Research On The Effects And Mechanism Of TLR7 In Invasive Pulmonary Aspergillosis

Background:Invasive pulmonary aspergillosis is a serious and often fatal disease.Patients with acute leukemia or undergoing hematopoietic stem cell transplantation have a distinctive elevated susceptibility to aspergillosis and infections in these patients are associated with a high mortality about 40%to 50%.Triazole is currently the main clinical antifungal drug,while emerging Triazole resistance among Aspergillus is one of the greatest challenges to clinical success.In order to improve the clinical efficacy of Aspergillus related infections,immunomodulation combined with antifungal therapy is considered to be the most effective disease improvement treatment.Toll-like receptor 7（TLR7）is a highly conserved pattern recognition receptor for innate immune pathogens located in cells which was originally identified to be a receptor that recognizes single-stranded RNA of viral origin.Studies have shown that in addition to viral single-stranded RNA,TLR7 can also recognize nucleic acids released by bacteria.At the same time,limited research shows that TLR7 is also involved in immune regulation during the pathogenic process of Candida albicans infection and clinical studies have shown that the expression level of TLR7 on the surface of patients’T lymphocytes was negatively correlated with the occurrence of fungal infection.Recent clinical studies have reported that TLR7 gene polymorphisms are associated with Aspergillus infection,but whether TLR7 is involved in the host immune response process against Aspergillus infection remains unknown.Objective:This study aims to clarify the role and the underlying mechanism of TLR7 in the anti–Aspergillus fumigatus host immune response,and provide novel approaches for the development of anti-A.fumigatus immunotherapy.Methods:A mouse model of invasive pulmonary aspergillosis was established using A.fumigatus strain AF293 via intratracheal administration of A.fumigatus conidia and the mRNA expression level of TLR7 in the lungs and spleens at indicated infection time points were detected using Q-PCR.Both non–immune-compromised and immune-compromised animal model were established in wild type（WT）and TLR7-deficient(TLR7^-/-)mice.Survival rate experiments and determination of fungal burden in the lungs were used to clarify the role of TLR7 in A.fumigatus infection and mice were also pretreated with TLR7 specific agonist imiquimod（R837）before A.fumigatus infection,and the survival rate of mice was then observed to verify the role of TLR7 in A.fumigatus infection.By HE staining,PAS staining of lung tissue pathological sections,detection of lung dry-wet ratio and total protein levels in bronchoalveolar lavage fluid（BALF）,the role of TLR7 in lung injury in mice after A.fumigatus infection was determined.Flow cytometry（FCM）was used to classify and count the inflammatory cells in BALF of WT and TLR7^-/-mice post A.fumigatus infection and the levels of inflammatory cytokines and chemokines in the lungs were also detected by ELISA to investigate the immunomodulatory role of TLR7 in A.fumigatus infection.Macrophages and neutrophils that play an important role in innate immune and antifungal defenses were isolated and cultured in vitro.Macrophages and bone marrow–derived neutrophils from WT and TLR7^-/-mice were compared for their fungal killing capacity to clarify whether TLR7 modulates the intrinsic antifungal functions of macrophages and neutrophils.Through macrophage depletion,the role of macrophages in the anti–A.fumigatus host immune defense was confirmed.Results:1.TLR7 is up-regulated in the lung and spleen tissues of mice infected with A.fumigatusCompared with the control group,the mRNA expressions of TLR7 in the lung and spleen tissues of mice were significantly up-regulated 12hours after A.fumigatus infection,and the difference was statistically significant（p<0.05 for lung,p<0.01 for spleen）.2.TLR7 plays a harmful role in invasive pulmonary aspergillosis1)Survival rate experiments show that under normal immunization,in the high-lethal dose invasive pulmonary aspergillosis model,the survival rate of TLR7^-/-mice is significantly improved with a survival rate of 32%compared with 100%mortality in WT mice（p<0.001）.In the mild-dose model,the survival rate of TLR7^-/-mice was also significantly improved with a survival rate of 90%,compared with 100%mortality in WT mice（p<0.001）.2)Non–immune-compromised WT mice were pretreated with or without R837 before A.fumigatus infection,in the low-lethal dose invasive pulmonary aspergillosis model,compared with the control group,survival rate experiments showed that R837 treated mice had a significantly reduced survival rate（reduced by 30%,p<0.05）.In