The RhoA GTPase regulates Type I 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-stimulated response element (ISRE)-luciferase reporter gene assays and Western blotting were conducted to asssess the biologic function of RhoA. An Enzyme-Linked Immunoassay (ELISA) measured C-X-C motif chemokine ligand 10(CXCL10)protein expression.

Results: Our studies demonstrated that the expression of RhoA in the PBMCs of SLE subjects was significantly higher than 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 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: Pathologensis; RhoA; RhoA/ROCK inhibitor; Systemic lupus erythematosus; type I IFN.

Figure 1.. Elevated RhoA expression correlates positively with type I IFN-inducible gene expression and IFN score in SLE patients.

(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) 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 IFN scores patients. The dotted line represents mean ± 2SD of the HC. (H) 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 relative expression of the gene of interest in comparison to GAPDH . *p < 0.05, ***p < 0.001 by Student’s t-test.

Figure 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 hours after transfection, the were left unstimulated (0 hours) or stimulated with type I IFN (1,000 units/mL, 6 hours). 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 and D) and THP1 cells(E and F) at 24 hours after transfection of the negative control (Ctrl) or siRNA (200 nM) stimulated with IFN-a(1,000 units/mL) for 6 hours. Bars show the mean ± SEM of 3 independent experiments. *p < 0.05, ***p < 0.001 by Student’s t-test.

Figure 3.. RhoA regulates type I IFN induced STAT-1 phosphorylation.

Western blot analysis (A and C) and densitometry histograms(B and D) of cell lysates of cultured HEK293T cells (5×10⁵ cells per well) transfected with siRNA at 200 nM targeting RhoA mRNA (A)or a RhoA expression plasmid vector at 4 ug/mL(C),and their controls(a negative control siRNA or pCMV6-NC) ,and then stimulated with IFN-a(1,000 units/mL) for different times.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 pCMV6-NC vectors (D) were set at a value of 1. *p < 0.05, **p < 0.01.by Student’s t-test.

Figure 4.. The RhoA/Rock Inhibitor Y27632 downregulates type I IFN signaling.

(A) HEK293T cells were cotransfected with ISRE-luciferase and Renilla reporter plasmids for 24 hours, then cultured with or without Y27632 (60 μM, 45 min) before the addition of IFα (1,000 U/ml, 6 hrs). Luciferase activity was measured by dual luciferase assay. (B) Peripheral blood mononuclear cells (PBMCs) from healthy controls were cultured in the presence or absence of Y27632 (60 μM, 45 min) and stimulated with IFNα (1,000 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 IFNα at 0 minute was set at 1. Bars show the mean±SEM of 3 individual healthy donors; *p < 0.05, **p < 0.01, versus vehicle by Student’s t-test. (D) Relative expression of OAS1 and (E) CXCL10 mRNA in PBMCs cultured with Y27632 (0–90 μ M, for 45 mins and stimulated with IFNα (1,000 U/mL) for 6 hrs. 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 PBMCs values from three different individuals. *p < 0.05 by Student’s t-test for IFNα stimulation in the absence or presence of Y27632.

Figure 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 hrs). (B) OAS1 and (C) CXCL10 mRNA measurements in lysates of lupus PBMCs (n = 6) cultured with or without Y27632 (60 μM, 45 mins) and stimulated with IFNα (1,000 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.