Altered miR-146a expression in Sjögren's syndrome and its functional role in innate immunity.

MicroRNAs (miRNAs), small non-coding RNA molecules that post-transcriptionally regulate gene expression, are known to play key roles in regulating immune responses and autoimmunity. We investigated miR-146a expression in Sjögren's syndrome (SjS) patients as well as in the SjS-prone C57BL/6.NOD-Aec1Aec2 mouse model, to elucidate its involvement in SjS pathogenesis. Expression of miR-146a was examined in the PBMCs of 25 SjS patients and ten healthy donors, as well as in PBMCs, salivary and lacrimal glands of SjS-prone mice and WT C57BL/6J mice. Functional assays using THP-1 human monocytes were conducted to determine the biological roles of miR-146a in innate immunity. Expression of miR-146a was significantly increased in SjS patients compared with healthy controls, and was upregulated in the salivary glands and PBMCs of the SjS-prone mouse at both 8 wk (prior to disease onset) and 20 wk (full-blown disease) of age. More importantly, functional analysis revealed roles for miR-146a in increasing phagocytic activity and suppressing inflammatory cytokine production while migration, nitric oxide production and expression of antigen-presenting/costimulatory molecules are not affected in human monocytic THP-1 cells. Taken together, our data suggest that abnormal expression/regulation of microRNAs in innate immunity may contribute to, or be indicative of, the initiation and progression of SjS.

A secretagogue-small interfering RNA conjugate confers resistance to cytotoxicity in a cell model of Sjögren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is characterized by xerophthalmia and xerostomia resulting from loss of secretory function due to immune cell infiltration in lacrimal and salivary glands. Current therapeutic strategies for SS use secretagogues to induce secretion via muscarinic receptor stimulation. The purpose of this study was to create a secretagogue-small interfering RNA (siRNA) conjugate to deliver siRNA into cells via receptor-mediated endocytosis, thereby altering epithelial cell responses to external cues, such as proinflammatory or death signals, while simultaneously stimulating secretion. METHODS: Based on our expertise with type 3 muscarinic receptor (M3), we used carbachol, a ligand specific for muscarinic receptor, as the secretagogue. Carbachol was synthesized with an active choline group and was conjugated with an siRNA that targets caspase 3. A human salivary gland (HSG) cell line was used to test the efficacy of this secretagogue-siRNA conjugate. RESULTS: Lipofectamine transfection of the conjugate into HSG cells resulted in a 78% reduction in the expression of the caspase 3 gene, while external conjugate treatment of HSG cells resulted in intracellular calcium release and induction of endocytosis at levels similar to those of carbachol stimulation, indicating that the siRNA and carbachol portions of the conjugate retained their function after conjugation. HSG cells treated with conjugate (without Lipofectamine transfection) exhibited a 50% reduction in caspase 3 gene and protein expression, indicating that our conjugate was effective in delivering functional siRNA into cells via receptor-mediated endocytosis. Furthermore, tumor necrosis factor α-induced apoptosis was significantly reduced in conjugate-treated cells. CONCLUSION: Our secretagogue-siRNA conjugate prevented cytokine-induced apoptosis in salivary gland epithelial cells, which is critical to maintaining fluid secretion and potentially reversing the clinical hallmark of SS.

miR-146a is critical for endotoxin-induced tolerance: IMPLICATION IN INNATE IMMUNITY.

The human toll-like receptor 4 (TLR4) pathway is activated in response to lipopolysaccharide (LPS), and subsequent signal transductions lead to the production of cytokines such as tumor necrosis factor-alpha (TNF-alpha) by innate immune cells. Defects in innate immune response may contribute to the overproduction of TNF-alpha leading to systemic inflammation and diseases. Thus, the innate immune response needs to be tightly regulated by elaborate mechanisms to control its onset and termination. LPS tolerance is a state of hyporesponsiveness to subsequent LPS challenge and is achieved by monocytic cells after prolonged exposure to LPS. In this report, kinetics of endotoxin-responsive microRNAs expression analysis revealed a unique pattern of gradual increase for miR-146a starting 4 h after LPS stimulation in THP-1 cells and continued up to 35-fold over 24 h. Conversely, TNF-alpha increased up to 4 h and then decreased gradually implicating a negative correlation with miR-146a progression. The characteristic up-regulation of miR-146a toward subsequent LPS challenge in THP-1 cells was studied. Strikingly, microRNA expression analysis during the tolerized state of THP-1 cells showed only miR-146a overexpression suggesting its important role in LPS tolerance. In addition, LPS tolerance was dependent on a LPS-priming dose and associated miR-146a up-regulation. LPS-tolerized cells were observed to regain responsiveness in TNF-alpha production 22 h after LPS removal correlating with a decrease in miR-146a level. Transfection of miR-146a into THP-1 cells mimicked LPS priming, whereas transfection of miR-146a inhibitor largely abolished LPS tolerance. Thus our studies demonstrated that miR-146a is critical for the in vitro monocytic cell-based endotoxin tolerance.

