Enhancement of Th1/Th17 inflammation by TRIM21 in Behçet's disease

Abstract

The etiology of Behçet's disease (BD), a chronic, multisystemic autoinflammatory and autoimmune disease, remains unknown; however, researchers have postulated that infectious agents, such as herpes simplex virus, are significant triggering factors of BD. Tripartite motif-containing (TRIM) proteins exhibit antiviral properties, mediating antiviral defense mechanisms. The purpose of this study was to investigate TRIM21 protein expression in the monocytes of BD patients and to identify the role of TRIM21 in immune dysregulation in BD. In this study, the expression of TRIM21 and related molecules, including interferon regulatory factor 8 (IRF8), was analyzed in monocytes from BD patients. Functional analyses using small interfering RNA and co-culture with responder T cells were performed to examine the pathological role of TRIM21 in BD. Peripheral blood monocytes from BD patients showed increased TRIM21 expression and decreased IRF8 expression compared with that in monocytes from healthy controls. TRIM21 was found to decrease IRF8 expression. BD monocytes facilitated Th1 and Th17 differentiation of co-cultured T cells, and knock-down of TRIM21 expression by small interfering RNA inhibited this differentiation. In conclusion, TRIM21 played a pivotal role in regulating the secretion of proinflammatory cytokines in monocytes of BD patients.

Figure 1

Monocytes from Behçet’s disease patients exhibited increased TRIM21 expression compared to those from healthy controls. (a and b) Human monocytes isolated from healthy volunteers and BD patients. TRIM21 protein levels in monocytes from 18 BD patients and five healthy control subjects, as determined by western blot (a). TRIM21 expression was quantified by densitometry following normalization to GAPDH expression as indicated in accompanying graph (b). Data are expressed as the mean ± SD. **P < 0.01. The cropped blots are displayed and full-length blots are shown in the Supplementary Information. (c) Endogenous TRIM21 was visualized by immunofluorescence using anti-TRIM21 (green) and DAPI (blue) and observed under confocal microscopy. Scale bar represents 20 μm.

Figure 2

The expression of IRF8, a representative target of TRIM21 ubiquitination, was also decreased in BD monocytes. (a) IRF8 protein levels in monocytes from 18 BD patients and five healthy control subjects, as determined by western blot. Cell lysates were from the same sample used in the analysis depicted in Fig. 1a and b. Primary monocytes (c) and THP-1 cells (d) were transfected with scrambled RNA or TRIM21 siRNA (10 nM) for 24 h. Expression of TRIM21 and IRF8 was detected by immunoblotting. TRIM21 and IRF8 expression was quantitated by densitometry following normalization to GAPDH expression as indicated in the accompanying graph. Data are represented as the mean ± SD. (e) Monocytes isolated from healthy controls or BD patients were stimulated with LPS (1 µg/ml) for 24 h. Supernatants were collected, and secretion of IL-12/23p40 was measured by ELISA. (f) Primary monocytes from healthy controls were transfected with scrambled RNA or TRIM21 siRNA (10 nM) for 24 h, and the transfected cells were stimulated with LPS (1 µg/ml) for 24 h, and the secretion of IL-12/23p40 was measured by ELISA. Data are represented as the mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. The cropped blots are displayed and full-length blots are shown in the Supplementary Information.

Figure 3

BD monocytes showed increased secretion of Th17-promoting cytokines, and knock-down of TRIM21 decreased cytokine secretion. (a) Monocytes isolated from healthy controls or BD patients were stimulated with LPS (1 µg/ml) for 24 h. Supernatants were collected, and secretion of IL-1β, IL-6, and IL-23 was measured by ELISA. (b) Primary monocytes from healthy controls were transfected with scrambled RNA or TRIM21 siRNA (10 nM) for 24 h, and the transfected cells were stimulated with LPS (1 µg/ml) for 24 h. Supernatants were collected; and secretion of IL-1β, IL-6, and IL-23 was measured by ELISA. Data are represented as the mean ± SD. *P < 0.05, **P < 0.01 compared with LPS stimulation or TRIM21 knock-down in monocytes in healthy monocytes.

Figure 4

LPS-stimulated BD monocytes exhibited enhanced nuclear translocation of NF-kB. (a and b) Monocytes isolated from healthy controls and BD patients were stimulated with LPS (1 µg/ml) for 1 h. Cell lysates were evaluated by western blot for levels of phosphorylated p65. β-actin was used as the loading control. A representative result from four independent experiments is shown in (a). (c and d) THP-1 cells were transfected with scrambled RNA or TRIM21 siRNA (10 nM) for 24 h, and the transfected cells were stimulated with LPS (1 µg/ml) for 1 h. (e) THP-1 cells were transfected with scrambled RNA or TRIM21 siRNA (10 nM) for 24 h and were incubated with pNF-kB-Luc vector (500 ng) and pβ-galactosidase (500 ng) for 24 h. The transfected cells were stimulated with LPS (1 µg/ml) for 6 h. The results represent NF-kB luciferase activity, normalized for β-galactosidase. Data are represented as the mean ± SD. The cropped blots are displayed and full-length blots are shown in the Supplementary Information.

Figure 5

BD monocytes promoted both Th1 and Th17 polarization after co-culture with effector T cells. (a and b) Primary monocytes from healthy controls or BD patients were co-cultured with allogeneic naïve CD4 + T cells in the presence of anti-CD3 (1 µg/ml) mAb and LPS (100 ng/ml) for 7 days. Cells were re-stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) for 6 h in the presence of GolgiPlug for 4 h, and then expression of IL-17 and IFN-γ were determined by intracellular flow cytometry. (c) Production of IL-17A in monocyte/T-cell co-culture supernatant was quantified by ELISA. Data are represented as the mean ± SD. **P < 0.01. (d) Knock-down TRIM21 in monocytes was co-cultured with allogeneic naïve CD4 + T cells in the presence of anti-CD3 (1 µg/ml) mAb and LPS (100 ng/ml) for 48 h. Total RNA was extracted and analyzed by RT-PCR for mRNA expression of IL17A, IFNG, IL4, and IL13. Data are represented as the mean ± SD.