Lang-Jing Zhu

Anti-TNF-α Therapies in Systemic Lupus Erythematosus

Tumor necrosis factor (TNF)-α is not just a proinflammatory cytokine. It has also been proposed to be an immunoregulatory molecule that can alter the balance of T regulatory cells. Anti-TNF-α therapies have been provided clinical benefit to many patients and introduced for treating moderate to severe rheumatoid arthritis, Crohns disease, and other chronic inflammatory disorders. However, their use also is accompanied by new or aggravated forms of autoimmunity, such as formation of autoantibodies, including antinuclear antibodies (ANAs), antidouble-stranded DNA (dsDNA) antibodies, and anticardiolipin antibodies (ACL). Systemic lupus erythematosus (SLE) is a disease with autoimmune disturbance and inflammatory damage. The role of TNF-α in human SLE is controversial. Here we review the role of TNF-α in the pathophysiological processes of SLE and the likely effects of blocking TNF-α in treatment of SLE.

Decreased expressions of the TNF-alpha signaling adapters in peripheral blood mononuclear cells (PBMCs) are correlated with disease activity in patients with systemic lupus erythematosus

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine. Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease. However, until now, the expression and pathophysiological role of TNF adapters in SLE have been poorly understood. This study aims to investigate the expression of mRNA for the TNF adapter proteins including TNF receptor-associated death domain (TRADD) protein, Fas-associated death domain (FADD) protein, receptor-interacting protein 1 (RIP-1), and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMCs) from patients with SLE and to explore the relationship between the expression of these adapters and the SLE disease activity. PBMCs were isolated from the venous blood of 51 SLE patients and 17 healthy subjects. The expression of mRNA for TNF adapter molecules such as TRADD, FADD, RIP-1, and TRAF-2 in PBMCs were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT–PCR) and quantitative real-time RT–PCR. There were constitutive expressions of mRNA for TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from healthy subjects. The expression of mRNA for all the adapter molecules significantly decreased in PBMCs from patients with SLE, which were 0.38-, 0.69-, 0.59-, and 0.55-fold, respectively, compared to those of control subjects (P < 0.05). The expression of Caspase 3 was significantly increased in SLE patients (P < 0.01); however, the expression of IL-1beta was not significantly different between SLE and control subjects. The expression of TRADD, FADD, RIP-1, and TRAF-2 in PBMCs from patients with SLE were negatively correlated with SLEDAI, the correlation coefficient of which was −0.285, −0.280, −0.307, and −0.298, respectively (P < 0.05). The expression of mRNA for TNF adapter molecules TRADD, FADD, RIP-1, and TRAF-2 decreased significantly in PBMCs from patients with SLE, and the expression of these adapters were negatively correlated with the SLE activity index. These abnormalities may be involved in the immunopathogenic injury mediated by the aberration TNF-alpha signaling pathway in SLE.

Altered Expression of TNF-α Signaling Pathway Proteins in Systemic Lupus Erythematosus

Objective. To investigate the expression of tumor necrosis factor receptors (TNFR1 and TNFR2) and adapter proteins (TRADD, RIP, and TRAF2) in peripheral blood mononuclear cell (PBMC) subsets from patients with systemic lupus erythematosus (SLE). Methods. PBMC were isolated from 45 SLE patients and 25 controls, and stained with labeled antibodies that enabled identification of various T cell, B cell, and monocyte subpopulations. Expression of TNF-related signaling molecules was measured by staining with labeled antibodies either directly or following fixation and permeabilization. Apoptosis was quantified using an anti-active caspase 3 antibody. RNA expression of TNF-related signaling molecules was assessed by quantitative RT-PCR and serum levels of TNF-α by ELISA. Results. SLE patients had increased levels of TNFR1, TNFR2, and TRAF2, together with decreased levels of RIP, on various B, CD4+ T, and CD8+ T cell subsets as compared to controls. This altered expression was seen in both naive and memory subpopulations, and reflected altered staining of the whole population rather than a subset of cells that were activated. The levels of these molecules were not significantly correlated with serum TNF-α levels or their RNA expression in whole peripheral blood. TNFR1 and TNFR2 expression was negatively correlated with disease activity. There was no association between the proportion of apoptotic cells in any of the subpopulations and serum TNF-α levels or expression of TNF-related signaling molecules. Conclusion. Patients with SLE had altered expression of TNF-related signaling molecules, suggesting that there may be an imbalance in TNF-α signaling favoring cellular activation as opposed to proapoptotic pathways.

