DNA microarray analysis of active- and inactive-adult Still disease (ASD)

Objective: Adult Still’s disease (ASD) is a systemic disorder of unknown etiology characterized by high spiking fever, rash and arthritis. The purpose of this study was to determine the pathogenic roles of specific genes in ASD. Methods: Differentially expressed genes (DEGs) were examined by DNA microarray and validated by quantitative PCR using monocytes isolated from patients with active-ASD, inactive-ASD and healthy controls. The correlation between validated DEGs and ASD activity was analyzed. After inflammasome activation with LPS and Nigericin, the production of IL-1β, IL-18, inflammasome and autophagy related proteins in DEGs-overexpressing THP-1 cells was carried out by ELISA or western blotting. DEGs-overexpressing THP-1 cells were treated with an inhibitor of autophagy followed by assessment of IL-1β and IL-18 production by ELISA and western blotting method.Conclusions: The overexpression of PLAC8 in monocytes might play a regulatory role in the production of IL-1β and IL-18 by the enhancement of autophagy, resulting in the suppression of ASD. Results:A total of 68 genes were highly expressed in monocytes isolated from active-ASD patients, relative to their expression in inactive-ASD patients and healthy controls. After validation of expression of 13 genes (CLU, FCGR1B, PLAC8, TLR1, S100A12, CD55, PIM1, BCL2A1, SOD2, PLSCR1, CYP1B1, STEAP4, IL1RN), the expression of PLAC8 was significantly higher in active-ASD patients than the other groups. In ASD, PLAC8 expression level correlated with serum levels of CRP, ferritin and IL-18. Stimulation of monocytes with lipopolysaccharide resulted in PLAC8 upregulation. LPS or Nigericin stimulation of PLAC8-overexpressing THP-1, but not THP-1 cells< was associated with significant decrease in IL-1β and IL-18 production. PLAC8 overexpressing in THP-1 cells was associated with enhanced autophagy and suppression of IL-1β and IL-18 production. Conclusions: PLAC8 upregulation in monocytes seemed to play a regulatory role in the production of IL-1β and IL-18 through enhanced autophagy, resulting in suppression of ASD. The results highlight the role of PLAC8 in the pathogenesis of ASD and suggest its potential suitability as a therapeutic target in ASD. All subjects were examined at the University of Tsukuba Hospital and each provided signed consent form for participation in the study. The study was approved by the ethics committee of the University of Tsukuba. In total, 9 active-ASD patients (3 males and 6 females, the mean±SD age; 51.2±17.0 years), 10 inactive-ASD patients (3 males and 7 females, the mean±SD age; 45.5±13.6 years), 8 rheumatoid arthritis (RA, 3 males and 5 females, the mean±SD age; 50.0±18.3 years) patients as a disease control and 8 healthy controls (HC, 3 males and 5 females, the mean±SD age; 50.1±8.4 years) were included in present prospective study. All active-ASD patients satisfied Yamaguchi’s criteria [Yamaguchi M, et al., J Rheumatol. 1992; 19: 424-430], and all RA patients satisfied the American College of Rheumatology classification criteria [Arnett FC, et al., Arthritis Rheum. 1988; 31:315-324]. Healthy control subjects matched for sex and age were recruited as the control group. Monocytes (CD14+ cells) were isolated from HC (n=3, 1 males and 2 females, the mean±SD age; 51.0±7.9 years), inactive-ASD (n=3, 1 males and 2 females, the mean±SD age; 48.7±10.7 years) and active-ASD patients (n=3, 1 males and 2 females, the mean±SD age; 48.7±10.7 years) by magnetic-activated cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). In ASD, monocytes were isolated from same patients on active and inactive stages.