Evaluation of cytokine profiles related to Mycobacterium tuberculosis latent antigens using a whole-blood assay in the Philippines.

Introduction: There is no useful method to discriminate between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB). This study aimed to investigate the potential of cytokine profiles to discriminate between LTBI and active PTB using whole-blood stimulation with Mycobacterium tuberculosis (MTB) antigens, including latency-associated antigens. Materials and methods: Patients with active PTB, household contacts of active PTB patients and community exposure subjects were recruited in Manila, the Philippines. Peripheral blood was collected from the participants and used for whole-blood stimulation (WBS) with either the early secretory antigenic target and the 10-kDa culture filtrate protein (ESAT-6/CFP-10), Rv3879c or latency-associated MTB antigens, including mycobacterial DNA-binding protein 1 (MDP-1), α-crystallin (Acr) and heparin-binding hemagglutinin (HBHA). Multiple cytokine concentrations were analyzed using the Bio-Plex™ multiplex cytokine assay. Results: A total of 78 participants consisting of 15 active PTB patients, 48 household contacts and 15 community exposure subjects were eligible. The MDP-1-specific IFN-γ level in the active PTB group was significantly lower than that in the household contact group (p < 0.001) and the community exposure group (p < 0.001). The Acr-specific TNF-α and IL-10 levels in the active PTB group were significantly higher than those in the household contact (TNF-α; p = 0.001, IL-10; p = 0.001) and community exposure (TNF-α; p < 0.001, IL-10; p = 0.01) groups. However, there was no significant difference in the ESAT-6/CFP-10-specific IFN-γ levels among the groups. Conclusion: The patterns of cytokine profiles induced by latency-associated MTB antigens using WBS have the potential to discriminate between LTBI and active PTB. In particular, combinations of IFN-γ and MDP-1, TNF-α and Acr, and IL-10 and Acr are promising. This study provides the first demonstration of the utility of MDP-1-specific cytokine responses in WBS.

Integrated plasma proteomics identifies tuberculosis-specific diagnostic biomarkers.

BACKGROUNDNovel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODSWe applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTSTB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSIONThis biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDINGMedical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.

Immunopathology in human pulmonary tuberculosis: Inflammatory changes in the plasma milieu and impaired host immune cell functions.

Host immune response is key for protection in tuberculosis, but the causative agent, Mycobacterium (M.) tuberculosis, manages to survive despite immune surveillance. Key mechanisms of immune protection have been identified, but the role of immunopathology in the peripheral blood of tuberculosis patients remains unclear. Tuberculosis immunopathology in the blood is characterised by patterns of immunosuppression and hyperinflammation. These seemingly contradictory findings and the pronounced heterogeneity made it difficult to interpret the results from previous studies and to derive implications of immunopathology. However, novel approaches based on comprehensive data analyses and revitalisation of an ancient plasma milieu in vitro assay connected inflammation with immunosuppressive factors in tuberculosis. Moreover, interrelations between the aberrant plasma milieu and immune cell pathology were observed. This review provides an overview of studies on changes in plasma milieu and discusses recent findings linking plasma factors to T-cell and monocyte/macrophage pathology in pulmonary tuberculosis patients.

Left Main Bronchus Stenosis Lesion, Neutrophil Count, and Platelet Count Are Predictors of Post-Tuberculosis Bronchomalacia.

BACKGROUND Post-tuberculosis bronchomalacia (PTBM) is one of the main conditions occurring in patients after tracheobronchial tuberculosis (TBTB), and is also associated with the recurrence of symptoms. The present study aimed to investigate the predictors of PTBM in patients who had been undergoing appropriate TB treatment. MATERIAL AND METHODS Clinical data of 104 patients with symptomatic airway stenosis after TBTB between January 01, 2019 and June 31, 2020 were recorded and analyzed. The association between baseline clinical characteristics, laboratory results, and PTBM was calculated with logistical regression. The time from onset of bronchoscopic intervention was examined by Kaplan-Meier estimates; differences between the 2 groups were tested by the log-rank test. RESULTS Fifty-seven patients (54.81%) had PTBM. In the multivariate logistical analysis, the left main bronchus stenosis lesion (odds ratio [OR]=3.763), neutrophil (NEUT) count (OR=1.527), and platelet (PLT) (OR=1.010) count were predictors of PTBM. During follow-up, patients with BM had a significantly longer duration from onset of bronchoscopic intervention than patients without BM (hazard ratio=2.412, P<0.0001). Further, all patients needing long-term bronchoscopic intervention therapy were subsequently identified as having PTBM. Additionally, blood PLT counts were significantly decreased to normal levels in the non-BM group (P<0.05), but not in the BM group (P>0.05). CONCLUSIONS PTBM is most likely to occur in the left main bronchus. The inflammatory and immune responses associated with NEUT and PLT may represent therapeutic targets of PTBM. Our study is the first to report that decreased blood PLT count has the potential to monitor the treatment response.

