Restoration of Follicular T Regulatory/Helper Cell Balance by OX40L-JAG1 Cotreatment Suppresses Lupus Nephritis in NZBWF1/j Mice.

Class-switched antinuclear autoantibodies produced by T follicular helper (TFH) cell-dependent germinal center (GC) B cell response play an essential pathogenic role in lupus nephritis (LN). The role of T follicular regulatory (TFR) cells, an effector subset of CD4+Foxp3+ T regulatory cells (Tregs), which are specialized in suppressing TFH-GC response and Ab production, remains elusive in LN. Contrasting reports have shown increased/reduced circulating TFR cells in human lupus that might not accurately reflect their presence in the GCs of relevant lymphoid organs. In this study, we report a progressive reduction in TFR cells and decreased TFR/TFH ratio despite increased Tregs in the renal lymph nodes of NZBWF1/j mice, which correlated with increased GC-B cells and proteinuria onset. Cotreatment with soluble OX40L and Jagged-1 (JAG1) proteins increased Tregs, TFR cells, and TFR/TFH ratio, with a concomitant reduction in TFH cells, GC B cells, and anti-dsDNA IgG Ab levels, and suppressed LN onset. Mechanistic studies showed attenuated TFH functions and diminished GC events such as somatic hypermutation and isotype class-switching in OX40L-JAG1-treated mice. RNA sequencing studies revealed inhibition of hypoxia-inducible factor 1-α (HIF-1a) and STAT3 signaling in T conventional cells from OX40L-JAG1-treated mice, which are critical for the glycolytic flux and differentiation into TFH cell lineage. Therefore, the increased TFR/TFH ratio seen in OX40L-JAG1-treated mice could involve both impaired differentiation of TFH cells from T conventional cells and expansion of TFR cells. We show a key role for GC-TFR/TFH imbalance in LN pathogenesis and how restoring homeostatic balance can suppress LN.

ß-Cell specific transcription factors in the context of diabetes mellitus and ß-cell regeneration.

All pancreatic cell populations arise from the standard gut endoderm layer in developing embryos, requiring a regulatory gene network to originate and maintain endocrine lineages and endocrine function. The pancreatic organogenesis is regulated by the temporal expression of transcription factors and plays a diverse role in the specification, development, differentiation, maturation, and functional maintenance. Altered expression and activity of these transcription factors are often associated with diabetes mellitus. Recent advancements in the stem cells and invitro derived islets to treat diabetes mellitus has attracted a great deal of interest in the understanding of factors regulating the development, differentiation, and functions of islets including transcription factors. This review discusses the myriad of transcription factors regulating the development of the pancreas, differentiation of ß-islets, and how these factors regulated in normal and disease states. Exploring these factors in such critical context and exogenous or endogenous expression of development and differentiation-specific transcription factors with improved epigenetic plasticity/signaling axis in diabetic milieu would useful for the development of ß-cells from other cell sources.

Post-translational modifications contribute to neoepitopes in Type-1 diabetes: Challenges for inducing antigen-specific tolerance.

Type-1 Diabetes (T1D) is the major autoimmune disease affecting the juvenile population in which insulin-producing pancreatic ß-cells are destroyed by self-reactive T-cells and B-cells. Emerging studies have identified the presence of autoantibodies and altered T-cell reactivity against several autoantigens in individuals who are at risk of developing T1D even before the clinical onset of diabetes. Whilst these findings could lead to the development of predictive biomarkers for early diagnosis, growing evidence on the generation of neoepitopes, epitope spreading and diverse antigen repertoire in T1D poses a major challenge for developing approaches to induce antigen-specific tolerance. Mechanisms of neoepitope generation include post-translational modifications of existing epitopes, aberrant translational products, peptide fusion, and differences in MHC binding registers. Here, we focus our discussion on how post-translational modifications can give rise to immunogenic neoepitopes in T1D and present our perspective on how it could affect the development of therapeutic approaches to induce antigen-specific tolerance.

TNF-α-mediated suppression of Leydig cell steroidogenesis involves DAX-1.

