S group）did not cause any change in its fluorescence intensity（P>0.05）.The fluorescence intensity of Galectin-3 in the N+S+P group was significantly higher than that in the N and N+S group（P<0.05）.Flow cytometry further confirmed the above results:After co-culture for 15 min,the expression of PSGL-1 on the surface of neutrophils was low in the N group,and the addition of spores caused a change in the fluorescence intensity of neutrophils（P<0.001）.The addition of the spores and platelets（N+S+P group）significantly increased the fluoresce-nce intensity of PSGL-1 expression on the surface of neutrophils,which was higher than that in the N and N+S groups.The difference was statistically significant（P<0.001）.After co-culture for 7 min in vitro,compared with the N group,there was no significant difference in the N+S group（P>0.05）.Platelets promoted the expression of CD62L on the surface of neutrophils significantly higher than that in the N and N+S groups（P<0.05）.After 2 minutes of co-culture,the expression of galectin-3 on the surface of neutrophils in the N+S+P group was significantly higher than that in the N and N+S group（P<0.05）,while there was no significant difference between the N and N+S group.Platelets promote the expression of pattern recognition receptors on the surface of neutrophils.When platelets were added for an co-reaction for 15 min in vitro,the results of immunofluorescence showed that the fluorescence intensity of Dectin-2 expressed by neutrophils stimulated by Fusarium solani spores was significantly higher than that in the N and N+S group（P<0.01）.Flow cytometry showed similar results:after 5 min of co-reaction in vitro,the expression of Dectin-2 on the surface of neutrophils in the N+S+P group was higher than that in the N+S group,and the difference was statistically significant（P<0.01）.When platelets were added to react in vitro for 5 min,the results of immunofluorescence showed that the fluorescence intensity of Mincle expressed by neutrophils stimulated by Fusarium solium spores was significantly higher than that in the N and N+S group（P<0.05）;After co-reaction for 2 minutes in vitro,Mincle expression on the surface of neutrophils in the N+S+P group was higher than that in the N+S group as detected by flow cytometry,and the difference was statistically significant（P<0.05）.Conclusions:During fungal infection,platelets stimulate the expression of adhesion factors PSGL-1,CD62L,and Galectin-3 on the surface of neutrophils,which causes a cascade of neutrophil and platelet aggregation,and promotes the migration and adhesion of neutrophils to endothelial cells;After entering the fungal infection site,platelets can induce the expression of pattern recognition receptors Mincle,Dectin-2 and adhesion factor galectin-3 on the surface of neutrophils to enhance neutrophil recognition of fungal pathogens and promote neutrophil migration to the fungal site.