| BACKGROUNDFungus is ubiquitous around our environment, and aspergillus accounts for 12% of fungus in the air. It can be inhaled commonly and clear effectively in immunocompetent people without developing into invasive fungal disease. In recent years, increasingly number of immune deficiency patients of tumor chemotherapy and organ transplantation, the widespread use of broad-spectrum antibiotics, corticosteroids and immunosuppressive agents, lead to invasive fungal infection incidence increased year by year. In china, a large multi center study showed, on the basis of the current invasive fungal infection diagnosis standard recognized at home and abroad, in non malignant blood disease patients ultimately confirmed in the top 7 for pulmonary fungal diseases were pulmonary aspergillosis(180 cases 37.9%), pulmonary candidiasis(162 cases 34.2%), pulmonary cryptococcosis(15.6%), pneumocystis carinii disease(23 cases 4.8%), pulmonary mucormycosis(10 cases 2.1%), penicilliosis mameffei(4 cases), histoplasmosis(2 cases). Invasive pulmonary aspergillosis(IPA) is characterized by hyphal invasion and necrosis and hemorrhage of lung tissue. IPA develops rapidly and with high mortality rate. Even with the continuous improvement of early diagnostic detection means to take preventive and empirical therapy treatment measures, the mortality rate of IPA is as high as 50%- 90%. The current immune therapy has become one of the important means of prevention and treatment of infectious diseases. Current clinical has started some immune regulation therapy to assist with antifungal therapy, including the administration of GM-CSF, IFN-γ, and obtained therapeutic effect. More attention given to role of natural immunity in aspergillus fumigatus infection will encourage more research on interventing host immune function to provide new ideas for the prevention and treatment of invasive aspergillosis.The innate immunity system recognizes a wide range of conserved fungal ’pathogen associated molecular patterns’(PAMPs), triggering a rapid, conserved response that activates inflammatory cells responsible for eliminating the intruder and initiating an appropriate adaptive immune response.t is achieved by engaging host pattern recognition receptor (PRR) that bind to fungal PAMPs. In addition to toll-like receptors(TLRs), The C-type lectin receptors(CLRs) of class of signaling PRR are involved in antifungal immunity. Dectin-1 is the best characterized signaling CLR and is expressed on the surface of bronchial epithelial cells and myeloid cells that binds β-glucan founded in fungal cell walls. Dectin-1 can stimulate a variety of cellular responses against A. fumigatus, including phagocytosis, cytokine production and the respiratory burst. Airway epithelial cells can upregulate dectin-1 expression and induce proinflammatory response and ROS generation in respond to A. fumigatus. Dectin-1-/- mice showed an increased mortality rate and an impairment in cytokine production, which results in poor neutrophil recruitment and fungal killing.The epithelium was usually thought to just serve as a barrier against invading pathogen. However, more and more studies have found that epithelial cells are capable of triggering an immune response similar to that of cells of the myeloid lineage. Corneal epithelium can significantly increase the expression of dectin-1 during the early period of Aspergillus fumigatus infection. Oral epithelial cells have the ability of inducing antimicrobial peptides, such as defensins. Human intestinal epithelial can secrete IL-8 and CCL2 in the present of β-glucan-consisting glycans curdlan and zymosan, and Syk inhibition significantly decreased β-glucan-induced chemokine secretion from intestinal epithelial cells. Human lung epithelial cell line A549 can increase the production of inflammatory mediators and antimicrobial peptides after stimulation molds. Bronchial epithelia play an important role in the innate defense against aspergillus fumigatus infection through the dectin-1 pathway, resulting in increased expression of TNF-a, GM-CSF, IL-8,HBD2 and HBD9, and induction of ROS generation.With a variety of animal studies and clinical trial studies on gene therapy successfully conducted, which makes the application of gene therapy in the treatment of diseases