Background: Tuberculosis (TB) continues to be a critical global health problem, which killed millions of lives each year. Certain circulating cell subsets are thought to differentially modulate the host immune response towards Mycobacterium tuberculosis (Mtb) infection, but the nature and function of these subsets is unclear. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from healthy controls (HC), latent tuberculosis infection (LTBI) and active tuberculosis (TB) and then subjected to single-cell RNA sequencing (scRNA-seq) using 10 £ Genomics platform. Unsupervised clustering of the cells based on the gene expression profiles using the Seurat package and passed to tSNE for clustering visualization. Flow cytometry was used to validate the subsets identified by scRNA-Seq. Findings: Cluster analysis based on differential gene expression revealed both known and novel markers for all main PBMC cell types and delineated 29 cell subsets. By comparing the scRNA-seq datasets from HC, LTBI and TB, we found that infection changes the frequency of immune-cell subsets in TB. Specifically, we observed gradual depletion of a natural killer (NK) cell subset (CD3-CD7+GZMB+) from HC, to LTBI and TB. We further verified that the depletion of CD3-CD7+GZMB+ subset in TB and found an increase in this subset frequency after anti-TB treatment. Finally, we confirmed that changes in this subset frequency can distinguish patients with TB from LTBI and HC. Interpretation: We propose that the frequency of CD3-CD7+GZMB+ in peripheral blood could be used as a novel biomarker for distinguishing TB from LTBI and HC.

Tuberculosis (TB) is a major cause of morbidity and mortality worldwide. The host-directed therapy is a promising strategy for TB treatment that synergize with anti-TB treatment drugs. In this study, we found that the anti-chronic lymphocytic leukemia drug, ibrutinib, inhibited the growth of intracellular Mtb in human macrophages. Mechanisms studies showed that ibrutinib treatment significantly decreased p62 and increased LC3b proteins in Mtb infected macrophages. In addition, ibrutinib increased LC3b colocalization with intracellular Mtb and auto-lysosome fusion. Furthermore, inhibition of autophagy by using siRNA targeting ATG7 abolished the effect of ibrutinib-mediated suppression of intracellular Mtb. Next, we found that ibrutinib induced autophagy was through inhibition of BTK/Akt/mTOR pathway. Finally, we confirmed that ibrutinib treatment significantly reduced Mtb load in mediastinal node and spleen of Mtb infected mice. In conclusion, our data suggest that ibrutinib is a potential host-directed therapy candidate against TB.

BackgroundThe diagnosis of active pulmonary tuberculosis (TB) remains a challenge in clinic, especially for sputum negative pulmonary TB. Bronchoalveolar lavage fluid (BALF) has higher sensitivity than sputum for detection of Mycobacterium tuberculosis (Mtb). However, bronchoscopy is invasive and costly, and not suitable for all patients. In order to make TB patients get more benefit from BALF for diagnosis, we explore which indicator might be used to optimize the choice of bronchoscopy.MethodsA total of 1539 sputum-smear-negative pulmonary TB suspects who underwent bronchoscopy were recruited for evaluation. The sensitivity, specificity and accuracy of Mtb detection in sputum and BALF were compared. Odds ratios and 95% confidence intervals were used to assess variables that associated with positive acid-fast bacilli (AFB) smear, Mtb culture and nucleic acid amplification test (NAAT) of BALF in sputum-negative and non-sputum-producing pulmonary TB suspects.ResultsBALF has significantly higher sensitivity (63.4%) than sputum (43.5%) for Mtb detection by culture and NAAT. 19.7% (122/620) sputum-negative and 40.0% (163/408) non-sputum-producing suspects had positive bacteriological results in BALF. Among sputum-negative and non-sputum-producing pulmonary TB suspects, the positivity of Mtb detection in BALF is associated with a younger age, the presence of pulmonary cavities and a positive result of interferon-gamma release assay (IGRA). Sputum-negative patients under 35 years old with positive IGRA and pulmonary cavity had 84.8% positivity of Mtb in BALF.ConclusionsOur study indicated that combination of age, the presence of pulmonary cavity, and the result of IGRA is useful to predict the positivity of Mtb detection in BALF among sputum-negative and non-sputum producing pulmonary TB suspects. Those who are under 35 years old, positive for the presence of pulmonary cavity and IGRA, should undergo bronchoscopy to collect BAFL for Mtb tests, as they have the highest possibility to get bacteriologically confirmation of TB.Electronic supplementary materialThe online version of this article (10.1186/s40249-018-0403-x) contains supplementary material, which is available to authorized users.

Orchestration of an effective T lymphocyte response at infection sites is critical for protection against Mycobacterium tuberculosis (Mtb) infection. However, the local T cell immunity landscape in human tuberculosis is poorly defined. Tuberculous pleural effusion (TPE), caused by Mtb, is characterized by an influx of leukocytes to the pleural space, providing a platform suitable for delineating complex tissue responses to Mtb infection. Using single-cell transcriptomics and T cell receptor sequencing, we analyzed mononuclear cell populations in paired pleural fluid and peripheral blood of TPE patients. While all major cell clusters were present in both tissues, their relative proportions varied significantly by anatomic location. Lineage tracking analysis revealed subsets of CD8 and CD4 T cell populations with distinct effector functions specifically expanded at pleural sites. Granzyme K–expressing CD8 T cells were preferentially enriched and clonally expanded in pleural fluid from TPE, suggesting that they are involved in the pathogenesis of the disease. The findings collectively reveal the landscape of local T cell immunity in tuberculosis.

Macrophage dysfunction is associated with increased tuberculosis (TB) susceptibility in patients with human immunodeficiency virus (HIV) infection. However, the mechanisms underlying how HIV infection impairs macrophage function are unclear. Here, we found that levels of autoantibodies against red blood cells (RBCs) were significantly elevated in patients with HIV as determined by direct antiglobulin test (DAT). DAT positivity was significantly associated with TB incidence in both univariate and multivariate analyses (odds ratio [OR] = 11.96 [confidence interval {CI}, 4.68 to 30.93] and 12.65 [3.33 to 52.75], respectively). Ex vivo analysis showed that autoantibodies against RBCs enhanced erythrophagocytosis and thus significantly impaired macrophage bactericidal function against intracellular Mycobacterium tuberculosis. Mechanistically, autoantibody-mediated erythrophagocytosis increased heme oxygenase-1 (HO-1) expression, which inhibited M. tuberculosis-induced autophagy in macrophages. Silencing ATG5, a key component for autophagy, completely abrogated the effect of erythrophagocytosis on macrophage bactericidal activity against M. tuberculosis. In conclusion, we have demonstrated that HIV infection increases autoantibody-mediated erythrophagocytosis. This process impairs macrophage bactericidal activity against M. tuberculosis by inhibiting HO-1-associated autophagy. These findings reveal a novel mechanism as to how HIV infection increases TB susceptibility. IMPORTANCE HIV infection significantly increases TB susceptibility due to CD4 T-cell loss and macrophage dysfunction. Although it is relatively clear that CD4 T-cell loss represents a direct effect of HIV infection, the mechanism underlying how HIV infection dampens macrophage function is unknown. Here, we show that HIV infection enhances autoantibody-mediated erythrophagocytosis, which dampens macrophage bactericidal activity against TB by inhibiting HO-1-associated autophagy. Our findings reveal a novel mechanism explaining how HIV infection increases susceptibility to TB. We propose that DAT could be a potential measure to identify HIV patients who are at high TB risk and who would be suitable for anti-TB chemotherapy preventive treatment.