Elevated matricellular proteins (MCPs), including osteopontin (OPN) and galectin-9 (Gal-9), were observed in the plasma of patients with Manila-type tuberculosis (TB) previously. Here, we quantified plasma OPN, Gal-9, and soluble CD44 (sCD44) by enzyme-linked immunosorbent assay (ELISA), and another 29 cytokines by Luminex assay in 36 patients with pulmonary TB, six subjects with latent tuberculosis (LTBI), and 19 healthy controls (HCs) from Japan for a better understanding of the roles of MCPs in TB. All TB subjects showed positive results of enzyme-linked immunospot assays (ELISPOTs). Spoligotyping showed that 20 out of 36 Mycobacterium tuberculosis (MTB) strains belong to the Beijing type. The levels of OPN, Gal-9, and sCD44 were higher in TB (positivity of 61.1%, 66.7%, and 63.9%, respectively) than in the HCs. Positive correlations between OPN and Gal-9, between OPN and sCD44, and negative correlation between OPN and ESAT-6-ELISPOT response, between chest X-ray severity score of cavitary TB and ESAT-6-ELISPOT response were observed. Instead of OPN, Gal-9, and sCD44, cytokines G-CSF, GM-CSF, IFN-α, IFN-γ, IL-12p70, and IL-1RA levels were higher in Beijing MTB-infected patients. These findings suggest immunoregulatory, rather than inflammatory, effect of MCPs and can advance the understanding of the roles of MCPs in the context of TB pathology.

Osteopontin (OPN) is a multifunctional matricellular protein produced by a broad range of cells including osteoclasts, macrophages, T cells, endothelial cells, and vascular smooth muscle cells. OPN modulates various physiological and pathological events such as inflammation, wound healing, and bone formation and remodeling. Dengue virus (DENV) infection causes an increase in plasma OPN levels, which is correlated with the severity of symptoms and coagulation abnormalities. DENV infection also induces OPN gene expression in human macrophages. This study investigated the inhibitory effects of brefelamide and its methyl ether derivative on DENV-3 by measuring changes in OPN levels in human THP-1 and 293T cell lines infected at different multiplicities of infection and post-infection time points. OPN mRNA expression and viral RNA were detected by reverse transcriptase quantitative real-time PCR, whereas protein level was determined by enzyme-linked immunosorbent assay. We found that viral copy number was higher in 293T than in THP-1 cells. However, THP-1 constitutively expressed higher levels of OPN mRNA and protein, which were enhanced by DENV-3 infection. Brefelamide and its derivative suppressed OPN production in DENV-3 infected THP-1 cells; the effective doses of these compounds had no effect on uninfected cells, indicating low cytotoxicity. These results suggest that brefelamide and its methyl ether derivative have therapeutic effects in preventing inflammation, coagulopathy, and fibrinolysis caused by OPN upregulation induced by DENV-3 infection.

In this study, we investigated the role of a matricellular protein galectin-9 (Gal-9) in pleural effusion related to tuberculosis (TB). Plasma and pleural fluid of a patient with extrapulmonary TB were analyzed for cytokine content by ELISA and Luminex. Peripheral blood mononuclear cells (PBMCs) and pleural fluid cells (PFCs) were examined for interferon-γ (IFN-γ) secretion by the enzyme-linked immunospot (ELISPOT) assay or IFN-γ ELISA, for apoptosis and necrosis by Cell Death Detection ELISA, and also underwent cell sorting. The results indicate that compared to plasma, pleural fluid had increased levels of IFN-γ (1.6 vs. 55.5 pg/mL), IL-10, IL-12p40, vascular endothelial growth factor (VEGF), and Gal-9 (3.0 vs. 936.0 pg/mL), respectively. PFCs culture supernatant exhibited higher concentration of Gal-9 compared to PBMCs in culture, consistent with enriched Gal-9 staining in the granuloma that is in closer vicinity to PFCs compared to PBMCs. PFCS displayed higher IFN-γ secretion after stimulation with TB antigens ESAT-6/CFP-10. Furthermore, in PFCs, Gal-9 alone could stimulate IFN-γ synthesis in culture or ELISPOT, which was inhibited by a Gal-9 antagonist lactose, and which may promote apoptosis and necrosis. These findings suggest that Gal-9 could modulate immune responses and participate in immunopathology of pleural effusion during TB.

Tuberculous glycolipid (TBGL) is a component of the Mycobacterium tuberculosis cell wall, and anti-TBGL antibodies are used for serodiagnosis of tuberculosis. Anti-TBGL IgG and IgA levels were measured in 45 pulmonary TB patients (PTB), 26 extra-pulmonary TB patients (ETB), 16 AIDS-TB patients, and 58 healthy controls (HC) including 39 health care workers (HW) and 19 newly enrolled students (ST). Anti-TBGL IgG measurements yielded 68.9% and 46.2% sensitivity in PTB and ETB, respectively, and 81.0% specificity. However, anti-TBGL IgA measurements were significantly less sensitive in detecting ETB than PTB (15.4% versus 46.7% sensitivity) but showed up to 89.7% specificity. Samples from AIDS-TB patients exhibited low reaction of anti-TBGL IgG and IgA with 6.3% and 12.5% sensitivity, respectively. Unlike anti-lipoarabinomannan (LAM) IgG that was found to elevate in sputum smearpositive subjects, anti-TBGL IgG and IgA elevated in those with cavitation and bronchiectasis, respectively. Anti-TBGL IgG in cavitary TB yielded 78.2% sensitivity compared to 57.1% in those otherwise. Meanwhile, higher anti-TBGL IgA titers were observed in HW than in ST, and increasing anti-TBGL IgG titers were observed in HW on follow-up. Therefore, higher anti-TBGL antibody titers are present in patients presenting cavities and bronchiectasis and subjects under TB exposure risk.

The Beijing genotype Mycobacterium tuberculosis (MTB), notorious for its virulence and predisposition to relapse, could be identified by spoligotyping based on genetic heterogeneity. The plasma samples from 20 cases of Beijing and 16 cases of non-Beijing MTB infected individuals and 24 healthy controls (HCs) were collected, and antibodies against 11 antigens (Rv0679c142Asn, Rv0679c142Lys, Ag85B, Ag85A, ARC, TDM-M, TDM-K, HBHA, MDP-1, LAM, and TBGL) were measured by ELISA. Compared to the HCs, the MTB infected subjects showed higher titers of anti-Ag85B IgG (positivity 58.2%) and anti-ACR IgG (positivity 48.2%). Of note, anti-ACR IgG showed higher titer in Beijing MTB infected tuberculosis (TB) patients than in HC (Kruskal–Wallis test, p < 0.05), while the levels of anti-Ag85B, anti-TBGL, anti-TDM-K, and anti-TDM-M IgG were higher in non-Beijing TB patients than in HC. Moreover, anti-Ag85B IgG showed higher response in non-Beijing TB patients than in Beijing TB patients (p < 0.05; sensitivity, 76.9% versus 44.4%). The sensitivity and specificity analysis showed that 78.8% Beijing infected individuals were negative in anti-TBGL-IgG or/and anti-Ag85B-IgG, while 75.0% of those were positive in anti-TBGL-IgA or/and anti-ACR-IgG tests. These results indicate the possibility of developing antibody-based test to identify Beijing MTB.