Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation.

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

Puerarin attenuates diabetic kidney injury through interaction with Guanidine nucleotide-binding protein Gi subunit alpha-1 (Gnai1) subunit.

Radix puerariae, a traditional Chinese herbal medication, has been used to treat patients with diabetic kidney disease (DKD). Our previous studies demonstrated that puerarin, the active compound of radix puerariae, improves podocyte injury in type 1 DKD mice. However, the direct molecular target of puerarin and its underlying mechanisms in DKD remain unknown. In this study, we confirmed that puerarin also improved DKD in type 2 diabetic db/db mice. Through RNA-sequencing odf isolated glomeruli, we found that differentially expressed genes (DEGs) that were altered in the glomeruli of these diabetic mice but reversed by puerarin treatment were involved mostly in oxidative stress, inflammatory and fibrosis. Further analysis of these reversed DEGs revealed protein kinase A (PKA) was among the top pathways. By utilizing the drug affinity responsive target stability method combined with mass spectrometry analysis, we identified guanine nucleotide-binding protein Gi alpha-1 (Gnai1) as the direct binding partner of puerarin. Gnai1 is an inhibitor of cAMP production which is known to have protection against podocyte injury. In vitro, we showed that puerarin not only interacted with Gnai1 but also increased cAMP production in human podocytes and mouse diabetic kidney in vivo. Puerarin also enhanced CREB phosphorylation, a downstream transcription factor of cAMP/PKA. Overexpression of CREB reduced high glucose-induced podocyte apoptosis. Inhibition of PKA by Rp-cAMP also diminished the effects of puerarin on high glucose-induced podocyte apoptosis. We conclude that the renal protective effects of puerarin are likely through inhibiting Gnai1 to activate cAMP/PKA/CREB pathway in podocytes.

The many lives of Myc in the pancreatic ß-cell.

Diabetes results from insufficient numbers of functional pancreatic ß-cells. Thus, increasing the number of available functional ß-cells ex vivo for transplantation, or regenerating them in situ in diabetic patients, is a major focus of diabetes research. The transcription factor, Myc, discovered decades ago lies at the nexus of most, if not all, known proliferative pathways. Based on this, many studies in the 1990s and early 2000s explored the potential of harnessing Myc expression to expand ß-cells for diabetes treatment. Nearly all these studies in ß-cells used pathophysiological or supraphysiological levels of Myc and reported enhanced ß-cell death, dedifferentiation, or the formation of insulinomas if cooverexpressed with Bcl-xL, an inhibitor of apoptosis. This obviously reduced the enthusiasm for Myc as a therapeutic target for ß-cell regeneration. However, recent studies indicate that "gentle" induction of Myc expression enhances ß-cell replication without induction of cell death or loss of insulin secretion, suggesting that appropriate levels of Myc could have therapeutic potential for ß-cell regeneration. Furthermore, although it has been known for decades that Myc is induced by glucose in ß-cells, very little is known about how this essential anabolic transcription factor perceives and responds to nutrients and increased insulin demand in vivo. Here we summarize the previous and recent knowledge of Myc in the ß-cell, its potential for ß-cell regeneration, and its physiological importance for neonatal and adaptive ß-cell expansion.

GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells.

GM-CSF (Csf-2) is a critical cytokine for the in vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103(+) DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103(+) and CD11b(+) DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8(+) T cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8(+) T cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in vivo.

BET N-terminal bromodomain inhibition selectively blocks Th17 cell differentiation and ameliorates colitis in mice.

T-helper 17 (Th17) cells have important functions in adaptor immunity and have also been implicated in inflammatory disorders. The bromodomain and extraterminal domain (BET) family proteins regulate gene transcription during lineage-specific differentiation of naïve CD4+ T cells to produce mature T-helper cells. Inhibition of acetyl-lysine binding of the BET proteins by pan-BET bromodomain (BrD) inhibitors, such as JQ1, broadly affects differentiation of Th17, Th1, and Th2 cells that have distinct immune functions, thus limiting their therapeutic potential. Whether these BET proteins represent viable new epigenetic drug targets for inflammatory disorders has remained an unanswered question. In this study, we report that selective inhibition of the first bromodomain of BET proteins with our newly designed small molecule MS402 inhibits primarily Th17 cell differentiation with a little or almost no effect on Th1 or Th2 and Treg cells. MS402 preferentially renders Brd4 binding to Th17 signature gene loci over those of housekeeping genes and reduces Brd4 recruitment of p-TEFb to phosphorylate and activate RNA polymerase II for transcription elongation. We further show that MS402 prevents and ameliorates T-cell transfer-induced colitis in mice by blocking Th17 cell overdevelopment. Thus, selective pharmacological modulation of individual bromodomains likely represents a strategy for treatment of inflammatory bowel diseases.