Formation of GW/P bodies as marker for microRNA-mediated regulation of innate immune signaling in THP-1 cells.

GW bodies (GWB or P bodies) are cytoplasmic foci thought to result from microRNA (miRNA) regulation of messenger RNA (mRNA) targets and subsequent mRNA degradation. The purpose of this study is to examine the effects of lipopolysaccharide (LPS) stimulation of human monocytes on GWB formation, miRNA induction, miRNA target regulation and downstream cytokine and chemokine expression. In response to LPS stimulation, the number of GWB consistently increased by twofold at 8 h after stimulation and this increase was abolished when the miRNA-effector proteins Rck/p54 or argonaute 2 were depleted. As the level of miR-146a increased from 19-fold up to 100-fold during LPS stimulation, the transfection of a miR-146a mimic into THP-1 cells was examined to determine whether miR-146a alone can induce similar changes in GWB. The results showed transfected miR-146a could produce a comparable increase in the number of GWB and this was accompanied by a reduction in major cytokines/chemokines induced by LPS. These data show that the increase in size and number of GWB may serve as a biomarker for miRNA-mediated gene regulation, and miR-146a has a significant role in the regulation of LPS-induced cytokine production in THP-1 cells.

RAGE expression and NF-kappaB activation attenuated by extracellular domain of RAGE in human salivary gland cell line.

The receptor for advanced-glycation-end-products (RAGE) has been implicated as a pro-inflammatory factor in chronic inflammatory conditions such as diabetes mellitus and rheumatoid arthritis. The aim of this study was to investigate the inhibitory effect of the soluble-RAGE (sRAGE), the extracellular domain of RAGE, on RAGE expression and NF-kappaB translocation in human-salivary gland-cell-lines (HSG). Cells were stimulated with agonist S100A4, fusion protein of RAGE encompassing the extracellular domain of RAGE (ex-RAGE), ex-RAGE followed by S100A4, or S100A4 followed by ex-RAGE. Our study indicates that RAGE expression was highest at 150 microg/microl of S100A4 and efficiently down-regulated by 1.8-fold (P < 0.05) when ex-RAGE was incubated prior to agonist S100A4. RAGE protein was also consistently down-regulated by 20-40% with pre-incubation of ex-RAGE. More importantly, nuclear translocation of p65 and p52 of NF-kappaB by S100A4 was inhibited in the presence of ex-RAGE, confirming anti-inflammatory function of ex-RAGE. In conclusion, ex-RAGE down-regulates RAGE expression and inhibits p65 and p52 activation in HSG, providing evidence that ex-RAGE functions as a "decoy" to RAGE-ligand interaction and thus potentially dampening inflammatory conditions.

MicroRNA in autoimmunity and autoimmune diseases.

MicroRNAs (miRNAs) are small conserved non-coding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3' untranslated region (UTR) of specific messenger RNAs (mRNAs) for degradation or translational repression. miRNA-mediated gene regulation is critical for normal cellular functions such as the cell cycle, differentiation, and apoptosis, and as much as one-third of human mRNAs may be miRNA targets. Emerging evidence has demonstrated that miRNAs play a vital role in the regulation of immunological functions and the prevention of autoimmunity. Here we review the many newly discovered roles of miRNA regulation in immune functions and in the development of autoimmunity and autoimmune disease. Specifically, we discuss the involvement of miRNA regulation in innate and adaptive immune responses, immune cell development, T regulatory cell stability and function, and differential miRNA expression in rheumatoid arthritis and systemic lupus erythematosus.

Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody.

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

RNAi Therapeutics in Autoimmune Disease.

Since the discovery of RNA interference (RNAi), excitement has grown over its potential therapeutic uses. Targeting RNAi pathways provides a powerful tool to change biological processes post-transcriptionally in various health conditions such as cancer or autoimmune diseases. Optimum design of shRNA, siRNA, and miRNA enhances stability and specificity of RNAi-based approaches whereas it has to reduce or prevent undesirable immune responses or off-target effects. Recent advances in understanding pathogenesis of autoimmune diseases have allowed application of these tools in vitro as well as in vivo with some degree of success. Further research on the design and delivery of effectors of RNAi pathway and underlying molecular basis of RNAi would warrant practical use of RNAi-based therapeutics in human applications. This review will focus on the approaches used for current therapeutics and their applications in autoimmune diseases, including rheumatoid arthritis and Sjögren's syndrome.

Autoantibodies against muscarinic type 3 receptor in Sjögren's syndrome inhibit aquaporin 5 trafficking.

Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS.

Animal models in autoimmune diseases: lessons learned from mouse models for Sjögren's syndrome.

The mouse model is the one of the most frequently used and well-established animal models, and is currently used in many research areas. To date, various mouse models have been utilized to elucidate underlying causes of multifactorial autoimmune conditions, including pathological immune components and specific signaling pathways. This review summarizes the more recent mouse models for Sjögren's syndrome, a systemic autoimmune disease characterized by lymphocytic infiltration in the exocrine glands, such as the salivary and lacrimal glands, and loss of secretory function, resulting in dry mouth and dry eyes in patients. Although every Sjögren's syndrome mouse model resembles the major symptoms or phenotypes of Sjögren's syndrome conditions in humans, the characteristics of each model are variable. Moreover, to date, there is no single mouse model that can completely replicate the human conditions. However, unique features of each mouse model provide insights into the roles of potential etiological and immunological factors in the development and progression of Sjögren's syndrome. Here, we will overview the Sjögren's syndrome mouse models. Lessons from these mouse models will aid us to understand underlying immune dysregulation in autoimmune diseases in general, and will guide us to direct future research towards appropriate diagnostic and therapeutic strategies.

miRNA-146a in rheumatoid arthritis: a new therapeutic strategy.

Evaluation of: Nakasa T, Shibuya H, Nagata Y, Niimoto T, Ochi M: The inhibitory effect of microRNA-146a expression on bone destruction in collagen-induced arthritis. Arthritis Rheum. 63(6), 1582-1590 (2011). miRNA-146a (miR-146a) has been shown to play an important role in the negative regulation of inflammatory innate immune responses, and is differentially expressed in a number of human diseases including rheumatoid arthritis. However, evidence for the potential therapeutic use of miR-146a in human disease has been lacking. The current paper demonstrates the potential therapeutic application of miR-146a for rheumatoid arthritis by demonstrating the inhibitory effect of miR-146a on osteoclastogenesis in vitro. Moreover, using a collagen-induced arthritis mouse model, they were able to demonstrate that intravenous administration of double-stranded miR-146a resulted in the suppression of cartilage and bone destruction, despite relatively unaffected immune cell infiltration of the synovium and inflammatory cytokine expression.

Presence of Porphyromonas gingivalis in gingival squamous cell carcinoma.

Periodontal disease has been recently linked to a variety of systemic conditions such as diabetes, cardiovascular disease, preterm delivery, and oral cancer. The most common bacteria associated with periodontal disease, Porphyromonas gingivalis (P. gingivalis) has not yet been studied in the malignant gingival tissues. The objective of this study was to investigate the presence of P. gingivalis in specimens from squamous cell carcinoma patients. We have performed immunohistochemical staining to investigate the presence of P. gingivalis and Streptococcus gordonii (S. gordonii), a non invasive oral bacteria, in paraffin embedded samples of gingival squamous cell carcinoma (n = 10) and normal gingiva (n = 5). Staining for P. gingivalis revealed the presence of the bacteria in normal gingival tissues and gingival carcinoma, with higher levels (more than 33%, P < 0.05) detected in the carcinoma samples. The staining intensity was also significantly enhanced in the malignant tissue by 2 folds (P < 0.023) compared to specimens stained for the non-invasive S. gordonii. P. gingivalis is abundantly present in malignant oral epithelium suggesting a potential association of the bacteria with gingival squamous cell carcinoma.

