Increased T-bet/GATA-3 and ROR-γt /Foxp3 Ratios in Cerebrospinal Fluid as Potential Criteria for Definite Neuro-Behçet's Disease

Abstract

When the central nervous system (CNS) is the primary affected site in an initial attack of Behçet's disease (BD), the differential diagnosis is particularly challenging. Some cases remain unclassified or qualified as probable neuro-Behçet's disease (NBD). Several cytokines are involved in the immunopathogenesis of this disease; however, studies establishing the differential cytokine pattern between probable and definite NBD are scarce. Twenty-eight parenchymal NBD patients, diagnosed according to the International Consensus Recommendation (ICR) criteria and classified into definite (D-NBD; n = 17) and probable (P-NBD; n = 11), were sampled at their first neurological symptoms, and compared with healthy control subjects (n = 20). Oligoclonal bands (OCB) of IgG were detected by isoelectric focusing on agarose, and immunoblotting of matched serum and cerebrospinal fluid (CSF) sample pairs. T cell cytokines (INF-γ, IL-4, IL-17, and IL-10) and transcription factors related to Th1, Th2, Th17, and T regulatory populations (respectively T-bet, GATA-3, ROR-γt, and Foxp3) were studied by quantitative RT-PCR in peripheral blood mononuclear cells (PBMCs) and CSF cells. Inflammatory cytokines such as IL-6, TNF-α, and IL-1β were also analyzed. CSF OCB pattern 2 was present in only 1 out of 28 neuro-Behçet's patients who belonged to the P-NBD group. Two D-NBD patients had OCB in CSF showing pattern 4. In the D-NBD CSF samples, IL-17 and IL-10 expressions were significantly elevated compared to P-NBD. Moreover, D-NBD patients had increased levels of T-bet/GATA-3 and ROR-γt/Foxp3 ratios compared to P-NBD. Furthermore, a significant increase of CSF IL-6 in D-NBD, compared to P-NBD and the controls, was found. In addition to the increased IL-6 level, the data obtained suggest the existence in D-NBD patients of a significantly disrupted balance between Th17 effector and T regulatory cells, as reflected by the enhanced ROR-γt/Foxp3 ratio. This could be considered as an additional criterion for definite neuro-Behçet's disease.

Keywords: PBMCs; ROR-γt/Foxp3; T-bet/GATA-3; cerebrospinal fluid; neuro-Behçet’s disease.

Figure 1

Flow chart depicting patient selection and grouping. DMT: disease-modifying therapy; CSF: cerebrospinal fluid; ISG: International Study Group.

Figure 2

Boxplots representation of PBMCs Th1, Th2, Th17 and Treg related gene expression: (a) Representative boxplots of INF-ɣ and T-bet expression in the PBMCs of patients and NIND; (b) Representative boxplots of IL-4 and GATA3 expression in the PBMCs of patients and NIND; (c) Representative boxplots of IL-17 and RoR-ɣt expression in the PBMCs of patients and NIND; (d) Representative boxplots of IL-10 and Foxp3 expression in the PBMCs of patients and NIND. Statistical significance between the two groups was assessed using the Wilcoxon–Mann–Whitney test ns = non significant, **** p < 0.0001.

Figure 3

Boxplots representation of CSF cells’ Th1, Th2, Th17 and Treg-related gene expression: (a) Representative boxplots of INF-ɣ and T-bet expression in the CSF of patients and NIND; (b) Representative boxplots of IL-4 and GATA3 expression in the CSF of patients and NIND; (c) Representative boxplots of IL-17 and RoR-ɣt expression in the CSF of patients and NIND; (d) Representative boxplots of IL-10 and Foxp3 expression in the CSF of patients and NIND. Statistical significance between the two groups was assessed using the Wilcoxon–Mann–Whitney test ns = non significant, * p ≤ 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001.

Figure 4

Boxplots representation of CSF ratios of the selected transcription factors: (a) Representative boxplots of RoR-ɣt/ Foxp3 ratio in the CSF of P-NBD and D-NBD; (b) Representative boxplots T-bet/GATA3 ratio in the CSF of P-NBD and D-NBD. Statistical significance between the two groups was assessed using the Wilcoxon–Mann–Whitney test * p ≤ 0.05.

Figure 5

Boxplots represention of inflammation-related genes expression in PBMCs and CSF cells: (a) Representative boxplots of IL-6 expression in the PBMCs and CSF of patients and NIND; (b) Representative boxplots of TNFα expression in the PBMCs and CSF of patients and NIND; (c) Representative boxplots of IL-1β expression in the PBMCs and CSF of patients and NIND. Statistical significance between the two groups was assessed using the Wilcoxon–Mann–Whitney test ns = non significant, * p ≤ 0.05, ** p < 0.01, **** p < 0.0001.