| Aspergillus fumigatus is recognized as the most prevalent airborne fungal pathogen and causes severe and fatal invasive pulmonary aspergillosis (IPA) in immunocompromised hosts. IPA has become one of the most common pulmonary infection diseases due to the increase of immunocompromised patients and wide application of immunosuppressive therapies. Despite the availability of new antifungal drugs, the deaths caused by invasive aspergillosis has increased steadily in the last decades because of the difficulty in diagnosing and its rapid progression. A better understanding of the mechanisms responsible for defense against invasive aspergillus infection is urgent required to develop strategies for the prevention and treatment of invasive aspergillosis.Innate immunity is the first component of microbial recognition and serves as primary host defense. The innate immune system could rapidly recognize a wide spectrum of pathogens by pattern recognition receptors (PRRs). The best studied innate immune receptors are the toll-like receptors (TLRs), which have been found to be involved in defense against aspergillus fumigatus. Another subset of pattern recognition receptors is the recently identified nucleotide-binding oligomerization domain (NOD) proteins, which were found intracelluar proteins and have been implicated in intracellular recognition of pathogens. The NOD family comprise more than 20 members, among which NOD1 and NOD2 are of the best studied. NOD1, also called caspase recruitment domain (CARD)-4 protein, has been found to recognize the peptidoglycans degradation product GlcNAc-MurACc-LAla-c-D-Glu-meso-DAP (GM-triDAP). Peptidoglycans containing meso-diaminopimelate acid are mainly found in Gram negativebacteria. NOD2 ,also called caspase recruitment domain (CARD)-15 protein , is mainly expressed by two cell types : epithelial cells and APCs. NOD2 mediates responsiveness to the muramyldipeptide MurNAc-L-Ala-D-iso-Gln (MDP) conserved in peptidoglycans degradation product contained in both gram negative and positive bacteria. Studies have demonstrated that NOD proteins are involved in host recognition of various bacteria. They stimulate NF-κB signal pathway by means of downstream receptor-interacting protein 2(RIP2) and promote the secretion of cytokine and proinflamantory molecules.The most frequent site of human aspergillus fumigatus infection is the lung. Once inhaled, spores reach distal areas of the lung by virtue of their small size. It has been shown that the lung epithelial cells and the alvelar macrophage cells can internalize and kill conidia, to secrete cytokine and proinflamantory molecules, and play an important role in defence against aspergillus fumigatus infection. There are a few reports about the expression of NOD proteins in the lung, The role of NOD proteins in the recognition of aspergillus fumigatus has not been reported yet. As aspergillus fumigatus are intracelluar infection pathogens, we hypothesized that NOD proteins might be the cytoplasmic pattern recognition receptors of aspergillus fumigatus, playing a role in innate immunity to defence against pulmonary aspergillus fumigatus infection. In this study we detected the expession of NOD Associated Proteins in Lungs of Mice infected with aspergillus fumigatus and to research the possible mechanism of the recognition of aspergillus fumigatus conidia by NOD proteins both in vivo and in vitro conditions.Part I The Expression of NOD Associated Proteins in Lungs of Mice Infected with Aspergillus FumigatusObjective: To observe the expression of nucleotide-binding oligomerization domain (NOD) proteins and it's downstream receptor-interacting protein 2 (RIP2) kinase in lungs of mice after infected with aspergillus fumigatus. Methods: Sixty male BALB/c mice were randomly divided into the infection group and the control group (n=30). The modelof mouse infected with aspergillus fumigatus was established by dropping aspergillus fumigatus conidia into the nares of each mouse in infection group. Equal volumes of normal saline was dropped into the nares of each mouse in control group .Aspergillus fumigatus burdence , pulmonary histopathology change were observed at different time points [before (0h) and after inoculated with aspergillus fumigatus conidia or normal saline 6h, 12h, 24h, 48h]. Semiquantitative RT-PCR was performed to observe the expression of NOD1,NOD2 and receptor-interacting protein 2 (RIP2) mRNA in lungs at different time points. Immunohistochemistry was also performed to detect the expression of NOD2 proteins in lungs of both two groups . Results : (1) The burdence of aspergillus fumigatus in lungs of infection group mice reached peak at 24h, and decreased rapidly at 48h. (2) In both groups there were same pathological changes such as peribronchitis. In infection group, the lesion was much severe than that in control group, the inflammatory cells infilitration was most obvious at 24 h, while clearance of inflammatory cells was observed at 48 h. (3) In infection group the level of NOD1 mRNA expression before(0h) and after droping aspergillus fumigatus conidia 12h, 24h, 48h was 0. 