Excess Salt Intake Activates IL-21-Dominant Autoimmune Diabetogenesis via a Salt-Regulated Ste20-Related Proline/Alanine-Rich Kinase in CD4 T Cells.

The fundamental mechanisms by which a diet affects susceptibility to or modifies autoimmune diseases are poorly understood. Excess dietary salt intake acts as a risk factor for autoimmune diseases; however, little information exists on the impact of salt intake on type 1 diabetes. To elucidate the potential effect of high salt intake on autoimmune diabetes, nonobese diabetic (NOD) mice were fed a high-salt diet (HSD) or a normal-salt diet (NSD) from 6 to 12 weeks of age and monitored for diabetes development. Our results revealed that the HSD accelerated diabetes progression with more severe insulitis in NOD mice in a CD4+ T-cell-autonomous manner when compared with the NSD group. Moreover, expression of IL-21 and SPAK in splenic CD4+ T cells from HSD-fed mice was significantly upregulated. Accordingly, we generated T-cell-specific SPAK knockout (CKO) NOD mice and demonstrated that SPAK deficiency in T cells significantly attenuated diabetes development in NOD mice by downregulating IL-21 expression in CD4+ T cells. Furthermore, HSD-triggered diabetes acceleration was abolished in HSD-fed SPAK CKO mice when compared with HSD-fed NOD mice, suggesting an essential role of SPAK in salt-exacerbated T-cell pathogenicity. Finally, pharmacological inhibition of SPAK activity using a specific SPAK inhibitor (closantel) in NOD mice ameliorated diabetogenesis, further illuminating the potential of a SPAK-targeting immunotherapeutic approach for autoimmune diabetes. Here, we illustrate that a substantial association between salt sensitivity and the functional impact of SPAK on T-cell pathogenicity is a central player linking high-salt-intake influences to immunopathophysiology of diabetogenesis in NOD mice.

Blimp-1 molds the epigenetic architecture of IL-21-mediated autoimmune diseases through an autoregulatory circuit.

Positive regulatory domain 1 (PRDM1) encodes B lymphocyte-induced maturation protein 1 (BLIMP1), also known as a master regulator of T cell homeostasis. We observed a negative relationship between Blimp-1 and IL-21 based on our previous data that Blimp-1 overexpression in T cells suppresses autoimmune diabetes while Blimp-1-deficient T cells contribute to colitis in NOD mice. Reanalysis of published data sets also revealed an inverse correlation between PRDM1 and IL21 in Crohn's disease. Here, we illustrate that Blimp-1 repressed IL-21 by reducing chromatin accessibility and evicting an IL-21 activator, c-Maf, from the Il21 promoter. Moreover, Blimp-1 overexpression-mediated reduction in permissive chromatin structures at the Il21 promoter could override IL-21-accelerated autoimmune diabetogenesis in small ubiquitin-like modifier-defective c-Maf-transgenic mice. An autoregulatory feedback loop to harness IL-21 expression was unveiled by the evidence that IL-21 addition induced time-dependent Blimp-1 expression and subsequently enriched its binding to the Il21 promoter to suppress IL-21 overproduction. Furthermore, intervention of this feedback loop by IL-21 blockade, with IL-21R.Fc administration or IL-21 receptor deletion, attenuated Blimp-1 deficiency-mediated colitis and reinforced the circuit between Blimp-1 and IL-21 in the regulation of autoimmunity. We highlight the translation of Blimp-1-based epigenetic and transcriptomic profiles applicable to a personalized medicine approach in autoimmune diseases.

Gut Microbiota-Modulated Metabolomic Profiling Shapes the Etiology and Pathogenesis of Autoimmune Diseases.

