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Objective To investigate the effect of the gene vaccine in anti-tuberculosis immunity by constructing immunity-related p47 GTPase/ezrin-radixin-moesin-binding phosphoprotein 50 (LRG47/EBP50) gene co-expression recombinant lentivirus targeting vector. Methods Recombinant lentiviral plasmid vector pLenti-EBP50-LRG47 was established by using molecular cloning and packaged as lentivirus LV-EBP50-LRG47 and H37Rv infect macrophages. Then their bactericidal ability was tested by colony-forming units while the cellular autophagy and apoptosis was detected by flow cytometry. iNOS protein was detected by Western blotting and the expression level of nitric oxide (NO) was detected by ultraviolet spectrophotometer. Results The recombinant lentivirus LV-EBP50-LRG47 successfully up-regulated the expression of EBP50 and LRG47 after infecting macrophages. Compared with the control group, LV-EBP50-LRG47 can significantly inhibit the growth of intracellular H37Rv. The autophagy and apoptosis levels of LV-EBP50-LRG47 infected macrophages increased significantly, and the expression levels of iNOS and NO were significantly up-regulated. Conclusion LRG47/EBP50 gene co-expression enhances macrophages autophagy and apoptosis, and increases generation of iNOS and NO, which significantly inhibites the growth of intracellular H37Rv.

Macrophages act as the first line of host immune defense against Mycobacterium tuberculosis (Mtb). Recent studies have demonstrated circular RNAs (circRNAs) are implicated in a variety of physiological and pathological processes; however, the role of circRNAs in macrophages response to Mtb infection remain unknown. To address this issue, here we characterized circRNAs expression profiles in human monocyte derived macrophages (MDMs) response to Mtb infection using microarray assay. Our results revealed that many circRNAs were differentially expressed in human MDMs after Mtb infection; of these, 32 circRNAs were up-regulated and 110 were down-regulated. Real time PCR results were generally consistent with the microarray data. Furthermore, we found that hsa_circ_0043497 and hsa_circ_0001204 may be effective diagnostic biomarkers for TB. This study provides the first evidence that circRNAs alterations are involved in human MDMs response to TB infection and reveal potential targets for diagnostics and the treatment of TB.

Foamy macrophages are present during the course of Mycobacterium tuberculosis (Mtb) infection and seems to be nutrient-rich reservoir and secure reservoir for the bacilli, which leads to bacterial persistence and infection transmission. Peroxisome proliferator activated receptor γ (PPARγ) is a key transcription factor for cholesterol metabolism in macrophages and its role in regulating atherosclerosis related foamy macrophages (FMs) formation has been well-studied. However, knowledge about the mechanism of PPARγ regulating Mtb infection induced FM formation remains very limited. In this study, we investigate the functional role of PPARγ in Mtb H37Ra infection-induced foamy macrophages formation. H37Ra infection induced a time-dependent decreased expression of PPARγ that paralleled the augmented lipid body formation in THP1-derived macrophages. PPARγ antagonist GW9662 significantly potentiate H37Ra induced lipid body formation and inhibit ABCG1 expression, overexpression of ABCG1 by transduced macrophages with lentivirus significantly reversed the promotion effect of GW9662 on FM formation. Moreover, Treatment with a TLR2 neutralizing antibody ameliorated the activation of ABCG1 by Mtb H37Ra without significantly effecting the suppression of PPARγ, suggesting a greater role for TLR2 to regulate ABCG1 compared to PPARγ. Overall, this study showed that PPARγ is involved in ameliorating FM formation by regulating ABCG1 expression, these observations expose a novel role of PPARγ in the Mtb infection induced FM formation. [ABSTRACT FROM AUTHOR]

