Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-beta1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.     

Interleukin‐21 Induces Proliferation and Proinflammatory Cytokine Profile of Fibroblast‐like Synoviocytes of Patients with Rheumatoid Arthritis

Fibroblast‐like synoviocytes (FLS) play a pivotal role in the pathogenesis of rheumatoid arthritis (RA) through aggressive proliferation and invasion, and certain proinflammatory cytokines may affect synoviocyte proliferation. To evaluate whether interleukin‐21 (IL‐21) could promote proliferation and proinflammatory cytokine production by RA‐FLS, immunohistochemistry and immunoblotting were performed to observe the expression of IL‐21 receptor (IL‐21R) in synovial tissues and FLS from RA and osteoarthritis (OA) patients. The MTS assay was used to analyse RA‐FLS proliferation. The concentrations of IL‐6 and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by enzyme‐linked immunosorbent assay (ELISA). The signalling pathways triggered by IL‐21 were characterized by immunoblotting. IL‐21R was upregulated in the synovial tissues and FLS of RA patients as compared with OA patients. IL‐21 stimulated RA‐FLS proliferation and promoted the production of TNF‐α and IL‐6 and blockade of IL‐21/IL‐21R pathway with IL‐21R.Fc attenuated IL‐21‐induced proliferation and secretion of TNF‐α and IL‐6. Moreover, IL‐21 induced activation of the ERK1/2, PI3K/AKT and STAT3 pathways, and blockade of these pathways attenuated IL‐21‐induced proliferation and secretion of TNF‐α and IL‐6. These results suggest that IL‐21 could promote RA‐FLS proliferation and production of proinflammatory cytokines. Therefore, therapeutic strategies targeting IL‐21 might be effective for the treatment of RA.     

Vitexin alleviates interleukin‐1β‐induced inflammatory responses in chondrocytes from osteoarthritis patients: Involvement of HIF‐1α pathway

It has been reported that vitexin has anti‐inflammatory effects in osteoarthritis (OA) rats. However, the effects of vitexin on interleukins‐1β (IL‐1β)‐stimulated OA patient‐derived chondrocytes have not been reported. The purpose of this study was to investigate the anti‐inflammatory effects of vitexin on IL‐1β‐stimulated human osteoarthritis chondrocytes and to reveal the involvement of hypoxia‐inducible factor 1α (HIF‐1α) pathway. Enzyme‐linked immunosorbent assay, quantitative real‐time PCR and Western blotting assays were employed. ELISA results demonstrated that the proinflammatory cytokine levels of interleukins‐6 (IL‐6) and tumour necrosis factor α (TNF‐α) in the serum and synovial fluid and HIF‐1α level in the synovial fluid were significantly elevated in OA patients compared to normal healthy subjects. Moreover, the Western blotting results indicated that the protein expression of HIF‐1α was significantly higher in the cartilage tissues of OA patients. OA patient‐derived chondrocytes were stimulated by IL‐1β and treated with different concentration of vitexin for 24 hours. Vitexin showed no cytotoxicity and increased the survival of chondrocytes under IL‐1β stimulation. Vitexin suppressed IL‐1β‐induced production of NO and prostaglandin E2 (PGE₂) in chondrocytes culture. The treatment of vitexin significantly inhibited IL‐1β‐induced expressions of proinflammatory cytokine levels of IL‐6, TNF‐α, matrix metalloproteinase (MMP)‐1, MMP‐3 and MMP‐13. Furthermore, Western blotting results demonstrated that HIF‐1α is involved in vitexin's protective effects on IL‐1β‐stimulated injuries in OA patient‐derived chondrocytes. Our study demonstrates that vitexin alleviates IL‐1β‐induced inflammatory responses in chondrocytes from osteoarthritis patients, which may be attributed partly to the inhibition of HIF‐1α pathway.     

Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt

Peripheral blood mononuclear cells taken from patients with scleroderma express increased levels of interleukin (IL)‐13. Moreover, the expression of matrix metalloproteinase‐1 (MMP‐1) from involved scleroderma skin fibroblasts is refractory to stimulation by tumour necrosis factor (TNF)‐α. To elucidate the mechanism(s) involved, we examined the effect of IL‐13 on TNF‐α‐induced MMP‐1 expression in normal and scleroderma human dermal fibroblast lines and studied the involvement of serine/threonine kinase B/protein kinase B (Akt) in this response. Dermal fibroblast lines were stimulated with TNF‐α in the presence of varying concentrations of IL‐13. Total Akt and pAkt were quantitated using Western blot analyses. Fibroblasts were treated with or without Akt inhibitor VIII in the presence of IL‐13 followed by TNF‐α stimulation. MMP‐1 expression was analysed by real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA). Statistical analysis was performed using analysis of variance (anova ) or Student's t‐test. Upon TNF‐α stimulation, normal dermal fibroblasts secrete more MMP‐1 than systemic sclerosis (SSc) fibroblasts. This increase in MMP‐1 is lost when fibroblasts are co‐incubated with IL‐13 and TNF‐α. IL‐13 induced a significant increase in levels of pAkt in dermal fibroblasts, while Akt inhibitor VIII reversed the suppressive effects of IL‐13 on the response of cultured fibroblasts to TNF‐α, increasing their expression of MMP‐1. We show that IL‐13 suppresses MMP‐1 in TNF‐α‐stimulated normal and scleroderma dermal fibroblast. Akt inhibitor VIII is able to reverse the suppressive effect of IL‐13 on MMP‐1 expression and protein synthesis. Our data suggest that IL‐13 regulates MMP‐1 expression in response to TNF‐α through an Akt‐mediated pathway and may play a role in fibrotic diseases such as scleroderma.     

CD11c+ macrophages and levels of TNF‐α and MMP‐3 are increased in synovial and adipose tissues of osteoarthritic mice with hyperlipidaemia

To understand more clearly the link between osteoarthritis and hyperlipidaemia, we investigated the inflammatory macrophage subsets and macrophage‐regulated matrix metalloprotease‐3 (MMP‐3) and A disintegrin and metalloprotease with thrombospondin motifs‐4 (ADAMTS4) in synovial (ST) and adipose tissues (AT) of osteoarthritic mice with hyperlipidaemia (STR/Ort). CD11c⁺F4/80⁺CD11b⁺ macrophage populations in the ST and AT of 9‐month‐old STR/Ort and C57BL/6J mice were characterized and compared by flow cytometry and real‐time polymerase chain reaction (PCR) analyses. Expression of tumour necrosis factor (TNF)‐α, MMP‐3 and ADAMTS4, and the response of these factors to anionic liposomal clodronate induced‐macrophage depletion were also evaluated by real‐time PCR. Expression of TNF‐α in CD11c⁺ cells, which were isolated by magnetic beads, was compared to CD11c^– cells. In addition, the effect of TNF‐α on cultured synovial fibroblasts and adipocytes was investigated. CD11c⁺F4/80⁺CD11b⁺ macrophages were increased in ST and AT of STR/Ort mice. The CD11c⁺ cell fraction highly expressed TNF‐α. Expression of TNF‐α and MMP3 was increased in ST and AT, and was decreased upon macrophage depletion. TNF‐α treatment of cultured synovial fibroblasts and adipocytes markedly up‐regulated MMP‐3. CD11c⁺F4/80⁺CD11b⁺ macrophages were identified as a common inflammatory subset in the AT and ST of STR/Ort mice with hyperlipidaemia. The induction of inflammation in AT and ST may be part of a common mechanism that regulates MMP3 expression through TNF‐α. Our findings suggest that increased numbers of CD11c⁺ macrophages and elevated levels of TNF‐α and MMP‐3 in AT and ST may explain the relationship between hyperlipidaemia and OA.     

Platelet-derived growth factor-AA increases IL-1beta and IL-8 expression and activates NF-kappaB in rheumatoid fibroblast-like synoviocytes

The effect of platelet-derived growth factor (PDGF)-AA on the inflammation in rheumatoid arthritis (RA) and osteoarthritis (OA) was investigated using cultured fibroblast-like synoviocytes (FLS) obtained from RA and OA patients as well as control nonarthritic (NA) individuals. PDGF-AA increased the mRNA and protein expressions of proinflammatory cytokines, interleukin (IL)-1beta and IL-8 in RA FLS. Biological activity of IL-1 in the culture supernatant of RA FLS was also increased by PDGF-AA stimulation. Interestingly, PDGF-AA synergized with tumour necrosis factor (TNF)-alpha to upregulate the protein expressions of IL-1beta and IL-8. PDGF-induced enhancement of the IL-1beta and IL-8 mRNA expressions was also observed in OA FLS. However, the expression of these proinflammatory cytokines in NA FLS did not change by PDGF treatment, suggesting that the inflammatory condition might have modified the biological effects of PDGF. In accordance with the enhanced expression of inflammatory cytokines, the activity of nuclear factor kappaB was also induced in response to PDGF-AA in RA FLS. These results suggest that PDGF-AA plays an important role in the progression of RA inflammation, and inhibiting PDGF activity may be useful for the effective RA treatment.     