T follicular helper cells restricted by IRF8 contribute to T cell-mediated inflammation.

The follicular helper T cell (TFH) are established regulators of germinal center (GC) B cells, whether TFH have pathogenic potential independent of B cells is unknown. Based on in vitro TFH cell differentiation, in vivo T cell transfer animal colitis model, and intestinal tissues of inflammatory bowel disease (IBD) patients, TFH and its functions in colitis development were analyzed by FACS, ChIP, ChIP-sequencing, WB, ELISA and PCR. Herein we demonstrate that intestinal tissues of patients and colon tissues obtained from Rag1-/- recipients of naïve CD4+ T cells with colitis, each over-express TFH-associated gene products. Adoptive transfer of naïve Bcl6-/- CD4+ T cells into Rag1-/- recipient mice abrogated development of colitis and limited TFH differentiation in vivo, demonstrating a mechanistic link. In contrast, T cell deficiency of interferon regulatory factor 8 (IRF8) resulted in augmentation of TFH induction in vitro and in vivo. Functional studies showed that adoptive transfer of IRF8 deficient CD4+ T cells into Rag1-/- recipients exacerbated colitis development associated with increased gut TFH-related gene expression, while Irf8-/-/Bcl6-/- CD4+ T cells abrogated colitis, together indicating that IRF8-regulated TFH can directly cause colon inflammation. Molecular analyses revealed that IRF8 suppresses TFH differentiation by inhibiting transcription and transactivation of the TF IRF4, which is also known to be essential for TFH induction. Our documentation showed that IRF8-regulated TFH can function as B-cell-independent, pathogenic, mediators of colitis suggests that targeting TFH could be effective for treatment of IBD.

A network medicine approach to build a comprehensive atlas for the prognosis of human cancer.

The Cancer Genome Atlas project has generated multi-dimensional and highly integrated genomic data from a large number of patient samples with detailed clinical records across many cancer types, but it remains unclear how to best integrate the massive amount of genomic data into clinical practice. We report here our methodology to build a multi-dimensional subnetwork atlas for cancer prognosis to better investigate the potential impact of multiple genetic and epigenetic (gene expression, copy number variation, microRNA expression and DNA methylation) changes on the molecular states of networks that in turn affects complex cancer survivorship. We uncover an average of 38 novel subnetworks in the protein-protein interaction network that correlate with prognosis across four prominent cancer types. The clinical utility of these subnetwork biomarkers was further evaluated by prognostic impact evaluation, functional enrichment analysis, drug target annotation, tumor stratification and independent validation. Some pathways including the dynactin, cohesion and pyruvate dehydrogenase-related subnetworks are identified as promising new targets for therapy in specific cancer types. In conclusion, this integrative analysis of existing protein interactome and cancer genomics data allows us to systematically dissect the molecular mechanisms that underlie unexpected outcomes for cancer, which could be used to better understand and predict clinical outcomes, optimize treatment and to provide new opportunities for developing therapeutics related to the subnetworks identified.

IFN regulatory factor 8 represses GM-CSF expression in T cells to affect myeloid cell lineage differentiation.

