Evaluation of IFIT3 and ORM1 as Biomarkers for Discriminating Active Tuberculosis from Latent Infection.

OBJECTIVE: Current commercially available immunological tests cannot be used for discriminating active tuberculosis (TB) from latent TB infection. To evaluate the value of biomarker candidates in the diagnosis of active TB, this study aimed to identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) between patients with active TB and individuals with latent TB infection by transcriptome sequencing. METHODS: The differentially expressed genes in unstimulated PBMCs and in Mycobacterium tuberculosis (Mtb) antigen-stimulated PBMCs from patients with active TB and individuals with latent TB infection were identified by transcriptome sequencing. Selected candidate genes were evaluated in cohorts consisting of 110 patients with TB, 30 individuals with latent TB infections, and 50 healthy controls by quantitative real-time RT-PCR. Receiver operating characteristic (ROC) curve analysis was performed to calculate the diagnostic value of the biomarker candidates. RESULTS: Among the differentially expressed genes in PBMCs without Mtb antigen stimulation, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) had the highest area under curve (AUC) value (0.918, 95% CI: 0.852-0.984, P<0.0001) in discriminating patients with active TB from individuals with latent TB infection, with a sensitivity of 91.86% and a specificity of 84.00%. In Mtb antigen-stimulated PBMCs, orosomucoid 1 (ORM1) had a high AUC value (0.833, 95% CI: 0.752-0.915, P<0.0001), with a sensitivity of 81.94% and a specificity of 70.00%. CONCLUSION: IFIT3 and ORM1 might be potential biomarkers for discriminating active TB from latent TB infection.

[Expression of CD160 in natural killer (NK) cells from patients with active tuberculosis and its relationship with cell functions].

Objective To investigate the relationship between the CD160 expression and anti-tuberculosis immunity. Methods Fluorescence quantitative real-time PCR was used to detect the expression of CD160 in peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze the expression of CD160 on main subtypes of PBMCs, such as T cells, B cells, NK cells and monocytes. The relationship among CD160 and perforin, granzyme B, granulysin, CD69, CD107 and IFN-γ in NK cells was analyzed by flow cytometry. Results CD160 mRNAs in the PBMCs from patients with active tuberculosis was significantly down-regulated, and the levels of CD160 expression in Mycobacterium tuberculosis (MTB)-positive patients was significantly lower than in MTB-negative patients. The expression of CD160 on B cells and monocytes was lower in patients with active tuberculosis as compared with normal controls, while no significant difference was observed on CD3+ T cells. NK cells from patients with active tuberculosis had significantly lower CD160 expression than those from normal controls. In vitro culture with MTB antigens led to down-regulated expression of CD160 on NK cells. The activation marker CD69 on NK in patients with active tuberculosis was significantly lower than that in normal controls. The expression of perforin, granzyme B, granulysin, CD69 and CD107 in CD160+ NK cells was significantly higher than that of CD160- NK cells. However, the expression of IFN-γ in CD160+ NK cells was significantly lower than that of CD160- NK cells. Conclusion The mRNA and protein expression of CD160 was significantly down-regulated in patients with active tuberculosis. CD160 promotes the activation and degranulation of NK cells associated with tuberculosis antigens, but suppresses the expression of IFN-γ of NK cells. CD160 may become a new target for the diagnosis and treatment of tuberculosis.

Mesenchymal-epithelial Transition Factor Regulates Monocyte Function during Mycobacterial Infection via Indoleamine 2,3-dioxygenase.

OBJECTIVE: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), causes an estimated 1.6 million human deaths annually, but the pathogenesis of TB remains unclear. Immunity plays a critical role in the onset and outcome of TB. This study aimed to uncover the roles of innate and adaptive immunity in TB. METHODS: The gene expression profiles generated by RNA sequencing from human peripheral blood mononuclear cells (PBMCs) stimulated with or without Mtb strain H37Rv antigens were analyzed. A total of 973 differentially expressed mRNAs were identified. RESULTS: The differentially expressed genes were enriched in innate immunity signaling functions. The mesenchymal-epithelial transition factor (MET) gene was significantly upregulated in CD14+ monocytes. A MET inhibitor improved the uptake of the BCG strain by monocytes and macrophages as well as inhibited the expression of indoleamine 2,3-dioxygenase (IDO). The expression of IDO was increased in PBMCs stimulated with Mtb antigens, and the IDO inhibitor promoted the expression of CD40, CD83, and CD86. CONCLUSION: Our results might provide clues regarding the immunomodulatory mechanisms used by Mtb to evade the host defense system.