TLR7^-/-mice with the same infection dose,the survival rates of mice in the R837-treated group and the control group were both 90%,and the survival rate of TLR7^-/-mice was significantly higher than that in WT mice（p<0.001）.And when TLR7 was deficient,R837 treatment did not reduce the survival rate of mice infected with A.fumigatus（p>0.05）.3)In an immune-compromised murine model,consistent with the results in non–immune-compromised model,survival rate experiments showed that after lethal dose of pulmonary A.fumigatus infection,compared with WT mice,the survival rate of TLR7^-/-mice was significantly improved（increased by 54%,p<0.01）.3.TLR7-deficient enhances the fungal clearance of mice1)Non–immune-compromised TLR7^-/-mice displayed a significant improvement in the clearance of A.fumigatus conidia compared with WT mice on day 1 and day 3 after A.fumigatus infection（p<0.01）.The result was confirmed by PAS staining of the lung sections which showed that TLR7^-/-mice had less A.fumigatus conidia in the lungs than WT mice.2)Immune-compromised TLR7^-/-mice also displayed a significantly improved clearance of A.fumigatus conidia compared with WT mice on day 3 after A.fumigatus infection（p<0.05）.4.Deficiency of TLR7 reduces lung injury in mice with A.fumigatus infection1)Histopathological analysis of the lungs of WT mice infected with A.fumigatus revealed more advanced signs of alveolar hemorrhage,protein exudate in focal alveolar cavity and severe inflammatory infiltrates when compared with those of TLR7^-/-mice on Days 1 and 3 post infection.2)The wet-to-dry lung tissue ratio,a measure of lung edema,was markedly decreased in TLR7^-/-mice on Days 1 post infection（p<0.01）.TLR7^-/-mice had decreased capillary leakage in the respiratory tract,leading to significantly decreased total protein levels in the BALF of these mice（p<0.05）.5.TLR7 deficiency reduces the recruitment of inflammatory cells in the lungs of mice with A.fumigatus infection1)There were significantly lower numbers of leukocytes in the BALF from TLR7^-/-mice compared with WT mice.2)The cell populations in the BALF were then determined by FCM and we found that the percentage of the CD11b⁺Ly6G⁺neutrophils was comparable between WT and TLR7^-/-mice both on Days 1 and 3 post infection.The percentage of another important type of phagocyte CD11b⁺F4/80⁺macrophages was comparable between WT and TLR7^-/-mice on Days 1 post infection while TLR7^-/-mice had an increased percentage of macrophages compared with WT mice on Days 3 post infection（23%vs 14%）.There was reduced number of neutrophils,which reached statistical significance in TLR7^-/-mice on Days 3 post infection compared with that in WT mice.6.TLR7 deficiency results in decreased secretion of inflammatory cytokines and increased CCL2 in the lungsSignificantly decreased levels of IL-6 and CXCL1 were observed in the lungs of TLR7^-/-mice when compared with WT mice on Days 1 post infection（p<0.01,p<0.05）.TLR7^-/-mice also had significantly decreased pulmonary IL-17A levels compared with WT mice on Days 3 post infection（p<0.05）.Importantly,TLR7^-/-mice had significantly increased level of pulmonary CCL2（p<0.05）,a chemokine that facilitates macrophage recruitment on Days 3 post infection.The pulmonary levels of other inflammatory mediators,including TNF-a,IL-1β,IL-4,IL-10,IL-27,IFN-a,and IFN-γwere comparable between TLR7^-/-and WT mice.7.TLR7 deficiency leads to increased phagocytosis and killing of A.fumigatus in macrophages,but not neutrophilsMacrophages and bone marrow–derived neutrophils from WT and TLR7^-/-mice were compared for their fungal killing capacity,and TLR7^-/-macrophages,but not neutrophils,proved to be more efficient at eradicating live Aspergillus conidia due to the enhanced capacity of phagocytosis（p<0.05）.8.Macrophages plays important role in host defense against A.fumigatusDepleting macrophages by clodronate-encapsulated liposomes dramatically impaired the survival of mice infected with A.fumigatus compared with mice treated with PBS-encapsulated liposomes as a control（p<0.05）.Conclusion:Our findings highlight a detrimental role for TLR7 in antifungal host defence against invasive pulmonary A.fumigatus infection.The expression of TLR7 was up-regulated during A.fumigatus infection and we here first showed that loss of TLR7 decreased fungal colonization and mortality of mice both in non–immune-compromised or immune-compromised state.The protection observed in TLR7^-/-mice was associated with attenuated lung inflammation and immunopathology following A.fumigatus infection which resulted in less lung injury and oedema.Our data provide in vivo proof of concept for TLR7 as promising complement to conventional anti-A.fumigatus agents in the treatment of invasive pulmonary aspergillosis.