HuR/CEBPD Regulates PTX3 Expression In Aspergillus Infection And The Study Of Clinical Diagnosis Of Pulmonary Aspergillosis

Chapter 1 Changes of CEBPD and PTX3 expression level during Aspergillus fumigatus infection Objective: To investigated the changes of CEBPD and PTX3 expression in Aspergillus fumigatus infection in vivo and in vitro.Methods: THP-1 cells and C57BL/6 mice were stimulated with Aspergillus fumigatus spores,and the expression of CEBPD and PTX3 protein were detected.Wax blocks of lung tissue from pulmonary aspergillosis patients were collected to detect CEBPD and PTX3 expression level.We predicted potential transcription factor binding sites for CEBPD in the PTX3 gene promoter sequence with bioinformatics database.Results:(1)Compared with the normal control group,both CEBPD and PTX3 proteins were significantly increased in THP-1 macrophages stimulated with Aspergillus fumigatus spores.(2)The lung tissue structure of the invasive pulomary aspergillosis(IPA)mouse was destroyed,large number of inflammatory cells infiltrated accompanied with bleeding and alveolar cavity collapse.Aspergillus fumigatus spores and hypha were observed in lung tissue of IPA mouse with PAS staining.(3)Compared with the NC mouse,CEBPD and PTX3 proteins in lung tissue and PTX3 expression in plasma and bronchoalveolar alveolar lavage fluid(BALF)were significantly increased in IPA mouse.(4)Immunohistochemistry staining showed that CEBPD and PTX3 expression in lung tissue of pulmonary aspergillosis patients were higher than those in normal lung tissue.(5)The presence of CEBPD binding sites on the PTX3 promoter region were predicted by bioinformatics results.Aspergillus fumigatus spore-infected macrophages m RNA sequencing showed that CEBPD,as a transcript of PTX3,was significantly over-expressed in Aspergillus fumigatus infection.Conclusions: Both in vivo and in vitro studies have shown that CEBPD and PTX3 proteins are significantly increased during the Aspergillus fumigatus infection.Chapter 2 Hu R/CEBPD regulates PTX3 expression during Aspergillus fumigatusinfection Objective: To investigate whether Hu R/CEBPD is involved in the regulation of PTX3 expression and affect the immunoprotective effects of PTX3.Methods:(1)After knockdown CEBPD expression by si RNA and over-expression CEBPD expression by lentiviral vector,detected Hu R,CEBPD and PTX3 protein expression level changed.(2)After knockdown Hu R expression by si RNA and over-expression Hu R expression by lentiviral vector,detected Hu R,CEBPD and PTX3 protein expression level changed.(3)Laser confocal microscope was used to observe phagocytosis ability of macrophages to Aspergillus fumigatus spores after changing expression of CEBPD or Hu R.(4)Chromosomal immunoprecipitation(Ch IP)method was used to detect whether CEBPD binds to the PTX3 gene promoter region.(5)The effect of Hu R on CEBPD m RNA stability was verified using actinomycin D to block cell transcription.Results:(1)Knockdown or over-expressed CEBPD resulted in reduced or increased PTX3 protein expression levels and macrophage phagocytosis ability,but Hu R expression was not significantly changed.(2)Both CEBPD and PTX3 protein expression levels were subsequently reduced or increased after knockdown or over-expressing Hu R,and the macrophage phagocytosis ability was also reduced or increased.(3)The total expression of Hu R was unaltered during Aspergillus fumigatus spores infection,whereas the expression increased in the cytoplasm,and the stability of CEBPD m RNA reduced or increased when knockdown or overexoression of Hu R.(4)Ch IP showed CEBPD dose bind to the PTX3 gene promoter region during Aspergillus fumigatus spores infection.(5)CEBPD m RNA stability increased after Hu R overexpression and decreased after Hu R knockdown.Conclusion: Hu R/CEBPD could regulate PTX3 expression during Aspergillus fumigatus infection.Hu R increased CEBPD expression by enhancing the stability of CEBPD m RNA through nucleus-cytoplasm transfer mechanism.CEBPD bound to the PTX3 gene promoter to increase PTX3 expression,and ultimately strengthened the process of phagocytose Aspergillus fumigatus spores by macrophages.Chapter 3 The performance of the BALF LFD test in the rapid diagnosis ofnon-neutropenic IPA Objective: We performed this study to evaluate the diagnostic potential of the Aspergillus-specific lateral-flow device(Asp LFD)test for invasive pulmonary aspergillosis in non-neutropenic patients.Methods: Clinical data and BALF were collected from suspected IPA patients without neutropenia,divided into IPA group and control group according to the final clinical diagnosis.The Asp LFD test was performed on all 136 BALF samples,of which 80 BALF samples were tested for galactomannan(GM)test.Results:(1)According to the guideline and clinical treatment,136 IPA suspected cases without neutropenia eventually diagnosed 41 cases IPA as a study group and 95 cases other diseases as a control group.(2)All 136 BALF samples had a sensitive of 69.7% and a specificity of 72.6% for Asp LFD diagnosis of pulmonary aspergillosis.(3)Asp LFD test had higher sensitivity(78.8% vs 69.7%,P<0.05),GM test had higher specificity(83.0% vs 70.2%,P<0.05),when comparing the GM and Asp LFD test results in 80 samples.(4)The sensitivity and specificity of the combination of the positive results of both the Asp LFD and GM tests were 63.6% and 93.6%,respectively.The sensitivity and specificity of either Asp LFD or GM positive results or both Asp LFD and GM positive results were 90.9% and 59.6%,respectively.Conclusion: Asp LFD has the potential to be an early and rapid diagnostic method for the patients with non-neutropenic invasive pulmonary aspergillosis.