Cigarette smoking (CS) triggers an intense and harmful inflammatory response in lungs mediated by alveolar and blood macrophages, monocytes, and neutrophils and is closely associated with prevalence of tuberculosis (TB). The risk of death in patients with long-term cigarette smoking-related pulmonary tuberculosis (LCS-PTB) is approximately 4.5 times higher than those with nonsmoking pulmonary tuberculosis (N-PTB). However, the mechanisms underlying the harmful inflammatory responses in the setting of LCS-PTB have not been well documented. 28 cases LCS-PTB patients, 22 cases N-PTB patients and 20 cases healthy volunteers were enrolled in this study. Monocytes were isolated from peripheral blood mononuclear cells. Differentiated human MDM and U937 cell were prepared with M-CSF and PMA stimulation, respectively. The miR-196b-5p, STAT1, STAT3, STAT4, STAT5A, STAT5B, STAT6, SOCS1 and SOCS3 mRNA expression were detected by qRT-PCR. Western blot was performed according to SOCS1, SOCS3, and pSTAT3 expression. The mycobacterial uptake by MDMs from different groups of patients after Bacillus Calmette–Guérin (BCG) infection and agomir-196b-5p or antagomir-196b-5p transfection were used by flow cytometry analysis. Human IL-6, IL-10 and TNF-α levels on the plasma and cell culture supernatant samples were measured using ELISA. For dual-luciferase reporter assay, the SOCS3 3′-UTR segments, containing the binding elements of miR-196b-5p or its mutant versions were synthesized as sense and antisense linkers. In this study, we found that IL-6, TNF-α production, SOCS3 mRNA expression were downregulated, while miR-196b-5p and STAT3 mRNA expression were upregulated in monocytes from LCS-PTB patients as compared to N-PTB patients. Meanwhile, we demonstrated that miR-196b-5p could target SOCS3 and activate STAT3 signaling pathway, which may possibly contribute to attenuation of BCG uptake and decrease in IL-6 and TNF-α production in macrophages. Our findings revealed that CS exposure regulates inflammatory responses in monocyte/macrophages from LCS-PTB patients via upregulating miR-196b-5p, and further understanding of the specific role of miR-196b-5p in inflammatory responses mightfacilitate elucidating the pathogenesis of LCS-PTB, thus leading to the development of new therapeutic strategies for PTB patients with long-term cigarette smoking.

Noninvasive and simple indicators for diagnosing latent tuberculosis (TB) infection (LTBI) and tracking progression from latent infection to active TB infection are still desperately needed. The aim of this study was to screen and identify possible biomarkers for diagnosing LTBI and monitoring the progression from latent infection to active TB infection, as well as to investigate the underlying processes and functions. To assess changes in metabolite composition associated with active tuberculosis infection in humans, whole blood supernatants were collected from patients with LTBI, drug-susceptible TB patients, drug-resistant TB patients, and healthy controls. The metabolites in all serum samples were extracted by oscillatory, deproteinization, and then detected by liquid chromatography-tandem mass spectrometry/MS analysis. Normalization by Pareto-scaling method, the difference analysis was carried out by Metaboanalyst 4.0 software, and 1-way analysis of variance analysis among groups showed that P-value < 0.05 was regarded as a different metabolite. To clarify the dynamic changes and functions of differential metabolites with disease progression, and explore its significance and mechanism as a marker by further cluster analysis, functional enrichment analysis, and relative content change analysis of differential metabolites. 65 metabolites were substantially different in four groups. Differential metabolites such as Inosine and Prostaglandin E1 may be important blood indicators for diagnosing mycobacterium tuberculosis latent infection, which were all tightly connected to amino acid metabolism, Biosynthesis of various secondary metabolites, Nucleotide metabolism, Endocrine system, Immune system, Lipid metabolism, and Nervous system. This study screened and identified Inosine, 16, 16-dimethyl-6-keto Prostaglandin E1, Theophylline, and Cotinine as potential serum biomarkers for diagnosing latent TB infection, and Cotinine as potential biomarkers for monitoring disease progression from healthy population to LTBI and then to active TB including drug-resistant TB infection and sensitive TB infection. Furthermore, this research provides a preliminary experimental basis to further investigate the development of metabolomics-based diagnosis of LTBI and monitoring the progress from latent infection to active TB infection.