| Background and objectiveAspergillus fumigatus(A.fumigatus)is an important ubiquitous mold that can lead to the infection of human,animals and poultry.The most common infection is the pulmonary infection.A.fumigatus can produce conidia which are only 2-3μm that can reach the alveoli when they were inhaled into the lung.Afterwards,they can germinate into hyphae which will invade the lung tissue.The styles of infection are determined according to the immune status,underlying lung diseases,and the amount of conidia and frequency of inhalation.The common styles are invasive pulmonary aspergillosis(IA),chronic pulmonary aspergillosis(CPA)and allergic bronchopulmonary aspergillosis(ABPA).Among these styles of infection,the pathogenesis and treatments of IA and ABPA have been studied more often,however,the pathogenesis and treatment of CPA have been seldom been studied due to the lack of animal model.CPA usually happens to the immune competent people with underlying lung diseases(tuberculosis)or patients with minor immune defect.CPA has been a disease that threatens the life of human for its high mortality.Therefore,it’s essential for us to establish the animal model of CPA.There has been a murine model of CPA,in which the researchers injected the conidia enwrapped in agar into the lung of mice so that the conidia would not be easily cleared from the lung tissue.The infection could last for 28 days.However,in this model,conidia and hyphae could not interact with the immunocytes and agar could also arise the immune reaction which lead to the inaccuracy of immune response they have detected.Since hypha is the main morphology colonizes in the lung during the CPA course,it is important to study the immune response for understanding the pathogenesis of CPA.In addition,our laboratory has reported a nonsporulating strain of A.fumigatus which was isolated from the lung of a CPA patient with history of tuberculosis.Besides,it has been proved that the immune responses to conidia and hyphae were different and there has been controversial over these.In our research,we established the chronic hyphae of A.fumigatus colonization in lung to mimic the human CPA.We studied the differences between the immune responses to conidia and hyphae with the infection of conidia as a comparison in order to study the pathogenesis of CPA.Besides,our research provided a murine model for human CPA caused by nonsporulating strain of A.fumigatus.MethodologyIn our research,we tagged the A.fumigatus wild-type strain Af293 with Green fluorescence protein(GFP)and made the hyphae into tightly-structured hyphae balls with the diameter of 150-250 μm.The hyphae balls were injected into the lung of immuno-competent mice.The ex vivo live imaging,histopathology(Periodic acid-Schiff staining,PAS staining)and fungal burden of lung tissues were performed to confirm existence time of infection.We also monitored the changes of body weight and general condition,such as urine,excrement,fur and pulmonary manifestations to differentiate the acute and chronic infection.Besides,flow cytometry was employed to detect the different immune response to conidia and hyphae infection.Results1.Af293 tagged with GFP(Af293-GFP)The gene sequence of GFP was transfected into Af293,which made the conidia and hyphae both had fluorescence and would not quench in the lung for 28 days.2.The hyphae balls of Af293The hyphae of Af293 were made into tightly-structured hyphae balls with the diameter of 150-250 win.3.The distribution of Af293 in the lung tissueThe fluorescence of the lungs in conidia group could be seen at day 1 and day 7 post-infection,and not after.Fluorescence intensity in hyphae balls group was strong at day 1 and day 7,kept being weaker at day 14 and day 21,while it became stronger and diffuse at day 28 post-infection.4.The histopathology of lung tissueHistopathology of lung tissues in conidia group showed the conidia phagocytized by the macrophages at day 1 post-infection and abundant magenta tracheal secretions in the airway lumen at day 7 post-infection.There was no abnormality in the lung tissue afterwards.In the hyphae group,the PAS-stained sections showed magenta hyphae at each time point.Histopathology in the H group showed that plenty of neutrophils and macrophages were recruited to the infection site at day 1 post-infection.The hyphae ball grew into a large hyphae block in which many neutrophils and macrophages-infiltrated at day 7 post-infection.At day 14 and day 21 post-infection.,the quantity of hyphae decreased compared to day 7 post-infection,and the airway wall was destroyed at day 14 post-infection.The infection sites formed circumscribed foci that were infiltrated by neutrophils,macrophages and lymphocytes at day 21 and 28 post-infection.There was lung consolidation around the infection sites at day 28 post-infection.There was no invasion of lung interstitial and blood vessel.The histopathology was similar to the lung tissue of human CPA.5.The pulmonary fungal burdenThe fungal burden of the mice infected with conidia decreased over time.Consistent with the distribution of hyphae in the lung,the fungal burden of H group did not change at the first 7 days post-infection,began to decrease at day 14 and remained the same until day 21,with a considerable increase at day 28 post-infection,which indicated that there was a recrudescence at day 28 post-infection that was correspondence to the high recurrence rate of human CPA.6.The body weight change and general condition There was no significant difference between the variation tendencies of body weights of mice between the infection groups and uninfected group.Mice in each group did not display any other clinical signs such as dark urine,wheezing,fur ruffling,or increased mortality.7.The innate immune responseWe mainly investigated the function of macrophages and neutrophils in the infection ofA.fumigatus.In the conidia group,an influx of macrophages and fewer neutrophils were noted in the lumen.The number of leukocytes reached their peak at day 7 post-infection.There was no increase of neutrophils and macrophages compared to the uninfected group all over the experiment,while a moderate increase of lymphocytes at day 28 post-infection infiltrated around the lumen.There was an infiltration of neutrophils,macrophages and lymphocytes around the lumen in the hyphae balls group.The number of neutrophils reached the highest value at day 7 post-infection,and decreased afterwards,and macrophages and lymphocytes began to increase at day 1 post-infection,decreasing at day 7 post-infection.Many neutrophils and macrophages flooded into the lumen and the amount of neutrophils and macrophages peaked day 7 post-infection8.The adaptive immune responseIn the conidia group,the amount of Thl,Th2 and Th17 cells began to increase at day 1,reached the greatest at day 7,decreased at day 14 post-infection,and remained higher afterwards compared with the control group.The Treg cell kept being high from day 1 to day 21 post-infection in conidia group.However,in the hyphae balls group,there was no statistically significant change of Thl cells over the process of infection.There was a transient increase of Th2 cells at day 7 post-infection and a rise of Th17 cells in the meanwhile.The number of Treg cell reached the greatest at day 7 and decreased afterwards in the hyphae balls group.ConclusionOur research established a new murine model of chronic infection of hyphae to mimic the formation of CPA,providing a murine model for the study pathogenesis and new treatment of CPA and a model for the study of CPA arising from nonsporulating strain of A.fumigatus.Besides,we also compared the different immune response to conidia and hyphae of A.fumigatus. |
PDF Full Text Request