Synovial Infiltration with CD79a-positive B Cells, But Not Other B Cell Lineage Markers, Correlates with Joint Destruction in Rheumatoid Arthritis

Objective. The efficacy of B cell depletion in the treatment of patients with rheumatoid arthritis (RA) has revitalized interest in the pathogenic role(s) of B cells in RA. We evaluated the distribution of synovial B lineage cells and their correlation with histologic disease activity and joint destruction in RA. Methods. Synovial tissue samples were obtained by closed-needle biopsy from 69 Chinese patients with active RA, from 14 patients with osteoarthritis (OA), and from 15 with orthopedic arthropathies (OrthA) as disease controls. Serial tissue sections were stained immunohistochemically for CD79a (pro-B cell to plasma cell), CD20 (B cells), CD38 (plasma cells), CD21 (follicular dendritic cells), CD68 (macrophages), CD3 (T cells), and CD34 (endothelial cells). Densities of positive-staining cells were determined and correlated with histologic disease activity (Krenn 3-component synovitis score) and radiographic joint destruction (Sharp score). Results. Mean sublining CD79a-positive cell density was significantly higher in RA than in OA (p <0.001) or OrthA (p = 0.003). Receiver operating characteristic curve analysis showed that CD79a-positive cell density differentiated RA well from OA [area under the curve (AUC) = 0.79] or OrthA (AUC = 0.75). Spearman’s rank order correlation showed significant correlations between sublining CD79a-positive cell density and the synovitis score (r = 0.714, p < 0.001), total Sharp score (r = 0.490, p < 0.001), and the erosion subscore (r = 0.545, p < 0.001), as well as the joint space narrowing subscore (r = 0.468, p = 0.001) in RA. Conclusion. Synovial CD79a-positive B cells may be a helpful biomarker for histologic disease activity in RA and may be involved in the pathogenesis of joint destruction in RA.

Elevated Serum Glucose-6-Phosphate Isomerase Correlates with Histological Disease Activity and Clinical Improvement After Initiation of Therapy in Patients with Rheumatoid Arthritis

Objective. To determine serum glucose-6-phosphate isomerase (GPI) concentrations in patients with rheumatoid arthritis (RA), and to test whether they correlate with objective measures of disease activity. Methods. Sera from 116 patients with RA, 69 patients with non-RA rheumatic diseases, and 101 healthy controls were analyzed. Levels of soluble serum GPI were measured by ELISA. Histological disease activity was determined with the synovitis score in synovial needle biopsies from 58 of the 116 patients with RA. Thirty-one of the 58 synovium samples were stained for CD68, CD3, CD20, CD38, CD79a, and CD34 by immunohistochemistry. Demographic data were collected, as well as serological and clinical variables that indicate RA disease activity, for Spearman correlation analysis. Results. Serum GPI level correlated positively with the synovitis score (r = 0.278, p = 0.034). Significantly higher soluble GPI levels were detected in the RA sera compared with sera from healthy controls and the non-RA disease controls (2.25 ± 2.82 vs 0.03 ± 0.05 and 0.19 ± 0.57 μg/ml, respectively; p < 0.0001). The rate of serum GPI positivity was significantly higher in the RA patients than in the non-RA disease controls (64.7% vs 10.1%; p < 0.0001). Spearman analysis showed no significant correlation between serum GPI level and Disease Activity Score in 28 joints at baseline. After initiation of antirheumatic treatments, GPI levels decreased significantly (2.81 ± 3.12 vs 1.44 ± 2.09 μg/ml; p = 0.016), paralleling improvement of the disease activity indices. Conclusion. Elevated serum GPI may be involved in the synovitis of RA and may prove useful as a serum marker for disease activity of RA.

Suppression of tumor necrosis factor receptor associated factor (TRAF)-2 attenuates the proinflammatory and proliferative effect of aggregated IgG on rat renal mesangial cells

Immune-complex (IC) mediated glomerulonephritis (GN) is a common cause of chronic kidney disease associated with increased levels of tumor necrosis factor (TNF)-alpha in renal cells. TNF-alpha signaling pathways involve complicated interactions between multiple proteins including TNF-receptor-associated factor (TRAF)-2. We have previously found markedly up-regulated expression of TRAF-2 in renal tissues from IC mediated lupus nephritis patients. Here we investigated the effect of TRAF-2 on inflammatory response in rat mesangial cells (MCs). The results showed that treatment with soluble aggregated IgG (AIgG) resulted in a time- and dose-dependent increase in the expression of interleukin (IL)-1beta and IL-6. Significant cell proliferation was also observed after the treatment with soluble AIgG. Knockdown TRAF-2 by siRNA significantly suppressed soluble AIgG induced up-regulation of TRAF-2, IL-1beta, and IL-6. Meanwhile the cell proliferation was inhibited and apoptotic cells were increased. It was concluded that TRAF-2 could induce the proinflammatory and proliferative effects of soluble AIgG on rat MCs. Thus, TRAF-2 may represent a future target for therapy of IC mediated GN.

