Evaluation of the role of cytokines in autoimmune disease: The importance of TNFα in rheumatoid arthritis

Cytokines and growth factors are involved in all important biological processes. Hence it is anticipated that they will be of importance in autoimmune disease. The pathogenesis of autoimmune diseases involves a number of stages, initiation, perpetuation and tissue damage, each of which involves different cell and molecular interactions. In this review, we will discuss an outline of the cytokine involvement in the various stages of autoimmune development, prior to focusing on the analysis of cytokines in rheumatoid arthritis. Cytokines exert their effect via high affinity cell surface receptors. Thus an understanding of cytokines involves the analysis of receptor expression, and also of cytokine inhibitors. Currently there is only adequate knowledge of these aspects in rheumatoid arthritis (RA), and as such the emphasis of this review is on RA. One of the major reasons for being interested in the role of cytokines in autoimmunity is to define possible therapeutic targets. There is now considerable evidence that TNFα is such a target in RA, and the effect of anti TNFα monoclonal antibody therapy in RA is discussed.     

Inactivation of TNF-α ameliorates diabetic neuropathy in mice

Tumor necrosis factor (TNF)-α is a potent proinflammatory cytokine involved in the pathogenesis of diabetic neuropathy. We inactivated TNF-α to determine if it is a valid therapeutic target for the treatment of diabetic neuropathy. We effected the inactivation in diabetic neuropathy using two approaches: by genetic inactivation of TNF-α (TNF-α(-/-) mice) or by neutralization of TNF-α protein using the monoclonal antibody infliximab. We induced diabetes using streptozotocin in wild-type and TNF-α(-/-) mice. We measured serum TNF-α concentration and the level of TNF-α mRNA in the dorsal root ganglion (DRG) and evaluated nerve function by a combination of motor (MNCV) and sensory (SNCV) nerve conduction velocities and tail flick test, as well as cytological analysis of intraepidermal nerve fiber density (IENFD) and immunostaining of DRG for NF-κB p65 serine-276 phosphorylated and cleaved caspase-3. Compared with nondiabetic mice, TNF-α(+/+) diabetic mice displayed significant impairments of MNCV, SNCV, tail flick test, and IENFD as well as increased expression of NF-κB p65 and cleaved caspase-3 in their DRG. In contrast, although nondiabetic TNF-α(-/-) mice showed mild abnormalities of IENFD under basal conditions, diabetic TNF-α(-/-) mice showed no evidence of abnormal nerve function tests compared with nondiabetic mice. A single injection of infliximab in diabetic TNF-α(+/+) mice led to suppression of the increased serum TNF-α and amelioration of the electrophysiological and biochemical deficits for at least 4 wk. Moreover, the increased TNF-α mRNA expression in diabetic DRG was also attenuated by infliximab, suggesting infliximab's effects may involve the local suppression of TNF-α. Infliximab, an agent currently in clinical use, is effective in targeting TNF-α action and expression and amelioration of diabetic neuropathy in mice.     

Comparative evaluation of the effects of treatment with tocilizumab and TNF-α inhibitors on serum hepcidin,anemia response and disease activity in rheumatoid arthritis patients

Introduction

Anemia of inflammation (AI) is a common complication of rheumatoid arthritis (RA) and has a negative impact on RA symptoms and quality of life. Upregulation of hepcidin by inflammatory cytokines has been implicated in AI. In this study, we evaluated and compared the effects of IL-6 and TNF-α blocking therapies on anemia, disease activity, and iron-related parameters including serum hepcidin in RA patients.

Methods

Patients (n = 93) were treated with an anti-IL-6 receptor antibody (tocilizumab) or TNF-α inhibitors for 16 weeks. Major disease activity indicators and iron-related parameters including serum hepcidin-25 were monitored before and 2, 4, 8, and 16 weeks after the initiation of treatment. Effects of tocilizumab and infliximab (anti-TNF-α antibody) on cytokine-induced hepcidin expression in hepatoma cells were analyzed by quantitative real-time PCR.