Inflammatory caspases are critical for enhanced cell death in the target tissue of Sjögren's syndrome before disease onset.

To date, little is known about why exocrine glands are subject to immune cell infiltrations in Sjögren's syndrome (SjS). Studies with SjS-prone C57BL/6.NOD-Aec1Aec2 mice showed altered glandular homeostasis in the submandibular glands (SMX) at 8 weeks before disease onset and suggested the potential involvement of inflammatory caspases (caspase-11 and -1). To determine whether inflammatory caspases are critical for the increased epithelial cell death before SjS-like disease, we investigated molecular events involving caspase-11/caspase-1 axis. Our results revealed concurrent upregulation of caspase-11 in macrophages, STAT-1 activity, caspase-1 activity and apoptotic epithelial cells in the SMX of C57BL/6.NOD-Aec1Aec2 at 8 weeks. Caspase-1, a critical factor for interleukin (IL)-1beta and IL-18 secretion, resulted in an elevated level of IL-18 in saliva. Interestingly, TUNEL-positive cells in the SMX of C57BL/6.NOD-Aec1Aec2 were not colocalized with caspase-11, indicating that caspase-11 functions in a noncell autonomous manner. Increased apoptosis of a human salivary gland (HSG) cell line occurred only in the presence of lipopolysaccharide (LPS-) and interferon (IFN)-gamma-stimulated human monocytic THP-1 cells, which was reversed when caspase-1 in THP-1 cells was targeted by siRNA. Taken together, our study discovered that inflammatory caspases are essential in promoting a pro-inflammatory microenvironment and influencing increased epithelial cell death in the target tissues of SjS before disease onset.

MicroRNAs and their emerging roles in immunology.

MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs that post-transcriptionally regulate gene expression by targeting specific messenger RNAs (mRNAs) for degradation or translational repression. Recent evidence indicates that miRNA-mediated gene regulation is critical for normal cellular functions, and as much as one-third of human mRNAs may be miRNA targets. Emerging evidence suggests that miRNAs play a key role in the regulation of immunological functions including innate and adaptive immune responses, development and differentiation of immune cells, and the prevention of autoimmunity. Here, we review the mechanisms of miRNA maturation and function, the roles of several miRNAs in immunological functions, and the involvement of miRNAs in disease pathogenesis.

Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients.

INTRODUCTION: MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis. METHODS: Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR. RESULTS: Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-alpha production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-alpha production. CONCLUSIONS: Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-alpha production in patients with rheumatoid arthritis.

The role of GW/P-bodies in RNA processing and silencing.

GW bodies, also known as mammalian P-bodies, are cytoplasmic foci involved in the post-transcriptional regulation of eukaryotic gene expression. Recently, GW bodies have been linked to RNA interference and demonstrated to be important for short-interfering-RNA- and microRNA-mediated mRNA decay and translational repression. Evidence indicates that both passenger and guide strands of short-interfering RNA duplexes can localize to GW bodies, thereby indicating that RNA-induced silencing complexes may be activated within these cytoplasmic centers. Formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Work over the past few years has significantly increased our understanding of the biology of GW bodies, revealing that they are specialized cell components that spatially regulate mRNA turnover in various biological processes. The formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Here, we propose a working model for GW body assembly in terms of its relationship to RNA interference. In this process, one or more heteromeric protein complexes accumulate in successive steps into larger ribonucleoprotein structures.

Formation of GW bodies is a consequence of microRNA genesis.

GW bodies (GWBs), or mammalian P bodies, proposed to be involved in messenger RNA storage and/or degradation, have recently been linked to RNA interference and microRNA (miRNA) processing. We report that endogenous let-7 miRNA co-precipitates with the GW182 protein complex. In addition, knockdown of two proteins, Drosha and its protein partner DGCR8, which are vital to the generation of mature miRNA, results in the loss of GWBs. Subsequent introduction of short interference RNA specific to lamin A/C is accompanied by reassembly of GWBs and concurrent knockdown of lamin A/C protein. Taken together, these studies show that miRNAs are crucial components in GWB formation.