Autoimmunity is a complex and multifaceted process that contributes to widespread functional decline that affects multiple organs and tissues. The pandemic of autoimmune diseases, which are a global health concern, augments in both the prevalence and incidence of autoimmune diseases, including type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. The development of autoimmune diseases is phenotypically associated with gut microbiota-modulated features at the molecular and cellular levels. The etiology and pathogenesis of autoimmune diseases comprise the alterations of immune systems with the innate and adaptive immune cell infiltration into specific organs and the augmented production of proinflammatory cytokines stimulated by commensal microbiota. However, the relative importance and mechanistic interrelationships between the gut microbial community and the immune system during progression of autoimmune diseases are still not well understood. In this review, we describe studies on the profiling of gut microbial signatures for the modulation of immunological homeostasis in multiple inflammatory diseases, elucidate their critical roles in the etiology and pathogenesis of autoimmune diseases, and discuss the implications of these findings for these disorders. Targeting intestinal microbiome and its metabolomic associations with the phenotype of autoimmunity will enable the progress of developing new therapeutic strategies to counteract microorganism-related immune dysfunction in these autoimmune diseases.

Adipokine-Modulated Immunological Homeostasis Shapes the Pathophysiology of Inflammatory Bowel Disease.

Inflammatory colon diseases, which are a global health concern, include a variety of gastrointestinal tract disorders, such as inflammatory bowel disease and colon cancer. The pathogenesis of these colon disorders involves immune alterations with the pronounced infiltration of innate and adaptive immune cells into the intestines and the augmented expression of mucosal pro-inflammatory cytokines stimulated by commensal microbiota. Epidemiological studies during the past half century have shown that the proportion of obese people in a population is associated with the incidence and pathogenesis of gastrointestinal tract disorders. The advancement of understanding of the immunological basis of colon disease has shown that adipocyte-derived biologically active substances (adipokines) modulate the role of innate and adaptive immune cells in the progress of intestinal inflammation. The biomedical significance in immunological homeostasis of adipokines, including adiponectin, leptin, apelin and resistin, is clear. In this review, we highlight the existing literature on the effect and contribution of adipokines to the regulation of immunological homeostasis in inflammatory colon diseases and discuss their crucial roles in disease etiology and pathogenesis, as well as the implications of these results for new therapies in these disorders.

Post-Translational Modifications of Transcription Factors Harnessing the Etiology and Pathophysiology in Colonic Diseases.

Defects in mucosal immune balance can lead to colonic diseases such as inflammatory bowel diseases and colorectal cancer. With the advancement of understanding for the immunological and molecular basis of colonic disease, therapies targeting transcription factors have become a potential approach for the treatment of colonic disease. To date, the biomedical significance of unique post-translational modifications on transcription factors has been identified, including phosphorylation, methylation, acetylation, ubiquitination, SUMOylation, and O-GlcNAcylation. This review focuses on our current understanding and the emerging evidence of how post-translational regulations modify transcription factors involved in the etiology and pathophysiology of colonic disease as well as the implications of these findings for new therapeutic approaches in these disorders.

Interplay between Cytokine Circuitry and Transcriptional Regulation Shaping Helper T Cell Pathogenicity and Plasticity in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic disorder manifested as Crohn's disease (CD) and ulcerative colitis (UC) characterized by intestinal inflammation and involves a dysregulated immune response against commensal microbiota through the activation of CD4 T helper cells. T helper cell differentiation to effector or regulatory phenotypes is controlled by cytokine networks and transcriptional regulators. Distinct polarized T helper cells are able to alter their phenotypes to adapt to diverse and fluctuating physiological environments. T helper cells exhibit intrinsic instability and flexibility to express cytokines of other lineages or transdifferentiate from one T helper cell type to another in response to various perturbations from physiological cytokine milieu as a means of promoting local immunity in response to injury or ensure tissue homeostasis. Furthermore, functional plasticity and diversity of T helper cells are associated with pathogenicity and are critical for immune homeostasis and prevention of autoimmunity. In this review, we provide deeper insights into the combinatorial extrinsic and intrinsic signals that control plasticity and transdifferentiation of T helper cells and also highlight the potential of exploiting the genetic reprogramming plasticity of T helper cells in the treatment of IBD.

