3-hydroxy butyrate dehydrogenase 2 deficiency aggravates systemic lupus erythematosus progression in a mouse model by promoting CD40 ligand demethylation

Abstract

The implications of the CD40-CD40 ligand (CD40L) signaling pathway in systemic lupus erythematosus (SLE) were well documented, due to its important role among immune cells. Previous research found that 3-hydroxy butyrate dehydrogenase 2 (BDH2), a modulator of intracellular iron homeostasis and iron transportation promoted the pathogenic process of SLE by regulating the demethylation of cd70, cd11a, and cd40l genes among CD4 + T cells. The purpose of this study was to explore the role of BDH2 in oxidative damage-induced SLE. First, CD4 + T cells treated with H₂O₂ were injected into the tail vein of mice to establish a lupus model. CD40L knockdown significantly decreased CD40L expression on CD4 + T cells in the spleen of SLE mice. Compared with SLE model mice, the levels of serum anti-dsDNA antibody and urinary protein in the CD40L interference group were significantly decreased. CD40L knockdown alleviated the immune complex glomerulonephritis in syngeneic SLE mice. Moreover, the levels of IFN-γ and IL-2 were decreased. However, IL-4 and IL-10 levels were significantly upregulated in the serum of CD40L knockdown SLE mice, compared with SLE model mice. Accordingly, CD40L knockdown reduced Th1/Th2 percentage in SLE mice. Inhibiting the expression of BDH2 of CD4 + T cells promoted the demethylation of CD40L, while it inhibited cell proliferation, elevated oxidative stress through increased expression of CD40L, and thus, promoted the progress of SLE. Our results demonstrate that BDH2 aggravates the pathologic progression of SLE in mice, by increasing the demethylation level of CD40L among CD4 + T cells.

Keywords: 3-hydroxy butyrate dehydrogenase 2; CD4+ T cells; CD40L; Systemic lupus erythematosus; demethylation.

Figure 1.

Identification of the role of CD40L in systemic lupus erythematosus mouse model (a) Quantitative PCR detects the CD40L mRNA level. Three shRNAs were designed targeting CD40L which were LV-CD40L-shRNA1 (KD-1), LV-CD40L-shRNA2 (KD-2), LV-CD40L-shRNA3 (KD-3). As showed in this part the knock down percentage of KD1 and KD2 was more than 50% and they were selected for the following experiment. ‘NC’ is the group transfected with lentivirus vector GV493. KD1, KD2, KD3 were GV493 insert with three shRNAs targeting CD40L. (b & c) Western blot detected the CD40L protein level. (d) Serum anti-dsDNA antibody level. (e) Urinary protein in mice was examined and CD40L alleviated the pathological changes in mouse kidneys. (f) HE staining detected the pathological changes in mouse kidneys. In (D)-(F) SLE oxidative model group, mice were injected with CD4 + T cells treated by H₂O₂; si-CD40L group, mice were injected with CD4 + T cells transfected with LV-CD40L-shRNA1 (KD-1). Control group, the negative control of transfection. Each group contained 6 mice and three independent repeats for each experiment. One-way ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

CD40L decreased the proportion of Th1/Th2 cells in systemic lupus erythematosus mice. (a) Cytokine level of IFN-γ, IL-2, IL-4 and IL-10 (p < 0.01). (b) Flow cytometry results Th1/Th2 cells. KD1 group, CD4 + T cells transfected with LV-CD40L-shRNA1 (KD-1); KD2 group, cells were treated with LV-CD40L-shRNA2 (KD-2). SLE oxidative model group, mice were injected with CD4 + T cells treated by H₂O₂, si-CD40L group, mice were injected with CD4 + T cells transfected with LV-CD40L-shRNA1 (KD-1). Control group, the negative control of transfection. Each group contained 6 mice and three independent repeats for each experiment. One-way ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

BDH2 upregulates the protein level of CD40L by inhibiting methylation. (a) Real-time PCR detected the expression of BDH2 and CD40L. Three shRNAs were designed targeting BDH2. They were LV-BDH2-shRNA1 (KD-1), LV-BDH2-shRNA2 (KD-2), LV-BDH2-shRNA3 (KD-3). As showed in this part the knock down percentage of KD1 was about 80%. And it was selected for the following experiment. ‘NC’ group, cells were transfected with lentivirus vector GV248. (b) Results of methylation specific PCR showed that the decrease of BDH2 resulted in the increase of demethylation level of CD40L. (C&D) Western blots showed that BDH2 upregulates the CD40L protein level by inhibiting CD40L methylation. Three independent repeats for each experiment and One-way ANOVA used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Effect of BDH2 on CD4 + T cell function and aggravation of the pathological process of systemic lupus erythematosus (a) Proliferation effects when BDH2 was solely or simultaneously interfered with CD40L interference among CD4 + T cells. (b&c) Results of flow cytometry showed that interfering with BDH2 expression among CD4 + T cells promoted level of ROS, while CD40L was simultaneously interfered with BDH2 interference among CD4 + T cells, the level of ROS was inhibited. (d) Data showed that interfering with BDH2’s expression promoted the levels of MDA, while after the interference with the combined expressions of both BDH2 and CD40L, cells fell to baseline levels. (f) Serum anti-dsDNA antibody level. (g) Urinary protein in mice was examined. ‘NC’ group was transfected with lentivirus vector GV248. ‘KD-BDH2’ group was transfected with LV-BDH2-shRNA1 (KD-1), ‘KD-BDH2-KD-CD40L’ group was transfected with both LV-BDH2-shRNA1 and LV-CD40L-shRNA1. Three independent repeats for each experiment and One-way ANOVA used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.