Effect of Health Education Combined with Dietary Guidance on Nutritional Indicator, Immune Level, and Quality of Life of Patients with Pulmonary Tuberculosis.

OBJECTIVE: To investigate the effects of health education combined with dietary guidance on nutritional indicators, immune level, and quality of life of patients with pulmonary tuberculosis. METHOD: A total of 123 patients with pulmonary tuberculosis who were hospitalized to our hospital between October 2019 and October 2020 were chosen for the study and were separated into 60 control cases and 63 observation cases based on the ward they were assigned to. Patients in the two groups were compared in terms of nutritional risk, nutritional indicator levels in serum, immunological function, treatment compliance, sputum culture conversion rate, and quality of life. RESULT: With the prolongation of patients' illness, the total NRS 2002 score gradually increased in both groups and the total NRS 2002 score of patients in the control group was higher than that of patients in the observation group at the same time point after discharge. The difference between the total NRS 2002 score of patients in both groups was significant at 3 and 6 months after discharge. After the intervention, the Hb, ALB, CD4+, and CD4+/CD8+ levels of patients in both groups were higher than those at the time of admission, and the CD8+ levels were lower than those at the time of admission. At 6 months after discharge, the Hb, ALB, CD4+, and CD4+/CD8+ levels of patients in the observation group were significantly higher than those in the control group, and the CD8+ levels were significantly lower than those in the control group. The treatment compliance rate of patients in the observation group (96.83%) was significantly higher than that of the control group (75%), and the negative sputum culture transfer rate (85.71%) was significantly higher than that in the control group (60%). The overall quality of life scores of patients in the observation group was significantly higher than that in the control group. CONCLUSION: Health education combined with dietary guidance for patients with pulmonary tuberculosis can deepen patients' understanding of disease and nutritional knowledge, improve treatment compliance, improve their nutritional status, enhance their immune function, accelerate sputum bacterial conversion, enhance treatment effect, and improve their quality of life.

Elevated Natural Killer Cell-Mediated Cytotoxicity Is Associated with Cavity Formation in Pulmonary Tuberculosis Patients.

Cavitation is a major pathological feature of pulmonary tuberculosis (TB). The study is aimed at investigating the mechanism of natural killer (NK) cells participating the cavity formation during Mycobacterium tuberculosis (MTB) infection. Human peripheral blood samples were donated by pulmonary TB patients with cavity or not. Real-time quantitative PCR and enzyme-linked immunosorbent assay were performed to analyze the expression of cytokines secreted by NK cells. And the cytotoxicity of NK cells was compared between two groups. Our data showed that NK cells were more abundant in cohorts of cavity. Increased abundance of granzyme A and granzyme B was observed in culture supernatants of NK cells isolated from cavitary TB patients, which also resulted in a higher level of nonviable MTB-infected monocytes. Our data firstly demonstrates that NK cells participate in cavity formation in pulmonary TB patients. The elevated level and increased cytotoxicity of NK cells accelerate the cavitary formulation.

The Frequency and Effect of Granulocytic Myeloid-Derived Suppressor Cells on Mycobacterial Survival in Patients With Tuberculosis: A Preliminary Report.