[The expression of T-bet is negatively correlated with the expressions of IFN-γ and TNF-α in CD4+ T cells of patients with active pulmonary tuberculosis].

Objective To investigate the relationship between T box expressed in T cells (T-bet) and the production of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) in CD4+T cells of patients with active pulmonary tuberculosis. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation. Individuals with latent Mycobacterium tuberculosis (MTB) infection were screened by enzyme-linked immunospot assay (ELISPOT). The expressions of T-bet, IFN-γ, TNF-α and IL-2 in CD4+T cells were detected by flow cytometry. Results The expression of IFN-γ significantly increased in PBMCs from the individuals with latent tuberculosis infection when stimulated with MTB H37Rv strain lysates. T-bet expression in CD4+IFN-γ+ cells from the patients with active pulmonary tuberculosis was significantly higher than that from the individuals with latent tuberculosis infection when stimulated with MTB H37Rv strain lysates. The expressions of IFN-γ and TNF-α in T-bet- MTB antigen-specific CD4+T cells were obviously higher than those in T-bet+ cells; however, the expression of IL-2 showed no significant difference between T-bet- cells and T-bet+ cells. Conclusion The expression of T-bet in MTB antigen-specific CD4+T cells from patients with active pulmonary tuberculosis is negatively correlated with IFN-γ and TNF-α.

Enhanced immune response of MAIT cells in tuberculous pleural effusions depends on cytokine signaling.

The functions of MAIT cells at the site of Mycobacterium tuberculosis infection in humans are still largely unknown. In this study, the phenotypes and immune response of MAIT cells from tuberculous pleural effusions and peripheral blood were investigated. MAIT cells in tuberculous pleural effusions had greatly enhanced IFN-γ, IL-17F and granzyme B response compared with those in peripheral blood. The level of IFN-γ response in MAIT cells from tuberculous pleural effusions was inversely correlated with the extent of tuberculosis infection (p = 0.0006). To determine whether cytokines drive the immune responses of MAIT cells at the site of tuberculosis infection, the role of IL-1ß, IL-2, IL-7, IL-12, IL-15 and IL-18 was investigated. Blockade of IL-2, IL-12 or IL-18 led to significantly reduced production of IFN-γ and/or granzyme B in MAIT cells from tuberculous pleural effusions. Majority of IL-2-producing cells (94.50%) in tuberculous pleural effusions had phenotype of CD3(+)CD4(+), and most IL-12p40-producing cells (91.39%) were CD14(+) cells. MAIT cells had significantly elevated expression of γc receptor which correlated with enhanced immune responses of MAIT cells. It is concluded that MAIT cells from tuberculous pleural effusions exhibited highly elevated immune response to Mtb antigens, which are controlled by cytokines produced by innate/adaptive immune cells.

Mucosal-associated invariant T cells from patients with tuberculosis exhibit impaired immune response.

