Innate Immune Function And Mechanism Of Transcription Factor PU.1 In Aspergillus Fumigatus Infection

BackgroundAspergillus fumigatus (A. fumigatus) is a common saprophytic mold that forms airborne spores (resting conidia) in our environment. Humans inhale this pathogen daily. In immunecompetent hosts, inhaled conidia are killed and cleared by the pulmonary immune system. However, in immunocompromised hosts, inhaled conidia can result in a variety of infectious diseases, including invasive pulmonary aspergillosis (IPA), aspergilloma, allergic bronchopulmonary aspergillosis (ABPA). PA is a lifethreatening disease that occurs mainly in patients with acquired immune deficiency syndrome (AIDS), cancer therapy, hematopoietic stem cell transplantation (HSCT), solid organ transplantation (SOT) and glucocorticosteroid therapy. In recent years, because of the increasing number of immunocompromised patients, the morbidity and mortality of IPA increases substantially. In addition, despite development of diagnostic techniques and antimicrobial therapies, patient outcomes remain poor. A better understanding of the mechanisms between host immune system and A. fumigatus may contribute to the prophylaxis of IPA.Innate immune system is the first line in human to defense against the invasion of pathogens. Alveolar macrophages (AMs) is the most important innate immune cell in respiratory system, which can recognize the pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs). Dendritic cell-associated C-type lectin receptor (Dectin-1), toll-like receptor (TLR)-2 and TLR-4 are three important PRRs expressed on the AMs membrane. They can specifically recognize different components of conidia cell wall, and activates macrophages to phagocytosis and kill the invading conidia, and further release inflammatory cytokines and chemokines. Several studies have shown that, both in human and mice, Dectin-1, TLR-2 and TLR-4 gene silence or knockout can result in increasing risk of invasive aspergillosis, decreasing inflammatory cytokine levels and higher fungal burden. Therefore, Dectin-1、TLR-2 and TLR-4 are essential for the innate defense against A. fumigatus.Transcription factor PU.1 (encoded by SPI1) belongs to the E26-transformation-specific (ETS) family. It has been demonstrated that PU.1 plays a critical role in the differentiation and development of hematopoietic system. In recent decades, increasing number of studies show that PU.1 involve in the function of innate and adaptive immunity. PU.1 is important for macrophages development and transcriptional control of inducible genes in mature macrophages, including Dectin-1, TLR-2 and TLR-4. Therefore, based on the fact that PU.1 is critical for the regulation of these three receptors in macrophages, we hypothesized that intervention of PU.1 may affect the innate defense against A. fumigatus. In this study, we successfully constructed a THP-1 derived macrophages model overexpressing PU.1 and silencing PU.1. And we further tested whether intervention of PU.1 would affect the innate immune functions in THP-1 derived macrophages during A. fumigatus stimulation, including cytokine release, phagocytosis and killing ability.Chapter I Overexpression and silencing of PU.1 in THP-1 derived macrophages ObjectionTo constructe a THP-1 derived macrophages model of overexpression and silencing of PU.1 via recombinant adenovirus and small interfering RNA. To further study on the changes of Dectin-1. TLR-2 and TLR-4 expression after intervention of PU.1.Methods① THP-1 derived macrophages were transfected with PU.1 small interfering RNA (siRNA) (siPU.1 group). Cells that transfected with negative control siRNA and untransfected were used as control groups (Mock group and siNC group). Forty-eight hours later, total RNA and protein were extracted. Transcriptional and translational expression of PU.1、Dectin-1、TLR-2 and TLR-4 were analysed by Real-time PCR and Western blot. ② To confirm the optimal multiplicity of infection (MOI) of adenovirus vector, THP-1 derived macrophages were transfected with adenovirus vector containing enhanced green fluorescent protein gene (Ad -EGFP) with a MOI of 500,1000,1500 and 2000, respectively. The fluorescence post-transfection with the Ad-EGFP at different time points was observed by fluorescence microscope. ③ THP-1 derived macrophages were transfected with PU.1 recombinant adenovirus at an optimal MOI (Ad-PU.1 group). Cells that transfected with empty adenovirus vectors and untransfected were used as control groups (Mock group and Ad group). Transcriptional and translational expression of PU.1、Dectin-1、TLR-2 and TLR-4 were analysed by Real-time PCR and Western blot.Results① Results of fluorescence microscope showed that fluorescence was higher than the other time points at 48 h with a MOI=1500, and could last for more than 72 h. ② THP-1 derived macrophages were transfected with Ad-PU.1 for 48 h. Compared with Mock group, Real-time PCR showed that PU.1, Dectin-1, TLR-2 and TLR-4 mRNA expression levels in Ad-PU.1 group increased 80.39-fold(p<0.001),8.31-fold (p<0.01),2.56-fold (p<0.001) and 4.28-fold (p<0.05), respectively, and 51.6-fold (p<0.001),3.05-fold(p<0.05),2.26-fold(p<0.001)and2.92-fold(p<0.05)compared with Ad group. ③ THP-1 derived macrophages were transfected with PU.1 siRNA for 48 h. Real-time PCR