Welcome to the CORA 2023
Interactive Program

Background and Aims

Plasmacytoid dendritic cells (pDCs) were previously thought to be the major source of type I interferon in SLE. BDCA-2 appears to be exclusively expressed on pDCs in fresh blood and has been investigated as a therapeutic target. However, we showed that pDCs lose their immunogenic functions prior to onset of SLE[PMID: 33262343]. Here, we aimed to characterise pDC marker expression on myeloid lineage cells.

Methods

Following density gradient separation, PBMCs were cultured in RPMI. At 0 and 24h, cells were stained for CD3, CD19, CD14, CD16, CD11c, HLA-DR, CD123, BDCA-2, BDCA-4 and ILT7 and analysed by flow cytometry. We next performed an in vitro M1 macrophage differentiation. Monocytes were purified by negative selection and cultured with GM-CSF for 5 days. On day 6, IFN-γ and LPS were added and cells analysed by flow cytometry.

Results

For PBMCs, at 0 hours, apart from pDCs, no monocyte subset or myeloid DCs expressed BDCA-2, BDCA-4, CD123 or ILT7. However, at 24h, each of these markers was expressed by at least one myeloid subset, predominantly by classical and intermediate monocytes. M1 macrophages (CD14+HLA-DR+CD80+CD206+) expressed all four pDC markers.

Conclusions

BDCA-2, CD123 and ILT7 are not specific to pDCs and are also expressed on monocyte subsets upon differentiation. Since monocyte subsets are key responder cells to IFN-I, with pathogenic roles in SLE, the efficacy of therapies targeting these markers may be explained by their effects on other myeloid cells and not pDCs. This is important for the safety of these therapies as well as their potential indications beyond the IFN-I mediated diseases.

Background and Aims

A major challenge in the field of chronic seronegative arthritis [e.g., negative for anti-citrullinated peptides autoantibodies (ACPA) and/or rheumatoid factors (RF)] is the research of potentially new circulating diagnostic and prognostic biomarkers. C-X-C motif chemokine 13 (CXCL13) is a promising molecule for this purpose, because of its association with active synovitis (Bechman et al. BMC Rheumatology 2020) and a poor prognosis in rheumatoid arthritis (RA) (Bugatti et al. Rheumatology 2014). We aimed to analyse CXCL13 serum levels in patients with peripheral psoriatic arthritis (PsA) in comparison to RA.

Methods

Cross sectional analysis of 81 patients with peripheral PsA [male/female=44/37; median age (25°-75°percentile)=54(46-62) years; 28-joint Disease Activity Score-C Reactive Protein (CRP-DAS28)=1.9 (1.6-2.5); active psoriasis=43%; Disease Activity in Psoriatic Arthritis (DAPSA) score=8(3-14)] and 143 RA [male/female=30/113; age=62(50-70) years; seropositive=67%; CRP-DAS28=2.1(1.5-2.8)] was performed. 100 sex and age-matched healthy controls (HC) were enrolled. CXCL13 levels were assessed using commercial ELISA test (R&D).

Results

CXCL13 levels (pg/mL) were higher in all subgroups [PsA:50.9(34.5-80.2), p<0.01; RA:77.2(52.9-107.7), p<0.01; RA/ACPA+:77.7(55.9-110.7), p<0.01; RA/ACPA-:69.5 (49.3-104), p<0.01] than in HC [22.3 (17.7-33.8)]. No significant differences were found among RA patients according with their seropositivity (p=0.378). CXCL13 was lower in PsA than in RA [vs ACPA+ RA, p<0.01; vs ACPA- RA, p=0.012] and positively correlated with CRP (r=0.30;p=0.008) but not with DAPSA score in PsA patients.

Conclusions

These results confirm the biomarker value of CXCL13 in the field of chronic arthritis. Higher levels in seronegative RA than in PsA suggest its potential value in the differential diagnosis of these two subsets.

Background and Aims

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of autoimmune disorders. Extracellular vesicles (EVs) are cell-derived nanoparticles involved in intercellular signaling convoying their cargo that act in autoimmune pathogenesis. The study aims to characterize circulating EVs surface markers and microRNA cargo investigating their potential role as biomarkers in IIM.

Methods

EVs were isolated from platelet-free plasma of IIM patients and healthy donors (HDs) through size exclusion chromatography and ultrafiltration. EVs were quantified by nanoparticles tracking analysis (NTA), immune-characterized by imaging-flow cytometry (IFC), and EVs-microRNA cargo investigated through Next-Generation Sequencing (NGS).

Results

NTA measurements reported higher mean EVs concentration in IIM patients (n=58) than in HDs (n=60) (1.75x10^10 ± 1.35x10^10 SD [EVs/mL] vs. 1.34x10^10 ± 7.35x10^9; p=0.0461). IFC characterization showed a prevalence of CD3-CD19+ EVs both in IIM (n=26) and HDs (n=25) (7.38x10^7 ± 3.67x10^7 vs. 6.20x10^7 ± 3.85x10^7, respectively) compared to CD3+ (2.64x10^6 ± 1.38x10^6 vs. 2.35x10^6 ± 1.78x10^6) (p<0.0001). NGS analysis detected 10 EVs-microRNA with different expression profile between IIM (n=21) and HDs (n=21). Hsa-miR-451a (p=0.0010), hsa-miR-15a-5p (p=0.0086), hsa-miR-486-5p (p=0.0012), hsa-miR-222-3p (p=0.0098), hsa-miR-32-5p (p=0.0038), hsa-miR-185-5p (p=0.0217) were up-regulated in IIM than HDs. Hsa-let-7b-5p (p=0.0046), hsa-let-7a-5p (p=0.0032), hsa-let-7e-5p (p=0.014), hsa-let-7f-5p (p=0.0123) were down-regulated in IIM.