Introduction: Protective host responses in those exposed to or infected with tuberculosis (TB) is thought to require a delicate balance between pro-inflammatory and regulatory immune responses. Myeloid-derived suppressor cells (MDSCs), regulatory cells that dampen T-cell function, have been described in cancer and other infectious diseases but there are limited data on their role in TB. Methods: Peripheral blood was obtained from patients with active pulmonary TB and participants with presumed latent TB infection (LTBI) from Cape Town, South Africa. MDSC frequency was ascertained by flow cytometry. Purified MDSCs were used to assess (i) their suppressive effect on T-cell proliferation using a Ki67 flow cytometric assay and (ii) their effect on mycobacterial containment by co-culturing with H37Rv-infected monocyte-derived macrophages and autologous pre-primed effector T-cells with or without MDSCs. Mycobacterial containment was measured by plating colony forming units (CFU). Results: MDSCs (CD15+HLA-DR-CD33+) had significantly higher median frequencies (IQR) in patients with active TB (n=10) versus LTBI (n= 10) [8.2% (6.8-10.7) versus 42.2% (27-56) respectively; p=0.001]. Compared to MDSC-depleted peripheral blood mononuclear and effector T cell populations, dilutions of purified MDSCs isolated from active TB patients suppressed T-cell proliferation by up to 72% (n=6; p=0.03) and significantly subverted effector T-cell-mediated containment of H37Rv in monocyte-derived macrophages (n=7; 0.6% versus 8.5%; p=0.02). Conclusion: Collectively, these data suggest that circulating MDSCs are induced during active TB disease and can functionally suppress T-cell proliferation and subvert mycobacterial containment. These data may inform the design of vaccines and immunotherapeutic interventions against TB but further studies are required to understand the mechanisms underpinning the effects of MDSCs.

Integrative Multi-Omics Reveals Serum Markers of Tuberculosis in Advanced HIV.

Tuberculosis (TB) accounts for disproportionate morbidity and mortality among persons living with HIV (PLWH). Conventional methods of TB diagnosis, including smear microscopy and Xpert MTB/RIF, have lower sensitivity in PLWH. Novel high-throughput approaches, such as miRNAomics and metabolomics, may advance our ability to recognize subclinical and difficult-to-diagnose TB, especially in very advanced HIV. We conducted a case-control study leveraging REMEMBER, a multi-country, open-label randomized controlled trial comparing 4-drug empiric standard TB treatment with isoniazid preventive therapy in PLWH initiating antiretroviral therapy (ART) with CD4 cell counts <50 cells/µL. Twenty-three cases of incident TB were site-matched with 32 controls to identify microRNAs (miRNAs), metabolites, and cytokines/chemokines, associated with the development of newly diagnosed TB in PLWH. Differentially expressed miRNA analysis revealed 11 altered miRNAs with a fold change higher than 1.4 or lower than -1.4 in cases relative to controls (p<0.05). Our analysis revealed no differentially abundant metabolites between cases and controls. We found higher TNFα and IP-10/CXCL10 in cases (p=0.011, p=0.0005), and higher MDC/CCL22 in controls (p=0.0072). A decision-tree algorithm identified gamma-glutamylthreonine and hsa-miR-215-5p as the optimal variables to classify incident TB cases (AUC 0.965; 95% CI 0.925-1.000). hsa-miR-215-5p, which targets genes in the TGF-ß signaling pathway, was downregulated in cases. Gamma-glutamylthreonine, a breakdown product of protein catabolism, was less abundant in cases. To our knowledge, this is one of the first uses of a multi-omics approach to identify incident TB in severely immunosuppressed PLWH.

Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69-CD8+ T cells expressed higher levels of CD45RO than CD103-CD69+CD8+ T cells did; CD103+CD69-CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103-CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69- memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-ß extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

Haematological profiles after Intensive phase of Anti Koch Treatment with special emphasis on bone marrow changes.