showed that, in siPU.l group, PU.1, Dectin-1, TLR-2 and TLR-4 mRNA expression levels were 19%(p<.001),53.7%(p<0.01),59.7% (p<0.Ol) and 56.6% (p<0.05) of that of Mock group, and were 17.7%(p<0.001), 47.1%(p<0.01),64.4%(p<0.05)和 61.2%(p<0.05)of that of siNC group. In addition, changes of PU.1, Dectin-1, TLR-2 and TLR-4 protein levels were consistent with that of mRNA levels.ConclusionTHP-1 derived macrophages model of overexpression and silencing of PU.1 were successfully constructed. In addition, overexpression of PU.1 results in the increased expression of Dectin-1, TLR-2 and TLR-4, and silencing of PU.1 decreases their expression.Chapter Ⅱ Role of PU.1 overexpression in THP-1 derived macrophages during A. fumigatus stimulationObjectionTo observe the effection of PU.1 overexpression in THP-1 derived macrophages during A. fumigatus stimulation.Methods①THP-1 derived macrophages were transfected with Ad-PU.1. Cells that transfected with empty adenovirus vectors and untransfected were used as control groups (Mock group and Ad group). Forty-eight hours later, cells were stimulated with A. fumigatus conidia for 0 h,4 h,8 h and 12 h, respectively. Total RNA and cell culture supernatants were extracted at indicated time points. IL-1β, TNF-a and IL-10 expression were measured by Real-time PCR and enzyme linked immunosorbent assay (ELISA). ② THP-1 derived macrophages were transfected with Ad-PU.1. Cells that transfected with empty adenovirus vectors and untransfected were used as control groups (Mock group and Ad group). Forty-eight hours later, cells were stimulated with FITC-labeled conidia for 4 h. Cells were gently washed with PBS and co-cultured with Dil and DAPI staining of the membrane and nucleus, respectively. Phagocytosis function was performed on a laser scanning confocal microscope. ③ THP-1 derived macrophages were transfected with Ad-PU.1. Cells that transfected with empty adenovirus vectors and untransfected were used as control groups (Mock group and Ad group). Forty-eight hours later, cells were stimulated with resting conidia for 0 h,4 h and 8 h, respectively. Killing ability was measured by killing experiment.Results① Real-time PCR showed thatTNF-a and IL-1β mRNA increased significantly in Ad-PU.1 group, compared with the control groups. But IL-10 mRNA increased without significance. In protein levels, ELISA showed that TNF-α increased significantly as well, and IL-10, without significance. ② Compared with control groups, confocal laser-scanning microscopy results demonstrated that the phagocytosis was enhanced in the Ad-PU.1 group. ③ Killing experiment further demonstrated that killing ability in THP-1 derived macrophages was significantly enhanced with overexpression of PU.1.ConclusionDuring A. fumigatus infection, inflammatory cytokine release, phagocytosis and killing ability were enhanced significantly with PU.1 overexpression. However, anti-inflammatory cytokine IL-10 increased without significance. Therefore, overexpression of PU.1 can enhance the innate defense against A. fumigatus in THP-1 derived macrophages.Chapter Ⅲ Role of PU.1 silencing in THP-1 derived macrophages during A. fumigatus stimulationObjectionTo explore the role of PU.1 silencing during A. fumigatus stimulation in THP-1 derived macrophagesMethods① THP-1 derived macrophages were transfected with PU.1 siRNA for 48 h. Cells that transfected with PU.1 negative control siRNA and untransfected were used as control groups (Mock group and siNC group). Cells were stimulated with conidia for 0 h,4 h,8 h and 12 h, respectively. Total RNA and cell culture supernatants were extracted at different time points. Expression of TNF-α, IL-1β and IL-10 were assessed by Real-time PCR and ELISA. ② THP-1 derived macrophages were transfected with PU.1 siRNA for 48 h. Cells that transfected with PU.1 negative control siRNA and untransfected were used as control groups (Mock group and siNC group). Cells were then co-cultured with FITC-labeled conidia. Four hours late, cells were co-cultured with Dil and DAPI staining of the membrane and nucleus, respectively. Phagocytosis function of THP-1 derived macrophages was performed on a laser scanning confocal microscope. ③ THP-1 derived macrophages were transfected with PU.1 siRNA for 48 h. Cells that transfected with PU.1 negative control siRNA and untransfected were used as control groups (Mock group and siNC group). Cells were stimulated with resting conidia for 0 h,4 h and 8 h, respectively. Killing ability of THP-1 derived macrophages was measured by killing experiment.Results① TNF-αand IL-1β mRNA decreased significantly in siPU.1 group, compared with the control groups (Mock group and siNC group). However, IL-10 mRNA increased significantly. In protein levels, results of ELISA were consistent with that of mRNA levels. ② Compared with control groups (Mock group and siNC group) phagocytosis was decreased in the siPU.1 group. ③ Killing ability in THP-1 derived macrophages was significantly decreased with silencing of PU.1.ConclusionDuring A. fumigatus infection, inflammatory cytokine release, phagocytosis and killing ability were decreased significantly with PU.1 silencing. However, anti-inflammatory cytokine IL-10 increased significanly. Therefore, silencing of PU.1 can decrease the innate defense against A. fumigatus in THP-1 derived macrophages.