The Experimental Research Of The Interaction Between Aspergillus Fumigatus Conidia And Pulmonary Alveolar Macrophage

Aspergillus is a kind of fungus capable of causing life-threatening opportunistic infections in immunocompromised patients. Patients with host defenses compromised by malignancy, granulocytopenia, neutrophil dysfunction, corticosteroid therapy, or immunosuppressive drugs are at great risk. Inhalation of airborne Aspergillus conidia in the alveoli primarily results in pulmonary infection, usually with dissemination to other organs. The incidence of invasive aspergillosis (IA) has increased considerably in the past decade, and this infection is a major cause of mortality in immunocompromised hosts. To a nomal immune function man, Aspergillus conidia can be cleared off by pulmonary alveolar macrophage (PAM).Unfortunately, the relarionship between PAM and Aspergillus conidia is still unclear. PAM is very important in the protection of lung infection. When it is activated by exotic matierials, the exotic cellular signals can change the protein mass spectra by activating or inactivating transcription factors, regulate-controlling sequences. It can not only boost the abilities of phagotosis, disposal and presentation of antigens but also excrete multiple cytokines and chemotaxis mediators such as interleukin 1β(IL-1β), tumor necrosis factor α(TNF-α), macrophage inflammatory protein(MIP) and leukotriene B4 (LTB4) , et al, promote T and B cells to proliferate and mature, collect neutrophil to lung, raise the immune ability of lung to resist the bad effect from exotic materials. It has already been confirmed by experiments that those inflammatory mediaters are very important factors in the protection of Aspergillus infection. NF-κB, a protein family consisting of complex sub-polypeptide units, exists ubiquitouly in Eukaryota. NF-κB is a central cell nuclear transcription factor in signal conducting pathway, relating with many important funtions such as immune, the occuring and developping of tumor, the regulating of cell units and embryo growth, et al. It is reported that the activation of NF-κB is very important during the Aspergullus infection. In our experiment, using PAM as target cells, we observed the morphologic changes, the releasing levels of inflammatory mediators of PAM and relating signal pathway research when stimulated by Aspergillus fumigatus conidia and its metalites. Firstly, we established and stablized the models of PAM and immunosupressive PAM in vitro, PAM was identified with Reiz coloration and immune histochemistry methods. Then we used scan and transmiss electron microscopy to observe the morphologic changes by incubating PAM and Aspergillus fumigatus conidia or metablites together or alone. The results suggest that exterior of nomal PAM has plenty of microruga, the surface area of PAM was increased so as to make it possible to conglutinate and englut more conidia when activated by Aspergillus fumigatus conidia; at the same time, it provides the histologic evidence of augmentation of lysosome and endoplasmic reticulum. Aspergillus fumigatus's metablites such as gliotoxin and Aspergillus fumigatus diffusate(AfD) have some cellular toxic functions on PAM and may do accelerating functions in the development of Aspergillus infection. Gliotoxin can induce the apotosis of PAM. We haven't found any toxic function of fumagillin and helvelic acid on PAM.The incidence of PAM rise greatly when the quantity of Aspergillus fumigatus conidia increased. Secondly, we checked up the levels of cytokines(IL-1βand TNF-α) in the culture supernatant by ELISA. We found that Aspergillus fumigatus conidia can stimulate nomal PAM to release much IL-1βand TNF-α, having quantity-effect and time-effect relationship. Immunosuppresive PAM releases less IL-1βand TNF-αthan nomal PAM does. AfD can inhibit the releasing of IL-1βand TNF-αpartly stimulated by Aspergillus fumigatus conidia. The results suggest that AfD do some toxic functions on PAM and may accelerate the development of Aspergillus infection. Because the well known materils of toxic Aspergillus fumigatus do not have obvious effect on PAM, it is necessary to make more research on the component of AfD. There are three methods to study transcription factors: electrophoretic mobility shift assay(EMSA), western blot(WB) and gene analysis, but these methods just can do semi-quantity, spend too much time and are lack of specificity and sensitivity. ELISA of checking NF-κB is a new method to check its energy. It can be more easily and spend less time to check up NF-κB quantificationally. Research had suggested that TLR may do some functions in the interaction between Aspergillus fumigatus conidia and PAM. In ourresearch, we used anti-TLR4 to block the TLR4 on the membrane of PAM and then observed the pathway activated by Aspergillus fumigatus conidia from cellular view. We confirmed that Aspergillus fumigatus conidia could activate PAM, improve the levels of IL-1βand TNF-αreleased by PAM which relating with activated of NF-κB, but its function could be blocked partly by TLR4(the cytokine turned less when TLR4 had been blocked ). Based on above, we conclude that the interaction beween Aspergillus fumigatus conidia and PAM is associated greatly with the activation of NF-κB. From our experiments, we confirm some relativity between the morphologic results and its functions. At the same time, Aspergillus fumigatus conidia could activate nomal PAM and stimulate it to release high levels of IL-1βand TNF-α, while immunosupressive PAM release less IL-1βand TNF-α, which suggest that high levels of IL-1βand TNF-αare in favor of the cleatance of Aspergillus fumigatus conidia. Morever, NF-κB is a central factor in PAM when it is activated by Aspergillus fumigatus conidia, but other signal pathway may participate in this activation of PAM.