Evaluation of: Vergadi E, Vaporidi K, Theodorakis EE et al. Akt2 deficiency protects from acute lung injury via alternative macrophage activation and miR-146a induction in mice. J. Immunol. 192, 394-406 (2013). Acute respiratory distress syndrome currently has limited effective treatments; however, recent evidence suggests that modulation of alveolar macrophage responses may be an effective method for protection or repair of lung injury. Vergadi et al. are the first to demonstrate that depletion of Akt2 kinase and microRNA-146a induction in mice resulted in polarization of alveolar macrophages towards an M2 activation phenotype and resulted in less severe injury following acid-induced lung injury. However, this M2 polarization also resulted in increased lung bacterial load following infection with Pseudomonas aeruginosa.
Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections.
Patients with Sjögren's syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia.
Although elevated interleukin-7 (IL-7) levels have been reported in patients with primary Sjögrens syndrome (SS), the role of IL-7 in this disease remains unclear. We undertook this study to characterize the previously unexplored role of IL-7 in the development and onset of primary SS using the C57BL/6.NOD-Aec1Aec2 (B6.NOD-Aec) mouse model, which recapitulates human primary SS.
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 Sjögrens syndrome.
Sjögrens 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 (?-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 ?-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.
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.
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.
Sjögrens 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.
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 Sjögrens 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.
Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral cavity resulting in severe morbidity and mortality. To date only few proteins have been suggested as potential biomarkers or targets for this type of cancer. Cancerous inhibitor of PP2A (CIP2A) is a protein expressed in epithelial tissues that stabilizes the oncogene c-Myc and causes cell transformation. This study was designed to investigate the expression of CIP2A in OSCC cell lines and tissues representing human normal, dysplasia and OSCC.
The receptor for advanced glycation end products (RAGE) is a multiligand receptor expressed in a number of cell types, including gingival epithelia. RAGE mediates inflammation and induces cellular oxidative stress. Upregulation of RAGE is associated with various diseases, such as periodontal and cardiovascular diseases. This study examines the hypothesis that the gingiva of rats fed a calorie-restriction (CR) diet expresses lower levels of RAGE than the gingiva of rats fed an ad libitum (AL) diet.
During the 10th International Symposium on Sjögrens Syndrome held in Brest, France, from October 1-3, 2009 (http://www.sjogrensymposium-brest2009.org), the creation of an international epigenetic autoimmune group has been proposed to establish gold standards and to launch collaborative studies. During this "epigenetics session", leading experts in the field presented and discussed the most recent developments of this topic in Sjögrens Syndrome research. The "Brest epigenetic task force" was born and has scheduled a meeting in Ljubljana, Slovenia during the 7th Autoimmunity congress in May 2010.The following is a report of that session.
Ample studies have reported on the association between periodontal diseases, a persistent inflammatory process, and other chronic ailments such as cardiovascular diseases, diabetes mellitus, Alzheimer disease, and cancer. Other conditions such as low birth weight and premature delivery due to chorioamnionitis are also known to be linked to poor periodontal health. Although much epidemiologic data support these associations, a cause-and-effect relationship has not been established. The receptor for advanced glycation end products (RAGE) is a multiligand receptor expressed on various cell membranes, including immune, endothelial, and epithelial, and cells of the central nervous system. This receptor, which is frequently associated with proinflammatory responses, has been shown to be activated by various ligands such as high mobility group box-1 (HMGB1/amphoterin), amyloid fibrils, transthyrein, Mac-1 (Integrin Mac-1), as well as advanced glycation end products (AGEs). Recent studies indicate that signaling through RAGE has been implicated as an underlying condition in diverse pathologies including periodontal disease, cardiovascular diseases, diabetes mellitus, Alzheimer disease, cancer, and neurologic conditions. Review of the literature supports the hypothesis that activation of RAGE by ligands in a variety of cell types and tissues may play a role in oral systemic associations. In addition, the ligand cell source and timing of RAGE stimulation may determine the disease produced by this axis. Understanding the distribution and functions of RAGE and its ligands would enhance clinicians knowledge on pathogenesis of the oral-systemic connection.
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.
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.
To date, little is known about why exocrine glands are subject to immune cell infiltrations in Sjögrens 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 (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.
Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sjögrens syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor ?/interferon ?-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C ?, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS.
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