Conclusions

The prevalence of B lymphocytes markers on circulating EVs surface might indicate their cell origin. Moreover, the increased EVs concentration in IIM than HDs and the dysregulated EVs-microRNA cargo expression suggest that EVs could potentially have a role in IIM, representing promising disease biomarkers.

Background and Aims

Objective of the study was to evaluate whether there are different biomarkers predicting response to two different Jak inhibitors with different selectivity (FIL,UPA) in patients with rheumatoid arthritis

Methods

We enrolled 52 RA patients with 48 females, 5 males. 33 patients were on therapeutic treatment with UPA15 mg / day, 19 with FIL 200 mg / day. Clinimetry with DAS28, CDAI, TJC, SJC, VAS, HAQ, and serological parameters ESR, CRP, APCA, RF, MRP (Myeloid Related Protein),classical pathway, MBL, complement alternative, TNF, IL-6, lymphocyte subpopulations CD3, CD4, CD8, CD19, CD56 , haematological parameters with ratio neutrophils / lymphocytes (N/L), monocytes / lymphocytes (M/L) and platelets / lymphocytes (P/L) were also evaluated at baseline, 12 and 24 weeks.

Results

Significant improvement were obtained for the following clinical and laboratory parameters: DAS28, CDAI, TJC, SJC, HAQ, VAS, ESR, CRP.

Significant differences in the two patient groups were: FIL P/L 176.53 vs 153.59 p = 0.049 and IL-6 p = 0.0039

For the UPA MRP group 5.72 vs 2.05 mcg / ml p = 0.029 and IL-6 p = 0.032. Differences in the UPA group were present for lymphocytes CD56 NK 259 vs 216 cells/mcl p = 0.044

Conclusions

our preliminary results show a different behavior of FIL and UPA on some parameters in patients with RA. While FIL demonstrates a reduction in IL-6 levels and P/L ratio, UPA demonstrates a reduction in IL-6 and MRP values. The reduction in the UPA group of CD56 / NK cells demonstrates the different selectivity on the IL-15 pathway.

Background and Aims

The Programmed cell death protein 1 (PD1) and its ligand (PDL1) are expressed on various subsets of cells including T and B cells and were successfully targeted for cancer immunotherapy. However, the role of these molecules in autoimmune diseases is vaguely known so far. In this study, we aimed to evaluate the regulation efficiency of PD1/PDL1 interaction in healthy and Rheumatoid Arthritis (RA) patients.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy and RA patients. T and B cells were negatively enriched by magnetic-activated cell sorting. Effector/memory helper T (Th) cells were sorted based on CD4 positivity and the differential expression of CD25 and CD127 using FACS aria III. T and B cells were stimulated by anti-CD3/CD28, and CpG/anti-CD40, respectively. The regulatory effects were assessed by monitoring cell proliferation and intracellular cytokines IFN-γ, TNF-α, and IL-21 using Flow cytometry.

Results

Effector/memory Th cells from healthy individuals showed an increased expression of activation markers CD25 and PD1 compared to RA patients upon stimulation. However, Th cell proliferation did not differ between healthy and RA patients either in monocultures or in co-cultures with PDL1+ Breg cells. The intracellular cytokines, IFN-γ, and IL-21 production were higher in RA compared to healthy sample monocultures, while TNF-α production was similar. Interestingly, PDL1+ Bregs downregulated all cytokines by approximately 50 % in both healthy and RA samples.

Conclusions

PD1/PDL1 cross-linking might not affect helper T cell proliferation, however, downregulates IFN-γ and IL-21 production, indicating that the disease progression can be manipulated by inducing PDL1+Breg cells in RA patients.

Background and Aims

With a prevalence of approximately 1%, rheumatoid arthritis (RA) is one of the most frequent autoimmune disorders. Treatment at early stages can significantly slow down disease progression or even prevent irreversible damages. However, an early diagnosis is often challenging. Up to 50% of early RA patients are negative for the standard serological markers rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) antibodies. A more comprehensive analysis of antigenic proteins and their underlying epitopes can provide the necessary information for innovative serological tests with higher sensitivity for early diagnosis of RA.

Methods

To identify novel biomarkers, we applied high-density peptide microarrays displaying large numbers of autoantigens/antigen candidates converted into >100.000 overlapping peptides including all citrullinated and carbamylated variants. Using this highly diverse library, we screened sera from different disease and control cohorts and identified various epitopes with a higher prevalence in previously seronegative early-stage patients.

Results

Bioinformatic analysis combined with machine learning resulted in a peptide biomarker combination, which allowed the diagnosis of early seronegative RA vs. healthy controls with an accuracy of 87% and early seropositive RA vs. healthy controls with an accuracy of 94%, respectively. The validation of the marker set using well-characterized patient samples (n=1027) by Aptiva particle-based multi-analyte technology (Werfen) revealed highly significant peptides for discriminating RA vs. controls. Random forest modeling demonstrated a sensitivity and specificity of 80 and 90%, respectively.

Conclusions

Hence, the novel marker set has the potential to improve the early diagnosis of RA.