Ultrasound Assessment of Entheseal Sites and Anterior Chest Wall in Ankylosing Spondylitis: A Cross-Sectional Study.

OBJECTIVES: The study was designed to evaluate entheseal sites and anterior chest wall (ACW) of patients with ankylosing spondylitis (AS) using ultrasound (US) and investigate the correlation between disease activity and US score. METHODS: This prospective cross-sectional study included 104 patients with AS and 50 control subjects. Each patient underwent US scanning of 23 entheses and 11 sites of the ACW. The US features, including hypoechogenicity, thickness, erosion, calcification, bursitis, and Doppler signal, were evaluated. Disease activity was assessed based on C reactive protein (CRP), erythrocyte sedimentation rate (ESR), disease activity score-C reactive protein (ASDAS-CRP), and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). RESULTS: The most commonly involved entheses on US were the Achilles tendon (AT) and quadriceps tendon (QT). The most involved site of ACW was the sternoclavicular joint (SCJ). Compared with the control group, significant differences were observed in the AS group in the rates of US enthesitis and ACW in AT (P = .01), SCJ (P = .00), and costochondral joint (CCJ) (P = .01). Patients with high or very high disease activity had a higher erosion score (P = .02). The erosion score was weakly positively associated with CRP, ESR, BASDAI, ASDAS-CRP, and ASDAS-ESR (correlation coefficient: 0.22-0.45). CONCLUSIONS: The most commonly involved entheseal sites on US were AT and QT, while the site of ACW was SCJ. The US assessment of AS should take the ACW into account. High disease activity might indicate erosion in AS.

Hypermethylation of Smad7 in CD4+ T cells is associated with the disease activity of rheumatoid arthritis.

Background: Smad7 is protective in a mouse model of rheumatoid arthritis. Here we investigated whether Smad7-expressing CD4+ T cells and the methylation of Smad7 gene in CD4+ T cells contribute to the disease activity of RA in patients. Methods: Peripheral CD4+ T cells were collected from 35 healthy controls and 57 RA patients. Smad7 expression by CD4+ T cells were determined and correlated with the clinical parameters of RA including RA score and serum levels of IL-6, CRP, ESR, DAS28-CRP, DAS28-ESR, Swollen joints and Tender joints. Bisulfite sequencing (BSP-seq) was used to determine the DNA methylation in Smad7 promoter (-1000 to +2000) region in CD4+ T cells. In addition, a DNA methylation inhibitor, 5-Azacytidine (5-AzaC), was added to CD4+ T cells to examine the possible role of Smad7 methylation in CD4+ T cell differentiation and functional activity. Results: Compared to the heath controls, Smad7 expression was significantly decreased in CD4+ T cells from RA patients and inversely correlated with the RA activity score and serum levels of IL-6 and CRP. Importantly, loss of Smad7 in CD4+ T cell was associated with the alteration of Th17/Treg balance by increasing Th17 over the Treg population. BSP-seq detected that DNA hypermethylation occurred in the Smad7 promoter region of CD4+ T cells obtained from RA patients. Mechanistically, we found that the DNA hypermethylation in the Smad7 promoter of CD4+ T cells was associated with decreased Smad7 expression in RA patients. This was associated with overreactive DNA methyltransferase (DMNT1) and downregulation of the methyl-CpG binding domain proteins (MBD4). Inhibition of DNA methylation by treating CD4+ T cells from RA patients with 5-AzaC significantly increased Smad7 mRNA expression along with the increased MBD4 but reduced DNMT1 expression, which was associated with the rebalance in the Th17/Treg response. Conclusion: DNA hypermethylation at the Smad7 promoter regions may cause a loss of Smad7 in CD4+ T cells of RA patients, which may contribute to the RA activity by disrupting the Th17/Treg balance.

C-Reactive Protein Promotes the Activation of Fibroblast-Like Synoviocytes From Patients With Rheumatoid Arthritis.

