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Increased Serum Levels of Brain-Derived Neurotrophic Factor Contribute to Inflammatory Responses in Patients with Rheumatoid Arthritis - PubMed

  • ️Fri Jan 01 2021

Clinical Trial

Increased Serum Levels of Brain-Derived Neurotrophic Factor Contribute to Inflammatory Responses in Patients with Rheumatoid Arthritis

Ning-Sheng Lai et al. Int J Mol Sci. 2021.

Abstract

The aim of this study is to investigate the role of brain-derived neurotrophic factor (BDNF) in the inflammatory responses in patients with rheumatoid arthritis (RA). Serum levels of BDNF and the precursor form of BDNF (proBDNF) from 625 RA patients and 40 controls were analyzed using enzyme-linked immunosorbent assay. Effects of BDNF on the mitogen-activated protein kinase pathway were analyzed by Western blotting. Microarray analysis was conducted to search BDNF regulated gene expression in Jurkat cells, and the differentially expressed genes were validated using T cells from patients with RA and controls. Serum BDNF levels were significantly elevated in patients with RA compared with the controls. Low serum BDNF levels were found in RA patients with anxiety or receiving biologics treatment. BDNF (20 ng/mL) enhanced the phosphorylation of ERK, JNK, and c-Jun, but suppressed the phosphorylation of p38, whereas BDNF (200 ng/mL) enhanced the phosphorylation of ERK and p38. After validation, the expression of CAMK2A, MASP2, GNG13, and MUC5AC, regulated by BDNF and one of its receptors, NGFR, was increased in RA T cells. BDNF increased the IL-2, IL-17, and IFN-γ expression in Jurkat cells and IL-2 and IFN-γ secretion in activated peripheral blood mononuclear cells.

Keywords: BDNF; JNK; T cells; anxiety; proinflammatory cytokines; rheumatoid arthritis.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1

Serum brain-derived neurotrophic factor (BDNF), the precursor form of BDNF (proBDNF) concentration, and proBDNF/BDNF ratio in healthy controls and patients with rheumatoid arthritis. Serum levels of (A) BDNF, and (B) proBDNF concentration and (C) proBDNF/BDNF ratio in 40 healthy controls and 625 patients with rheumatoid arthritis. Serum levels of BDNF and proBDNF were measured by enzyme-linked immunosorbent assay (ELISA). After adjusting for age and sex in the multiple linear regression analysis, the BDNF levels remained significantly (p = 0.033) elevated in serum from patients with rheumatoid arthritis compared with those from controls. (ns not significant; * p < 0.05).

Figure 2
Figure 2

Expression of BDNF receptor in Jurkat cells and T cells from healthy controls and patients with rheumatoid arthritis. The expression of BDNF receptors, including neurotrophic receptor tyrosine kinase 2 (NTRK2) and nerve growth factor receptor (NGFR) in Jurkat cells, T cells from healthy controls and patients with rheumatoid arthritis analyzed using flow cytometry.

Figure 3
Figure 3

Effect of different concentrations of BDNF in mitogen-activated protein kinase (MAPK) phosphorylation and cell proliferation. (A) The phosphorylation ratio of p38, extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK) in Jurkat cells after cocultured in culture medium with a low concentration BDNF (20 ng/mL) or a high concentration BDNF (200 ng/mL) for 48 h. (B) a representative case. (C) The viability and proliferation of Jurkat cells after cocultured in culture medium with a low and a high concentration of BDNF for 48 h analyzed by WST-1 cell proliferation assay. (D) The phosphorylation ratio of c-Jun in Jurkat cells after cocultured in culture medium with a low and a high concentration BDNF for 48 h (ns, not significant; * p < 0.05; ** p < 0.01).

Figure 4
Figure 4

Identification and validation of BDNF regulated gene and its expression levels in T cells from patients with rheumatoid arthritis and healthy controls. (A) Expression profiles of mRNAs in Jurkat cells cocultured with BDNF 200 ng/mL or culture medium for 48 h were evaluated using microarray analysis. Each scatter spot represents the mean raw signal of mRNA in three repeats of each treatment. (B) Six genes, including CD40, CAMK2A, STARD13, MASP2, GNG13, and MUC5AC, which are related to the inflammatory pathway, were validated using real-time PCR. The expression levels of the BDNF receptors: NTRK2 and NGFR were compared in Jurkat cells cocultured with BDNF 200 ng/mL or culture medium for 48 hours. (C) The expressed levels of CD40, CAMK2A, STARD13, MASP2, GNG13, MUC5AC, NTRK2, and NGFR were compared in T cells from healthy controls and patients with rheumatoid arthritis. (ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5
Figure 5

Functional studies of BDNF and its receptors in Jurkat cells and normal peripheral blood mononuclear cells (PBMCs). (A) The mRNA expression of IL-2, IL-17 and IFN-γ in Jurkat cells with BDNF (0, 20 or 200 ng/mL) for 4 hours. (B) The mRNA expression of IL-2, IL-17, and IFN-γ in Jurkat stimulated with phorbol 12-myristate 13-acetate (PMA; 20 ng/mL) + ionomycin (Iono; 1000 ng/mL) plus BDNF (0, 20 or 200 ng/mL) for 4 h. (C) Normal PBMCs (1 × 106/mL) were stimulated with 1 μg/mL anti-human CD3 and 1 μg/mL anti-human CD28 plus different concentrations of BDNF (0, 20 or 200 ng/mL) for 24 hours. (D) The mRNA expression of NTRK2 and NGFR in Jurkat cells after stimulated with PMA and ionomycin for 4 hours. (E) The concentration of BDNF in culture soup of Jurkat cells after cocultured with culture medium or PMA (20 ng/mL) + Iono (1000 ng/mL) for 24 hours (ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

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