SUMO-defective c-Maf preferentially transactivates Il21 to exacerbate autoimmune diabetes.

SUMOylation is involved in the development of several inflammatory diseases, but the physiological significance of SUMO-modulated c-Maf in autoimmune diabetes is not completely understood. Here, we report that an age-dependent attenuation of c-Maf SUMOylation in CD4+ T cells is positively correlated with the IL-21-mediated diabetogenesis in NOD mice. Using 2 strains of T cell-specific transgenic NOD mice overexpressing wild-type c-Maf (Tg-WTc) or SUMOylation site-mutated c-Maf (Tg-KRc), we demonstrated that Tg-KRc mice developed diabetes more rapidly than Tg-WTc mice in a CD4+ T cell-autonomous manner. Moreover, SUMO-defective c-Maf preferentially transactivated Il21 to promote the development of CD4+ T cells with an extrafollicular helper T cell phenotype and expand the numbers of granzyme B-producing effector/memory CD8+ T cells. Furthermore, SUMO-defective c-Maf selectively inhibited recruitment of Daxx/HDAC2 to the Il21 promoter and enhanced histone acetylation mediated by CREB-binding protein (CBP) and p300. Using pharmacological interference with CBP/p300, we illustrated that CBP30 treatment ameliorated c-Maf-mediated/IL-21-based diabetogenesis. Taken together, our results show that the SUMOylation status of c-Maf has a stronger regulatory effect on IL-21 than the level of c-Maf expression, through an epigenetic mechanism. These findings provide new insights into how SUMOylation modulates the pathogenesis of autoimmune diabetes in a T cell-restricted manner and on the basis of a single transcription factor.

The Modulatory Roles of N-glycans in T-Cell-Mediated Autoimmune Diseases.

Glycosylation is a ubiquitous posttranslational modification of proteins that occurs in the endoplasmic reticulum/Golgi. N-glycans and mucin-type O-glycans are achieved via a series of glycohydrolase- and glycosyltransferase-mediated reactions. Glycosylation modulates immune responses by regulating thymocyte development and T helper cell differentiation. Autoimmune diseases result from an abnormal immune response by self-antigens and subsequently lead to the destruction of the target tissues. The modification of N-glycans has been studied in several animal models of T-cell-mediated autoimmune diseases. This review summarizes and highlights the modulatory effects of N-glycosylation in several autoimmune diseases, including multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and type 1 diabetes mellitus.

Peripheral Autoimmune Regulator Induces Exhaustion of CD4+ and CD8+ Effector T Cells to Attenuate Autoimmune Diabetes in Non-Obese Diabetic Mice.

Autoimmune regulator (Aire) is one of the most crucial genes expressed in the thymus, where it orchestrates the promiscuous expression and presentation of tissue-specific antigens during thymocyte selection. The presence of Aire-expressing cells outside the thymus points toward its plausible extrathymic functions; however, the relative contribution of Aire-expressing cells of hematopoietic origin and their role in the modulation of autoimmune diseases are still obscure. Here, we report that non-obese diabetic mice with transgenic Aire expression under the control of the CD11c (integrin alpha X) promoter were significantly protected from autoimmune diabetes compared with their non-transgenic littermates. The protective effect of Aire transgene was mediated primarily by an increase in the "exhausted" populations of CD4+ and CD8+ T cells, both demonstrating poor expressions of interferon-γ and tumor necrosis factor-α. Both CD4+ and CD8+ effector T cells in transgenic mice displayed distinctive and differential expression of T-bet and Eomesodermin, respectively, in conjunction with high expression of programmed cell death protein-1 and other exhaustion-associated markers. Importantly, transgenic Aire expression did not result in any detectable changes in the population of Foxp3+ regulatory T (Treg) cells. Co-transfer experiments also demonstrated that Aire transgenic dendritic cells, as a "stand-alone" cell population, had the potential to suppress effector T cells in vivo without the support of Treg cells, but eventually failed to prevent the diabetogenesis in recipient mice. In conclusion, our study suggests that apart from its role in clonal deletion of autoreactive T cells or clonal diversion to Treg lineage, Aire can also contribute to tolerance by forcing effector T cells into a state of exhaustion with poor effector functions, thereby effectively containing autoimmune diseases.