Objective: To evaluate the biological effect and mechanisms of C-reactive protein (CRP) on the activation of fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). Study design: To understand if CRP is involved in RA, expression of CRP and its receptors CD32/64 was examined in synovial tissues from RA patients and normal controls. In vitro, the potential role and mechanisms of CRP in FLS proliferation and invasion, expression of pro-inflammatory cytokines, and activation of signaling pathways were investigated in both RA - FLS and a normal human fibroblast-like synoviocyte line (HFLS). Results: Compared to normal controls, synovial tissues from 21 RA patients exhibited highly activated CRP signaling, particularly by FLSs as identified by 65% of CRP-expressing cells being CRP+vimentin+ and CD32/64+vimentin+ cells. In vitro, FLSs from RA patients, but not HFLS, showed highly reactive to CRP by largely increasing proliferative and invasive activities and expressing pro-inflammatory cytokines and chemokines, including CCL2, CXCL8, IL-6, and MMP2/9. All these changes were blocked largely by a neutralizing antibody to CD32 and, to a less extent by the anti-CD64 antibody, revealing CD32 as a primary mechanism of CRP signaling during synovial inflammation. Further studies revealed that CRP also induced synovial inflammation differentially via CD32/CD64- NF-κB or p38 pathways as blockade of CRP-CD32-NF-κB signaling inhibited CXCL8, CCL2, IL-6, whereas CRP induced RA-FLS invasiveness through CD32-p38 and MMP9 expression via the CD64-p38-dependent mechanism. Conclusions: CRP signaling is highly activated in synovial FLSs from patients with RA. CRP can induce synovial inflammation via mechanisms associated with activation of CD32/64-p38 and NF-κB signaling.

Frequencies of the LILRA3 6.7-kb Deletion Are Highly Differentiated Among Han Chinese Subpopulations and Involved in Ankylosing Spondylitis Predisposition.

Introduction: Leukocyte immunoglobulin-like receptor A3 (LILRA3) belongs to the LILR family with unique feature of a 6.7-kb deletion variation among individuals. Frequencies of the 6.7-kb deletion vary widely across populations, but so far it has not been carefully investigated among Han Chinese subpopulations. Furthermore, we previously identified the non-deleted (functional) LILRA3 as a novel genetic risk for multiple autoimmune diseases. The current study aimed to investigate (i) whether frequencies of the LILRA3 6.7-kb deletion differ within Han Chinese subpopulations and (ii) whether the functional LILRA3 is a novel genetic risk for ankylosing spondylitis (AS). Methods: The LILRA3 6.7-kb deletion was genotyped in two independent cohorts, including 1,567 subjects from Shenzhen Hospital and 2,507 subjects from People's Hospital of Peking University. Frequencies of the 6.7-kb deletion were first investigated in combined healthy cohort according to the Chinese administrative district divisions. Association analyses were performed on whole dataset and subsets according to the geographic regions. Impact of the functional LILRA3 on AS disease activity was evaluated. Results: Frequencies of LILRA3 6.7-kb deletion were highly differentiated within Han Chinese subpopulations, being gradually decreased from Northeast (80.6%) to South (47.4%). Functional LILRA3 seemed to be a strong genetic risk in susceptibility to AS under almost all the alternative genetic models, if the study subjects were not geographically stratified. However, stratification analysis revealed that the functional LILRA3 was consistently associated with AS susceptibility mainly in Northern Han subgroup under the alternative genetic models, but not in Central and Southern Hans. Functional LILRA3 conferred an increased disease activity in AS patients (P < 0.0001 both for CRP and ESR, and P = 0.003 for BASDAI). Conclusions: The present study is the first to report that the frequencies of LILRA3 6.7-kb deletion vary among Chinese Hans across geographic regions. The functional LILRA3 is associated with AS susceptibility mainly in Northern Han, but not in Central and Southern Han subgroups. Our finding provides new evidence that LILRA3 is a common genetic risk for multiple autoimmune diseases and highlights the genetic differentiation among different ethnicities, even within the subpopulations of an ethnic group.

Potential utility of anti-TNF drugs in synovial chondromatosis associated with ankylosing spondylitis.

We describe a previously unreported association of ankylosing spondylitis with synovial chondromatosis, and briefly review previously reported cases and treatment of synovial chondromatosis in patients with other immune-mediated inflammatory arthritides. A 20-year-old man with ankylosing spondylitis whose axial disease was in remission with nonsteroidal anti-inflammatory drugs and oral disease-modifying anti-rheumatic drugs developed recurrent right knee pain and swelling. Magnetic resonance imaging of his right knee revealed calcified loose bodies, suggestive of synovial chondromatosis. While waiting for the surgical intervention and other invasive therapy previously reported in patients with synovial chondromatosis, a trial of etanercept eliminated the pain and swelling of the knee; however, the loose bodies have persisted during the 2-year follow-up. Thus, synovial chondromatosis should be considered in the differential diagnoses of a refractory monoarticular pain and swelling in patients with otherwise controlled inflammatory arthritis. Our report advocates a trial of anti-tumor necrosis factor drugs, which might delay the need for invasive therapy in patients with synovial chondromatosis.