BACKGROUND: Tuberculosis remains a major public health problem in various parts of the world. It leads to various haematological changes. Study of these haematological changes will help better patient management. OBJECTIVE & METHODS: It is to evaluate haematological changes in tuberculosis patients and compare the result with special emphasis to bone marrow changes as active case search is sharply decreasing the miliary tuberculosis. It is also to evaluate the patients with before and after the Intensive Phase of Anti Koch Treatment. Sputum positive and sputum negative tuberculosis patients confirmed by other ancillary techniques were included into this study. It is conducted at a tertiary level hospital in rural area. RESULT: In this study bone marrow hypercellularity was of erythroid series with only 1.92% patients showed granuloma in bone marrow aspiration. In addition to bone marrow changes, significant changes were evident in haemoglobin level, Erythrocyte Sedimentation Rate (ESR) Total White Blood Cell count and RBC count. DISCUSSION: In majority cases this study showed Erythroid Hyperplasia. It is sharp contrast with other study where myeloid hyperplasia was evident. This study also differs from other study where high number of bone marrow granuloma was reported. In this study only 1.92% cases showed bone marrow granuloma. This study also documented higher number of anaemic cases mostly because of the institute serves poor and tribal population. CONCLUSION: In our study the cases showing granuloma and hyperplasia of myeloid series were limited. With introduction of Directly Observed Treatment and house to house active case search helped to sharply decrease bone marrow granuloma by limiting multi-organ spread. This study showed, ESR level may be considered as prognostic parameters of tuberculosis.

Diagnostic value of microRNA­125b in peripheral blood mononuclear cells for pulmonary tuberculosis.

Pulmonary tuberculosis (TB) is a chronic respiratory infectious disease. Certain microRNAs (miRNAs or miRs) are reported to be involved in regulating TB progression. The present study aimed to evaluate the diagnostic potential of miR­125b in pulmonary TB. The expression levels of miR­125b and Raf1 proto­oncogene serine/threonine protein kinase (RAF1) were analyzed via reverse transcription­quantitative (RT­q)PCR in patients with TB. The correlation between miR­125b and the clinical indicators was investigated, and a receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of miR­125b. The relationship between miR­125b and RAF1 was examined using the dual luciferase reporter gene assay. IL­6, TNF­α, NF­κB and IFN­Î³ levels were detected using ELISA kits, and then the correlation between miR­125b expression and the levels of IL­6, TNF­α, NF­κB, IFN­Î³ and RAF1 in peripheral blood mononuclear cells (PBMCs) was analyzed. Moreover, RAF1 mRNA and protein expression levels were detected via RT­qPCR and western blotting. The results demonstrated that miR­125b expression was decreased in patients with TB, while RAF1 expression was increased. Furthermore, miR­125b expression was associated with sputum acid­fast bacillus smear. The area under the ROC curve of miR­125b was 0.9413, and the sensitivity and specificity of miR­125b expression for TB were 90 and 92.5%, respectively. IL­6, TNF­α, NF­κB and IFN­Î³ levels were negatively correlated with miR­125b expression, and were inhibited by miR­125b in PBMCs. Moreover, miR­125b targeted RAF1 to negatively regulate its expression levels. RAF1 reversed the role of miR­125b in attenuating IL­6, TNF­α, NF­κB and IFN­Î³ levels in PBMCs. The present study demonstrated that the levels of IL­6, TNF­α, NF­κB and IFN­Î³ were negatively correlated with miR­125b expression in PBMCs. Thus, it was suggested that miR­125b served important roles in the occurrence and development of TB by decreasing the levels of IL­6, TNF­α, NF­κB and IFN­Î³ by inhibiting RAF1.

Serum Creatinine as a Potential Biomarker for the Diagnosis of Tuberculous Pleural Effusion.

BACKGROUND: Previous studies have revealed the disadvantages of traditional methods for the diagnosis of tuberculous pleural effusions (TPEs) and have created interest in exploring other effective biomarkers. Many studies have focused on the correlation between pulmonary diseases and serum creatinine (Cr), a representative biomarker of renal function, but little is known about the direct relationship between Cr and TPE. Our study aimed to explore whether Cr can act as a biomarker for the diagnosis of TPE and to evaluate the correlation between Cr and TPE. MATERIALS AND METHODS: Patients with pleural effusions (PEs) were enrolled in this study. By comparing the concentrations of Cr and adenosine deaminase (ADA) in patients with TPEs and non-TPEs, we determined the sensitivity, specificity, Youden index, and area under the curve for these biomarkers. We generated receiver operating characteristic curves and quantifications to evaluate the diagnostic accuracy. RESULTS: In total, 86 patients (44 with TPE, 25 with malignant pleural effusion (MPE) and 17 with non-tuberculosis infectious PE (NTIPE)) were enrolled in the study. The concentrations of Cr in TPE were significantly higher than those in non-TPE. However, a similar trend was not observed for NTIPE and MPE. The levels of ADA in TPE were significantly higher than those in NTIPE and MPE. CONCLUSION: Cr has the potential for the diagnosis of TPE to some extent though its accuracy is not as good as that of ADA. Further studies are necessary for Cr to be applied in clinical practice for the diagnosis of TPE.