more attractive. At present, gene therapy vector include viral vectors and non viral vector. Compared with non viral vector, viral vector has higher transfection efficiency. Adenovirus, a double-stranded DNA virus, which has been widely used as a non-integrating vector in lung, transduces a wide variety of proliferating and non-proliferating cells and shows tropism for airway cells.Some clinical trials about adenoviral vectors for treatment of pulmonary diseases are being conducted.Gaven that Dectin-1 play a crucial role in aspergillus-induced immune responses, we constructed a murine model of upregulation of Dectin-1 in the airway by intratracheal administration of a recombinant replication deficient adenovirus encoding murine dectin-1 (Ad-mDectin-1) and studied its beneficial effect in a murine model of IPA.Part I study on identification and security of the murine model administrated with Ad-mDectin-1ObjectionTo constructe a murine model of upregulation of Dectin-1 in the airway by intratracheal administration of Ad-mDectin-1 and to study on identification and security of the murine model.MethodsBALB/c mice were divided into PBS group, Ad-EGFP group and Ad-mDectin-1 group, intratracheal administrated with PBS, Ad-EGFP and Ad-mDectin-1 respectively. At day 3 after administration, the levels of dectin-1 mRNA in lung tissue were tested by real-time PCR, the level and distribution of dectin-1 protein in lung tissue was detected by immunohistochemical, the levels of IL-6 in lung homogenate and serum were detected by ELISA, distribution of adenovirus vector in lung and liver was detected by confocal. At day 5,7,14 after administration, immunohistochemical was used to detect the change of level of dectin-1 in each group. The average optical density of dectin-1 staining on bronchial wall was analyzed with Image-Pro Plus 6.0.ResultsAt day 3 after administration of Ad-mDectin-1,the Dectin-1 mRNA expression was obviously increase in mice treated with Ad-mDectin-1, and was resulted in nearly eighteen fold increase compared with mice treated by Ad-EGFP or PBS(Ad-mDectin-1 group vs Ad-EGFP group, p=0.012; Ad-mDectin-1 group vs PBS group, p=0.011). There was no statistical differences of dectin-1 mRNA expression between controlled group(PBS group vs Ad-EGFP group, p=0.994). The distribution of expression of Dectin-1 in lungs of each mice at day 3,5,7 and 14 after administration was examined by immunohistochemistry. For mice treated with Ad-mDectin-1, results showed strong immunostaining in the airways epithelium in bronchioles and nearly negative immunostaining in alveolar walls, the immunostaining of Dectin-1 can last for two weeks, the positive particles were mainly located in cytoplasm and membrane of epithelial cells. In contrast, mice treated with PBS or Ad-EGFP did not detected immunostaining in the lungs. The average optical density of dectin-1 staining on bronchial wall in mice treated with Ad-mDectin-1 at different time points was examined and results showed time-dependent change with peak reach at day 5 after administration. Because Ad-mDectin-1 and Ad-EGFP can all cause infected cells expressed green fluorescent protein, we used confocal microscopy to detect the green fluorescent protein which can emit green fluorescence under excitation of 488nm laser. We found green fluorescence in lung but not in liver. Results of ELISA showed no significantly differences of levels of IL-6 were founded compared with mice treated with Ad-EGFP or PBS. Pathology of lung tissue of mice treated with Ad-mDectin-1 showed mild inflammation reaction, characterized by polymorphonuclear cells infiltration at 3 day post-infection and peribronchial lymphocytes hyperplasia after 3 days. Necrosis and structure changes were not found.ConclusionAfter intratracheal administration of Ad-mDectin-1,the expression of Dectin-1 in the lungs of mice were successful upregulated, and location of Dectin-1 was mainly on airways epithelium in bronchioles. The peak of expression of Dectin-1 was at day 5 after administration of Ad-mDectin-1, expression of dectin-1 can last for more than two weeks. After intratracheal of administration of Ad-mDectin-1, no significant adverse effect and obvious inflammatory reaction were observed. Adenovirus vector was mainly located in lung and did not