Glucosamine Modulates T Cell Differentiation through Down-regulating N-Linked Glycosylation of CD25.

Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through down-regulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both down-regulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis.

Targeting tumour necrosis factor receptor 1 assembly reverses Th17-mediated colitis through boosting a Th2 response.

OBJECTIVE: The soluble preligand assembly domain (PLAD) of tumour necrosis factor receptor 1 (TNFR1) interferes with receptor trimerisation to block downstream signalling, and mediates Th17 suppression. We explored the therapeutic potential of recombinant PLAD.Fc protein on a spontaneous experimental colitis. DESIGN: A T-cell-specific BLIMP-1 knockout mouse model with mixed Th1/Th17 responses, resembling human Crohn's disease (CD) was established, and its colitogenic phenotype was characterised. Mice, 9 weeks old, were treated with PLAD.Fc protein at 5 mg/kg of body weight twice per week for 16 weeks, and presence of colitis was monitored by the appearance of diarrhoea, weight loss, and by histological colonic scoring. Activation status, cytokine profiles, and transcription factors in T cells were further analysed. RESULTS: The colitogenic phenotype in BLIMP-1 knockout mice was alleviated when an interleukin (IL)-23 knockdown transgene was introduced, indicating a therapeutic potential by downregulating IL-23-Th17 axis in these knockout mice. In PLAD.Fc-treated group, the mouse body weight remained stable and only mild disease scores were revealed. The percentage of naive CD4 T cells was increased and that of effector/memory CD4 T cells was decreased after PLAD.Fc-treatment. Moreover, the levels of IFN-γ, IL-17, IL-21, IL-22, IL-23R, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α were diminished. Strikingly, Th2-associated cytokines (IL-4, IL-13 and IL-10) in sera, as well as percentages of Th2 cells, were increased in PLAD.Fc-treated mice. However, PLAD.Fc-mediated suppression of effector phenotypes in Th1/Th17 was abrogated after neutralising IL-10. CONCLUSIONS: The Th2 cytokine milieu induced by PLAD.Fc rebalanced T-helper cell subsets and conferred a protection against colitis in BLIMP-1 knockout mice.

Triptolide ameliorates autoimmune diabetes and prolongs islet graft survival in nonobese diabetic mice.

OBJECTIVES: Triptolide (TPL) possesses profound immunosuppressive effects and has potential in allograft transplantation. We investigated whether TPL treatment prevents autoimmune diabetes in nonobese diabetic (NOD) mice and prolongs the survival of islet grafts against autoimmune attack or allograft rejection. METHODS: Diabetic incidence was monitored in TPL-treated NOD mice. Nonobese diabetic or BALB/c islets were transplanted into diabetic recipients treated with TPL. Different T-cell subsets in grafts or spleen were analyzed. The proliferation, apoptosis, cytokines, and activities of AKT, NFκB, and caspases 3, 8, and 9 of T cells were determined. RESULTS: Diabetic incidence was reduced and inflammatory cytokines were decreased in islets and spleen under TPL treatment. T-cell proliferation was reduced and the survival of syngeneic or allogeneic grafts was significantly increased in TPL-treated mice. The populations of CD4, CD8, CD4CD69, CD8CD69, and interferon-γ-producing T cells in islet grafts and spleen were reduced. Triptolide treatment increased the apoptosis of T cells in the spleen of recipients. Levels of phosphorylated protein kinase B and phosphorylated inhibitor of kappa B in splenocytes were reduced and caspases 3, 8, and 9 were increased in TPL-treated mice. CONCLUSIONS: Triptolide treatment not only reduced the diabetic incidence in NOD mice but also prolonged the survival of syngeneic or allogeneic grafts.

Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells.