Identification of potential lipid biomarkers for active pulmonary tuberculosis using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Early diagnosis of active pulmonary tuberculosis (TB) is the key to controlling the disease. Host lipids are nutrient sources for the metabolism of Mycobacterium tuberculosis. In this research work, we used ultra-high-performance liquid chromatography-tandem mass spectrometry to screen plasma lipids in TB patients, lung cancer patients, community-acquired pneumonia patients, and normal healthy controls. Principal component analysis, orthogonal partial least squares discriminant analysis, and K-means clustering algorithm analysis were used to identify lipids with differential abundance. A total of 22 differential lipids were filtered out among all subjects. The plasma phospholipid levels were decreased, while the cholesterol ester levels were increased in patients with TB. We speculate that the infection of M. tuberculosis may regulate the lipid metabolism of TB patients and may promote host-assisted bacterial degradation of phospholipids and accumulation of cholesterol esters. This may be related to the formation of lung cavities with caseous necrosis. The results of receiver operating characteristic curve analysis revealed four lipids such as phosphatidylcholine (PC, 12:0/22:2), PC (16:0/18:2), cholesteryl ester (20:3), and sphingomyelin (d18:0/18:1) as potential biomarkers for early diagnosis of TB. The diagnostic model was fitted by using logistic regression analysis and combining the above four lipids with a sensitivity of 92.9%, a specificity of 82.4%, and the area under the curve (AUC) value of 0.934 (95% CI 0.873 - 0.971). The machine learning method (10-fold cross-validation) demonstrated that the model had good accuracy (0.908 AUC, 85.3% sensitivity, and 85.9% specificity). The lipids identified in this study may serve as novel biomarkers in TB diagnosis. Our research may pave the foundation for understanding the pathogenesis of TB.

Ultra-low Dose Aerosol Infection of Mice with Mycobacterium tuberculosis More Closely Models Human Tuberculosis.

Tuberculosis (TB) is a heterogeneous disease manifesting in a subset of individuals infected with aerosolized Mycobacterium tuberculosis (Mtb). Unlike human TB, murine infection results in uniformly high lung bacterial burdens and poorly organized granulomas. To develop a TB model that more closely resembles human disease, we infected mice with an ultra-low dose (ULD) of between 1-3 founding bacteria, reflecting a physiologic inoculum. ULD-infected mice exhibited highly heterogeneous bacterial burdens, well-circumscribed granulomas that shared features with human granulomas, and prolonged Mtb containment with unilateral pulmonary infection in some mice. We identified blood RNA signatures in mice infected with an ULD or a conventional Mtb dose (50-100 CFU) that correlated with lung bacterial burdens and predicted Mtb infection outcomes across species, including risk of progression to active TB in humans. Overall, these findings highlight the potential of the murine TB model and show that ULD infection recapitulates key features of human TB.

Soluble Transferrin Receptor (sTfR) Identifies Iron Deficiency Anemia (IDA) in Pulmonary Tuberculosis Patients.

BACKGROUND: iron deficiency in pulmonary tuberculosis (TB) patients may weaken their immune system, causing difficulty in overcoming the infection. Accurate diagnosis of iron deficiency anemia (IDA) in pulmonary TB patients is essential. In order to prove the iron deficient state, diagnosis should focus on inflammatory factors, which could highly affect the outcome of iron status, such as measurement of serum ferritin (SF). Soluble Transferrin Receptor (sTfR) is the best parameter to diagnose iron deficiency in the inflammatory condition. This study aimed to understand the role of sTfR to identify IDA in TB patients. METHODS: cross-sectional study were applied to 3 study groups: anemic pulmonary TB (68 subjects), IDA (7 subjects), and non-anemic pulmonary TB (15 subjects). The test averages and correlations between sTfR, SF, and other hematological parameters were measured and analyzed. RESULTS: significant differences of sTfR were found in the anemic TB group compared to the IDA group and also between the IDA and non-anemic TB groups (p<0.0001). However, there was no significant difference (p>0.05) between TB anemic and non-anemic groups. We also found no significant difference between the TB anemic sub-group with normal levels of sTfR compared with the non-anemic group. There was no significant difference of sTfR levels between sub-group of increasing sTfR and group IDA (p>0.05). However, there was strong correlation between sTfR and SF in the IDA group (r=-0.89; p=0.007). CONCLUSION: the findings suggest verifying the sTfR amount in anemic patients with pulmonary TB suffering from inflammation, so that the iron deficiency could be more accurately identified and properly treated.

Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection.

Silencing miR-125b-5p attenuates inflammatory response and apoptosis inhibition in mycobacterium tuberculosis-infected human macrophages by targeting DNA damage-regulated autophagy modulator 2 (DRAM2).

Tuberculosis is one of the most important infectious diseases worldwide and macrophage apoptosis is the major host defense mechanism against TB. We attempted to characterize the role of miRNA (miR)-125b-5p on mycobacterium tuberculosis (Mtb) infection and macrophages behaviors in vitro. According to fluorescence-activated cell separation (FACS), primary monocytes (CD14+) in TB patients were accumulated, and apoptotic monocytes were decreased. Peripheral blood mononuclear cells (PBMCs)-derived macrophages (MDMs) and monocytic cells THP-1-derived macrophage-like cells (TDMs) in vitro were used to be infected with H37Rv. After infection, colony-forming units assay revealed the increase of bacterial activity, FACS demonstrated the decrease of apoptosis rate of MDMs and TDMs, as well as promoted levels of IL-6, TNF-α, Bax, and Bim and suppressed levels of IL-10 and Bcl-2, examined by enzyme-linked immunosorbent assay (ELISA) and western blot assay. Expression of miR-125b-5p and DNA damage-regulated autophagy modulator 2 (DRAM2) was examined, and real-time PCR and western blot assay showed that miR-125b-5p was upregulated, whereas DRAM2 was downregulated in primary monocytes and H37Rv-infected macrophages (MDMs and TDMs). Moreover, blocking miR-125b-5p could attenuated H37Rv-induced bacterial activity and inflammatory response of MDMs and TDMs, accompanied with apoptosis inhibition. Whereas these effects of miR-125b-5p knockdown were abolished by downregulating DRAM2. In mechanism, DRAM2 was a downstream target of miR-125b-5p, as evidenced by dual-luciferase reporter assay. Collectively, silencing miR-125b-5p could protect human macrophages against Mtb infection through promoting apoptosis and inhibiting inflammatory response via targeting DRAM2, suggesting a novel target for Mtb eliminating. Abbreviations: TB: tuberculosis; PBMCs: peripheral blood mononuclear cells; Mtb: mycobacterium tuberculosis; AFB: acid fast bacilli; FITC: fluorescein isothiocyanate; MDMs: monocytes-derived macrophages; TDMs: THP-1-derived macrophage-like cells; ERFP: Mtb-enhanced red fluorescent protein; CFU: colony-forming units; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell separation; PI: propidium iodide; DRAM2: DNA damage-regulated autophagy modulator 2; Real-time PCR: real-time polymerase chain reaction; in-miR-125b-5p: miR-125b-5p inhibitor; si-DRAM2: siRNA against DRAM2.

The mRNA expression of visfatin and lipocalin-2 in peripheral blood mononuclear cells from patients with pulmonary tuberculosis.

BACKGROUND: In this study, we aimed to assess mRNA expressions of visfatin and lipocalin-2 in peripheral blood mononuclear cells (PBMCs) from patients with pulmonary tuberculosis (PTB). METHODS: Overall, 79 PTB patients and 71 healthy controls were enrolled. In PBMCs, mRNA expressions of visfatin and lipocalin-2 were detected using real-time quantitative polymerase chain reaction (qRT-PCR), and the diagnostic value of these adipokine mRNAs in PTB patients was calculated through receiver operating characteristic (ROC) analysis. RESULTS: In PBMCs from PTB patients, the visfatin mRNA level was significantly higher than in healthy controls (P < .001), with no significant association between the lipocalin-2 mRNA level and PTB patients (P = .933). In PTB patients, lipocalin-2 mRNA expression positively correlated with the erythrocyte sedimentation rate (ESR) (P = .010). However, the visfatin mRNA level was not associated with any major clinical and laboratory parameter in PTB patients. The ROC curve demonstrated that visfatin could help distinguish PTB patients from healthy controls, with an optimal cutoff value of 0.645 and a corresponding sensitivity of 79.7%. CONCLUSIONS: The altered visfatin mRNA expression indicated that this adipokine might play a role in PTB and could be an auxiliary biomarker for PTB diagnosis.

