Screening Of The Novel Potential Biomarkers For Pulmonary Tuberculosis Diagnosis And The Mechanism Of Two Important Molecules Regulating The Inflammatory Response Of Macrophages

Tuberculosis(TB)as a zoonotic disease poses a serious threat to human health,caused by Mycobacterium tuberculosis(MTB).Compared with other countries,China shows a high incidence trend.With the environmental pollution,the emergence of multi-drug resistant strains and the spread of AIDS,the incidence of tuberculosis become more and more severe.There is an urgent need to diagnose,treat tuberculosis early and control the epidemic with high sensitivity and low-cost way,based on rapid and specific molecular target.Besides,it will be useful for early and accurate diagnosis and control of TB.Currently,proteomics and transcriptomics have been widely used in the research of various body fluids,such as blood and urine,and have become the omics tools to use in various diseases research.Our research screened biomarkers for pulmonary tuberculosis from urine: mannose-binding lectin 2(MBL2),a 35 k Da fragment of inter-?-trypsin inhibitor H4(ITIH4-35k),retinol binding protein 4(RBP4)and miR-625-3p as potential biomarkers,and then established the combinational diagnostic model.And the specific mechanism of regulating Mycobacterium tuberculosis mediated inflammatory response was discussed in detail according to the blood candidate biomarker miRNA-362-5p.Moreover,confirmed the pathogenesis of Mycobacterium tuberculosis protein YrbE3A by activating innate immunity in vivo and in vitro,to reveal the pathogenic mechanism of Mycobacterium tuberculosis and the identification of RD region protein as the specific diagnostic antigen of tuberculosis.The main contents are as follows:(1)Screening and identification of novel TB diagnostic biomarkersThe urine proteomic profiles of 45 pulmonary tuberculosis patients before initiating anti-TB treatment and 45 healthy controls were analyzed and compared using two-dimensional electrophoresis with matrix-assisted laser desorption/ionization time of flight mass spectrometry.The 19 differentially expressed proteins were preliminarily identified and then confirmed with q RT-PCR and western blotting at the translational and transcriptional levels,respectively,on samples from additional 122 pulmonary tuberculosis patients and 73 healthy controls.Two proteins,mannose-binding lectin 2 and a 35 k Da fragment of inter-?-trypsin inhibitor H4 were identified as being highest differentially expressed.Also,we constructed a protein-micro RNA interaction network mainly involved with complement and inflammatory responses.A total of 11 micro RNAs from micro RNA-target protein interactions were screened and validated using q RT-PCR as part of the above samples including 97 pulmonary tuberculosis patients and 48 healthy controls.Only miR-625-3p displayed significantly differential expression(p <0.05).Coincidently this miR-625-3p was predicted to target mannose-binding lectin two protein.A binary logistic regression model based on miR-625-3p,mannose-binding lectin 2,and inter-?-trypsin inhibitor H4 was further established.This three-biomarker combination showed better performance in tuberculosis diagnosis than individual biomarkers or any two-biomarker combination by generating a diagnostic sensitivity of 85.87%(95%CI 77.05%–92.26%)and a specificity of 87.50%(95%CI 74.75%–95.27%).These novel urine biomarkers could play a significant role in improving tuberculosis diagnosis.(2)Effection of miR-362-5p on immune response of Mycobacterium tuberculosis escaping hostMiRNA-362-5p expression patterns were analyzed by using qRT-PCR in MTB-infected human THP-1 cells and mouse RAW246.7 cells.The results revealed that miRNA-362-5p levels were significantly declined in a time-dependent manner.We also investigated the expression of miRNA-362-5p in PBMC of 75 pulmonary TB patients,39 differential diagnosis groups and 40 healthy controls.The expression of miRNA-362-5p in pulmonary TB patients was significantly lower than lung disease patients and healthy controls(p<0.05).These results strongly demonstrate that miRNA-362-5p expression is downregulated after MTB infection in vitro and in vivo.In addition,we found miRNA-362-5p can positively regulate the expression of inflammatory cytokines induced by MTB by activity p38 MAPK and NF-?B signaling pathway,so that MTB evades host immune,increase intracellular survival,and protects themselves by inhibiting the viability of intracrllular bacteria.(3)MTB YrbE3A positively regulates the expression of inflammatory cytokines by activity MAPK and NF-?B signaling pathwayTo further elucidate the mechanism of interaction of YrbE3A protein with macrophages,in this current study,we constructed recombinant Mycobacterium smegmatis and Pasteur-BCG(MS_YrbE3A and BCG_YrbE3A)to infect macrophages(RAW246.7 cells).We found that MTB YrbE3A positively regulates the expression of inflammatory cytokines(TNF-? and IL-6)by activity Jnk MAPK and NF-?B signaling pathway,to activity host innate immune response to kill and clean MTB.Furthermore,in the animal model,C57BL/6 mice were infected by MS_YrbE3A,from the result of CFU counts,HE,immunohistochemistry and multiplex immunoassay,we found that YrbE3A induced high-level inflammatory cytokines and activity host innate immune response to promote host to kill and clean MTB.In summary,our research screened biomarkers for pulmonary tuberculosis for MBL2,ITIH4-35 K,RBP4 and miR-625-3p as potential biomarkers,and then established combinational a diagnostic model.We also confirmed the mechanism of blood candidate biomarker miRNA-362-5p regulating MTB mediated inflammatory response and the pathogenesis of MTB protein YrbE3A activating innate immunity for providing a theoretical basis for the diagnosis and treatment of pulmonary tuberculosis.