Results

Anemia at base line was present in 66% of patients. Baseline serum hepcidin-25 levels were correlated positively with serum ferritin, C-reactive protein (CRP), vascular endothelial growth factor (VEGF) levels and Disease Activity Score 28 (DAS28). Significant improvements in anemia and disease activity, and reductions in serum hepcidin-25 levels were observed within 2 weeks in both groups, and these effects were more pronounced in the tocilizumab group than in the TNF-α inhibitors group. Serum hepcidin-25 reduction by the TNF-α inhibitor therapy was accompanied by a decrease in serum IL-6, suggesting that the effect of TNF-α on the induction of hepcidin-25 was indirect. In in vitro experiments, stimulation with the cytokine combination of IL-6+TNF-α induced weaker hepcidin expression than did with IL-6 alone, and this induction was completely suppressed by tocilizumab but not by infliximab.

Conclusions

Hepcidin-mediated iron metabolism may contribute to the pathogenesis of RA-related anemia. In our cohort, tocilizumab was more effective than TNF-α inhibitors for improving anemia and normalizing iron metabolism in RA patients by inhibiting hepcidin production.     

NIK prevents the development of hypereosinophilic syndrome-like disease in mice independent of IKKα activation

Immune cell-mediated tissue injury is a common feature of different inflammatory diseases, yet the pathogenetic mechanisms and cell types involved vary significantly. Hypereosinophilic syndrome (HES) represents a group of inflammatory diseases that is characterized by increased numbers of pathogenic eosinophilic granulocytes in the peripheral blood and diverse organs. On the basis of clinical and laboratory findings, various forms of HES have been defined, yet the molecular mechanism and potential signaling pathways that drive eosinophil expansion remain largely unknown. In this study, we show that mice deficient of the serine/threonine-specific protein kinase NF-κB-inducing kinase (NIK) develop a HES-like disease, reflected by progressive blood and tissue eosinophilia, tissue injury, and premature death at around 25-30 wk of age. Similar to the lymphocytic form of HES, CD4(+) T cells from NIK-deficient mice express increased levels of Th2-associated cytokines, and eosinophilia and survival of NIK-deficient mice could be prevented completely by genetic ablation of CD4(+) T cells. Experiments based on bone marrow chimeric mice, however, demonstrated that inflammation in NIK-deficient mice depended on radiation-resistant tissues, implicating that NIK-deficient immune cells mediate inflammation in a nonautonomous manner. Surprisingly, disease development was independent of NIK's known function as an IκB kinase α (IKKα) kinase, because mice carrying a mutation in the activation loop of IKKα, which is phosphorylated by NIK, did not develop inflammatory disease. Our data show that NIK activity in nonhematopoietic cells controls Th2 cell development and prevents eosinophil-driven inflammatory disease, most likely using a signaling pathway that operates independent of the known NIK substrate IKKα.     

Periodontal disease,Porphyromonas gingivalis,and rheumatoid arthritis: what triggers autoimmunity and clinical disease?

