Expression Of Toll-like Receptors In Immunosuppressed Hosts And Immunue Response To Aspergillus Fumigatus Infections

Since 1990s of last century, the incidence of invasive aspergillosis(IA) has been increased continuously, mainly because of an expanding population of immunocompromised hosts. The overall incidence of IA has actually surpassed invasive candidiasis and has emerged as the major life-threatening infectious complication in the immunocompromised hosts. IA is difficult to diagnose in the early stage because of its rapid progression. The options of powerful antifungals are restricted. The efficacy of antifungal agents is suboptimal. The mortality rate of IA is about 40~65%. Individuals with apparently normal immune defense mechanisms even the individuals with compromised immune system may rarely be infected by A. fumigatus when they are exposed to a few A. fumigatus. Infection of IA mainly occurs in the individuals whose immune defensive function is decreased with variousconditions such as prolonged neutropenia, hematologic malignancy and long period steroid treatment, allogeneic bone marrow transplantation or solid organ transplantation and acquired immunodeficiency syndrome. The host immune system to fungal infections includes innate immunity and adaptive immunity. Both Europhiles and macrophages are considered as the first lines of innate immunity while dendritic cells and Th cells usually mediate adaptive immunity. The on set of the disease, its development and outcome are determined by host immune system. Recent studies have demonstrated that TLR signaling pathway plays a crucial role in innate and Th1-cell-mediated resistance to the fungal pathogens. The TLR4-deficient mice are susceptible to Aspergillus conidia and died shortly after infection. The experimental studies might elucidate pathogenesis of the infection and molecular mechanisms especially in immunosuppressed hosts. The information collected from the study may provide a new way for finding strategies of diagnosis and preventive measures of IA. Our present studies include three parts as follow:1. Establishment of animal model of invasive pulmonary aspergillosis in immunosuppressed mice and pathological analysis: Immunosuppressed mice were induced by intraperitoneal injections of prednisolone. The quantity of peritoneal macrophages and spleen cells were counted at day 3. The Aspergillus fumigatus conidia were droped into nares of each mouse to cause invasive pulmonary aspergillosis. All the mice were killed at the different time points and the lung tissue was cultured and histopathologically analyzed. RESULTS: the peritoneal macrophages of the mice decreased sharply after prednisolone injections. The number of spleen cells and lymphocytes of PBMC was also reduced significantly (p<0.01). A great deal of Aspergillus fumigatus and tissue necrosis were observed in the lung after infected withaspergillus fumigatus in immunosuppressed mice.2. The changes of toll-like receptor mRNA expression in peritoneal macrophages of immunosuppressed mice: The expression of TLRl-13 mRNA of the peritoneal macrophages of normal mice and mouse monocyte/ macrophages cell strain RAW264.7 were determined by RT-PCR technique. Immunosuppressed mice were induced by prednisolone intraperitoneal injections. The expression of TLRs mRNA of the peritoneal macrophages of immunosuppressed mice were tested with RT-PCR. RESULTS: all thirteen of the TLRs reported to date of the macrophages in normal mice and RAW264.7 were expressed. Some of TLRs were up-regulated in mRNA level when the mice and RAW264.7 were treated with glucocorticoids. The results have shown that TLRs are important component parts of innate immunity to fungal infections. Prednisolone has toxical effect to macrophages and decreases the peritoneal macrophages of the mice (6. 33 ± 1. 76×106 vs 3.18 ± 0. 57×106 p<0. 001) . It can inhibite the growth even induces the cell death of the RAW264.7 cells.3. RAW264.7 immune response to inactivated Aspergillus fumigatus conidia: RAW264.7 cells were cultured in RPMI1640 added with 10% FCS. These cells were stimulated in two different conditions: the cells in the first group were stimulated with inactivated Aspergillus fumigatus conidia directly while the cells in the second group were firstly stimulated by prednisolone 24h followed by inactivated Aspergillus fumigatus conidia. After 8h incubation with inactivated Aspergillus fumigatus conidia in both groups, cells were harvested and total RNA was isolated. The expression of TLR1-9 mRNA were examined by RT-PCR RESULTS: co-cultivation of RAW264.7 cells with inactivated Aspergillus fumigatus conidia resulted in increase in some of TLRs...