S100A8 and S100A12 Proteins as Biomarkers of High Disease Activity in Patients with Rheumatoid Arthritis That Can Be Regulated by Epigenetic Drugs.

Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease that is still not well understood in terms of its pathogenesis and presents diagnostic and therapeutic challenges. Monocytes are key players in initiating and maintaining inflammation through the production of pro-inflammatory cytokines and S100 proteins in RA. This study aimed to test a specific DNA methylation inhibitor (RG108) and activator (budesonide) in the regulation of pro-inflammatory mediators-especially the S100 proteins. We also searched for new biomarkers of high disease activity in RA patients. RNA sequencing analysis of healthy controls (HCs) and RA monocytes was performed. Genes such as the S100 family, TNF, and IL-8 were validated by qRT-PCR following DNA-methylation-targeted drug treatment in a monocytic THP-1 cell line. The concentrations of the S100A8, S100A11, and S100A12 proteins in the sera and synovial fluids of RA patients were tested and correlated with clinical parameters. We demonstrated that RA monocytes had significantly increased levels of S100A8, S100A9, S100A11, S100A12, MYD88, JAK3, and IQGAP1 and decreased levels of IL10RA and TGIF1 transcripts. In addition, stimulation of THP-1 cells with budesonide statistically reduced the expression of the S100 family, IL-8, and TNF genes. In contrast, THP-1 cells treated with RG108 had increased levels of the S100 family and TNF genes. We also revealed a significant upregulation of S100A8, S100A11, and S100A12 in RA patients, especially in early RA compared to HC sera. In addition, protein levels of S100A8, S100A11, and S100A12 in RA synovial fluids compared to HC sera were significantly increased. Overall, our data suggest that the S100A8 and S100A12 proteins are strongly elevated during ongoing inflammation, so they could be used as a better biomarker of disease activity than CRP. Interestingly, epigenetic drugs can regulate these S100 proteins, suggesting their potential use in targeting RA inflammation.

Impact of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Rheumatic Disease Patients on T Helper Cell Differentiation.

Complex pathogenesis of systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) is associated with an imbalance of various Th-cell subpopulations. Mesenchymal stem cells (MSCs) have the ability to restore this balance. However, bone marrow-derived MSCs of SLE and SSc patients exhibit many abnormalities, whereas the properties of adipose derived mesenchymal stem cells (ASCS) are much less known. Therefore, we examined the effect of ASCs obtained from SLE (SLE/ASCs) and SSc (SSc/ASCs) patients on Th subset differentiation, using cells from healthy donors (HD/ASCs) as controls. ASCs were co-cultured with activated CD4+ T cells or peripheral blood mononuclear cells. Expression of transcription factors defining Th1, Th2, Th17, and regulatory T cell (Tregs) subsets, i.e., T-bet, GATA3, RORc, and FoxP3, were analysed by quantitative RT-PCR, the concentrations of subset-specific cytokines were measured by ELISA, and Tregs formation by flow cytometry. Compared with HD/ASCs, SLE/ASCs and especially SSc/ASCs triggered Th differentiation which was disturbed at the transcription levels of genes encoding Th1- and Tregs-related transcription factors. However, we failed to find functional consequences of this abnormality, because all tested ASCs similarly switched differentiation from Th1 to Th2 direction with accompanying IFNγ/IL-4 ratio decrease, up-regulated Th17 formation and IL-17 secretion, and up-regulated classical Tregs generation.

Direct anti-proliferative effect of adipose-derived mesenchymal stem cells of ankylosing spondylitis patients on allogenic CD4+ cells.

