toll like receptor 3 stimulation promotes ro52 trim21 synthesis and nuclear redistribution in salivary gland epithelial cells partially via type i interferon pathway

Up-regulated expression of Ro52/tripartite motif-containing protein 21 (TRIM21), Ro60/TROVE domain family, member 2 (TROVE2) and lupus LA protein/Sjogren’s syndrome antigen B (La/SSB) Autoantigens has been described in the salivary gland epithelial cells (SGEC) of patients with Sjogren’s syndrome (SS). SGECs, the key regulators of autoimmune SS responses, express high levels of surface functional Toll-like receptor (TLR)-3, whereas Ro52/TRIM21 negatively regulates TLR-3-mediated inflammation. Herein, we investigated the effect of TLR-3-signalling on the expression of Ro52/TRIM21, as well as Ro60/TROVE2 and La/SSB Autoantigens, by SGECs. The effect of TLR-3 or TLR-4 stimulation on Autoantigen expression was evaluated by polyI:C or lipopolysaccharide (LPS) treatment, respectively, of SGEC lines (10 from SS patients, 12 from non-SS controls) or HeLa cells, followed by analysis of mRNA and protein expression. PolyI:C, but not LPS, resulted in a two-step induction of Ro52/TRIM21 mRNA expression by SGECs, a 12-fold increment at 6 h followed by a 2·5-fold increment at 24–48 h, whereas it induced a late two-fold up-regulation of Ro60/TROVE2 and La/SSB mRNAs at 48 h. Although protein expression levels were not affected significantly, the late up-regulation of Ro52/TRIM21 mRNA was accompanied by protein redistribution, from nucleolar-like pattern to multiple coarse dots spanning throughout the nucleus. These late phenomena were mediated significantly by interferon (IFN)-β production, as attested by cognate secretion and specific inhibition experiments and associated with IFN regulatory factor (IRF)3 degradation. TLR-3-signalling had similar effects on SGECs obtained from SS patients and controls, whereas it did not affect the expression of these Autoantigens in HeLa cells. TLR-3 signalling regulates the expression of Autoantigens by SGECs, implicating innate immunity pathways in their over-expression in inflamed tissues and possibly in their exposure to the immune system.

Toll‐like receptor 3 stimulation promotes Ro52/TRIM21 synthesis and nuclear redistribution in salivary gland epithelial cells, partially via type I interferon pathway

Up-regulated expression of Ro52/tripartite motif-containing protein 21 (TRIM21), Ro60/TROVE domain family, member 2 (TROVE2) and lupus LA protein/Sjogren’s syndrome antigen B (La/SSB) Autoantigens has been described in the salivary gland epithelial cells (SGEC) of patients with Sjogren’s syndrome (SS). SGECs, the key regulators of autoimmune SS responses, express high levels of surface functional Toll-like receptor (TLR)-3, whereas Ro52/TRIM21 negatively regulates TLR-3-mediated inflammation. Herein, we investigated the effect of TLR-3-signalling on the expression of Ro52/TRIM21, as well as Ro60/TROVE2 and La/SSB Autoantigens, by SGECs. The effect of TLR-3 or TLR-4 stimulation on Autoantigen expression was evaluated by polyI:C or lipopolysaccharide (LPS) treatment, respectively, of SGEC lines (10 from SS patients, 12 from non-SS controls) or HeLa cells, followed by analysis of mRNA and protein expression. PolyI:C, but not LPS, resulted in a two-step induction of Ro52/TRIM21 mRNA expression by SGECs, a 12-fold increment at 6 h followed by a 2·5-fold increment at 24–48 h, whereas it induced a late two-fold up-regulation of Ro60/TROVE2 and La/SSB mRNAs at 48 h. Although protein expression levels were not affected significantly, the late up-regulation of Ro52/TRIM21 mRNA was accompanied by protein redistribution, from nucleolar-like pattern to multiple coarse dots spanning throughout the nucleus. These late phenomena were mediated significantly by interferon (IFN)-β production, as attested by cognate secretion and specific inhibition experiments and associated with IFN regulatory factor (IRF)3 degradation. TLR-3-signalling had similar effects on SGECs obtained from SS patients and controls, whereas it did not affect the expression of these Autoantigens in HeLa cells. TLR-3 signalling regulates the expression of Autoantigens by SGECs, implicating innate immunity pathways in their over-expression in inflamed tissues and possibly in their exposure to the immune system.

