Diminished expression of the ubiquitin-proteasome system in early treatment-naïve patients with rheumatoid arthritis and concomitant type 2 diabetes may be linked to IL-1 pathway hyper-activity; results from PEAC cohort

Abstract

Objective: Based on the recent evidence of IL-1 inhibition in patients with rheumatoid arthritis (RA) and concomitant type 2 diabetes (T2D), we evaluated the synovial tissue expression of IL-1 related genes in relationship to the ubiquitin-proteasome system and the effects of insulin on ubiquitinated proteins in fibroblast-like synoviocytes (FLSs).

Methods: The synovial expression of IL-1 pathway genes was compared in early (< 1 year) treatment-naïve RA patients with T2D (RA/T2D n = 16) and age- and sex-matched RA patients without T2D (n = 16), enrolled in the Pathobiology of Early Arthritis Cohort (PEAC). The synovial expression of ubiquitin in macrophages and synovial lining fibroblasts was also assessed by Immunohistochemistry/immunofluorescence and correlated with synovial pathotypes. Finally, FLSs from RA patients (n = 5) were isolated and treated with human insulin (200 and 500 nM) and ubiquitinated proteins were assessed by western blot.

Results: Synovial tissues of RA/T2D patients were characterised by a consistent reduced expression of ubiquitin-proteasome genes. More specifically, ubiquitin genes (UBB, UBC, and UBA52) and genes codifying proteasome subunits (PSMA2, PSMA6, PSMA7, PSMB1, PSMB3, PSMB4, PSMB6, PSMB8, PSMB9, PSMB10, PSMC1, PSMD9, PSME1, and PSME2) were significantly lower in RA/T2D patients. On the contrary, genes regulating fibroblast functions (FGF7, FGF10, FRS2, FGFR3, and SOS1), and genes linked to IL-1 pathway hyper-activity (APP, IRAK2, and OSMR) were upregulated in RA/T2D. Immunohistochemistry showed a significant reduction of the percentage of ubiquitin-positive cells in synovial tissues of RA/T2D patients. Ubiquitin-positive cells were also increased in patients with a lympho-myeloid pathotype compared to diffuse myeloid or pauci-immune-fibroid. Finally, in vitro experiments showed a reduction of ubiquitinated proteins in RA-FLSs treated with a high concentration of insulin (500 nM).

Conclusions: A different IL-1 pathway gene expression was observed in the synovial tissues of early treatment-naïve RA/T2D patients, linked to decreased expression of the ubiquitin-proteasome system. These findings may provide a mechanistic explanation of the observed clinical benefits of IL-1 inhibition in patients with RA and concomitant T2D.

Keywords: IL-1β, ubiquitin; Of Early Arthritis Cohort study (PEAC); Pathobiology; Rheumatoid arthritis; Synovial biopsy; Type 2 diabetes.

Fig. 1

RNA sequencing assessment of RA patients with and without T2D. A Volcano plot is shown reporting differential expressed genes analysis between RA/T2D and RA patients using nominal p values. Ubiquitin genes (UBB, UBC, and UBA52) were significantly lower in T2D/RA patients than RA patients. Furthermore, some genes codifying proteasome subunits were significantly lower in RA/T2D patients (PSMA2, PSMA6, PSMA7, PSMB1, PSMB3, PSMB4, PSMB6, PSMB8, PSMB9, PSMB10, PSMC1, PSMD9, PSME1, and PSME2). Additionally, Additionally, some positive gene regulators of the ubiquitin–proteasome system (RACK1, RBX1, RPS27A, SEM1, S100A12, S100B, and SAA1) were significantly downregulated in RA/T2D patients than others. In addition, some additional genes resulted to be downregulated in RA/T2D patients in comparison with RA patients (CILP, CASP1, FGF23, IL31RA, and IL1B). On the contrary, some genes regulating fibroblast functions resulted to be upregulated (FGF7, FGF10, FRS2, FGFR3, and SOS1). Furthermore, APP, IRAK2, and OSMR were upregulated in RA/T2D patients than others. Similarly, RA/T2D patients showed an increased synovial expression of IL6ST IL18R1, and LIF. B, C, and D Individual differences in ubiquitin genes (UBB, UBC, and UBA52) are reported between RA and RA/T2D. E, F, G, H, I, and J Representative individual differences of genes for proteasome subunits (PSMA2, PSMB1, and PSMC1) and positive gene regulators of the ubiquitin proteasome system (RACK1, RBX1, and RPS27A) are shown between RA and RA/T2D. K, L, and M Representative individual differences of genes regulating fibroblast functions (FGF7, FGF10, and SOS1) are described. N, O, and P Individual genes differences for APP, IRAK2, and OSMR are reported between RA and RA/T2D

Fig. 2

Histologic assessment of synovial tissues of RA patients with or without T2D. A and B Representative images are reported of immunohistochemistry for ubiquitin on synovial tissues of patients with early treatment naïve RA patient without T2D (out of a total of n = 11) (A) and with T2D (out of a total of n = 15) (B); BROWN: DAB positive staining for ubiquitin. In the 10 × magnification: VIOLET circles show the nuclei detected by QuPath, RED circles the positive cell detection for ubiquitin by QuPath. C Representative image is shown of Immunofluorescence on synovial tissue (out of n = 4 stainings); DAPI-blue for nuclei, CD55-yellow for lining synovial fibroblasts, CD68-green for macrophages, ubiquitin-red. D, E, and F Summary of differences are shown in the assessment of ubiquitin-positive cells between RA and RA/T2D, considering percentage of cells (D) or number per mm.² (E), and according to different pathotype (F)

Fig. 3

The effect of insulin and glucose on RA-FLSs. Western blot analysis of total ubiquitin levels of RA-FLS treated with insulin at concentrations of 200 and 500 nM and glucose for 24 h. A Blot shown is representative of three independent experiments performed using RA-FLS samples from different patients. B Densitometry analysis of total ubiquitin levels relative to β-actin is also reported. Values are expressed as mean ± sd. * p = 0.02