Roles Of PU.1 In Innate Immunity Against Aspergillus Fumigatus And Regulation Of Dectin-1 Expression

Advances in medical technologies such as chemotherapy or organ transplantation and the growing appearance of AIDS patients have led to a noted increase in the incidence of invasive Aspergillosis(IA)in the last several decades.IA as the most common type of the invasive fungal infections usually came along with sophisticated clinical mani-festations,difficulty in diagnosis and poor prognosis.Despite the development of new diagnostic tests and treatments,the mortality of IA remains high.Aspergillus fumiga-tus(A.fumigatus)accounted for 90%cases of infections.Innate immune system are the first line of defence against fungal infection,being able to recognise and initiate an effective response to eliminate invading A.fumigatus,but our knowledge is limited in the regulation and detailed mechanism.Thus,further investigation of the mecha-nism during A.fumigatus infection would help develop effective immunoprophylaxis to make great improvement in patients' diagnosis andprognosis.Pattern recognition receptors(PRRs)recognise a variety of pathogen-associated mol-ecules(pathogen-associated molecular patterns,PAMPs)expressed by an invading microorganism and then induce downstream events including phagocytosis,respira-tory burst,and releasing cytokine and chemokine which polarize adaptive immune response crucial for clearance of infection.Dectin-1 is a well-defined C-type lec-tin-like receptor(CLR)that recognises ?-glucans presenting in the cell wall of A.fu-migatus and initate antifungal immunity.The transcription factor PU.l as a member of Ets family is essential for hematopoietic stem cell development and regulation of im-mune response.In Pneumocystis-infected mice,the expression of Dectin-1 and PU.1 is decreased in AMs.However,dectin-1 is up-regulated in human bronchial epithelial cells(HBE)infected with A.fumigatus.Nevertheless,the role of PU.l regulating the transcription and expression of Dectin-1 remains unclear during A.fumigatus infection.We demonstrated the expression of PU.1 in THP-1 cells infected with A.fumigatus for the first time,characterized the transcription factor binding sites(TFBSs)in hDectin-1 promoter,and further deter-mine the expression change of Dectin-1 following up-regulation or down-regulation of PU.1.Chapter 1 Roles of PU.1 and Dectin-1ininnate immunity against Aspergillus fumigatusObjective:Recent studies have shown that the expression of PU.1 and Dectin-1 are decreased in AMs in pneumocystis-infected mice,however,Dectin-1 is up-regulated in human bronchial epithelial cells infected with A.fumigatus.Thus,we intended to determine the expression and subcellular localization of PU.l and dectin-1 in THP-1 cells infected A.fumigatus.Methods:To determine the expression and subcellular localization of PU.1 and Dec-tin-1 in THP-1 cells infected with A.fumigatus conidia using quantitative real-time PCR,Western Blot and immunofluorescence.Results:The expression of PU.1 protein increased post infection and translocated from cytoplasm to neucleus.The expression of Dectin-1 mRNA increased from2 h.p.i.to 8 h.p.i.,and the expression of Dectin-1 protein increased from 2 h.p.i.to 12 h.p.i.Conclusion:The increased expression of PU.l protein translocated from cytoplasm to neucleus in THP-1 infected with A.fumigatus conidia,and the expression of Dectin-1 mRNA and protein increased.Chapter 2 Mechanism of PU.1 in regulation ofhDectin-1 transcriptionObjective:To investigate the transcription factor binding sites of PU.1 in hDectin-1 promoter sequence.Methods:To predict the potential transcription factor binding sites of PU.1 in hDec-tin-1 promoter sequence utilizing bio informatics database and validate it with lucifer-ase reporter gene assay,chromatin immunoprecipitation(ChIP),and electrophoretic mobility shift assay(EMSA).Results:The expression of the PU.1 in HEK293T cells was found to enhance the lu-ciferase activity in a dose-dependent manner;ChIP and EMSA showed that PU.l pro-tein bound hectin-1 promoter at-1053?-1047,-719?-713 and-527?-521 sites.Conclusion:Transcription factor PU.lcanactive hDectin-1 transcription;PU.1 protein bound hDectin-1 promoter at-1053?-1047,-719?-713 and-527?-521 sites.Chapter 3 Roles of PU.1 in regulationof Dectin-1 expressionObjective:To determine the expression change of Dectin-1 following up-regulation or down-regulation of PU.1.Methods:To determine the expression of Dectin-1 in THP-1 cells when up-regulating and down-regulating the expression of PU.1 with Ad-PU.1 and siPU.1.Results:The expression of the Dectin-1 increased in THP-1 cells infected with Ad-PU.1 virus at 48 h.p.i..The upregulated Dectin-1 was reduced in PU.1 silence THP-1 cells at 12h post-infection with A.fumigatus compared to those transfected with siNS.Conclusion:PU.1 expression overexpression or knockdown by adenovirus or siRNA caused a increase or decrease in Dectin-1 in protein level in THP-1 cells.