Rheumatoid arthritis, currently regarded as a complex multifactorial disease, was initially characterized as such at the turn of the 19th century. Ever since, multiple lines of investigation have attempted to elucidate the etiological factor(s) involved in disease incidence. Genes – including those risk alleles within HLA-DR4 – have been implicated but are insufficient to explain the vast majority of cases. Several environmental factors, therefore, are being studied. Among them, the role of periodontal disease and Porphyromonas gingivalis in the pathogenesis of rheumatoid arthritis has attracted both clinical and bench interest given supportive epidemiologic and mechanistic data.The notion that rheumatoid arthritis (RA) is a polygenic autoimmune disorder that requires environmental factors in order to become clinically apparent is not novel. Since the very beginning, infectious agents have been implicated. This includes early theories such as the ‘oral sepsis’ hypothesis, which supported the notion that periodontal infections were the true etiologic factors behind many chronic diseases. Soon after, dental extraction became a central part of the RA therapeutic armamentarium.Over the last decade, an ever-increasing body of literature has been devoted to study the association between periodontal disease, Porphyromonas gingivalis, and RA. In this issue of Arthritis Research & Therapy, Arvikar and colleagues [1] demonstrate a positive correlation between P. ginigivalis antibody responses and presence/levels of anti-cyclic citrullinated peptide antibodies in a subset of patients with early RA. Moreover, subjects with serological reactivity toward P. ginvgivalis also tended to have higher RA disease activity as measured by Disease Activity Score in 28 Joints and Clinical Disease Activity Index. This occurred both at baseline – that is, in recently diagnosed patients who have not previously been treated with disease-modifying anti-rheumatic drugs (DMARDs) – and 12 months after initiation of therapy.The study had several strengths. First, the authors enrolled early RA patients who were DMARD-naïve at the time of antibody measurements and clinical assessments. Second, patients were followed for a period of 1 year to address biologic and phenotypic alterations after initiation of therapy. This is of utmost importance since very few studies have addressed clinical and immunologic changes at the very onset of disease [2,3]. Too often, however, the natural history of RA is studied without considering the confounding effects of long-standing systemic inflammation or immunosuppressive therapies or both. It is almost certain that the use of DMARDs and biologics alters the quantification and behavior of multiple immune cells and proteins (including auto- and alloantibody responses), thus distorting the true understanding of disease pathogenesis. Efforts to elucidate the earliest changes in pre-clinical and clinical RA are underway [2,4].An accumulating body of evidence suggests a role for clinical periodontal diseases in RA pathogenesis. Periodontitis was more common and severe in patients with RA compared with osteoarthritis [5], and subjects with RA had an increased likelihood of periodontitis compared with controls [6]. Multiple recent studies have specifically implicated P. gingivalis, a periodontopathic bacterium, as a possible triggering factor. This microorganism has gained scientific attention given its ability to citrullinate peptides via unique enzymatic properties conferred by peptydil arginine deiminase (PAD), which reportedly promotes the generation of neoantigens and the subsequent production of antibodies to citrullinated protein antigens (ACPAs). Experimentally, P. gingivalis-PAD is capable of citrullinating human peptides [7] and ACPAs have proven pathogenic in murine models of arthritis [8].A decade ago, it was postulated that a specific humoral immune response to P. gingivalis was the actual stimulus for the development of RA [9]. Since then, multiple reports used serological methods [10,11] to correlate the generation of antibodies to P. gingivalis with autoimmunity (that is, ACPA antibodies) and clinical RA. Several lessons can be learned from these types of approaches. First, as in the case of reports by Arvikar and colleagues [1] and others [2,10,11], the methodology and antigens used to quantify anti-P. gingivalis antibodies have been heterogeneous. Prior studies used antibodies against whole-cell, bacterial lipopolysaccharide, or P. gingivalis-specific chaperone protein. The sensitivity and specificity of each one of these antibodies (and their measurements through different phases of disease) add to the complexity of correlating P. gingivalis serologic responses to RA pathogenesis. A concerted effort toward standardization is warranted in the interest of scientific validation and replication. Second, very few studies have reported the direct presence of P. gingivalis (or other periodontopathic bacteria) in subgingival biofilms of patients with RA. This can now be achieved without the need for laborious, classic microbiologic culture techniques. The advent of high-throughput, bacterial DNA sequencing has allowed taxonomic classification of multiple bacterial species within hundreds of samples (that is, microbiome analysis) in a matter of days.Finally, and perhaps more importantly, virtually all studies consistently reported only a small fraction of RA patients as being exposed to P. gingivalis (serologically, microbiologically, or both). This can have several (and possibly complementary) explanations. It is conceivable that the overabundance of other, non-measured, periodontopathic bacteria (or the lack of protective flora or both) contributes to disease initiation. Moreover, exposure to bacterial antigenic burden at other body sites, such as the lung or the gut, may represent triggering factors for RA. The intestinal microbiome, for example, is vast and diverse. It contains 100 times more protein-coding genes than the human genome and harbors 100 trillion cells (10-fold the amount of total host human cells). Studies in animal models support the notion that the oral, lung, or intestinal microbiome (or a combination thereof) is required to develop inflammatory arthritis. This is based on the fact that rodents do not develop joint inflammation under germ-free conditions or when treated with antibiotics. It is plausible, therefore, that an alteration in the bacterial taxa of several mucosal sites (including oral, lung, and intestinal microbiomes) is required for the transition from a pre-clinical, autoimmune phase of RA into clinically classifiable disease.Novel and comprehensive approaches for the study of the microbiome and the initiation of RA are now possible. Immunologic and microbiome analyses in prospective cohorts of subjects with periodontal disease and other risk factors for the development of RA (for example, first-degree relatives, discordant twins, or asymptomatic individuals with circulating autoantibodies or a combination thereof) may help elucidate some of these questions and ultimately target these organisms (or their components) as a diagnostic or even preventive strategy for RA.     