OBJECTIVES: T-cell-mediated adaptive immunity contributes to the development and persistence of ankylosing spondylitis (AS). Mesenchymal stromal/stem cells (MSCs) have immunomodulatory potential and are able to inhibit T-cell proliferation, but their functionality in AS patients is relatively unknown. The aim of the study was to assess the direct anti-proliferative effects of MSCs isolated from subcutaneous abdominal adipose tissue of AS patients (AS/ASCs) on allogeneic T lymphocytes, using commercially available ASC lines from healthy donors (HD/ASCs) as a control. MATERIAL AND METHODS: CD3+CD4+ T-cells were isolated from peripheral blood of healthy blood donors, activated with anti-CD3/CD28 beads, and co-cultured for 5 days with untreated and TNF+IFN-γ pre-stimulated HD/ASCs (5 cell lines) and AS/ASCs, obtained from 11 patients (6F/5M). The proliferative response of T-cells was analysed by flow cytometry, while the concentrations of kynurenines, prostaglandin E2 (PGE-2), interleukin 10 (IL-10), and interleukin 1 receptor antagonist (IL-1Ra) were measured spectrophotometrically or using a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: HD/ASCs and AS/ASCs similarly reduced the T-cell proliferation response, i.e. the percentage of proliferating cells, the proliferation, and replication indices, and these effects were dependent mostly on soluble factors. In the co-cultures of activated CD4+ T-cells with HD/ASCs and AS/ASCs significant increases of kynurenines, PGE-2, and IL-1Ra, but not IL-10, production were observed. The release of these factors was dependent either on cell-to-cell contact (IL-10, IL-1Ra) or soluble factors (kynurenines, PGE-2). There was a moderate to strong negative correlation between T-cell proliferative response, and the concentrations of kynurenines, PGE-2, and IL-10, but not IL-1Ra. This association was more evident in the case of TI-treated AS/ASCs than HD/ASCs. CONCLUSIONS: AS/ASCs, similar to HD/ASCs, exert a direct effective anti-proliferative impact on CD4+ T cells, acting via soluble factors that are released in cell contact-dependent (IL-10) and independent (kynurenines, PGE-2) pathways. Thus, our results suggest that AS/ASCs are potentially useful for therapeutic application.

Survival of lymphocytes is not restricted by IDO-expressing fibroblast from rheumatoid arthritis patients.

Objective: Rheumatoid arthritis (RA) is characterized by expansion of fibroblast-like synoviocytes (FLS) in inflamed joints and activation of lymphocytes. Tryptophan (trp) is an essential amino acid indispensable for the biosynthesis of proteins and critical for survival of lymphocytes. Indoleamine 2,3-dioxygenase (IDO) that initiates the degradation of trp and tryptophanyl-tRNA synthetase (TTS) essential for tryptophan synthesis, regulate trp bioavailability. Here, we tested the hypothesis that triggered by cytokines, enhanced IDO activity modulate regulatory function of otherwise non-tolerogenic FLS isolated from RA patients. Materials and methods: IDO and TTS mRNA expression were evaluated by RT-PCR. IDO enzymatic activity was confirmed using HPLC. Resting or PHA-activated PBMC from healthy volunteers and RA patients were co-cultured with IDO expressing untreated (FLSC) or IFNγ-treated (FLSIFNγ) RA FLS. Lymphocyte survival and proliferation were evaluated by flow cytometry analysis and tritiated thymidine incorporation, respectively. Results: RA FLSIFNγ produce functionally active IDO and constitutively express TTS. RA FLSC and FLSIFNγ increased survival of resting lymphocytes in both studied groups, and decreased proliferation of healthy, but not RA, PBMC. Only FLSIFNγ diminished survival of activated CD3+CD4-, but not CD3+CD4+, healthy T cells and similar tendency was observed in rheumatoid cells. Importantly, IDO inhibitor, 1-methyl-DL-tryptophan (1-MT), failed to reverse this effect. PBMC, irrespective of their state (resting versus activated) or origin (healthy or RA), expressed high level of TTS mRNA. Conclusions: We suggest that RA FLS express functionally active IDO but control survival and expansion of healthy cells in IDO-independent mechanism and exert weaker, if any, suppressive effect on rheumatoid cells.

Basic Properties of Adipose-Derived Mesenchymal Stem Cells of Rheumatoid Arthritis and Osteoarthritis Patients.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are destructive joint diseases, the development of which are associated with the expansion of pathogenic T lymphocytes. Mesenchymal stem cells may be an attractive therapeutic option for patients with RA or OA due to the regenerative and immunomodulatory abilities of these cells. The infrapatellar fat pad (IFP) is a rich and easily available source of mesenchymal stem cells (adipose-derived stem cells, ASCs). However, the phenotypic, potential and immunomodulatory properties of ASCs have not been fully characterised. We aimed to evaluate the phenotype, regenerative potential and effects of IFP-derived ASCs from RA and OA patients on CD4+ T cell proliferation. The MSC phenotype was assessed using flow cytometry. The multipotency of MSCs was evaluated on the basis of their ability to differentiate into adipocytes, chondrocytes and osteoblasts. The immunomodulatory activities of MSCs were examined in co-cultures with sorted CD4+ T cells or peripheral blood mononuclear cells. The concentrations of soluble factors involved in ASC-dependent immunomodulatory activities were assessed in co-culture supernatants using ELISA. We found that ASCs with PPIs from RA and OA patients maintain the ability to differentiate into adipocytes, chondrocytes and osteoblasts. ASCs from RA and OA patients also showed a similar phenotype and comparable abilities to inhibit CD4+ T cell proliferation, which was dependent on the induction of soluble factors The results of our study constitute the basis for further research on the therapeutic potential of ASCs in the treatment of patients with RA and OA.

