The limited role of interferon-γ in systemic juvenile idiopathic arthritis cannot be explained by cellular hyporesponsiveness

Abstract

Objective: Systemic juvenile idiopathic arthritis (JIA) is an autoinflammatory syndrome in which the myelomonocytic lineage appears to play a pivotal role. Inflammatory macrophages are driven by interferon-γ (IFNγ), but studies have failed to demonstrate an IFN- induced gene signature in active systemic JIA. This study sought to characterize the status of an IFN-induced signature within affected tissue and to gauge the integrity of IFN signaling pathways within peripheral monocytes from patients with systemic JIA.

Methods: Synovial tissue from 12 patients with active systemic JIA and 9 with active extended oligoarticular JIA was assessed by real-time polymerase chain reaction to quantify IFN-induced chemokine gene expression. Peripheral monocytes from 3 patients with inactive systemic JIA receiving anti-interleukin-1β (anti-IL-1β) therapy, 5 patients with active systemic JIA, and 8 healthy controls were incubated with or without IFNγ to gauge changes in gene expression and to measure phosphorylated STAT-1 (pSTAT-1) levels.

Results: IFN-induced chemokine gene expression in synovium was constrained in active systemic JIA compared to the known IFN-mediated extended oligoarticular subtype. In unstimulated peripheral monocytes, IFN-induced gene expression was similar between the groups, except that lower levels of STAT1, MIG, and PIAS were observed in patients with active disease, while higher levels of PIAS1 were observed in patients with inactive disease. Basal pSTAT-1 levels in monocytes tended to be higher in systemic JIA patients compared to healthy controls, with the highest levels seen in those with inactive disease. Upon stimulation of monocytes, the fold increase in gene expression was roughly equal between groups, except for a greater increase in STAT1 in patients with inactive systemic JIA compared to controls, and a greater increase in IRF1 in those with active compared to inactive disease. Upon stimulation, the fold increase in pSTAT-1 was highest in monocytes from patients with inactive systemic JIA.

Conclusion: Monocytes in patients with active systemic JIA retain the ability to respond to IFNγ, suggesting that the lack of an IFN-induced gene signature in patients with active disease reflects a limited in vivo exposure to IFNγ. In patients with inactive systemic JIA who received treatment with anti-IL-1β, hyperresponsiveness to IFNγ was observed.

Figure 1

Expression levels of IFN-γ-induced genes, relative to GAPDH, within inflamed synovial tissue (A) and freshly isolated CD14+ peripheral monocytes (B) as determined by real-time PCR. A, Relative expression levels of IFN-γ-induced chemokine genes in inflamed synovial tissue from SJIA and extended oligoarticular JIA patient groups. B, Confirmation of the relative lack of IFN-γ-induced gene expression in unstimulated peripheral monocytes. () Healthy controls; () Extended oligoarticular JIA () Active SJIA patients; () Inactive SJIA patients. Results shown as mean + SD. JIA = juvenile idiopathic arthritis; SJIA = systemic JIA; IFN-γ = interferon-γ; STAT1 = signal transducer and activator of transcription 1; IRF-1 = interferon regulatory factor 1; IP-10 = interferon gamma-induced protein 10; MIG = monokine induced by interferon-γ; I-TAC = Interferon-inducible T-cell alpha chemoattractant. * = p < 0.05, ** = p < 0.01.

Figure 2

STAT1 phosphorylation within peripheral monocytes upon IFN-γ stimulation. Monocytes were left unstimulated or stimulated with 100 units/mL of IFN-γ for 30 minutes to assess the degree of STAT1 phosphorylation by intracellular flow cytometry. A, Representative example of P-STAT1 experiment on unstimulated peripheral monocytes. B, MFI of P-STAT1 within unstimulated peripheral monocytes. C, Representative example of P-STAT1 experiment on peripheral monocytes stimulated with IFN-γ. D, Fold-change increase in P-STAT1 MFI following stimulation with IFN-γ. Active SJIA patients (gray-line histogram); inactive SJIA patients on anti-IL-1 β therapy (black-line histogram); healthy controls (dotted-line histogram). Lines within dot-plots represent mean values. STAT1 = signal transducer and activator of transcription 1; IFN-γ = interferon-γ, P-STAT1 = phosphorylated STAT1, MFI = mean fluorescence intensity; IL-1β = interleukin-1β; SJIA = systemic juvenile idiopathic arthritis. † = p < 0.05, †† = p < 0.01.

Figure 3

SJIA peripheral blood monocyte responsiveness to IFN-γ stimulation. Monocyte responsiveness expressed as fold-change in expression levels of IFN-γ-induced genes upon stimulation as determined by real-time RT-PCR. Monocytes stimulated with 100 units/mL of IFN-γ for 3 hours. () Healthy controls; () Active SJIA patients; () Inactive SJIA patients. Results shown as mean + SD. SJIA = systemic JIA; IFN-γ = interferon-γ; STAT1 = signal transducer and activator of transcription 1; IRF-1 = interferon regulatory factor 1; IP-10 = interferon gamma-induced protein 10; MIG = monokine induced by interferon-γ; I-TAC = Interferon-inducible T-cell alpha chemoattractant. * = p < 0.05, ** = p < 0.01.

Figure 4

Gene expression levels of negative-regulators of IFN-γ signaling relative to GAPDH expression within peripheral monocytes (A and B, unstimulated and C, post-stimulation) as determined by real-time RT-PCR. A and B, Unstimulated gene expression of SOCS3, PIAS1, and SOCS1 in peripheral monocytes. C, Fold-change in expression levels of indicated genes after stimulation with 100 units/mL of IFN-γ for 3 hours. () Healthy controls; () Active SJIA patients; () Inactive SJIA patients. Results shown as mean + SD. IFN-γ = interferon-γ; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; SOCS1/3 = suppressor of cytokine signaling 1/3; PIAS1 = protein inhibitor of activated STAT1; IL-1β = interleukin-1β. * = p < 0.05, ** = p < 0.01.