Dysregulation of RasGRP1 in rheumatoid arthritis and modulation of RasGRP3 as a biomarker of TNFα inhibitors

Abstract

Background: B and T cells play a key role in rheumatoid arthritis (RA) pathophysiology. RasGRP1 and RasGRP3 are involved in T and B cell receptors signaling, and belong to gene combination able to predict infliximab responsiveness, leading to the question of RasGRP1 and RasGRP3 involvement in RA.

Methods: RasGRP1 and RasGRP3 expression levels were measured by qRT-PCR and/or western-blot in peripheral blood mononuclear cells (PBMCs), in T and B cells from untreated RA patients and in RA patients treated by TNFα inhibitors. T and B cells from healthy controls (HC) were cultured with TNFα, and TNFα receptors neutralizing antibodies to highlight the TNFα effects on RasGRP1 and RasGRP3 pathways. MAPK pathways and apoptosis were respectively analyzed using the Proteome Profiler arrays and flow cytometry.

Results: In PBMCs from RA patients, gene expression levels of RasGRP1 were invariant while RasGRP3 was downregulated under TNFα inhibitors and upregulated under TNFα. In T cells from RA patients, RasGRP1 was decreased and its gene expression level was correlated with disease activity. In T cells from HC, TNFα stimulation increased RasGRP1 gene expression level while it reduced RasGRP1 protein expression level. Bryostatin-1 experiments have confirmed that the TNFα effect observed on T cells proliferation was due to the decrease of RasGRP1 expression. Besides, RasGRP3 expression level increased in PBMCs from RA patients under TNFα and in B cells from HC leading us to conclude that RasGRP3 in B cells was modulated by TNFα.

Conclusion: This study demonstrates RasGRP1 dysregulation in RA patients while RasGRP3 is characterized as a biomarker linked to TNFα inhibitors. After binding to TNFR1, TNFα reduced RasGRP1 protein expression resulting in inhibition of T cell activation.

Trial registration: Clinicaltrials.gov NCT00234234 , registered 04 November 2008; NCT00767325 , registered 05 October 2005.

Figures

Fig. 1

RasGRP1 and RasGRP3 gene expression fluctuations in periperhal blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA) treated with biologic agents at baseline and after treatment or after in vitro TNFα stimulation. a PBMCs were isolated from whole blood from RA patients treated with adalimumab (n = 21; 4 male and 17 female; 52 ± 3 years old) and etanercept (n = 9; 4 male and 5 female; 54 ± 5 years old) before treatment (V0) and 3 months later (V2) or abatacept (n = 30; 3 male and 27 female; 61 ± 1 years old) before treatment (V0) and 6 months later (V2). b PBMCs were isolated from whole blood of RA patients untreated with biologics (n = 6; 1 male and 5 female; 54 ± 10 years old) and cultured with or without TNFα for 24, 48 or 72 hours. Quantitative PCR analysis was performed to measure RasGRP1 and RasGRP3 gene expression levels. The relative expression levels (in arbitrary units (AU)) of RasGRP1 and RasGRP3 were normalized with 18S RNA abundance. Mean ± standard error of the mean were compared using Student’s t test or the Wilcoxon paired test: *p <0.05; **p <0.01; ***p <0.001

Fig. 2

RasGRP1 and RasGRP3 gene expression levels in B and T cells from healthy controls and patients with rheumatoid arthritis (RA). After T and B cell negative selection, quantitative PCR analysis of RasGRP1 and RasGRP3 gene expression was performed. aRasGRP1 and RasGRP3 gene expression levels in B and T cells from healthy controls (HC) (n = 20; 6 male and 14 female; 32 ± 9 years old). bRasGRP1 and cRasGRP3 gene expression levels respectively in T and B cells from HC (n = 20; 6 male and 14 female; 32 ± 9 years old) and patients with RA (n = 26; 8 male and 18 female; 53 ± 15 years old). d Correlation between RasGRP1 gene expression level in T cells and RA disease activity (disease activity score in 28 joints (DAS28)) was assessed using Pearson’s rank correlation coefficient. The relative expression levels (in arbitrary units (AU)) of RasGRP1 and RasGRP3 were normalized with 18S RNA abundance. Mean ± standard error of the mean were compared using the Wilcoxon paired test or one-way analysis of variance followed by Dunnett post-hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 3

