To Explore The Effect And Mechanism Of TLR2-mediated Regulatory T Cell Differentiation On Aspergillus Fumigatus Lung Injury

Background:Aspergillus fumigatus is a conditional pathogenic fungus that has been ubiquitous in the natural environment.In recent years,due to the wide application of solid organ transplantation,glucocorticoids,and broad-spectrum antibiotics,the number of patients with invasive aspergillosis has been increasing year by year.Faced with the harsh living environment in the host,fungi have formed a series of corresponding mechanisms to facilitate their own growth and development,weaken the host’s immune response and avoid degradation.This is one reason why the antifungal resistance of A.fumigatus has been increasing year by year in recent years.Regulatory T cells（Tregs）have a negative immunomodulatory effect.However,this effect may also become a factor that limits host inflammation and promotes immune tolerance and immune escape.Toll-like receptor 2（TLR2）recognizes A.fumigatus cell wall components and regulates host immune response in A.fumigatus infection.Evidence has shown that TLR2 is a costimulatory receptor on T cells’surface and can participate in regulating the differentiation and proliferation of Treg cells.However,Treg cells’role after A.fumigatus infection and the mechanism of TLR2 mediated Treg cells under normal immune function are still unclear.Objective:To investigate the regulatory effect of TLR2 on regulatory T cells and its mechanism in lung injury induced by A.fumigatus.Methods:The standard A.fumigatus strain AF293（Aspergillus fumigatus Fresenius 293）was cultured,and the wild type（WT）and TLR2-deficient(TLR2^-/-)mouse models of A.fumigatus pulmonary infection were constructed by intraperitoneal injection using the tracheal drip method.CD25 antibody inhibited Treg cells in mice.CD4⁺T cells were isolated and extracted from mouse spleen cells,treated with TLR2 inhibitor C16H15NO4（C29）,and cultured for 72h.The fungal infection and pathological changes in the lung tissues of mice were observed under a microscope.Plate colony count determined the fungal load in the lung tissue.ELISA kit detected the expression levels of TNF-α,IL-6,and CCL2in the lung tissue homogenate.The lung was detected by q-PCR.The expressions of Foxp3 and TLR2 were detected by Western blot and q-PCR.The proportion of CD4⁺CD25⁺Foxp3⁺Treg cells was detected by FCM.Results:H&E staining showed that inflammatory cell infiltration appeared in the lung tissue of the mouse trachea after instillation of A.fumigatus spores,and the histopathological score was significantly increased（p＜0.0001）.TNF-α（p＜0.0001）,IL-6（p＜0.0001）and CCL2（p＜0.001）expression levels after infection were significantly higher than those in the control group.Compared with the control group,the proportion of CD4⁺CD25⁺Foxp3⁺Treg cells in the spleen of mice infected with A.fumigatus increased（p＜0.0001）,and the expression levels of Foxp3m RNA（p＜0.01）and protein（p＜0.05）in the lungs were up-regulated.After intraperitoneal injection of CD25 antibody,the proportion of Treg cells in the mice’s spleen decreased（p＜0.0001）.In the Treg cell suppression group24 hours after A.fumigatus infection,the blood vessels’inflammatory cells decreased.Compared with the control group,the histopathological score decreased（p＜0.05）,and the lung burden of A.fumigatus was slightly reduced（p＜0.05）.Compared with the control group,the expression of TLR2 was up-regulated in the lung tissue of WT mice 72 hours after A.fumigatus infection（p＜0.01）.Under normal immune conditions,the lung injury of TLR2^-/-mice was reduced after 72 hours of infection（p＜0.05）,and the fungal load in the lung tissue was slightly reduced（p＜0.05）.Compared with WT mice,the proportion of CD4⁺CD25⁺Foxp3⁺Treg cells in the spleen tissue of TLR2-deficient mice decreased（p＜0.0001）,and the expression level of Foxp3 in the lungs was decreased（p＜0.05）;while TLR2^-/-.The proportion of Treg cells in the spleen of mice is still lower than that in the WT infection group（p＜0.01）,and the expression of Foxp3in the lungs is reduced（p＜0.05）.In in vitro experiments,after C29intervention,the proportion of CD4⁺T cells that differentiated into CD25⁺Foxp3⁺Treg cells decreased（p＜0.0001）.Conclusion:Aspergillus fumigatus mediated the proliferation and differentiation of CD4⁺CD25⁺Foxp3⁺Treg cells by activating TLR2,causing continuous aggravation of lung injury in mice infected with Aspergillus pulmonary,which may be a potential mechanism for evading the host defense of A.fumigatus.