Autoimmune response and target Autoantigens in Sjogren’s syndrome

Eur J Clin Invest 2010; 40 (11): 1026–1036

Abstract

Background  Primary Sjogren’s syndrome (pSS) is characterized by the presence of autoantibodies targeting mainly the Ro/La ribonucleoprotein complex. It is now appreciated that the production of autoantibodies is an antigen-driven immune response.

Design  In this review, candidate mechanisms for Autoantigen presentation and perpetuation of the autoimmune response within the autoimmune tissue lesion of pSS are discussed.

Results  Several studies have shown that the epithelial cell in labial salivary glands of patients with Sjogren’s syndrome is activated, bearing characteristics of an antigen-presenting cell, as suggested by inappropriate expression of class II HLA and co-stimulatory molecules. Other studies have confirmed that in salivary glands, there is an increased Autoantigen presentation via apoptotic blebs and bodies, exosomes and heat shock protein-mediated cross-priming. There is also an increased expression of interferon (IFN)-induced genes, such as the Autoantigen Ro52, which provide negative feedback regulation in inflammation. Ro60 and La Autoantigens also appear to play a major role in the local autoimmune response in Sjogren’s syndrome. In this regard, La and Ro60 the messenger RNA (mRNA) expression is upregulated in the affected salivary glands with different isoforms of La Autoantigen mRNA to be expressed in patients with pSS. At the protein level, La/SSB in pSS salivary glands is found to be post-translationally modified.

Conclusions  Autoantigen alterations in a microenvironment of local inflammation with increased in situ apoptosis, Toll-like receptor (TLR) signalling and antigen presentation may drive the autoimmune response and local autoantibody production in pSS.

Autoantigens as partners in initiation and propagation of autoimmune rheumatic diseases

Systemic autoimmune diseases are characterized by specific targeting of a limited group of ubiquitously expressed Autoantigens by the immune system. This review examines the mechanisms underlying their selection as immune targets. Initiation of autoimmune responses likely reflects the presentation of antigens with a distinct structure not previously encountered by the immune system, in a proimmune context (injury, malignancy, or infection). Causes of modified structure include somatic mutation and posttranslational modifications (including citrullination and proteolysis). Many Autoantigens are components of multimolecular complexes, and some of the other components may provide adjuvant activity. Propagation of autoimmune responses appears to reflect a bidirectional interaction between the immune response and the target tissues in a mutually reinforcing cycle: Immune effector pathways generate additional Autoantigen, which feeds further immune response. We propose that this resonance may be a critical prin…

immune mediated pore forming pathways induce cellular hypercitrullination and generate citrullinated Autoantigens in rheumatoid arthritis

Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated Autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination. Although histone citrullination is a common event during neutrophil activation and death induced by different pathways including apoptosis, NETosis, and necroptosis/autophagy, hypercitrullination is not induced by these stimuli. However, marked hypercitrullination is induced by two immune-mediated membranolytic pathways, mediated by perforin and the membrane attack complex (MAC), which are active in the RA joint and of importance in RA pathogenesis. We further demonstrate that perforin and MAC activity on neutrophils generate the profile of citrullinated Autoantigens characteristic of RA. These data suggest that activation of peptidylarginine deiminases during complement and perforin activity may be at the core of citrullinated Autoantigen production in RA. These pathways may be amenable to monitoring and therapeutic modulation.

peptidylarginine deiminase 2 3 and 4 have distinct specificities against cellular substrates novel insights into Autoantigen selection in rheumatoid arthritis

Objective To define the relationship between Autoantigen citrullination and different peptidylarginine deiminase (PAD) enzymes in rheumatoid arthritis (RA). Methods Citrullinated Autoantigens were identified by immunoblotting control and ionomycin-activated human primary neutrophil lysate with RA sera. Autoantigen identity and citrullination sites were defined by mass spectrometry. PAD isoenzyme expression in human neutrophils was determined by immunoblotting. PAD substrate specificity was addressed in HL-60 cell lysates co-incubated with human recombinant PAD2, PAD3 and PAD4. Results Although prominent protein citrullination is observed in ionomycin-activated neutrophils, RA sera only recognised a limited number of these citrullinated molecules. Among these, the authors identified that β and γ-actins are citrullinated on at least 10 arginine residues, generating a novel 47 kDa species that is frequently recognised by RA autoantibodies. Interestingly, the authors showed that the PAD enzymes expressed in human neutrophils (ie, PAD2, PAD3 and PAD4) have unique substrate specificities, independent of their subcellular distribution. Thus, only PAD2 was able to citrullinate native β/γ-actin, while histone H3 was only citrullinated by PAD4. Conclusion These studies identified β and γ-actins as novel citrullinated Autoantigens in RA, allowing enzyme specificity against intracellular substrates to be addressed. The studies provide evidence that PAD enzymes have the intrinsic capacity to select unique protein targets. The authors propose that unique PAD specificity may play a role in Autoantigen selection in RA.