RasGRP1 and RasGRP3 protein expression levels in T and B cells in healthy controls (HC) and rheumatoid arthritis (RA) patients. Western blot analysis was performed to measure RasGRP1 and RasGRP3 protein expression in T and B cells, respectively. RasGRP1 protein expression was measured in T cells from HC (n = 4) and RA patients (n = 5). RasGRP3 protein expression was measured in B cells from HC (n = 4) and RA patients (n = 3). A value of 1 was arbitrarily assigned to control conditions to which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was reported and expressed as fold change. Mean ± standard error of the mean were compared using Student’s t test

Fig. 4

TNFα influences RasGRP1 and RasGRP3 gene expression levels in T and B cells respectively via TNFR1 and TNFR2. a Quantitative PCR analysis was performed to measure RasGRP1 and RasGRP3 gene expression levels in T and B cells, respectively, obtained from three buffy coats. In each condition, cells were cultured with or without TNFα for 24 and 48 hours. b TNF receptor (TNFR)1 and TNFR2 expression in B (CD19) and T cells (CD3) was checked by flow cytometry. c Quantitative PCR analysis was performed to measure RasGRP1 and RasGRP3 gene expression levels in T and B cells, respectively, from three buffy coats. In each condition, T or B cells, previously exposed to anti-TNFR1 or anti-TNFR2 neutralizing antibodies, were cultured with TNFα for 48 hours. The relative expression levels (in arbitrary units (AU)) of RasGRP1 and RasGRP3 were normalized with 18S RNA abundance. Mean ± standard error of the mean were compared using one-way analysis of variance followed by Bonferroni post-hoc test: *p <0.05; **p <0.01; ***p <0.001

Fig. 5

TNFα influences RasGRP1 and RasGRP3 protein expression levels respectively in T and B cells via TNF receptor (TNFR)1 and TNFR2. Western blot analysis was performed to measure RasGRP1 protein expression in T cells from five healthy controls (HC) (a) and RasGRP3 protein expression in B cells from three HC (b). In the control condition, cells were cultured without TNFα treatment. Cells were cultured with TNFα for 48 hours and with or without antibodies targeting TNFR1 or TNFR2. A value of 1 was arbitrarily assigned to control conditions (without TNFα treatment) to which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was reported and expressed as fold change. Mean ± standard error of the mean were compared using one-way analysis of variance followed by the Dunnett post-hoc test: *p <0.05; **p <0.01

Fig. 6

Functional effects of TNFα in B and T cells. a Cell proliferation was evaluated with (3H) thymidine incorporation by B and T cells with or without TNFα (n = 3). b Analysis of mitogen-activated protein kinase (MAPK) phosphorylation in B and T cells with or without TNFα for 48 hours was performed with a human phospho-kinase array kit to measure Lyn, Lck, PLCγ1, MEK, ERK and RSK phosphorylation (n = 2). The dotted line represents the control condition without TNFα. c Western blot analysis was performed to measure ERK1/2 and phospho-ERK1/2 protein expressions in T cells from five healthy controls (HC). Cells were cultured with or without TNFα for 48 hours and with or without antibodies targeting TNFR1 or TNFR2. Histogram represents ratio of phosphorylated ERK1/2 to total ERK. A value of 1 was arbitrarily assigned to control conditions (without TNFα treatment) to which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was reported and expressed as fold change. d After incubation with 1 ng/ml of TNFα for 48 hours, cells were stimulated with 50nM Bryostatin-1 for 5 hours. CD69 expression in T cells (CD3) was assessed using flow cytometry (one representative result of three independent experiments is presented). Mean ± standard error of the mean were compared using Student’s t test or one-way analysis of variance followed by Dunnett post-hoc test. *p <0.05; **p <0.01