Polymorphisms in the TNF-α and IL-10 gene promoters and risk of psoriasis and correlation with disease severity

Several cytokines were assumed to play an essential role in the induction and the pathogenesis of psoriasis. The aim of this work was to investigate the role of TNF-α-308 and IL-10-1082 polymorphisms and their serum levels in the pathogenesis of psoriasis and determine their relation to disease severity. 110 Psoriasis patients and 120 healthy volunteers were genotyped for TNF-α-308 and IL-10-1082 polymorphism by polymerase chain reaction. Serum level of TNF-α and IL-10 were measured by ELISA. Our study demonstrated an association of IL-10-1082 polymorphism and psoriasis and between TNF α-308 polymorphism and psoriasis disease and severity. Serum TNF α increased in patients, while serum IL-10 decreased in patients with significant correlation between serum TNF-α and psoriasis severity. These results indicated that TNF-α-308 and IL-10-1082 polymorphisms imparted significant risk towards the development of psoriasis.     

Citrullination of autoantigens: upstream of TNFα in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by synovial inflammation and destruction of joints. Over 20 years ago, tumour necrosis factor alpha (TNFα) was identified as a key player in a cytokine network, whose multifunctional effects could account for both the inflammation and destruction in RA. The remarkable efficacy of TNF inhibitors in the treatment of RA has resulted in extensive research addressing the regulation of TNFα production responsible for this excessive production. The discovery of autoimmunity to citrullinated protein/peptide antigens (ACPA) has led the concept that ACPA may be the essential link between disease susceptibility factors and the production of TNFα, which ultimately accounts for the disease phenotype. In this review we will consider (1) the mechanisms of citrullination, both physiological and pathological, (2) how known genetic and environmental factors could drive this peculiar form of autoimmunity and (3) how the immune response could lead to excessive production of TNFα by the synovial cells and ultimately to the disease phenotype (Fig. 1).     

Gene expression of thrombomodulin,TNF-α and NF-KB in coronary artery disease patients of Pakistan

Thrombomodulin (THBD) is an endothelial surface glycoprotein receptor, having a pivotal role in maintaining laminar blood flow. It functions to protect endothelial integrity by exhibiting anti-coagulation and anti-inflammatory properties thereby playing a key role in cardiovascular disease (CVD) pathology. Cholesterol lowering drugs have shown to alter the anti-inflammatory effects of cytokines. Understanding the molecular aspects of THBD gene and its relation to inflammatory cytokines is important to identify new prognostic and therapeutic targets for the CVD treatments. The present study was conducted to measure the expression of THBD, TNF-α and NF-kB genes in coronary artery disease patients (CAD) in Pakistani population. Lipid profile and BMI was compared both on fifty CAD patients and fifty healthy individuals. Expression analysis for THBD, TNF-α and NF-kB was carried out using real time PCR. The effect of lipid lowering drugs on cardiometabolic risk variables especially gene expression was analyzed. Our results indicated that the difference in BMI was marginal; however LDL-cholesterol and triglycerides levels in CAD patients were significantly higher than healthy individuals. THBD gene was significantly up-regulated whereas TNF-α and NF-kB were significantly down regulated in CAD individuals. Further exploration revealed that these variations were accounted to the use of statins by the patients. The use of statins by CAD patients up-regulated the mRNA expression of THBD by down-regulation of inflammatory mediators. The enhanced expression of endothelial THBD in response to cholesterol lowering drugs establishes a novel pleiotropic target that can be of clinical significance in thromboembolic and inflammatory disorders.

