The Role Of Vitamin D3in The Infection Of Respiratory Tract With Aspergillus Fumigatus

Invasive pulmonary aspergillosis (IPA) is an important cause of death ofimmunocompromised patients. Aspergillus spores suspended in the air, after beinginhaled airway and contacting with airway epithelial cells, are expelled out by airwayepithelial cells through physical barriers and promoting cilia swinging. In addition,airway epithelial cells activate the immune response through innate immune recognition,and thereby promote the body’s clearance of fungi, which is a key initial part of thebody’s immune defense. Pulmonary macrophages are the first line of defense of hostimmune for inhaled spores, which can make use of pattern recognition receptors torecognize and engulf Aspergillus spores, and secrete inflammatory cytokines, thenrecruit peripheral blood mononuclear cells and neutrophils and start downstreamimmune response. It was found that by the research group through the experiment ofAspergillus fumigatus infecting HBE, THP-1macrophages, after macrophages and HBEcells receiving Aspergillus fumigatus spores stimulation, the upregulation of patternrecognition receptor expression was started, pathogens were recognized and engulfed,and the subsequent inflammatory response was started.Excessive inflammatory response occurred during the immune reconstitution is animportant mechanism leading to the death of patients with IPA. In the recent10years,the important role of vitamin D3in the immune response continues to receive attention.Vitamin D3can be synthesized in epithelial and endothelial cells of the mucosal barrier(including lung), and in macrophages by1-α-hydroxylase, and plays a role in the formof autocrine and paracrine. The regulation of Vitamin D involved in the immune andinflammatory includes two aspects, on the one hand, it can involve in pathogenelimination by increasing the secretion of antimicrobial peptides and defensins; on theother hand, it can also induce immune tolerance and keep immunity stable throughpromoting TH cell differentiation and inhibiting the secretion of proinflammatoryfactors. Lack of vitamin D can lead to reduced immunity and excessive inflammation.Some studies have shown that the regulation of vitamin D on alveolar macrophages andHBE cells plays an important role in the process ofAspergillus fumigatus infection.,Part1: Comparison of three different methods to transfect THP-1cellsPurpose: Through three methods of adenovirus, lentivirus and liposomes2000totransfect THP-1macrophages, we explore the best way to transfect THP-1macrophages,and then provide an initial basis for the research group experiments to interfere therelevant process ofAspergillus fumigatus infecting THP-1by VD.Methods: when THP-1macrophages were transfected using adenovirus, lentivirus andliposomes2000, the fluorescence expression of the transfected cells was observed byfluorescence microscope, the transfection efficiency of THP-1macrophages wasdetected by flow cytometer, and cell mortality rate was detected by trypan blue staining.Results: Liposomes2000for THP-1macrophages had bigger toxicity, higher cellmortality, and extremely low transfection efficiency, so that it was not suitable fortransfection. The transfection efficiency of adenovirus could always reach a higher levelin the case of small cell damage. However, due to THP-1macrophage was immune cell that had the ability to engulf and kill the virus, so compared with other cells, the viralMOI to achieve the desired transfection efficiency was significantly increased.Nevertheless, a large dose of virus transfection seriously affects cells immune status, soit will very likely affect subsequent experiments. As for lentivirus, its low yield andhuge experiment consumes impacts its application.Conclusions: In conclusion, THP-1macrophags are not the first choice as therespiratory model with VD interferingAspergillus fumigatus infection.Part2: Vitamin D3function of Aspergillus fumigatus infecting airways epithelialcellsPurpose: To investigate vitamin D3function, the ways to play the role of vitamin D3and the impact on HBE immune status when airway epithelial cell (HBE) was infectedbyAspergillus.Methods:1. Set up the control group, swelling spores (SC) group, resting spores (RC) group,infect HBE cells and then detect the expression of1-α-hydroxylase and vitamin Dreceptor. When vitamin D substrate25(OH) D3was added to above experiment groups,vitamin D expression was detected.2. Set up the control group, swelling spores (SC) group,25(OH) D3group, restingspores (RC) group, swelling spores (SC)+25(OH) D3group, resting spores (RC)+25(OH) D3group, infect HBE cells and detect the expression of its pattern recognitionreceptors Dectin-1receptor, antimicrobial peptide LL-37and pro-inflammatorycytokines IL-1β, TNF-α, IL-8’s.Results: In the basal state, HBEs express only trace amounts of1α-hydroxylase(CYP27B1) and VDR. Aspergillus spores stimulation significantly increases theexpression of VDR and CYP27B1in HBE cells and swelling spores (SC) was moreobvious compared with resting spores (RC). Provided a substrate25(OH)D3, theAspergillus stimulating HBE cells synthesize1,25(OH)2D3, and the effect in SC wasmore obvious than that in RC. A.conidia stimulus makes HBEs transcript antibacterialpeptides LL-37and proinflammatory factor (IL-1β, TNF-α, IL-8) and increases Dectin-1receptor expression in HBEs significantly. And the effect of stimulated swelling spores(SC) was more obvious than that of resting spore (RC); the VD substrate cansignificantly enhance LL-37transcription in HBEs stimulated byA.conidia.But at the same time, it significantly inhibits the proinflammatory factor(IL-1β,TNF-α,IL-8) expression, and makes Dectin-1synthetic significantly reduce.Conclusion: In the process of aspergillus fumigatus infecting airway epithelial cells,vitamin D3plays the role to make immune stability by raising the antibacterial peptideLL-37in the pathogen removal and inhibiting the expression of the pattern recognitionreceptors and proinflammatory factor.