Rheumatoid arthritis fibroblast‐like synoviocytes co‐cultured with PBMC increased peripheral CD4+CXCR5+ICOS+ T cell numbers

‘Circulating’ T follicular helper cells (Tfh), characterized by their surface phenotypes CD4⁺chemokine receptor 5 (CXCR5)⁺ inducible co‐stimulatory molecule (ICOS)⁺, have been identified as the CD4⁺ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast‐like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA‐FLS co‐cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co‐cultured RA‐FLS with or without anti‐CD3/CD28‐stimulated PBMC. The results showed that RA‐FLS co‐cultured with stimulated PBMC could increase the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells of RA PBMC possibly via the production of interleukin (IL)‐6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co‐culture system of RA‐FLS and PBMC. The percentage of CD4⁺CXCR5⁺ICOS⁺ T cells was decreased when ROS production was inhibited by N‐acetyl‐L‐cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)‐α and IL‐1β in the co‐culture system and the blocking of TNF receptor 2 (TNF‐R2) and IL‐1β receptor (IL‐1βR) both decreased the numbers of CD4⁺CXCR5⁺ICOS⁺ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA‐FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA.     

Role of cathepsin B in regulating migration and invasion of fibroblast‐like synoviocytes into inflamed tissue from patients with rheumatoid arthritis

Cathepsin B (CB), an important proteinase that participates in joint destruction in rheumatoid arthritis (RA), exhibits higher expression in fibroblast‐like synoviocyte (FLS) of abnormal proliferative synovial tissues. Whether and how it affects the biological behaviours of RA‐FLS, such as migration and invasion, are poorly understood. In the present study, CB expression in synovial tissues of patients with RA and ostearthritis (OA) were measured by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC), respectively. Stable depletion of endogenous CB was achieved by small interfering RNA (siRNA) transfection, and decrease of CB activity was acquired by using its specific inhibitor (CA074Me). The effects of CA074Me and RNA interference (RNAi) treatments on proliferation, migration, invasion, matrix metalloproteinase (MMP)‐2/‐9 expression, focal adhesion kinase (FAK) activation, and mitogen‐activated protein kinases (MAPKs) phosphorylation of FLS were analysed. In RA synovial tissues, CB was expressed at elevated levels compared with OA synovial tissues. CA074Me could inhibit invasion of FLS obtained from RA patients in an ex‐vivo invasion model. CA074Me and siRNA treatments suppressed the migration and invasion of FLS, reduced the activity, expression and mRNA level of MMP‐2, restrained the activation of FAK and reduced the expression of F‐actin. Moreover, CA074Me decreased the phosphorylation of P38 MAPK and c‐Jun N‐terminal kinase (JNK) in FLS, while siCB treatment reduced the phosphorylation of P38 but not JNK. CB substantially contributes to the invasive phenotype of FLS that leads to joint destruction in RA. This proteinase may show promise as a therapeutic target in inflammatory arthritis.     

LILRA5 is expressed by synovial tissue macrophages in rheumatoid arthritis,selectively induces pro‐inflammatory cytokines and IL‐10 and is regulated by TNF‐α, IL‐10 and IFN‐γ

Leukocyte immunoglobulin‐like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane‐bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross‐linking of LILRA5 on monocytes induced production of pro‐inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common γ chain of the FcR and LILRA5 cross‐linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro‐inflammatory cytokines as well as IL‐10. LILRA5 mRNA and protein expression was tightly regulated by TNF‐α, IL‐10 and IFN‐γ. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.     