     

Antibacterial protection and the microbiological status of the oral cavity in Sj?gren's disease

The microflora of the oral cavity was studied with a view to the evaluation of the microbiological status and the content of lysozyme in mixed saliva samples from 14 patients with Sj?gren's syndrome and the control group of 19 persons. Disturbances in the biocenosis of the oral cavity of the patients, characterized by the increased occurrence of rod-shaped forms of lactobacilli, yeast-like fungi of the genus Candida and cariogenic streptococci (S. mutans) in the cultures obtained by the inoculation of oral smears, was detected. This "cariogenic situation" was confirmed by clinical data on the stomatological status. In patients with Sj?gren's syndrome the intensity of caries, determined by the ratio of carious, filled and extracted teeth, was high and reached 27.4 +/- 1.0 in comparison with 15.3 +/- 0.7 in the control group (P less than 0.05). A decrease in the level of mixed saliva secretion and in the content of lysozyme in secreted saliva was noted in the patients in comparison with the control group (P less than 0.05). The results thus obtained indicate that in Sj?gren's syndrome the use of the preparations of eubiotic microorganisms with a view to the correction of the microflora of the oral cavity, as well as the application of 0.1% lysozyme solution to the mucous membrane of the oral cavity, may be recommended among other therapeutic measures.     

Altered redox status in the blood of psoriatic patients: involvement of NADPH oxidase and role of anti-TNF-α therapy

Abstract

Background

Psoriasis is a chronic hyperproliferative inflammatory skin disease, characterized by a generalized redox imbalance. Anti-tumor necrosis factor (TNF)-α therapy is widely used for the treatment of this disease, but its effect on blood redox status hasn't been explored.

Objective

To investigate the effects of anti-TNF-α therapy on blood redox status in psoriatic patients.

Methods

Twenty-nine psoriatic patients (PSO) were divided into two groups: one remained untreated (NRT) and to another the anti-TNF-α therapy was prescribed (TR). The levels of main oxidative stress markers and total antioxidant capacity (TAC) in plasma, levels of total reactive oxygen species (ROS) production, lipoperoxidation, TAC, glutathione content, and activity of NADPH oxidase in white blood cells (WBC) were evaluated in PSO, in NTR and TR after 6 months of the study.

Results

Plasma levels of malondialdehyde (MDA) and protein carbonyl content (PCO), ROS production, lipoperoxidation, and glutathione content in WBC were increased, while TAC in both plasma and WBC was decreased in PSO with respect to controls. In the plasma of TR, levels of MDA and PCO were significantly lower with respect to PSO and NTR. The activity of NADPH oxidase was significantly increased in WBC of PSO and NTR but not in TR versus controls.

Discussion

Our results represent novel data about the redox status of WBC in psoriatic patients. A significant redox-balancing effect of anti-TNF-α therapy, probably associated with the normalization of NADPH oxidase activity in WBC, was demonstrated.     

Phosphorylation of 4EBP by oral leucine administration was suppressed in the skeletal muscle of PGC-1α knockout mice

Leucine is known to increase mTOR-mediated phosphorylation of 4EBP. In this study, leucine was administered to skeletal muscle-PGC-1α knockout mice. We observed attenuated 4EBP phosphorylation in the skeletal muscle, but not in the liver, of the PGC-1α knockout mice. These data suggest that skeletal muscle-PGC-1α is important for leucine-mediated mTOR activation and protein biosynthesis.