Increased B cell activating factor is associated with B cell class switching in patients with tuberculous pleural effusion.

B cell activating factor (BAFF), a member of the tumor necrosis factor family, is a key cytokine for B cell survival, a function that is essential for B cell maturation and memory. The expression levels of BAFF and its potential contribution to B cell maturation remain elusive in patients with tuberculous pleural effusion (TPE). The present study enrolled 40 healthy controls (HC) and 45 TPE patients, and investigated the levels of BAFF in the plasma and pleural effusion. Concomitantly, B cell subsets including naïve B cell (CD19+IgD+CD27­), unswitched B cell (CD19+IgD+CD27+), switched B cell (CD19+IgD­CD27+), total memory B cell (CD19+CD27+), plasma B cell (CD19+IgD­CD38+CD27+) and transitional B cell (CD19+IgDdim CD38+) in peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) were assessed using multicolor flow cytometry. Finally, the associations between BAFF and each sub­group of B cells in TPE patients were analyzed. Compared with HC cases, an increased BAFF level and elevated frequency of switched B cell were observed in the blood and pleural effusion from patients with TPE. The proportions of naïve B cell, plasma B cell and transitional B cell were lower in the PFMCs of TPE patients. Furthermore, a significant correlation was observed between the level of BAFF, and the proportion of switched B cell in the peripheral blood and pleural effusion of TPE patients. These findings indicated that the B cell profile may be different in the pleural effusion, and BAFF may activate switched B cells to enhance the humoral immune responses in patients with TPE. Further studies are required to elucidate the underlying mechanisms and determine the potential immunotherapy of the BAFF­switched B cell axis.

Profiling dendritic cell subsets in the patients with active pulmonary tuberculosis.

Dendritic cell (DC) plays an important role in the immune response against pulmonary tuberculosis. However, the phenotypic profile of DC subsets in peripheral blood in individuals with active pulmonary tuberculosis (APT) is still inconclusive. Here, we demonstrated that the absolute numbers of total DC (tDC), myeloid DC (mDC) and plasmacytoid DC (pDC) in individuals with APT were decreased compared to healthy controls (HCs). The decreased number of DCs, especially of pDC, seems to be a useful diagnostic marker of APT. Meanwhile, the number of DCs was associated with the prolonged/complicated TB, ATD treatment effect and lymphocyte immune reactions, as manifested that relapsed APT patients with a higher number of tDC and lower number of pDC compared to newly diagnosed patients. Interestingly, mDC from APT patients displayed high expressions of CD83 and CCR7, but pDC displayed low expressions of CD83 and CCR7. Moreover, DCs from APT patients expressed lower levels of HLA-DR and CD80, but expressed a higher level of CD86 than those from HCs. However, the antigen uptake capacity of DC subsets was not different between APT and HCs, despite the antigen uptake capacity of pDC was much lower than that of mDC in both APT patients and HCs. Our data represent a systematic profile of DC subsets in the blood of APT patients, and would represent a useful biomarker for APT.

The circular RNA of peripheral blood mononuclear cells: Hsa_circ_0005836 as a new diagnostic biomarker and therapeutic target of active pulmonary tuberculosis.

It has been reported that circular RNA (circRNA) is associated with human cancer. However, few studies have been reported in active pulmonary tuberculosis (APTB). The global circRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) of APTB patients (n=5) and health controls (HC) (n=5) by using high-throughput sequencing. According to the systematical bioinformatics analysis, the basic content of circRNAs and their fold changes in the two groups were calculated. We selected 6 significant differentially expressed circRNAs, hsa_circ_0005836, hsa_circ_0009128, hsa_circ_0003519, hsa_circ_0023956, hsa_circ_0078768, and hsa_circ_0088452 and validated the expression in PBMCs from APTB (n=10) and HC (n=10) by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Further, the verification of these specific circRNAs (hsa_circ_0005836 and hsa_circ_0009128) between APTB (n=34) and HC (n=30) in PBMCs was also conducted by qRT-PCRs. The RNA-seq data showed the significant differential expression of the 523 circRNAs between the APTB and HC groups (199 circRNAs were significantly up-regulated and 324 circRNAs were down-regulated). Hsa_circ_0005836 and hsa_circ_0009128 expression was significantly down-regulated in the PBMCs of APTB (P<0.05) in the samples of APTB compared to HC in our study. The gene ontology based enrichment analysis of the circRNA-miRNA-mRNAs network showed that cellular catabolic process (P=7.10E-08), regulation of metabolic process (P=2.10E-06), catalytic activity (P=3.67E-08), protein binding (P=1.71E-07), cell part (P=3.46E-06), intracellular part (P=1.71E-07), and intracellular (P=3.67E-08) were recognized in the comparisons between APTB and HC. Based on KEGG analysis, HTLV-I infection, regulation of actin cytoskeleton, neurotrophin signaling pathway and mTOR signaling pathway were relevant during tuberculosis bacillus infection. We found for the first time that hsa_circ_0005836 and hsa_circ_0009128 were significantly down-regulated in the PBMCs of APTB compared with HC. Our findings indicate hsa_circ_0005836 might serve as a novel potential biomarker for TB infection.

Development and characterization of monoclonal antibody against human IL-37b.

IL-37 has been described as a natural inhibitor of immune responses. Monoclonal antibody (mAb) against human IL-37b with high affinity and specificity can serve as a molecular probe to detect IL-37 and study IL-37 functions, mechanisms and related signal pathways in inflammatory diseases. However, there are very few such mAbs against human IL-37 commercially available so far. In the current study, monoclonal antibodies against human IL-37b were developed by fusing splenocytes from immunized mouse with SP2/0 myeloma cells and polyethylene glycol. Then the antibodies were screened with prokaryotic expressed human IL-37b protein and eukaryotic expressed human IL-37b protein subsequently. Western blot and flow cytometry analysis revealed that selected mAb clons were able to recognize human IL-37 with high specificity. And more importantly, the IL-37b mAbs were fluorescently labeled and can be directly used in flow cytometry and immunohistochemistry. In conclusion, the current study developed new mAbs against human IL-37b, which are applicable in flow cytometry and immunohistochemistry.

Tuberculosis-sensitized monocytes sustain immune response of interleukin-37.

Roles of human IL-37 in infections remain poorly characterized. Although plasma IL-37 is elevated in patients with tuberculosis (TB), IL-37 source and immune correlate in TB have not been investigated. It is also unknown whether and how TB can influence the ability of immune cells to mount innate responses of IL-37 and pre-inflammatory cytokines. Here, we demonstrated that IL-37b-producing monocytes coincided with a source of elevated plasma IL-37b in TB patients. While IL-37b production in TB was associated with prolonged/complicated TB, TB burdens and inflammatory reactions, it negatively correlated with immune responses of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α or IL-10. Interestingly, mycobacterial re-infection of monocytes from TB patients, but not healthy BCG-vaccinated controls, enhanced or sustained IL-37b production by cultured monocytes. TB-sensitized monocytes from TB patients mounted more robust immune responses of IL-37b than those of pre-inflammatory cytokines during mycobacterial re-infection in culture. Our data represent new findings in terms of IL-37b responses, immune correlates and potential mechanisms in TB patients.