Mechanism of Fibroblast‐Like Synoviocyte Apoptosis Induced by Recombinant Human Endostatin in Rats with Adjuvant Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by pronounced synovial hyperplasia, in which there may be an imbalance between the growth and death of fibroblast‐like synoviocytes (FLS). The present study was undertaken to examine the effect of recombinant human endostatin (rhEndostatin) on FLS apoptosis in experimental RA. Adjuvant arthritis (AA) was induced in male Sprague Dawley (SD) rats. Using cultured AA FLS obtained from these rats, the apoptosis process was measured by terminal deoxyribonucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) as well as Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. In addition, the expression levels of the Fas, c‐jun, NFκB, and caspase‐3 gene products in synovial tissues were quantified by quantitative real‐time polymerase chain reaction (qPCR) and/or Western blotting assays. Our data revealed that rhEndostatin induced apoptosis in AA FLS. The number and signal density of TUNEL‐positive cells were significantly increased in rats treated with rhEndostatin (2.5 mg/kg). The percentage of Annexin V‐FITC‐positive cells was 6.67% after treatment with rhEndostatin at 25 μg/mL for 48 hr, compared with only 3.32% among untreated control cells. There were significant increases in Fas mRNA, c‐jun mRNA, c‐Jun protein, and caspase‐3 (p20) protein in AA synovial tissues treated with rhEndostatin (2.5 mg/kg), whereas no significant difference in NFκB expression was detected between treated and untreated tissues. These findings indicate that rhEndostatin has a therapeutic effect on RA by inducing FLS apoptosis, which is strongly associated with increased expression of Fas, c‐jun, and caspase‐3, but not NFκB. Anat Rec, 291:1029–1037, 2008. © 2008 Wiley‐Liss, Inc.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

Hypoxia‐inducible factor‐1α perpetuates synovial fibroblast interactions with T cells and B cells in rheumatoid arthritis

Synovial fibroblast hyperplasia, T‐cell hyperactivity, B‐cell overactivation, and the self‐perpetuating interactions among these cell types are major characteristics of rheumatoid arthritis (RA). The inflamed joints of RA patients are hypoxic, with upregulated expression of hypoxia‐inducible factor‐1α (HIF‐1α) in RA synovial fibroblasts (RASFs). It remains unknown whether HIF‐1α regulates interactions between RASFs and T cells and B cells. We report here that HIF‐1α promotes the expression of inflammatory cytokines IL‐6, IL‐8, TNF‐α, and IL‐1β, and cell–cell contact mediators IL‐15, vascular cell adhesion molecule (VCAM)‐1, thrombospondin (TSP)‐1, and stromal cell‐derived factor (SDF)‐1 in RASFs. Furthermore, HIF‐1α perpetuates RASF‐mediated inflammatory Th1‐ and Th17‐cell expansion while differentially inhibiting regulatory B10 and innate‐like B cells, leading to increased IFN‐γ, IL‐17, and IgG production and decreased protective natural IgM secretion. Our findings suggest that HIF‐1α perpetuates the interactions between RASFs and T cells and B cells to induce inflammatory cytokine and autoantibody production, thus exacerbating the severity of RA. Targeting HIF‐1α may provide new therapeutic strategies for overcoming this persistent disease.     

TRIM32 promotes inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is a worldwide autoimmune disease. The study of its aetiology and mechanism has always been a focus topic in medicine. This research was designed to investigate the effect of E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) in rheumatoid arthritis (RA). We found in fibroblast-like synoviocytes (FLS) of RA patients, the expression of TRIM32 was significantly increased compared with its expression in osteoarthritis (OA) patients FLS. A widely used pro-inflammatory stimuli tumour necrosis factor-alpha (TNF-α) was found to promote TRIM32 expression in a time-dependent manner. Furthermore, we observed that overexpression of TRIM32 aggravated the production of pro-inflammatory cytokines in FLS, silencing of TRIM32 showed the consistent results. In addition, TRIM32 was found to activate nuclear factor κB (NF-κB) signalling pathway, and TRIM32 could interact with TNF receptor-associated factor 2 (TRAF2) to promote the K63-linked polyubiquitination of TRAF2 in RA-FLS. In conclusion, we suggested that TRIM32 as a positive regulator of inflammatory responses in RA-FLS.     

sFRP3 and DKK1 Regulate Fibroblast-Like Synoviocytes Markers and Wnt Elements Expression Depending on Cellular Context

Context: Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) display pathogenic behavior. Various members of the Wnt pathway, especially the canonical Wnt/β-catenin cascade, may contribute to autonomous RA FLS activation. It has been shown that the two Wnt inhibitors: sFRP3 and DKK1 contribute to several critical aspects of joint biology. However, their effects on RA FLS are poorly characterized. The aim of our study was to investigate the effects of sFRP3 and DKK1 on FLS markers, Wnt components, and target oncogenes expression by RA FLS and compare the findings to osteoarthritic (OA) FLS.