34 ± 0. 05, 0.41 ± 0.08, 0.46 ± 0.07, 0.47 ± 0.04, 0. 47 ± 0. 48 respectivly; The level of NOD2 mRNA expression was 0. 30 ± 0. 03, 0. 36 ± 0. 06, 0. 45±0.10, 0. 60 ± 0. 06, 0. 73 ± 0. 08 respectivly and the level of RIP2 mRNA expression was 0. 44 ± 0. 10, 0. 56±0. 09,0. 62±0. 07,0. 64±0. 07,0. 63 ± 0. 06 respectivly. Comparing with that at 0h, the expression of NOD1, NOD2 and RIP2 mRNA were upregulated significantly at 12h, 24h, 48h after droping aspergillus fumigatus conidia (P<0. 05) .There were no statisticly significant changes in expression of NOD1, NOD2 and RIP2 mRNA in control group at different time points. (4) The location of NOD2 proteins was mainly in alveolar epithelial cells . The immunohistochemistry positive index of NOD2 protein in infection group mice lung was 117582.7 ± 20189.58, 163854. 4 ±17185. 61, 341363.9 ± 35102. 07,289114. 5 ± 38424. 35 at 0h, 12h, 24h,48h time point respectivly. Comparing with that at 0h , the expression of NOD2 proteins was upregulated significantly at 24h after droping aspergillus fumigatus conidia (P< 0. 05) . There were no statisticly significant changes in expression of NOD2 proteins in control group at different time points. There were significantdifference in expression of NOD2 proteins between two groups at 24h, 48h time point (P<0. 05) . Conclusion: NOD proteins , especially NOD2, have relationship to pulmonary infection with aspergillus fumigatus, and may participate in the innate resistance to pulmonary aspergillus fumigatus infection .Part II The Relationship among the Expression of NOD2 Protein ,the Stimulation of Aspergillus Fumigatus Conidia and the Secretion of TNF-α in A549,THP1 cellsObjective: To observe the expression of NOD2 protein and RIP2 in human alveolar epithelial cell line A549 cells and human monocyte cell line THP1 cells stimulated with aspergillus fumigatus conidia under different conditions. Methods: (1) The cell line A549 and THP1 cells were incubated with aspergillus fumigatus conidia for 10h,24h,48h, then the phagocytosis were examined with a transmission electron microscope(TEM). (2) Real-time PCR was performed to detect the expression of NOD2,RIP2 and tumor necrosis factor-α (TNF-α ) mRNA in A549,THP1 cells before and after incubated with aspergillus fumigatus conidia for different time. (3)A549,THP1 cells were exposed to conidia with different cell-fungi ratio for 10 hours, real -time PCR was performed to detect the expression of NOD2 mRNA. (4) Immunofluorescence was also performed to observe the expressin of NOD2 protein in A549 cells after exposed to conidia for 24 h . (5) Immunosedimentation and western blot methods were performed to detect the experssion of NOD2 protein in A549, THP1 cells after exposed to conidia with different cell-fungi ratio for 24 hours. (6) The cells were divided into 4 groups, stimulated with none(control group) , MDP alone , aspergillus fumigatus conidia alone , aspergillus fumigatus conidia combinated with MDP respectivly. Specific capture enzyme-linked immunosorbent assays (ELISA) was performed to quantify TNF-α secretion for evaluating the influence of MDP (specific ligand of NOD2) on the cytokine secretion by cells stimulated with heat-killed conidia. Results: (1) The internalized heat-killed aspergillus fumigatus conidia could be found in both A549,THP1 cells after inculbation with conidia for 10h.Partly digested conidia could be seen in lysosome of A549, THP1 cells after incubation with conidia for 24h. (2) The expression of NOD2 , RIP2, TNF-α mRNA were upregulated significantly after incubated with aspergillus fumigatus conidia for 24h compared with that before incubation (P< 0. 05) . (3) The expression of NOD2 mRNA was upregulated with the increase of cell-fungi ratio, and there were significant difference between the groups (P<0. 05) . (4) The expression of NOD2 protein was upregulated after exposed to aspergillus fumigatus conidia for 24h. (5) The expression of NOD2 protein was upregulated after inoculated with different cell-fungi ratio aspergillus fumigatus conidia for 24h, and the upregulation is dose-depended, there were significant difference between the groups (P <0. 05) . (6). The level of TNF-α secretion in 4 group of A549 cells (control group , MDP group , aspergillus fumigatus conidia group, aspergillus fumigatus conidia combinated with MDP group) were 10.46 ± 0.70, 11. 36±1. 20, 12.63 ± 2.76, 13. 94 ± 3. 42 pg/ml respectivly and 27.40 ± 5.72, 27.93 ± 9.11, 32.63 ± 4.71, 82.13 ± 10. 76 pg/ml respectivly in 4 group of THP1 cells. MDP combinated with heat-killed conidia could significantly (P <0. 05) increase the secretion of TNF-α. Conclusion: The expression of NOD2 protein respond to the digestion of aspergillus fumigatus conidia, and play a role in TNF-α secretion. It may serves as PRRs to activate the downstream singal pathway through RIP2 kinase by recognizing the degradation product of aspergillus fumigatus conidia.
|
PDF Full Text Request