The Kinetics of Humoral and Cellular Responses after the Booster Dose of COVID-19 Vaccine in Inflammatory Arthritis Patients.

Impaired immunogenicity of COVID-19 vaccinations in inflammatory arthritis (IA) patients results in diminished immunity. However, optimal booster vaccination regimens are still unknown. Therefore, this study aimed to assess the kinetics of humoral and cellular responses in IA patients after the COVID-19 booster. In 29 IA patients and 16 healthy controls (HC), humoral responses (level of IgG antibodies) and cellular responses (IFN-γ production) were assessed before (T0), after 4 weeks (T1), and after more than 6 months (T2) from the booster vaccination with BNT162b2. IA patients, but not HC, showed lower anti-S-IgG concentration and IGRA fold change at T2 compared to T1 (p = 0.026 and p = 0.031). Furthermore, in IA patients the level of cellular response at T2 returned to the pre-booster level (T0). All immunomodulatory drugs, except IL-6 and IL-17 inhibitors for the humoral and IL-17 inhibitors for the cellular response, impaired the immunogenicity of the booster dose at T2. Our study showed impaired kinetics of both humoral and cellular responses after the booster dose of the COVID-19 vaccine in IA patients, which, in the case of cellular response, did not allow the vaccination effect to be maintained for more than 6 months. Repetitive vaccination with subsequent booster doses seems to be necessary for IA patients.

Comparison of rheumatoid articular adipose and synovial tissue reactivity to proinflammatory stimuli: contribution to adipocytokine network.

OBJECTIVES: (1) To compare spontaneous and stimuli-induced adipocytokine secretion by articular adipose tissue (AAT) and synovial membrane (SM) explants obtained from patients with rheumatoid arthritis (RA). (2) To investigate the biological activity of AAT and SM released factors. METHODS: Tissues were obtained from patients undergoing joint replacement surgery. Tissue explants were treated with proinflammatory cytokines relevant to RA pathogenesis (interleukin 1ß (IL-1ß), tumour necrosis factor (TNF), interferon γ, IL-15, IL-17, IL-23). Selected adipocytokine (TNF, IL-6, IL-8, IL-1ß, IL-1Ra, adiponectin, leptin) concentrations were measured in culture supernatants using ELISA. The biological activity of tissue-conditioned media was evaluated by measuring production of selected factors (IL-6, IL-8, Dickkopf-1, osteoprotegerin) by fibroblast-like synoviocytes (FLS). RESULTS: Spontaneous cytokine release from AAT was ≤12% of that produced by SM, while leptin was secreted in similar amounts. AAT was highly reactive to proinflammatory cytokines (IL-1ß>TNF). AAT treated with IL-1ß released four times more leptin, similar amounts of IL-6 and IL-8 and about 20% of TNF, as compared with SM. Upon activation, the IL-1 receptor antagonist (IL-1Ra)/IL-1ß ratio was higher in AAT than in SM cultures. Irrespective of activation status, SM produced twice as much adiponectin as AAT. Conditioned media from AAT and SM cultures similarly upregulated IL-6, IL-8, Dickkopf-1 and osteoprotegerin production by rheumatoid FLS. CONCLUSION: Rheumatoid AAT is highly reactive tissue which upon stimulation secretes considerable amounts of proinflammatory (IL-6, IL-8, TNF) and anti-inflammatory (IL-1Ra) cytokines and classical adipokines. This tissue releases biologically active factors that intensify pathogenic activities of rheumatoid FLS. Thus, AAT should be considered an important contributor to the pathological processes taking place in the RA joint.

Circulating miRNA Correlates with Lipid Profile and Disease Activity in Psoriatic Arthritis, Rheumatoid Arthritis, and Ankylosing Spondylitis Patients.