Materials and methods: RA and OA FLS were treated with sFRP3 and DKK1 for 6 days. Wnt signaling components (Wnt5a, LRP5 and β-catenin), Wnt target oncogenes (cyclin E1 and WISP1), and FLS markers (fibronectin and MMP3) were analyzed using western blotting and/or qRT-PCR.

Results: Our data indicated that sFRP3 down-regulated the key gene β-catenin in RA FLS. sFRP3 decreased fibronectin, a well-known downstream effectors gene of Wnt/β-catenin pathway, and LRP5 expression in both RA and OA FLS. In OA FLS, sFRP3 induced increased expression of Wnt5a and MMP3 but did not affect their levels in RA FLS. On the other hand, DKK1 increased fibronectin expression in RA FLS and decreased its expression in OA FLS.

Conclusion: Our results confirm the involvement of Wnt signaling in FLS transformation and show that two inhibitors of the same cascade can regulate differently the same elements and that a single inhibitor can initiate signaling depending on cellular context.

Abbreviations: FLS: fibroblast-like synoviocytes; RA: rheumatoid arthritis; Wnt: Wingless; Fz: frizzled; LRP: Fz/low-density lipoprotein receptor protein; WISP1: Wnt1 inducible signaling pathway protein 1; sFRP: secreted Fz-related proteins; DKK: Dickkopf; OA: osteoarthritis; DMEM: Dulbecco’s modified Eagle’s medium; FBS: fetal bovine serum; PBS: phosphate buffered saline; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ECL: enhanced chemiluminescence detection solution; MMP3: metaloproteinase 3; qRT-PCR: quantitative real-time polymerase chain reaction; S.D: standard deviation; CRD: cysteine-rich domain; MeCP2: methyl-CpG-binding protein; RANKL: nuclear factor-kappa B ligand.     

Expression of miRNA‐146a,miRNA‐155, IL‐2, and TNF‐α in inflammatory response to Helicobacter pylori infection associated with cancer progression

miRNAs appear to play an important role in controlling the expression of several genes, and they are a potential biomarker and prognostic tool in gastric diseases. We analyzed 53 controls, 86 patients with gastritis, and 19 patients with gastric cancer. Real‐time‐PCR was used to determine the expression levels of miRNA‐146a, miRNA‐155, IL‐2, and TNF‐α. The subsequent analysis of the target genes was performed using the bioinformatics approach. There was no difference in IL‐2 expression between the groups. However, there was a significant increase in TNF‐α expression in the gastritis group relative to the control and a significant decrease in the gastric cancer group relative to the control. There was also a statistically significant increase in miRNA‐146a and miRNA‐155 expression in the gastritis group relative to the control, but not in the gastric cancer group. Similar results were found when the presence of H. pylori was considered. The data revealed an increase in miRNA‐146a and miRNA‐155 expression but not enough to control the expression of TNF‐α. The presence of H. pylori was found to affect increases in TNF‐α and microRNA expression, and miRNA‐146a and miRNA‐155 alone were not able to eliminate bacteria or restore tissue homeostasis.     

Differential effects of decoy receptor‐ and antibody‐mediated tumour necrosis factor blockage on FoxP3 expression in responsive arthritis patients

Our aim was to clarify if anti‐tumour necrosis factor (TNF) drugs have effect on expression of three splice forms of FoxP3 mRNA in blood CD4+ T cells from rheumatoid arthritis (RA) patients compared with healthy controls. Forty‐five rheumatoid arthritis patients treated with anti‐TNF therapy were investigated in a 12‐week prospective cohort study. FoxP3 isoforms, CD25 and CTLA‐4 mRNA in blood CD4+ T cells were measured with quantitative real‐time PCR. Patients benefitting from the treatment, based on changes in DAS28 scores, revealed a significant decrease in expression of full‐length FoxP3 following 12 weeks treatment with TNF receptor 2 fusion protein (Etanercept), but not following treatment with anti‐TNF antibodies (Adalimumab or Infliximab). A partial normalization of the CTLA‐4/FoxP3fl ratio and a correlation between clinical improvement and change in FoxP3 mRNA expression were also seen in Etanercept responders. These changes were not observed in responsive patients treated with the antibody therapies. Our data suggest that TNF decoy receptor and anti‐TNF antibodies differ in their effect on FoxP3 expression in responsive patients. As Etanercept binds both TNF‐α and Lymphotoxin‐α (LT‐α), whereas the antibodies only target TNF‐α, LT‐α may regulate FoxP3 expression in a subset of RA patients. Our findings support the view that anti‐TNF treatment is mainly symptomatic.