OBJECTIVES: To identify factors which regulate MAIT cell response to Mycobacterium tuberculosis antigens, and to investigate the role of MAIT cells in patients with active tuberculosis. METHODS: Immune response of MAIT cells to M. tuberculosis antigens were compared between patients with active TB and healthy controls by flow cytometry and RNA sequencing. RESULTS: IFN-γ response of MAIT cells to M. tuberculosis lysates was dramatically improved by signal 3 cytokine IL-15 (p = 0.0002). Patients with active TB exhibited highly reduced IFN-γ production in MAIT cells stimulated with M. tuberculosis lysates/IL-15 compared with healthy controls (p < 0.0001) and individuals with latent TB infection (p = 0.0008). RNA sequencing of flow-sorted MAIT cells from patients with TB and healthy controls identified numerous differentially expressed genes, and the expression of genes that encode IFN-γ, TNF-α, IL-17F, granulysin and granzyme B were all down-regulated in patients with TB. MAIT cells from patients with TB has significantly lower expression of γc receptor than those from healthy controls under condition of Mtb lysates/IL-15 stimulation (p = 0.0028). Blockade of both γc and IL-2Rß receptors resulted in highly reduced frequency of IFN-γ-producing MAIT cells (79.4%) (p = 0.0011). CONCLUSIONS: MAIT cells from patients with active TB exhibited impaired cytokine and cytotoxic response to M. tuberculosis antigens.

[Down-regulated expression of c-Jun in peripheral blood mononuclear cells of patients with severe secondary pulmonary tuberculosis].

OBJECTIVE: To compare the expression of c-Jun in severe versus mild secondary pulmonary tuberculosis (TB) and understand the relationship of the c-Jun expression with the inflammation and immune injury of severe secondary TB patients. METHODS: Differentially expressed genes were screened in patients with severe TB, the ones with mild TB and healthy controls using Affymetrix human gene expression chips. Bioinformatic analysis was performed on the results of the gene chip screening. The relative transcript level of c-Jun was detected by real-time quantitative PCR (qRT-PCR). The protein expression of c-Jun in peripheral blood mononuclear cells was detected by ELISA. RESULTS: Patients with severe pulmonary TB exibited a large number of differentially expressed genes compared with healthy controls and patients with mild secondary TB, and these differential expressed genes involved complicated pathways of immue response and inflammation. C-Jun was down-regulated 2.27 times in the patients with severe secondary TB compared with the ones with mild TB, and it is involved in 61 pathways. The qRT-PCR verified that c-Jun was down-regulated significantly in the patients with severe secondary TB compared with the mild ones. ELISA confirmed the trend of down-regulation of c-Jun in the patients with severe secondary TB. The results of qRT-PCR and ELISA were consistent with gene chip analysis. CONCLUSION: C-Jun was down-regulated in the patients with severe secondary TB compared with the patients with mild TB, and it is involved in many pathways of immue response and inflammation. Its down-regulation might be related to the immune injury of severe secondary TB.

Mucosal-associated invariant T-cell function is modulated by programmed death-1 signaling in patients with active tuberculosis.

RATIONALE: Mucosal-associated invariant T (MAIT) cells have been proven to play an important role in host defense against mycobacterial infection in animal infection models; however, the functional role of MAIT cells in patients with active tuberculosis (TB) is still largely unknown. OBJECTIVES: To understand the clinical features and functions of MAIT cells in patients with active TB. METHODS: MAIT cells were analyzed in patients with pulmonary TB, tuberculous pleurisy, and tuberculous peritonitis by flow cytometry. The functions of MAIT cells were compared between patients with active TB and healthy control subjects. MEASUREMENTS AND MAIN RESULTS: The frequency of MAIT cells was significantly reduced both in peripheral blood from patients with active pulmonary TB (P < 0.0001) and in tuberculous pleural effusions compared with healthy control subjects but not in ascitic fluids from patients with tuberculous peritonitis. A comparison of bacillus Calmette-Guérin (BCG)-stimulated cytokine production showed that patients with active TB had significantly higher production of IFN-γ (P = 0.0034) and tumor necrosis factor (TNF)-α (P = 0.0399) compared with healthy control subjects. In contrast, when MAIT cells were stimulated with Escherichia coli, patients with active TB had significantly lower production of IFN-γ (P = 0.0007) and TNF-α (P = 0.0032). MAIT cells in patients with active TB exhibited elevated expression of programmed death-1 (PD-1) (P = 0.0015), and blockade of PD-1 signaling resulted in a significantly higher frequency of BCG-stimulated IFN-γ production in MAIT cells (P = 0.0178). CONCLUSIONS: MAIT-cell immune response to antigen stimulation in patients with active TB is regulated by PD-1, which could be a potential target for TB immunotherapy.