| Aspergillus fumigatus, widespread fungi in nature, can cause severe opportunistic infection in the immune-defecient patients. With the development of hematopoietic stem cell transplantation(HSCT) technology, more and more patients appear accompanied by an increase in the chance of infection after the HSCT in recent 20 years. On one hand, some risk factors such as malignancies, neutropenia or dysfunction, and use of glucocorticoid, steroids, or immunosuppressants cause immune dysfunction in patients. On the other hand, these risk factors also result in an increased incidence of invasive aspergillosis, which becomes one of the main reasons for death in the patients with immune suppression after the HSCT.For people with normal immune function, the swallowed aspergillus fumigatus is easily to be killed and then cleared by polymorphonuclear leukocyte(PMN). PMN is an important part of the body’s defense system, and it resists and destroys invading pathogens, which is associated with non-specific anti-infection by phagocytosis and production of antibodies. This type of cell has strong phagocytosis and bactericidal effects. It plays its role via chemotaxis, recognizing, binding, and swallowing substances and then uptaking intracellularly, ultimately injuring and degradating pathogens. However, the interaction between aspergillus fumigatus and human PMN has not been fully understood.Therefore in this study, the peripheral blood neutrophils from healthy people and patients with hematopoietic stem cell transplantation(4-6 weeks) were under the culture condition of aspergillus spores and their metabolic products after stimulation in vitro, In this experiment, human PMN is selected as a target cell. After the PMN mixed with aspergillus fumigatus spores and their metabolites under certain conditions, the morphology of the PMN were observed under an electron microscopy and the volume size of the PMN, superoxide ion(O2-) production by respiratory burst, CD64 expression on the surface of the PMN and variations of phagocytic rate and phagocytic index were determined by flow cytometry. Based on this, we expect to provide some experimental evidences for the pathogenesis in aspergillus fumigatus susceptible patients after the HSCT.Firstly, the PMNs were isolated and purified from the healthy people and the patients received HSCT. The PMNs purity was confirmed higher than 95% by giemsa staining dye and the cell viability exceeded 98% by trypan blue exclusion method. And we found that the neutral grain of nuclei from healthy people are rods or 2 ~ 5 leaf, leaf and leaf have filaments are linked together, while which from HSCT patients increases neutrophils cytoplasm and nuclear by light microscopy; After added the spore mixture we found that the PMN from patients were adhesion and spore number less than which from healthy people by electron microscopy, Patients with PAS staining also found that after adding Af D HSCT phagocytosis rate and index of PMN were significantly decreased, and the influence of PMN consuming rate and index of healthy people.The results show that PMN in the HSCT the patient’s blood more likely at immature state.Secondly, the aspergillus fumigatus spores and aspergillus fumigatus diffusate(AfD) were cocultured with human PMN for 2 hours. After that, electron microscopy and periodic acid-schiff(PAS) staining technologies were used to observe relationship between morphological changes and phagocytosis. Meanwhile, scanning electron microscopy and transmission electron microscopy were used to explore morphological changes in internal and external structure of the PMN prior to and post the interaction. After 2 hours of administrated the spore mixture(spores + AfD) stimulation the PMN found that healthy peoplemorphology and size of the PMN of the normal people is different from the patients received HSCT, and with a high phagocytosis rate and index of aspergillus spores in the Af D, while in the patients with HSCT, the the morphology and size of the PMN of the normal people is different from the patients received HSCT. Projections and filopodia formations are on the surface of the normal PMN. In addition, density of normal cytoplasm and cytoplasmic is relatively uniform and mitochondrial structure is complete. Meanwhile, in the process of endocytosis, intracellular lysosomes and endoplasmic reticulum significantly increased. After the HSCT, cytoplasmic nuclear of the PMN is markedly enlarged, and part surface of immature to lobocytes eosinophils is significantly elevated. Once activated by aspergillus fumigatus spores, the surface area of the PMN significantly increases. The PMN from the patients received HSCT adhere and swallow less spores compared to the normal ones, accompanied by significantly swelling mitochondria, expanded endoplasmic reticulum, and part of visible intracytoplasmic focal necrosis. Further, O2-level by respiratory burst, carboxyfluorescein succinimidyl amino ester(CFSE)-positive cell number were significantly lower in the PMNs of the patients received HSCT by spores mixture than those by spores suspension. The CD64 expression was also lower in the patients received HSCT than that in the normal ones. And the phagocytic rate and the phagocytic index of the PMN of the patients received HSCT were significantly reduced compared to the normal ones.To further explore mechanism of the effect on hematopoietic stem cell transplantation patients with PMN phagocytic function, the O2- levels and CD64 were measured by flow cytometry analysis of ROS in PMN. The results showed that the level of O2- and CD64 PMN from HSCT patients with a mixture of spores(Aspergillus fumigatus spores + AfD) PMN were significantly lower than in healthy people. And the reduced ability of induced cytotoxic Af D O2- production levels may be associated with HSCT patients PMN anti-Aspergillus reduce closely related. HSCT patients PMN surface CD64 expression in front of Aspergillus fumigatus spores + AfD late stimulate expression was not significantly changed, but the background HSCT patients PMN CD64 expression levels were significantly lower than in healthy PMN cells, which may break out level with HSCT neutrophil respiratory depression related.In summary, the patients received HSCT are susceptible to aspergillus fumigatus, which is associated with significantly decreased levels in O2- by respiratory burst and low levels of CD64 expression in the PMNs. AfD inhibits PMN cell phagocytosis function in patients with hematopoietic stem cell transplantation, which is associated with reduced generation of reactive oxygen species. Furthermore, it reveals the correlation between the morphology and construction changes of the PMN swallowing aspergillus fumigatus and the changes in the function in the patients received HSCT. |
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