spread to liver of distant organ.Part Ⅱ experimental study on Ad-mDectin-1 for immune protection of invasive pulmonary aspergillosis in miceObjectionTo study the role of Ad-mDectin-1 in immune protection of invasive pulmonary aspergillosis in mice.MethodsBALB/c mice were divided into PBS group, Ad-EGFP group and Ad-mDectin-1 group, intratracheal administrated with PBS, Ad-EGFP and Ad-mDectin-1 respectively.1 day after administration, mice were immunocompromised by intraperitoneal injection of 200mg/kg of cyclophosphamide.4 days later, mice were challenged with conidia of 3×105. At day 2 after inoculation of conidia, lungs and bronchoalveolar lavage fluid (BALF) were harvested. Levels of TNF-a and GM-CSF in lungs and BALF were examined by ELISA. The expressions of Dectin-1 mRNA, IL-1β mRNA and IL-10 mRNA in lungs were detected by real-time PCR. Lungs for histologic examination were stained with haematoxylin and eosin(H&E), Gomori methanamine silver(GMS). Lungs for fungal load were homogenized and inoculation on Sabouraud dextrose agar for 24h at 37℃, the results were presented as the numbers of colony forming unit (CFU) per gram of lung tissues. Lung MPO activity was measured as a maker of neutrophil sequestration. For survival analysis, mice were challenged with conidia of 5×106, mice were monitored daily for death post-A. fumigatus challenge.ResultsFor mice treated with Ad-mDectin-1, the levels of TNF-a in the lungs and BALF had no significantly different with those treated with PBS or Ad-EGFP(lung: p=0.215; BALF:p=0.906). However, Ad-mDectin-1 significantly increased the levels of GM-CSF in the lungs and BALF compared with control groups(lung:Ad-mDectin-1 group vs PBS group, p<0.001; Ad-mDectin-1 vs Ad-EGFP group, p<0.001. BALF: Ad-mDectin-1 group vs PBS group, p=0.002; Ad-mDectin-1 vs Ad-EGFP group, p=0.003.). Although it was already 1 week after administration of Ad-mDectin-1, we still found increased Dectin-1 mRNA compared with control groups(Ad-mDectin-1 group vs PBS group, p=0.043; Ad-mDectin-1 vs Ad-EGFP group, p=0.031). we found mice treated with Ad-mDectin-1 obviously elevated IL-1β mRNA expression in the lungs, resulting in nearly three folds increase compared with control groups(Ad-mDectin-1 group vs PBS group, p<0.001;Ad-mDectin-1 vs Ad-EGFP group, p<0.001).Results showed lung samples from control groups that were administrated with PBS or Ad-EGFP revealed less evidence of inflammation. In contrast, evidence of obviously inflammation was observed in mice treated with Ad-mDectin-1.Hematoxylin and eosin(H&E)-staining staining of lung tissue sections manifested profound inflammatory response in mice treated with Ad-mDectin-1, including inflammatory cells infiltration, interstitial inflammation and alveolar septal thickening. Grocott’s methenamine silver (GMS) staining of lung tissue revealed fewer germinating conidia and relatively fewer hyphae were detected in mice treated with Ad-mDectin-1 compared with controlled groups. Fungal levels for Ad-mDectin-1-treated mice were significantly lower than the levels detected in mice that received PBS or Ad-EGFP(Ad-mDectin-1 group vs PBS group, p=0.007; Ad-mDectin-1 vs Ad-EGFP group, p=0.007). Fungal burden of mice receiving Ad-mDectin-1 than that of mice receiving PBS or Ad-EGFP more than halved. For mice receiving AdmDectin-1, there was a significant increase in lung MPO activity compared with control groups that were treated with PBS or Ad-EGFP(Ad-mDectin-1 group vs PBS group, p=0.03; Ad-mDectin-1 vs Ad-EGFP group, p=0.036). For results of effect of Ad-mDectin-1 on survival in immunocompromised mice inoculated with A. fumigatus conidia, The mortality was 50% in mice treated with PBS and 40% in mice treated received Ad-EGFP, compared with no deaths in mice administrated with Ad-mDectin-1 (Ad-mDectin-1 group vs PBS group, p=0.012; Ad-mDectin-1 vs Ad-EGFP group, p=0.029; PBS group vs Ad-EGFP group, p=0.796).ConclusionTransient adeno virus-mediated Dectin-1 transgene expression in immunocompromised mice can augment innate immune response against A. fumigatus, resulting in elevated production of proinflammatory cytokines that were essential for neutrophil mobilization to lung, increased aspergillus clearance and improved survival rate. |
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