This study aimed to investigate the associations of microRNA (miRs) signatures with cytokines, serum lipids, and disease activity in patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA). In total, 65 patients (PsA n = 25, AS n = 25, RA n = 15) and 25 healthy controls (HC) were enrolled into the study. The expression of miR-223-5p, miR-92b-3p, miR-485-3p, miR-10b-5p, let-7d-5p, miR-26a-2-3p, miR-146b-3p, and cytokines levels were measured in sera. DIANA-mirPath analysis was used to predict pathways targeted by the dysregulated miRs. Disease activity scores were calculated. Lipid profile, uric acid, glucose level, and C-reactive protein (CRP) concentrations were determined in the blood. Based on lipid profiles, the PsA group had hypertriglyceridaemia, and RA patients revealed mixed dyslipidaemia, while in AS, no specific changes were found. miR expression analysis revealed upregulation of miR-26a-2-3p and miR-10b-5p in PsA, miR-485-3p in AS, and let-7d-5p in RA. Several correlations between disease activity indexes, metabolites levels, and expression of miRs were observed in PsA, RA, and AS patients. Finally, in ROC analysis, miR-26a-2-3p/miR-485-3p, and let-7d-5p/miR-146b-3p tandems revealed high sensitivity and specificity in distinguishing between PsA, AS, and RA. Our study illustrates the superiority of miR expressions in distinguishing between RA, PsA, and AS. In PsA, a unique regulatory pathway exists through miR-26a-2-3p, miR-223-5p, miR-10b-5p, and miR-92b-3p that converges proatherogenic metabolism and disease activity.

Humoral and cellular immunogenicity of COVID-19 booster dose vaccination in inflammatory arthritis patients.

Introduction: Previous studies have shown a reduction in the effectiveness of primary COVID-19 vaccination in patients with rheumatic diseases. However, limited data is available regarding the effectiveness of the COVID-19 vaccine booster dose, especially on cellular response. The study aimed to assess the humoral and cellular immunogenicity of a booster dose in patients with inflammatory arthritis (IA). Patients and methods: 49 IA and 47 age and sex-matched healthy controls (HC) were included in a prospective cohort study. Both groups completed primary COVID-19 vaccination and after more than 180 days received a BNT162b2 booster shot. Humoral responses (level of IgG antibodies) and cellular responses (IFN-γ production) were assessed before and after 4 weeks from the booster dose of the vaccine. Results: After the booster dose, all participants showed an increased humoral response, although significantly reduced antibody levels were observed in IA patients compared to HC (p=0.004). The cellular response was significantly lower both before (p<0.001) and after (p<0.001) the booster dose in IA patients as compared to HC. Among the immunomodulatory drugs, only biological and targeted synthetic drugs lowered the humoral response after booster vaccination. However, the cellular response was decreased after all immunomodulatory drugs except IL-17 inhibitors and sulfasalazine. Conclusion: Our data indicate that patients with rheumatic diseases present lower humoral and cellular responses after the COVID-19 booster vaccine in comparison to HC. This may translate into a recommendation for subsequent booster doses of the COVID-19 vaccine for rheumatic patients.

Inhibition of Allogeneic and Autologous T Cell Proliferation by Adipose-Derived Mesenchymal Stem Cells of Ankylosing Spondylitis Patients.

BACKGROUND: In ankylosing spondylitis (AS), accompanied by chronic inflammation, T cell expansion plays a pathogenic role; the immunoregulatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) are impaired, while functional characteristics of their adipose tissue-derived counterparts are (ASCs) unknown. METHODS: We evaluated the antiproliferative activity of AS/ASCs, obtained from 20 patients, towards allogeneic and autologous T lymphocytes, using ASCs from healthy donors (HD/ASCs) as the reference cell lines. The PHA-activated peripheral blood mononuclear cells (PBMCs) were cocultured in cell-cell contact and transwell conditions with untreated or TNF + IFNγ- (TI-) licensed ASCs, then analyzed by flow cytometry to identify proliferating and nonproliferating CD4+ and CD8+ T cells. The concentrations of kynurenines, prostaglandin E2 (PGE2), and IL-10 were measured in culture supernatants. RESULTS: In an allogeneic system, HD/ASCs and AS/ASCs similarly decreased the proliferation of CD4+ and CD8+ T cells and acted mainly via soluble factors. The concentrations of kynurenines and PGE2 inversely correlated with T cell proliferation, and selective inhibitors of these factors synthesis significantly restored T cell response. AS/ASCs exerted a similar antiproliferative impact also on autologous T cells. CONCLUSION: We report for the first time that despite chronic in vivo exposure to inflammatory conditions, AS/ASCs retain the normal capability to restrain expansion of allogeneic and autologous CD4+ and CD8+ T cells, act primarily via kynurenines and PGE2, and thus may have potential therapeutic value. Some distinctions between the antiproliferative effects of AS/ASCs and HD/ASCs suggest in vivo licensing of AS/ASCs.

CD4+FOXP3+ T Cells in Rheumatoid Arthritis Bone Marrow Are Partially Impaired.

