In Vitro Study On The Effect Of Anti-Aspergillus Fumigatus Biofilm And MicroRNA-146a Regulates Aspergillus Fumigatus-induced Inflammatory Responses In Macrophages

Aspergillus fumigatus(A.fumigatus)is the most common pathogen of Aspergillus infection,A.fumigatus biofilm is one of the most important virulence factors.Macrophages play essential roles in innate immune system.Our study mainly discusses the effect of anti-aspergillus biofilm and microRNA-146a regulates A.fumigatus-induced inflammtory in macrophages.The first chapter mainly discusses the mechanism of the thermal effects on the inhibition of A.fumigatus biofilm.Our previous study have shown that heat stress has anegative regulation effect on polar growth of hyphae which will reduce the depth of biofilm.In order to explore the effect of thermal effects on the polar growth of A.fumigatus biofilm,we constructed a CaM-RFP A.fumigatus strain using the alcohol dehydrogenase promoter-fusion PCR technique to dynamically observe the localization of CaM during the polar growth of A.fumigatus.It was found that in the budding stage of spores,red fluorescence mainly focus on the budding.As the hyphae lengthen,the red fluorescence is still at the top of the growth of the hyphae.At 41℃,the CaM was distributed at the top of the hyphae and below.It is speculated that the excessive branching of the hyphae and the inability to maintain the polar growth of the hyphae may be related to the CaM distribution.We used q-RT-PCR to detect the expression levels of CchA and MidA mRNA,were up-regulated under thermal effect.The results revealed that the Ca2+signal pathway participates in the inhibition of the thermal effect on the A.fumigatus biofilm.The second chapter mainly discusses the i effect of ALA-PDT on A.fumigatus biofilm.The ALA incubation time was 2 h and the ALA concentration was 20 mM as the experimental parameters.Next,we investigated the effects of different light doses of ALA-PDT on A.fumigatus biofilm.When the light dose is 25 J/cm2,the biofilm structure is basically no significant difference from the normal biofilm.When the light dose is 50 J/cm2,the biofilm structure begins to loosen and the hyphae shorten.When the light dose is 100 J/cm2,the biofilm structure is more loose and the hyphae gradually break.We found that the thickness of the biofilm also decreased with the increase of the light dose,and the biofilm structure of the light group and the photosensitizer group remained basically unchanged.It is indicated that ALA-PDT has a certain destructive effect on the structure of biofilm.The third chapter of this study mainly discusses the role of macrophages in the immune response to A.fumigatus.The expression of pro-inflammatory factors such as TNF-α and IL-6 was detected and showed a time-dependent effect.The signal molecules such as p38 MAPK,and NF-κB were activated.The expression of Dectin-1,TLR2 and TLR4 receptors was down-regulated by flow cytometry.Next,we investigate the function of miR-146a in inflammatory responses in macrophages after A.fumigatus stimulation in this study.We found that TNF-αand IL-6 were increased in THP-1 macrophage-like cells treated with Aspergillus fumigatus.In Aspergillus fumigatus challenged THP-1 macrophage-like cells,overexpression of miR-146a by miR-146a mimics decreased TNF-α and IL-6 production,whereas downregulation of miR-146a by anti-miR-146a significantly enhanced TNF-α and IL-6.Our study demonstrates that the cross-talk between miR-146a and the inflammation regulatory pathways p38 MAPK and NF-κB might be a fine-tune mechanism in modulating the inflammatory response in macrophages with Aspergillus fumigatus infection.