PURPOSE: To investigate the effects and mechanisms of glucosamine (GlcN) on the proliferation of retinal pigment epithelial cells in response to epidermal growth factor (EGF). METHODS: Cell proliferation was measured in the human retinal pigment epithelial cell line (ARPE-19) cells with the 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay and cell counting. The results were confirmed in human donor cells with the carboxyfluorescein diacetate succinimidyl ester cell proliferation assay (CFSE) cell proliferation assay. In ARPE-19 cells, cell-cycle progression was determined by flow cytometry; the protein levels of cell cycle regulators and heat shock protein 90 (Hsp90) were measured by western blotting; the levels and branching of N-glycans were assessed using the L-Phaseolus vulgaris agglutinin lectin-binding assay; and the modulation of N-glycans on EGF receptor (EGFR) was examined by western blotting. RESULTS: GlcN inhibited retinal pigment epithelium (RPE) proliferation in a dose-dependent manner. During cell-cycle progression induced by EGF, GlcN caused delays at the G(1)-S and G(2)-M transitions without affecting cell viability. GlcN modulated the level and branching of N-glycans on EGFR, suppressed phosphorylation of EGFR, and reduced phosphorylation of extracellular signal-regulated kinases, erine/threonine protein kinase, and the signal transducer and activator of transcription 3 (STAT3). GlcN had only minor effects on the expression of Hsp90, Grp78, and transcription factor CHOP/GADD 153 markers of nonspecific stress in the endoplasmic reticulum. CONCLUSIONS: GlcN effectively suppressed proliferation of RPE cells in vitro. This effect appeared to be achieved through modification of N-glycans on EGFR. Further research into the role of GlcN as a potential agent for the prevention and treatment of RPE-mediated ocular proliferative disorders, such as proliferative vitreoretinopathy, and other EGF-dependent proliferative cell-growth disorders, is warranted.

Melatonin prolongs islet graft survival in diabetic NOD mice.

Islet transplantation has been established as a potential therapy for type 1 diabetes. However, inflammation, allorejection, and on-going autoimmune damage contribute to early graft loss and failure of islet transplantation. Melatonin is the major secretory product of the pineal gland during the dark period of each day and displays multifunctional properties including the regulation of circadian and seasonal rhythms, antioxidation reactions and immune modulation. Based on the immunosuppressive properties of melatonin, we investigated whether melatonin treatment prolonged the survival of islet grafts in non-obese diabetic (NOD) mice. The mean islet graft survival time was 7.33 +/- 1.51 and 7.75 +/- 2.66 days in untreated controls and in the solvent-treated animals, respectively. Strikingly, the mean survival time of islet grafts in recipients treated with melatonin (200 mg/kg/bw) was 17 +/- 7.76 days. Moreover, melatonin treatment reduced the proliferation of splenocytes in NOD mice. Using a T1 and T2 double transgenic mouse model, we found that T helper 1 (Th1) cells in mice treated with melatonin were significantly decreased. The reduction of Th1 cells and T cell proliferation may result from an increase in the immunosuppressive cytokine IL-10. Our results indicate that melatonin treatment suppresses autoimmune recurrence by inhibiting the proliferation of Th1 cells in NOD mice and thus prolongs the survival of syngeneic islet grafts.

Inhibitory effects of glucosamine on endotoxin-induced uveitis in Lewis rats.