Identification of candidate host serum and saliva biomarkers for a better diagnosis of active and latent tuberculosis infection.

In our work, we aim to identify new candidate host biomarkers to discriminate between active TB patients (n = 28), latent infection (LTBI; n = 27) and uninfected (NoTBI; n = 42) individuals. For that, active TB patients and their contacts were recruited that donated serum and saliva samples. A multiplex assay was performed to study the concentration of different cytokines, chemokines and growth factors. Proteins with significant differences between groups were selected and logistic regression and the area under the ROC curve (AUC) was used to assess the diagnostic accuracy. The best marker combinations that discriminate active TB from NoTBI contacts were [IP-10 + IL-7] in serum and [Fractalkine + IP-10 + IL-1α + VEGF] in saliva. Best discrimination between active TB and LTBI was achieved using [IP-10 + BCA-1] in serum (AUC = 0.83) and IP-10 in saliva (p = 0.0007; AUC = 0.78). The levels of TNFα (p = 0.003; AUC = 0.73) in serum and the combination of [Fractalkine+IL-12p40] (AUC = 0.83) in saliva, were able to differentiate between NoTBI and LTBI contacts. In conclusion, different individual and combined protein markers could help to discriminate between active TB and both uninfected and latently-infected contacts. The most promising ones include [IP-10 + IL-7], [IP-10 + BCA-1] and TNFα in serum and [Fractalkine + IP-10 + IL-1α + VEGF], IP-10 and [Fractalkine+IL-12p40] in saliva.

Plasma levels of tumor necrosis factor-alpha, interferon-gamma, inducible nitric oxide synthase, and 3-nitrotyrosine in drug-resistant and drug-sensitive pulmonary tuberculosis patients, Ibadan, Nigeria.

Background: Nigeria is one of the countries with a high burden of tuberculosis (TB) in the world. TB associated inflammation is reported to be central to progression from latent TB to active TB or drug sensitive TB (DSTB) to drug resistant TB (DRTB). Inflammatory cytokines, especially interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), act synergistically in the control of TB infection. They activate macrophages to produce effector molecules such as inducible nitric oxide synthase (iNOS), nitric oxide, and ultimately 3-nitrotyrosines(3-NTs), which are involved in the control of TB. This study investigated the potential involvement of TNF-α, IFN-γ, iNOS, and 3-NT in differentiating DRTB and DSTB in Ibadan, Nigeria. Methods: One hundred participants above 18 years were recruited into this study and were grouped as follows: 32 DRTB, 34 DSTB, and 34 apparently healthy controls. Plasma from the patients was used for the analyses of inflammatory (TNF α and IFN-γ) and oxidative stress (iNOS and 3-NT) biomarkers using the ELISA. Mann-Whitney test was applied for the statistical test. Results: Mean levels of plasma TNF-α, IFN-γ, iNOS, and 3-NT were higher in DRTB (19.74 ± 3.62 pg/mL, 4.41 ± 0.96 pg/mL, 1791.07 ± 419.42 pg/mL, and 20.27 ± 1.80 ng/mL, respectively) and DSTB (17.02 ± 1.84 pg/mL, 5.59 ± 1.40 pg/mL, 2823.42 ± 685.32 pg/mL, and 25.06 ± 2.15 ng/mL, respectively) compared with controls (12.18 ± 0.92 pg/mL, 1.58 ± 0.21 pg/mL, 1275.86 ± 166.12 pg/mL, and 19.98 ± 1.23 ng/mL, respectively). In addition, higher plasma levels of IFN-γ (P > 0.05), iNOS (P > 0.05), and 3-NT (P < 0.05) were observed in DSTB compared with DRTB patients. Conclusion: The 3-NT may be used as differentiating markers of DSTB from DRTB.