Heparin and aspirin combination therapy restores T-cell phenotype in pregnant patients with antiphospholipid syndrome-related recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is the most common manifestation of anti-phospholipid syndrome (APS), and activated CD4+ T cells are involved in its pathogenesis. Treatment with low-molecular weight heparin (LMWH) and aspirin combination improves pregnancy outcome, however, its mechanism of action is unclear. We investigated the effect of this therapy on Th1/Th2 cells in 89 patients with APS-RPL. The results showed that serum cytokine levels, T cell phenotypes, and transcription factors' gene expression levels representing Th1 responses were higher, whereas those representing Th2 responses were lower in patients with APS-RPL at the time of early pregnancy. This Th1-bias was reversed in patients who had live birth after receiving the combination therapy at the time of delivery. Patients with miscarriages continued to exhibit Th1-bias. In conclusion, these data support a role of Th1-bias in the pathogenesis of APS-RPL and suggest restoring T-cell phenotype as a new immunomodulatory mechanism of LMWH/aspirin combination.

Loss of Smad7 Promotes Inflammation in Rheumatoid Arthritis.

Objective: Smad7 is an inhibitory Smad and plays a protective role in many inflammatory diseases. However, the roles of Smad7 in rheumatoid arthritis (RA) remain unexplored, which were investigated in this study. Methods: The activation of TGF-ß/Smad signaling was examined in synovial tissues of patients with RA. The functional roles and mechanisms of Smad7 in RA were determined in a mouse model of collagen-induced arthritis (CIA) in Smad7 wild-type (WT) and knockout (KO) CD-1 mice, a strain resistant to autoimmune arthritis induction. Results: TGF-ß/Smad3 signaling was markedly activated in synovial tissues of patients with RA, which was associated with the loss of Smad7, and enhanced Th17 and Th1 immune response. The potential roles of Smad7 in RA were further investigated in a mouse model of CIA in Smad7 WT/KO CD-1 mice. As expected, Smad7-WT CD-1 mice did not develop CIA. Surprisingly, CD-1 mice with Smad7 deficiency developed severe arthritis including severe joint swelling, synovial hyperplasia, cartilage damage, massive infiltration of CD3+ T cells and F4/80+ macrophages, and upregulation of proinflammatory cytokines IL-1ß, TNFα, and MCP-1. Further studies revealed that enhanced arthritis in Smad7 KO CD-1 mice was associated with increased Th1, Th2 and, importantly, Th17 over the Treg immune response with overactive TGF-ß/Smad3 and proinflammatory IL-6 signaling in the joint tissues. Conclusions: Smad7 deficiency increases the susceptibility to autoimmune arthritis in CD-1 mice. Enhanced TGF-ß/Smad3-IL-6 signaling and Th17 immune response may be a mechanism through which disrupted Smad7 causes autoimmune arthritis in CD-1 mice.

Curcumin modulation of the activation of PYK2 in peripheral blood mononuclear cells from patients with lupus nephritis.

INTRODUCTION: Proline-rich tyrosine kinase 2 (PYK2) provides important signals during the activation of lymphocytes, which is essential in autoimmune diseases. Systemic lupus erythematosus (SLE) is a representative autoimmune disease, and lupus nephritis (LN) is one of its most severe complications. Although glucocorticoid-binding immuno-suppression is the first-line therapy for patients with LN, the common and severe side effects of such treatment call for new strategies to improve long-term prognosis and life quality for these patients. Curcumin has been used to treat autoimmune disease with good curative effect, but little is known about the effect of curcumin on LN patients. Our aim was to investigate the mechanism of curcumin for management of LN, specifically regarding the PYK2 pathways. MATERIAL AND METHODS: Freshly isolated peripheral blood mononuclear cells (PBMCs) from 20 LN patients and 20 healthy individuals were cultured and stimulated with either PMA, PMA+TyrA9 (PYK2 specific inhibitor), or PMA+Curcumin, and with PBS as control. After 48 hours of incubation, cells were harvested and the expression of PYK2, p-PYK2, CD40L, CTLA-4, and PBMCs proliferation were measured. Then the expression and activation of PYK2 was evaluated using Western blot, the expression of costimulatory molecules CD40L and CTLA-4 protein was evaluated using flow cytometry, and PBMC proliferation was assessed using a [3H]-thymidine incorporation assay. RESULTS: Curcumin inhibited the expression and activation of PYK2 in PBMCs in patients with LN in vitro. The inhibition rate of curcumin was negatively correlated with the level of serum complement, but positively correlated with 24-h proteinuria. Curcumin also suppressed the expression of costimulatory molecules CD40L and CTLA-4, as well as PBMC proliferation. Interestingly, these effects were not reproduced on PBMC cultures of healthy subjects. CONCLUSIONS: The inhibition of PYK2 signalling protein may be one of the mechanisms underlying the action of curcumin in LN treatment.