PURPOSE: Glucosamine sulfate (GS) is a naturally occurring sugar that exerts immunosuppressive effects in vitro and in vivo. The authors investigated whether GS modulates the inflammatory reaction in endotoxin-induced uveitis (EIU) of rats and the mechanisms by which it exerts its effects. METHODS: Two-hundred micrograms of lipopolysaccharide (LPS) was injected subcutaneously into Lewis rats to induce EIU. Doses of GS (10, 100, or 1000 mg/kg) were divided into three aliquots and administered intraperitoneally 30 minutes before LPS injection, concurrently with LPS injection, and 30 minutes after LPS injection. Twenty-four hours after LPS injection, aqueous humor was collected for cell counting and measurement of protein concentration. Immunohistochemical staining of the iris-ciliary body was performed to evaluate the effects of GS on intercellular adhesion molecule (ICAM)-1 and nuclear factor (NF)-kappaB activation and to demonstrate macrophage infiltration. The effects of various doses of GS pretreatment were also examined using a mouse macrophage cell line (RAW264.7 cells) and LPS stimulation. Levels of prostaglandin (PG)-E2 and nitric oxide (NO) were determined. Expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were measured using Western blot analysis. The effect of GS on LPS-induced NF-kappaB activation in RAW cells was also examined. RESULTS: Cell counting and analysis of protein concentration in aqueous humor revealed that GS suppressed EIU in rats treated with a high dose of GS (1000 mg/kg). Immunohistochemistry showed that treatment with GS reduced ICAM-1 expression and suppressed activation of NF-kappaB in the iris-ciliary body. The main inflammatory cells in the iris-ciliary body during EIU were macrophages. In LPS-stimulated macrophage RAW cell culture, GS inhibited the production of NO and PG-E2, the expression of iNOS and COX-2, and the activation of NF-kappaB. CONCLUSIONS: GS suppresses EIU in rats by blockading the NF-kappaB-dependent signaling pathway and the subsequent production of ICAM-1 and proinflammatory mediators. This study has extended the authors' previous observation that GS is a potentially important compound for reducing ICAM-1-mediated inflammatory effects in the eye.

Glucosamine sulfate inhibits leukocyte adhesion in response to cytokine stimulation of retinal pigment epithelial cells in vitro.

Glucosamine is an amine-containing sugar that exhibits immunosuppressive effects in vitro and in vivo, although its mechanism of action is unknown. We investigated whether glucosamine sulfate (GS) modulates the proinflammatory cytokine interleukin (IL)-1beta-induced expression and production of intercellular adhesion molecule (ICAM)-1, the mechanism responsible for this effect, and whether GS inhibits leukocyte adhesion to the monolayer of retinal pigment epithelial (RPE) cells stimulated with various cytokines. We used flow cytometry and an ARPE-19 cell model to determine the effect of GS on the production of ICAM-1 in response to IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha plus IL-1beta, TNF-alpha plus IL-6, and TNF-alpha plus interferon (IFN)-gamma. We also used semiquantitative RT-PCR to determine the effect of GS on IL-1beta-induced expression of the ICAM-1 gene, and immunocytochemistry and western blotting to measure the effect of GS on the activation and nuclear translocation of the nuclear factor NF-kappaB and the degradation of cytoplasmic IkappaB. The functionality of GS-modulated ICAM-1 on leukocyte adhesion was demonstrated in an RPE cell-neutrophil adherence assay. IL-1beta increased the expression of ICAM-1 at the mRNA and protein levels in ARPE-19 cells. GS downregulated the production of ICAM-1 induced by IL-1beta, IL-6, TNF-alpha, and IFN-gamma at the protein level in a dose-dependent manner. GS also inhibited the nuclear translocation of NF-kappaB subunit p65 and partially prevented the degradation of cytoplasmic IkappaB in IL-1beta-stimulated ARPE-19 cells. GS significantly decreased the number of neutrophils adhering to the RPE monolayer in response to cytokines IL-1beta, IL-6, TNF-alpha, and IFN-gamma. GS inhibits the expression of the ICAM-1 gene in ARPE-19 cells stimulated with IL-1beta by blocking NF-kappaB subunit p65 translocation and by partially preventing IkappaB degradation. GS also decreases leukocyte adhesion to the monolayer of ARPE-19 cells stimulated with various cytokines by decreasing ICAM-1 production. Our study demonstrates a potentially important property of GS in reducing ICAM-1-mediated inflammatory mechanisms in the eye.

Glucosamine sulfate inhibits TNF-alpha and IFN-gamma-induced production of ICAM-1 in human retinal pigment epithelial cells in vitro.