α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells

Endothelial cells (ECs) express fibroblast growth factor (FGF) receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs) were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS) expression, nitric oxide (NO) production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

Differential expression patterns of housekeeping genes increase diagnostic and prognostic value in lung cancer

Background Using DNA microarrays, we previously identified 451 genes expressed in 19 different human tissues. Although ubiquitously expressed, the variable expression patterns of these “housekeeping genes” (HKGs) could separate one normal human tissue type from another. Current focus on identifying “specific disease markers” is problematic as single gene expression in a given sample represents the specific cellular states of the sample at the time of collection. In this study, we examine the diagnostic and prognostic potential of the variable expressions of HKGs in lung cancers. Methods Microarray and RNA-seq data for normal lungs, lung adenocarcinomas (AD), squamous cell carcinomas of the lung (SQCLC), and small cell carcinomas of the lung (SCLC) were collected from online databases. Using 374 of 451 HKGs, differentially expressed genes between pairs of sample types were determined via two-sided, homoscedastic t-test. Principal component analysis and hierarchical clustering classified normal lung and lung cancers subtypes according to relative gene expression variations. We used uni- and multi-variate cox-regressions to identify significant predictors of overall survival in AD patients. Classifying genes were selected using a set of training samples and then validated using an independent test set. Gene Ontology was examined by PANTHER. Results This study showed that the differential expression patterns of 242, 245, and 99 HKGs were able to distinguish normal lung from AD, SCLC, and SQCLC, respectively. From these, 70 HKGs were common across the three lung cancer subtypes. These HKGs have low expression variation compared to current lung cancer markers (e.g., EGFR, KRAS) and were involved in the most common biological processes (e.g., metabolism, stress response). In addition, the expression pattern of 106 HKGs alone was a significant classifier of AD versus SQCLC. We further highlighted that a panel of 13 HKGs was an independent predictor of overall survival and cumulative risk in AD patients. Discussion Here we report HKG expression patterns may be an effective tool for evaluation of lung cancer states. For example, the differential expression pattern of 70 HKGs alone can separate normal lung tissue from various lung cancers while a panel of 106 HKGs was a capable class predictor of subtypes of non-small cell carcinomas. We also reported that HKGs have significantly lower variance compared to traditional cancer markers across samples, highlighting the robustness of a panel of genes over any one specific biomarker. Using RNA-seq data, we showed that the expression pattern of 13 HKGs is a significant, independent predictor of overall survival for AD patients. This reinforces the predictive power of a HKG panel across different gene expression measurement platforms. Thus, we propose the expression patterns of HKGs alone may be sufficient for the diagnosis and prognosis of individuals with lung cancer.

Tumor necrosis factor receptor-associated factor (TRAF) 6 inhibition mitigates the pro-inflammatory roles and proliferation of rheumatoid arthritis fibroblast-like synoviocytes

HighlightsTRAF6 was expressed obviously in CD55+ cells and some other CD55− cells in RA synovium.Inhibition of TRAF6 in RA‐FLSs mitigated secretion of IL‐1&bgr;, IL‐8, IL‐6, TNF‐&agr;, MMP‐3, and MMP‐13.TRAF6 suppression in RA‐FLSs decreased cell proliferation, but not facilitated apoptosis.TRAF6 plays important roles in the pro‐inflammatory effects and proliferation of RA‐FLSs.TRAF6 may serve as a potential treatment target in RA. Background: Rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLSs) play a crucial role in RA through producing inflammatory cytokines and proteases which could cause cartilage destruction. We showed previously that elevated expression of tumor necrosis factor receptor‐associated factor 6 (TRAF6) in RA synovium correlated significantly with the severity of synovitis and the number of infiltrated inflammatory cells. The aims of this study are to detect the roles of TRAF6 in RA‐FLSs. Methods: Synovium were collected by closed needle biopsy from inflamed knees of active RA patients, and FLSs were isolated by modified tissue culture method. Expression of TRAF6 and CD55 in RA synivium was tested by double immunofluorescence (IF) staining. TRAF6 in RA‐FLSs was inhibited using Lentiviral‐TRAF6‐shRNA transfection. Real‐time PCR and ELISA were used to detect the mRNA expression and secretion of matrix metalloproteinase (MMP) and pro‐inflammatory cytokines. Cell Counting Kit‐8 was used to detect cell proliferation, flow cytometry was used to detect cell cycle, and Annexin V assay was used to detect cell apoptosis. Results: We showed that in the intimal and subintimal area of RA synovium, TRAF6 was expressed obviously not only in CD55+ cells, but also in some other CD55− cells. TRAF6 expression in RA‐FLSs was suppressed effectively by Lentiviral‐TRAF6‐shRNA transfection. Inhibition of TRAF6 in RA‐FLSs mitigated the mRNA levels and secretion of pro‐inflammatory cytokines and MMPs, such as IL‐1&bgr;, IL‐8, IL‐6, TNF‐&agr;, MMP‐13, and MMP‐3. In addition, it decreased the proliferation of RA‐FLSs, blocked RA‐FLSs in G0/G1‐phase, and inhibited the cells to go into S‐phase and G2/M‐phase, but not facilitated apoptosis of RA‐FLSs. Conclusion: TRAF6 plays direct roles in the pro‐inflammatory effects and proliferation of RA‐FLSs. TRAF6 may serve as a potential treatment target in RA.