Background: T-SPOT TB (T-SPOT) assay is widely used for detection of Mycobacterium tuberculosis infection that is based on the detection of M. tuberculosis -specific interferon-γ-secreting T cells (ISCs) in peripheral blood mononuclear cells (PBMCs). Recently, high frequencies of low-density granulocytes (LDGs) were found in the PBMCs of tuberculosis patients. Whether these LDGs affect the detection of T-SPOT has not been investigated. The impact of LDGs on T-SPOT assay and related mechanism were investigated in this study. Methods: The correlations between the frequencies of LDGs and the results of T-SPOT were analyzed. T-SPOT with LDG-removed PBMCs and PBMCs with exogenous addition of LDGs were performed. The possible mechanism was explored by detecting the levels of negative immune regulatory molecules on LDGs. The impact of programmed death ligand 1 (PD-L1) on T-SPOT was evaluated and confirmed by function blocking with neutralizing antibody. Results: The positive rates of T-SPOT and ISCs in tuberculosis patients with low LDGs frequency (n = 22) were significantly higher than those with high LDGs frequency (n = 39). Removal or exogenous addition of LDGs significantly increased or decreased the ISCs and the positive rate of T-SPOT. The frequencies of interferon-γ-producing T cells were negatively correlated with the frequencies of LDGs. The expression of PD-L1 was significantly elevated on LDGs. Pretreatment of LDGs with anti-PD-L1 antibody significantly counteracted the impact of LDGs on T-SPOT. Treatment of PBMCs with anti-PD-L1 antibody resulted in comparable ISCs with that of LDG removal. Conclusion: LDGs can inhibit the production of interferon-γ in T cells and decrease the positive rated of T-SPOT assay via highly expressed PD-L1. [ABSTRACT FROM AUTHOR]

The roles and characteristics of low-density granulocytes (LDGs) have recently attracted attention; however, the mechanism of the formation of LDGs is yet unclear. In one of our previous studies, the frequency of LDGs was significantly elevated in the peripheral blood of tuberculosis patients, and in situ activation contributed to the generation of LDGs upon Mycobacterium tuberculosis infection. However, the underlying molecular mechanisms are yet to be elucidated. In the present study, the release of neutrophil extracellular traps (NETs) and the levels of ROS were regulated before the normal-density granulocytes (NDGs) to be infected with M. tuberculosis , and the conversion of NDGs to LDGs was monitored subsequently as well. The results showed that tuberculosis-related LDGs spontaneously released high levels of NETs. Promoting the release of NETs led to increase in the conversion of NDGs to LDGs in M. tuberculosis infection, while inhibiting the release of NETs suppressed this conversion after the infection. The M. tuberculosis infection significantly increased the ROS levels in neutrophils and the conversion of NDGs to LDGs. Scavenging ROS or blocking the ROS generation of M. tuberculosis -infected NDGs significantly suppressed the release of NETs and blocked the generation of LDGs. Moreover, inhibiting the formation of NETs without affecting the levels of ROS significantly decreased the conversion of NDGs to LDGs after M. tuberculosis infection. Overall, this study demonstrated that M. tuberculosis could induce the generation of LDGs by promoting the release of NET via ROS pathway. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated that circular RNAs (circRNAs) could serve as potential molecular markers for disease diagnosis; however, little is known about their diagnostic value in active tuberculosis (TB). This study first performed a microarray screening of circRNA changes in plasma samples from 3 patients with active pulmonary TB and 3 healthy controls. Then, candidate circRNAs were selected for validation on a quantitative real-time PCR system. Of the 61 differentially expressed circRNAs recorded, 43 and 18 were upregulated and downregulated in the TB group, respectively. Validation assays demonstrated that plasma levels of 6 circRNAs, including hsa_circ_0009024, hsa_circ_0001953, hsa_circ_0008297, hsa_circ_0003528, hsa_circ_0003524 and hsa_circ_0015879 were remarkably increased in TB patients. Plasma levels of hsa_circ_0001953 and hsa_circ_0009024 were correlated with TB severity. Next, hsa_circ_0001953 and hsa_circ_0009024 were assessed in an independent cohort consisting of 120 TB patients and 100 control individuals. An area under the receiver operating characteristic (ROC) curve of 0.915 (95% confidence interval 0.880-0.951; P < 0.001) was obtained for detecting TB, with hsa_circ_0001953 and hsa_circ_0009024 used in combination. Additionally, plasma levels of hsa_circ_0001953 and hsa_circ_0009024 were reduced significantly in patients after treatment (P < 0.001). The present findings indicate that the circRNAs hsa_circ_0001953 and hsa_circ_0009024 may represent novel plasma biomarkers for active TB diagnosis. [ABSTRACT FROM AUTHOR]