PURPOSE: Glucosamine sulfate (GS) is a naturally occurring sugar that possesses some immunosuppressive effects in vitro and in vivo, but its mechanism is unknown. We investigated whether GS could modulate the proinflammatory cytokine-induced expression of the gene for intercellular adhesion molecule (ICAM)-1, an inflammatory protein in human retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used as a model to determine the effects of GS on the expression of the ICAM-1 gene upregulated by TNF-alpha or IFN-gamma, by Western blot analysis and semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The activation and nuclear translocation of the nuclear factors NF-kappaB and STAT1 were evaluated by immunocytochemistry, Western blot analysis, and electrophoretic mobility shift assay (EMSA). RESULTS: Both TNF-alpha and IFN-gamma increased the expression of ICAM-1 at the mRNA and protein levels in a time- and dose-dependent manner in ARPE-19 cells. GS effectively downregulated the TNF-alpha- or IFN-gamma-induced expression of ICAM-1 in the protein and mRNA level in a dose-dependent manner. GS further inhibited the nuclear translocation of p65 proteins in TNF-alpha and phosphorylated STAT1 in IFN-gamma-stimulated ARPE-19 cells. CONCLUSIONS: GS inhibits the expression of the ICAM-1 gene in ARPE-19 cell stimulated with TNF-alpha or IFN-gamma through blockade of NF-kappaB subunit p65 and nuclear translocation of STAT1. This study has demonstrated a potentially important property of GS in reducing ICAM-1 mediated inflammatory mechanisms in the eye.

Glucosamine sulfate inhibits proinflammatory cytokine-induced icam-1 production in human conjunctival cells in vitro.

PURPOSE: We investigated whether glucosamine sulfate modulates the production of ICAM-1 induced by proinflammatory cytokines and whether glucosamine sulfate inhibits leukocyte adhesion to a monolayer of human conjunctival epithelial cells stimulated with proinflammatory cytokines. METHODS: We used flow cytometry and either primary cultured human conjunctival cells or the Chang conjunctival cell model to determine the effects of glucosamine sulfate on the production of ICAM-1 in response to tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, TNF-alpha plus IFN-gamma, or TNF-alpha plus IL-1beta. The effects of glucosamine sulfate on the expression of the ICAM-1 gene, upregulated by various cytokines, were determined by semiquantitative reverse transcription-polymerase chain reaction. The activation and nuclear translocation of the nuclear factors NF-kappaB and STAT1 were evaluated by the transient transfection of reporter gene systems and immunocytochemistry. The influence of glucosamine-sulfate-modulated ICAM-1 on neutrophil adhesion was demonstrated in a model that measures the adherence of conjunctival cells and neutrophils. RESULTS: TNF-alpha, IFN-gamma, and IL-1beta significantly increased the production of ICAM-1 by both primary cultured human conjunctival cells and Chang conjunctival cells. Glucosamine sulfate effectively downregulated the production of ICAM-1 induced by TNF-alpha, IFN-gamma, IL-1beta, TNF-alpha plus IFN-gamma, or TNF-alpha plus IL-1beta. This downregulation occurred through the interferon-stimulated response element, IFN-gamma activation sequence, and binding sequence of NF-kappaB at the mRNA and protein levels. Glucosamine sulfate further inhibited the nuclear translocation of p65 protein in TNF-alpha- and IL-1beta-stimulated Chang conjunctival cells and phosphorylated STAT1 in IFN-gamma-stimulated Chang conjunctival cells. Glucosamine sulfate also significantly reduced the number of neutrophils adhering to a conjunctival monolayer in response to TNF-alpha, IFN-gamma, or IL-1beta. CONCLUSIONS: Our results suggest that glucosamine sulfate inhibits ICAM-1 production in conjunctival epithelial cells in vitro. Therefore, glucosamine sulfate might be valuable in the treatment of inflammatory ocular-surface conditions.

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells.

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.