Immune therapy for type 1 diabetes mellitus—what is unique about anti-CD3 antibodies?

Immune therapies that induce or, in the case of established type 1 diabetes mellitus (T1DM), restore immune tolerance to target Autoantigens hold potential to cure T1DM whilst avoiding the need for chronic immunosuppression. This Review discusses different immune therapies for T1DM and, in particular, focuses on two promising strategies in clinical development—the use of candidate Autoantigens and anti-CD3 monoclonal antibodies—that aim to restore immune tolerance. Type 1 diabetes mellitus (T1DM) is a prototypic organ-specific autoimmune disease that results from selective destruction of insulin-secreting β-cells by immune-mediated inflammation (insulitis), that is, the infiltration of pancreatic islets by autoreactive CD4^+ and CD8^+ T lymphocytes. Current treatment is substitutive—chronic use of exogenous insulin—which, in spite of considerable advances, is still associated with constraints and lack of effectiveness over the long-term in relation to the prevention of vascular and neurological complications. Finding a cure for T1DM is an important medical health challenge, as the disease’s incidence is steadily increasing in industrialized countries and projections of future prevalence are alarming. Crucially, as T1DM mainly affects children and young adults, any candidate immune therapy must be safe and avoid chronic use of immunosuppressants that promote sustained depression of immune responses. The ideal approach would, therefore, involve induction or, in the case of established T1DM, restoration of immune tolerance to target Autoantigens. This Review presents, in particular, two strategies that are still in clinical development but hold great promise. These strategies are focused on the use of candidate Autoantigens and anti-CD3 monoclonal antibodies. Type 1 diabetes mellitus (T1DM) is a prototypic autoimmune disease whose pathophysiology involves Autoantigen-presenting cells (dendritic cells, macrophages and B lymphocytes), T lymphocytes and target insulin-secreting β cells Immune destruction of β cells in humans starts months to years before the advent of overt hyperglycemia The incidence of T1DM has steadily increased in developed countries over the past few decades; prevalence in children

Proinsulin: a unique Autoantigen triggering autoimmune diabetes

In healthy individuals the immune system does not react aggressively toward host cells, a phenomenon defined as self tolerance. If self tolerance is broken autoimmune disease can develop, during which autoreactive lymphocytes are directed to a variety of Autoantigenic epitopes. However, researchers have yet to determine whether immune responses to multiple Autoantigens develop independently of each other or are the result of the response “spreading” from one Autoantigen to another. In a study of NOD mice in this issue of the JCI, Krishnamurthy et al. show that the autoreactive T cell response to the Autoantigen proinsulin lies upstream of that to islet-specific glucose-6-phosphatase catalytic subunit–related protein, suggesting that the pathogenic autoimmune response to proinsulin subsequently spreads to other antigens (see the related article beginning on page 3258). These data support the current view that this pancreatic β cell hormone is the first Autoantigen targeted by the immune response in autoimmune diabetes.

tolerance to islet Autoantigens in type 1 diabetes

Tolerance to β cell Autoantigens represents a fragile equilibrium. Autoreactive T cells specific to these Autoantigens are present in most normal individuals but are kept under control by a number of peripheral tolerance mechanisms, among which CD4+ CD25+ CD62L+ T cell–mediated regulation probably plays a central role. The equilibrium may be disrupted by inappropriate activation of Autoantigen-specific T cells, notably following to local inflammation that enhances the expression of the various molecules contributing to antigen recognition by T cells. Even when T cell activation finally overrides regulation, stimulation of regulatory cells by CD3 antibodies may reset the control of autoimmunity. Other procedures may also lead to disease prevention. These procedures are essentially focused on Th2 cytokines, whether used systemically or produced by Th2 cells after specific stimulation by Autoantigens. Protection can also be obtained by NK T cell stimulation. Administration of β cell antigens or CD3 antibodie…