FRI0370 Over-Expression of Sublining Bcl6 Correlates with Hyperplasia of Synovitis in Rheumatoid Arthritis

Background The pathological basis in RA is synovitis characterized by hyperplasia of synovial lining and synovial stroma with neovascularisation and inflammatory infiltration. B-cell lymphoma (Bcl) 6 is a nuclear sequence-specific transcriptional suppressor and its target genes, such as p53, p27, have been reported overexpressed in RA synovium. However, little is known about Bcl6 expression and its significance in RA synovium. Objectives To explore the correlation of synovial Bcl6 expression with synovitis and its components. Methods Synovial tissue was obtained by arthroscopy or closed-needle biopsy from 45 active RA patients fulfilled the 1987 revised ACR criteria and 12 OA patients. Serial tissue sections were stained with H&E and IHC for Bcl6, CD3, CD20, CD38, CD68 and CD34. Percentage of lining Bcl6(+) cells and densities of sublining Bcl6(+) cells per mm2 were calculated. Three synovitis subscores (lining hyperplasia, inflammatory infiltration and stroma activation) were summarized to yield total synovitis score (≤4: low-grade synovitis; >4: high-grade synovits). Results 1. Among 45 RA patients, 82% were female, median age was 55 years (range 23∼80), median disease duration was 36 months (range 2∼480), median DAS28 was 5.2 (interquartile range, IQR 4.4∼6.3) and the median synovitis score was 5 (IQR 4∼6). 2. In RA synovium, Bcl6 expressed strongly in the nuclei of synovial lining cells and sublining stroma cells including fibroblast, fibrocytes and endothelial cells (Fig.1). The median of lining Bcl6(+) cells percentage and sublining Bcl6(+) density was significantly higher than that in OA synovium (lining: 56% vs 25%, sublining: 1529 cells/mm2 vs 129 cells/mm2, both p<0.001). 3. Twenty-five patients had high-grade histological rheumatoid synovits and sublining Bcl6(+) density in high-grade synovits was significantly higher than that in low-grade synovits (median 1978 cells/mm2 vs 1297 cells/mm2, p<0.05). No significant difference of lining Bcl6 between these two groups (p>0.05). 4. Spearmans rank correlation test showed significant positive correlation of sublining Bcl6(+) cells density in RA synovium with total histological synovitis score, lining hyperplasia subscore, stroma activation subscore, CD34+ total vessels and microvessels densities (r=0.354∼0.412, all p<0.05), but not with inflammatory infiltration subscore, sublining CD3+T cells, CD20+ B cells, CD38+ plasmocytes and CD68+ macrophages (all p>0.05). 5. The Receiver operating characteristic (ROC) curve showed sublining Bcl6(+) density≥1666 cells/mm2 could discriminate high-grade synovitis from low-grade synovitis with sensitivity of 60% and specificity of 70% (AUC=0.694, P<0.05). RA synovium were divided into Bcl6 high-expression group (n=21) and Bcl6 low-expression group (n=24) according to this cutoff value. Lining hyperplasia subscore, stroma activation subscore, but not inflammatory infiltration subscore, in synovium with high-expression Bcl6 was higher than that with low-expression Bcl6 (all p>0.05). Conclusions Our results showed over-expression of sublining Bcl6 correlated with hyperplasia of synovitis in RA. Together with similar reported expression of p53, it is speculated that Bcl6 may regulate the proliferation and apoptosis of synovial stromal and lining cells by regulating its downstream target genes. Acknowledgements It was supported by Specialized Research Fund for the Doctoral Program of Higher Education (no.20130171110075), Guangdong Natural Science Foundation (no.S2013010014396), Medical Scientific Research Foundation of Guangdong Province, China (no. A2013201). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3497