G-CSF Regulates Suppression Of Aspergillus Fumigatus By Neutrophils

Aspergillosis is a series of diseases caused by infection of aspergillus spp.including invasive aspergillosis and other allergic diseases.Despite decades of research into the development of anti-fungal treatment,Aspergillus spp.infection still posts a major thread especially among hospitalized patients,it is still a huge burden on our health system.Latest research showed that invasive aspergillosis(IA)is still the second most common invasive fungal infections diseases only after candidiasis.Among the immune-compromised patients,including stem organ transplant recipients,patients with inherited immunodeficiency or acquired immune deficiency syndrome,the invasive aspergillosis is still associated with an unacceptable mortality rate of 50%.So,the research into the mechanisms of aspergillosis to develop new treatment plans is still in urgent need.Aspergillus fumigatus is the most common cause of aspergillosis,which could find in the soil,air and carbon-rich substrates.People usually inhaled hundreds of conidia of A.fumigatus each day,but only in immunocompromised patients it could cause diseases,indicating that immune system could recognized inhaled mold and avoid the germination.The mucosa-epithelium barrier can eliminate most inhaled conidia of A.fumigatus in healthy individuals.Recent studies showed that both alveolar macrophages and epithelial cells played an essential role in the elimination of conidia of A.fumigatus during early stages,and dysfunctions of these two cell types could result in inflammation in the lung or even angioinvasion of A.fumigatus which lead to infections in other organs and tissues.The innate immune response,which is the first line of defense in our body against invading pathogens,will be initiated if conidia of A.fumigatus colonize the lower airways while the mucosa-epithelium barrier fails to eliminate them.Neutrophil is one of the most important cells in innate immune system,is also the first responder during infections,it will be recruited into the infection site at very early stage after the barriers were penetrated.Recent studies showed that during the infection of A.fumigatus,neutrophil plays a more important role than macrophage as neutrophil can attack both conidia and hyphae while macrophage mainly attaches to the conidia.Neutrophil is the most abundant cell among leukocytes,it counts for 50-70 percent of white blood cells,and it plays an essential role during fungal infections including A.fumigatus infection.Neutrophils can kill pathogens directly via phagocytosis,releasing antimicrobial peptides,reactive oxygen species(ROS),and neutrophil extracellular traps(NETs),or indirectly via recruiting and activating other immune cells to initiate a full-size immune response by secreting cytokines and chemokines.But over-activation or prolonged activation of neutrophils could lead to detrimental consequences since these neutrophils could cause damage to self-tissue.So,a fine-tuned activation and regulation of neutrophils mediated immune responses is required for the successful elimination of invading pathogens while keeping the self-damages to a minimum.A ?20k Da glycoprotein called granulocyte colony-stimulating factor(G-CSF)which is encoded by CSF3 is one of the cytokines that regulate the development,survival,recruitment and activation of neutrophils.G-CSF has been shown to play essential roles during the development,proliferation,and survival of neutrophils,as well as neutrophils functions.There are extensive research into the protective role of G-CSF during infections,but the exact role of G-CSF played during A.fumigatus infection is still not clear,previous reports showed controversial or even opposite results.So,further research into the role of G-CSF in immune response against A.fumigatus would help bring more knowledge about how our immune system especially neutrophils performed its anti-fungal functions.After the conidia of A.fumigatus were inhaled,they were first in contact with stromal cells like epithelial cells and endothelial cells,and neutrophils required cytokines and chemokines to leave the capillary to go into the infection site and start performing its functions.So,in order to study the interactions among conidia of A.fumigatus,neutrophils and stromal cell-like human umbilical vein endothelial cells(HUVECs),we performed the following experiments and observed the following results as well.1.Neutrophil' involvement in aspergillosisBy analyzed lung tissue samples from patients with aspergillus spp.infections,we observed increased infiltration of neutrophils into the infection loci,but blood results showed that the neutrophils numbers and percentage in the peripheral blood remains in the normal ranges.2.Effects in the germination of conidia of A.fumigatus of HUVECsBy co-culture of conidia of A.fumigatus and HUVECs,we observed that HUVECs could prevent most of the conidia from germinating,then we analysed the gene expression profile of HUVECs upon stimulation of conidia,the results showed that CSF3 is among the most up-regulated genes.We next over-expressed or knockdown CSF3 in the HUVEC,the abilities of HUVECs in the prevention of germination of conidia were not changed by the different expressions of CSF3.3.Effects on the different expression of CSF3 of HUVECs in the neutrophils' suppression of germination of conidia of A.fumigatusAfter co-culture of conidia of A.fumigatus,HUVECs and neutrophils,we observed that neutrophils could limit the germination of conidia.After this ability was further enhanced by the over-expression of CSF3 and inhibited by CSF3 knockout.4.Cellular mechanisms underline the neutrophils' suppression of germination of conidia of A.fumigatusWe used western blotting to analyzed the phosphating of some key proteins in the activation and survival of neutrophils.We found out that phosphorylation of p38 MAPK,ERK and AKT were enhanced by G-CSF,and reduced in absence of G-CSF.In summary,this work revealed that the G-CSF and neutrophils it activated did have a role in preventing the conidia of A.fumigatus germinated into hyphae.But the inflammatory cytokines produced by neutrophils and the reduced apoptosis rate of neutrophils themselves rose more questions that need further investigations.We believe the further detailed understanding of the role of G-CSF and neutrophils play in fungal infections may help develop more treatment strategies against fungal infection-induced diseases.