Potential role of RhoA GTPase regulation in type interferon signaling in systemic lupus erythematosus

Abstract

Objective: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by abnormal activation of the type I interferon (IFN) pathway, which results in tissue inflammation and organ damage. We explored the role of the RhoA GTPase in the type I IFN activation pathway to provide a potential basis for targeting GTPase signaling for the treatment of SLE.

Methods: Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls, and the mRNA expression levels of RhoA and IFN-stimulated genes were measured by SYBR Green quantitative reverse transcriptase-polymerase chain reaction. IFN-a-stimulated response element (ISRE)-luciferase reporter gene assays and Western blotting were conducted to assess the biologic function of RhoA. An enzyme-linked immunoassay (ELISA) measured C-X-C motif chemokine ligand 10 (CXCL10) protein expression.

Results: Our studies demonstrate that the expression of RhoA in the PBMCs of SLE subjects was significantly higher than in healthy controls and positively correlated with type I IFN scores and type I IFN-stimulated gene (ISGs) expression levels. SiRNA-mediated knockdown of RhoA and the RhoA/ROCK inhibitor Y27632 reduced the activity of the type I IFN-induced ISRE, the signal transducer and activator of transcription 1 (STAT-1) phosphorylation, and the expression of CXCL10 and 2'-5'-oligoadenylate synthetase 1 (OAS1). Finally, we verified that Y27632 could significantly down-regulate the OAS1 and CXCL10 expression levels in the PBMCs of SLE patients.

Conclusion: Our study shows that RhoA positively regulates the activation of the type I IFN response pathway. Reducing the expression level of RhoA inhibits the abnormal activation of the type I IFN system, and the RhoA/ROCK inhibitor Y27632 decreases aberrant type I IFN signaling in SLE PBMCs, suggesting the possibility of targeting the RhoA GTPase for the treatment of SLE.

Keywords: Autoimmunity; RhoA; RhoA/ROCK inhibitor; Systemic lupus erythematosus; Type I IFN.

Fig. 1

Elevated RhoA expression correlates positively with type I IFN-inducible gene expression and IFN scores. A Significantly higher expression of RhoA mRNA in the PBMCs of SLE patients (n = 36) than in healthy controls (HC, n = 60). B–E Correlations between RhoA expression and the type I IFN-inducible genes OAS1, CXCL10, IFIT3, and MX1 in SLE patients. F Type I IFN scores calculated by integrating the relative expression of the OAS1, CXCL10, IFIT3, and MX1 genes in the SLE and HC groups. G RhoA was highly expressed in high-type I IFN-scoring patients. The dotted line represents the mean ± 2SD of the HC values. H Positive correlation between type I IFN scores and SLEDAI values. I Positive correlation between RhoA expression levels and SLEDAI values. Bars in A and G show the mean ± SEM. Each symbol represents an individual sample. Expression is defined as the relative expression of the gene of interest in comparison to GAPDH. *p < 0.05, ***p < 0.001 A and G by Mann–Whitney U test and B-F, H, and I by Spearman rank correlation test

Fig. 2

RhoA regulates the type I IFN-stimulated response element (ISRE) and downstream gene expression. A ISRE activity of HEK293T cells transfected with ISRE-luciferase and a Renilla reporter plasmid together with a negative control (Ctrl) or RhoA-targeted siRNA (each at 200 nM). B ISRE activity of HEK293T cells transfected with RhoA-overexpression or control plasmids (each at 100 ng). At 24 h after transfection, the cells were left unstimulated (0 h) or stimulated with IFN-a (1000 U/mL, 6 h). Data in A and B are expressed as fold-change based on relative luciferase activities (ratio of firefly luciferase to Renilla luciferase). C–E The relative expression of OAS1, IFIT3, and CXCL10 mRNAs were determined by quantitative PCR. F The CXCL10 levels in culture supernatants were determined by enzyme-linked immunosorbent assay. Data were assessed in HEK293T (C, D) and THP1 cells (E, F) at 24 h after transfection of the negative control (Ctrl) or siRNA (200 nM) stimulated cells with IFN-a (1000 U/mL) for 6 h. Bars show the mean ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by Student’s t-test

Fig. 3

RhoA regulates type I IFN-induced STAT-1 phosphorylation. Western blot analysis (A, C) and densitometry histograms (B, D) of cell lysates of cultured HEK293T cells (5 × 10⁵ cells per well) transfected with siRNA (200 nM) targeting RhoA mRNA (A) or a RhoA expression plasmid vector (4 µg/mL) (C), and their controls (a negative control siRNA or pCMV-NC) stimulated with IFN-a (1000 U/mL) for different times, as shown. The staining density histograms are from three independent experiments and values are expressed as the ratios of phosphorylated STAT-1 protein to total STAT-1. The immunoblot of cells transfected with a negative control siRNA (B) or the pCMV-NC vectors (D) were set at a value of 1. *p < 0.05, **p < 0.01.by Student’s t-test

Fig. 4

The RhoA/Rock Inhibitor Y27632 downregulates type I IFN signaling. A HEK293T cells were co-transfected with ISRE-luciferase and Renilla reporter plasmids for 24 h, then cultured with or without Y27632 (60 μM, 45 min) before the addition of IFN-a (1000 U/mL, 6 h). Luciferase activity was measured by dual luciferase assay. B PBMCs from healthy controls were cultured in the presence or absence of Y27632 (60 μM, 45 min) and stimulated with IFN-a (1000 U/mL) for different times. Western blots show whole-cell lysates harvested at the indicated times. C Histograms show the ratios of phosphorylated to total STAT-1 at the indicated times. The ratio of pSTAT-1/STAT-1 in the absence of Y27632 and IFN-a at 0 min was set at 1. D Relative expression of OAS1 and E CXCL10 mRNA in PBMCs cultured with Y27632 (0, 30, 60, and 90 μM, for 45 min and stimulated with IFN-a (1000 U/mL) for 6 h. Results are the relative expression levels of OAS1 and CXCL10 mRNA normalized to endogenous GAPDH mRNA levels. F CXCL10 levels in PBMC culture supernatants quantified by ELISA. Bars show the mean ± SEM of 3 individual healthy donors; *p < 0.05, **p < 0.01, A and C versus vehicle by Student’s t-test

Fig. 5

The RhoA/ROCK inhibitor Y27632 reduces type I IFN signaling in lupus PBMCs. A Relative expression of OAS1 and CXCL10 mRNA in PBMCs obtained from SLE patients (n = 6) and incubated in medium alone or with Y27632 (60 μM, 6 h). B OAS1 and C. CXCL10 mRNA measurements in lysates of lupus PBMCs (n = 6) cultured with or without Y27632 (60 μM, 45 min) and stimulated with IFN-a (1000 U/mL). Results are the relative expression levels of OAS1 and CXCL10 mRNA normalized to endogenous GAPDH mRNA levels. D CXCL10 levels in culture supernatants quantified by ELISA. Bars show the mean ± SEM of 6 individual PBMC samples. *p < 0.05 by Student’s t-test