There is evolving evidence that dysregulation of immune homeostasis in the bone marrow (BM) adjacent to the inflamed joints is involved in the pathogenesis of. In this study, we are addressing the phenotype and function of regulatory T cells (Tregs) residing in the BM of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). BM and peripheral blood samples were obtained from RA and OA patients undergoing hip replacement surgery. The number and phenotype of Tregs were analyzed by flow cytometry and immunohistochemistry. The function of Tregs was investigated ex vivo, addressing their suppressive activity on effector T cells. [3H]-Thymidine incorporation assay and specific enzyme-linked immunosorbent assay were used for quantification of cell proliferation and pro-inflammatory (TNF, IFN-γ) cytokine release, respectively. Significantly lower numbers of CD4+FOXP3+ T cells were found in the BM of patients with RA compared to control patients with OA. High expression of CD127 (IL-7 receptor) and relatively low expression of CXCR4 (receptor for stromal cell-derived factor CXCL12) are characteristics of the CD4+FOXP3+ cells residing in the BM of RA patients. The BM-resident Tregs of RA patients demonstrated a limited suppressive activity on the investigated immune response. Our results indicate that the reduced number and impaired functional properties of CD4+FOXP3+ T cells present in the BM of RA patients may favor the inflammatory process, which is observed in RA BM.

Different Secretory Activity of Articular and Subcutaneous Adipose Tissues from Rheumatoid Arthritis and Osteoarthritis Patients.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are characterized by joint and systemic high- or low-grade inflammation, respectively. Adipose tissue (AT) may contribute to the pathogenesis of these diseases. To address this issue, we investigated whether basal and pro-inflammatory cytokine (IL-1ß)-triggered release of adipocytokines (TNF, IL-6, IL-10, IL-1Ra, TGFß, CCL2/MCP-1, CCL5/RANTES, MMP-3) from subcutaneous (ScAT) and intraarticular (AAT) adipose tissues of RA and OA patients mirror differences between these diseases in an intensity of systemic and local inflammation. We found that in both diseases basal adipocytokine release was usually higher from AAT than ScAT, reflecting stronger local than systemic inflammation. However, ScAT secreted considerable amounts of pro- and anti-inflammatory factors as well. Spontaneous secretion of some adipocytokines (MMP-3 and/or TNF, CCL2/MCP-1, IL-1Ra) was higher in osteoarthritis than rheumatoid ATs and probably caused by weaker anti-inflammatory treatment of OA patients. By contrast, reactivity of ATs to IL-1ß was significantly lower in OA than RA and IL-1ß antagonist (IL-1Ra) could be responsible for this because we found its overproduction in OA ATs. Interestingly, higher reactivity of ScAT than AAT to IL-1ß was a characteristic for OA while reactivity of rheumatoid ScAT and AAT to this stimulus was equal. We conclude that differences between OA and RA in reactivity of AAT and ScAT to pro-inflammatory stimulus mimicking in vivo condition reflect dissimilarity in an intensity of disease-specific inflammation and thus support contribution of ATs to these pathological processes. Moreover, we propose that more efficient anti-inflammatory mechanism(s) are preserved in ATs of OA than RA patients.

Rheumatoid arthritis bone marrow environment supports Th17 response.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic, autoimmune disease leading to joint destruction and ultimately disability. Bone marrow (BM) is an important compartment in RA, where pathological processes from "outside the joint" can occur. IL-17 is a cytokine that exerts proinflammatory effects and participates in the process of bone destruction. It is believed that IL-17 is involved in pathogenesis of RA. However, little is known about the biology of this cytokine in BM. In the present study we investigated Th17-related cytokines in RA BM. METHODS: BM samples were obtained from RA and osteoarthritis (OA) patients during total hip replacement surgery. Levels of IL-17AF, IL-17AA, IL-17FF, IL-1ß, IL-6, IL-23, TGF-ß and CCL20 in BM plasma were determined by specific enzyme-linked immunosorbent assay tests. Percentage of IL-17-producing cells in BM was evaluated by flow cytometry. The effect of IL-15 stimulation on IL-17 production by BM mononuclear cells was examined in vitro. RESULTS: Increased levels of IL-17AF were observed in BM plasma of RA patients in comparison to OA patients. Increased concentrations of IL-1ß, IL-6 and CCL20 were observed in RA compared to OA BM plasma. Concordant with these findings, significantly increased percentages of CD3+CD4+IL-17+ and CD3+CD4+IL-17+IFN-γ+ cells were present in RA BM in comparison to OA BM samples. Finally, abundant in RA BM, IL-15 increased IL-17 production by cultured BM mononuclear cells. CONCLUSIONS: In the course of RA, the BM microenvironment can promote the development of Th17 cell responses and overproduction of IL-17AF that may lead to increased inflammation and tissue destruction in RA BM.