OBJECTIVE AND DESIGN: The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has an inhibitory role in gonadal functions particularly in the steroidogenesis of Leydig cells. A detailed understanding of the mechanisms by which TNF-α regulates testicular steroidogenesis will be helpful in the design of novel clinical interventions for the treatment and prevention of male reproductive disorders. Here, we report that TNF-α-mediated activation of DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is involved in the inhibition of Leydig cell steroidogenesis. MATERIALS AND METHODS: Rat testis Leydig tumor cells (LC-540) were treated with TNF-α (10 ng/ml) for different time intervals. To elucidate the pathways of intracellular signal transduction that regulate DAX-1 expression, we utilized specific inhibitors. The siRNA transfection of DAX-1 into LC-540 cells was performed by electroporation. The mRNA and protein levels were determined by RT-PCR and Western blotting, respectively. RESULTS: We found that the mRNA and protein levels of DAX-1 were increased by threefold approximately in TNF-α-treated cells when compared to controls. Staurosporine, JNK inhibitor SP600125 and ERK inhibitor PD98059 significantly decreased DAX-1 expression in TNF-α-treated Leydig cells when compared to their respective controls. Further, a siRNA-mediated knockdown of DAX-1 restores the expression of steroidogenic proteins in TNF-α-treated Leydig cells. CONCLUSIONS: These findings provide valuable information that TNF-α activates DAX-1 through JNK/ERK MAP kinase pathway which regulates the expression of steroidogenic enzyme genes in Leydig cells.

HDAC7 modulates TNF-α-mediated suppression of Leydig cell steroidogenesis.

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) has an inhibitory role in gonadal functions particularly in the steroidogenesis of Leydig cells. In the present study, we demonstrate that TNF-α activates histone deacetylases 7 (HDAC7), which regulates the expression of steroidogenic enzyme genes in Leydig cells. LC-540 Leydig cells were treated with TNF-α (10 ng/ml) for different time intervals. TNF-α treatment significantly suppressed histone H3 acetylation and methylation and, concomitantly, increased the total histone deacetylases activity in LC-540 Leydig cells. RT-PCR and western blot analysis revealed that HDAC7 was up-regulated in TNF-α-treated cells. Our results also demonstrated that an siRNA-mediated knockdown of HDAC7 restores the expression of steroidogenic proteins in TNF-α-treated Leydig cells. These findings provide valuable information that TNF-α-mediated suppression of steroidogenesis involves HDAC7 in Leydig cells.

The role of phosphoenolpyruvate carboxykinase in neuronal steroidogenesis under acute inflammation.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key gluconeogenic enzyme found in many tissues throughout the body including brain. In the present study, we have investigated the effect of bacterial lipopolysaccharide (LPS) on PEPCK and its role in neuronal steroidogenesis. Adult female albino rats were administered LPS (5mg/kg body weight) to induce acute inflammation. LPS administration resulted in a significant increase of PEPCK mRNA expression with concomitant increase in mRNA levels of steroidogenic acute regulatory (StAR) protein and other steroidogenic enzymes including 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and aromatase in brain tissue. Further, the inhibition of PEPCK expression by glipizide significantly decreased the mRNA expression of steroidogenic proteins and concurrently increased the mRNA levels of proinflammatory cytokines under LPS administration. The results of this study suggest a novel finding that PEPCK may have an important role in neuronal steroidogenesis; which serves as an adaptive response under inflammation.

Glucose modulates Pax6 expression through the JNK/p38 MAP kinase pathway in pancreatic beta-cells.

AIM: The paired and homeodomain-containing transcription factor, paired box 6 (Pax6), has shown to play pivotal roles in beta-cell function, including cell survival, insulin biosynthesis and secretion. The present study investigates the signaling events that regulate the modulation of Pax6 expression by glucose and the role of this modulation in cell survival in rat insulinoma-1E (INS-1E) cells. MAIN METHODS: INS-1E cells were incubated on 1mM (low) or 25 mM (high) glucose overnight. To elucidate the signaling pathways that regulate Pax6 expression, we utilized specific inhibitors. The siRNA transfection of Pax6 into INS-1E cells was performed by electroporation. The mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. KEY FINDINGS: We found that the mRNA and protein levels of Pax6 were reduced by approximately 4-fold in high, compared to low, glucose-treated cells. Staurosporine, the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 significantly increased Pax6 levels in high glucose-treated INS-1E cells compared to their respective controls. However, neither calcium ionophore nor the extracellular signal-regulated kinase (ERK) inhibitor U0126 resulted in any alteration in Pax6 protein expression. Further, a siRNA-mediated knockdown of Pax6 significantly decreased the expression of tumor-suppressor phosphatase with tensin homology (PTEN) while increasing cell viability in low glucose-treated INS-1E cells. SIGNIFICANCE: This study addresses the signaling events that regulate the glucose-dependent expression of Pax6 and the role of these events in cell survival in pancreatic beta cells.