During hematopoiesis, hematopoietic stem cells constantly differentiate into granulocytes and macrophages via a distinct differentiation program that is tightly controlled by myeloid lineage-specific transcription factors. Mice with a null mutation of IFN regulatory factor 8 (IRF8) accumulate CD11b(+)Gr1(+) myeloid cells that phenotypically and functionally resemble tumor-induced myeloid-derived suppressor cells (MDSCs), indicating an essential role of IRF8 in myeloid cell lineage differentiation. However, IRF8 is expressed in various types of immune cells, and whether IRF8 functions intrinsically or extrinsically in regulation of myeloid cell lineage differentiation is not fully understood. In this study, we report an intriguing finding that, although IRF8-deficient mice exhibit deregulated myeloid cell differentiation and resultant accumulation of CD11b(+)Gr1(+) MDSCs, surprisingly, mice with IRF8 deficiency only in myeloid cells exhibit no abnormal myeloid cell lineage differentiation. Instead, mice with IRF8 deficiency only in T cells exhibited deregulated myeloid cell differentiation and MDSC accumulation. We further demonstrated that IRF8-deficient T cells exhibit elevated GM-CSF expression and secretion. Treatment of mice with GM-CSF increased MDSC accumulation, and adoptive transfer of IRF8-deficient T cells, but not GM-CSF-deficient T cells, increased MDSC accumulation in the recipient chimeric mice. Moreover, overexpression of IRF8 decreased GM-CSF expression in T cells. Our data determine that, in addition to its intrinsic function as an apoptosis regulator in myeloid cells, IRF8 also acts extrinsically to repress GM-CSF expression in T cells to control myeloid cell lineage differentiation, revealing a novel mechanism that the adaptive immune component of the immune system regulates the innate immune cell myelopoiesis in vivo.

Regulation of pathogenic Th17 cell differentiation by IL-10 in the development of glomerulonephritis.

Although it is clear that T helper (Th)17 cells play a pathologic role in the pathogenesis of several inflammatory diseases, the contribution and regulation of pathogenic Th17 cells in the development of glomerulonephritis are still not fully understood. Herein, we show that IL-10-deficient mice exhibit exacerbation of glomerulonephritis after induction with anti-glomerular basement membrane globulin, with enhanced pathogenic Th17 immune responses. We further demonstrate that Rag1(-/-) mice reconstituted with IL-10(-/-) CD4(+) T cells develop more severe glomerulonephritis after induction of anti-glomerular basement membrane disease, with more infiltration of inflammatory cells into the kidneys. Finally, IL-17 and interferon γ double-positive cells were significantly increased in IL-10(-/-) CD4(+) T-cell cultures under pathogenic Th17 conditions compared with wild-type cell cultures. These findings suggest that T-cell-derived IL-10 plays a critical suppressive role in the control of pathogenic Th17 cell differentiation and highlights the importance of IL-10 as protection against glomerulonephritis development.

Harmine and exendin-4 combination therapy safely expands human ß cell mass in vivo in a mouse xenograft system.

Five hundred thirty-seven million people globally suffer from diabetes. Insulin-producing ß cells are reduced in number in most people with diabetes, but most individuals still have some residual ß cells. However, none of the many diabetes drugs in common use increases human ß cell numbers. Recently, small molecules that inhibit dual tyrosine-regulated kinase 1A (DYRK1A) have been shown to induce immunohistochemical markers of human ß cell replication, and this is enhanced by drugs that stimulate the glucagon-like peptide 1 (GLP1) receptor (GLP1R) on ß cells. However, it remains to be demonstrated whether these immunohistochemical findings translate into an actual increase in human ß cell numbers in vivo. It is also unknown whether DYRK1A inhibitors together with GLP1R agonists (GLP1RAs) affect human ß cell survival. Here, using an optimized immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) protocol in mouse kidneys bearing human islet grafts, we demonstrate that combination of a DYRK1A inhibitor with exendin-4 increases actual human ß cell mass in vivo by a mean of four- to sevenfold in diabetic and nondiabetic mice over 3 months and reverses diabetes, without alteration in human α cell mass. The augmentation in human ß cell mass occurred through mechanisms that included enhanced human ß cell proliferation, function, and survival. The increase in human ß cell survival was mediated, in part, by the islet prohormone VGF. Together, these findings demonstrate the therapeutic potential and favorable preclinical safety profile of the DYRK1A inhibitor-GLP1RA combination for diabetes treatment.

LGR4 is essential for maintaining ß-cell homeostasis through suppression of RANK.

Pancreatic ß-cell stress contributes to diabetes progression. This study demonstrates that Leucine-rich repeat-containing G-protein-coupled-receptor-4 (LGR4) is critical for maintaining ß-cell health and is modulated by stressors. In vitro , Lgr4 knockdown decreases proliferation and survival in rodent ß-cells, while overexpression protects against cytokine-induced cell death in rodent and human ß-cells. Mechanistically, LGR4 suppresses Receptor Activator of Nuclear Factor Kappa B (NFκB) (RANK) and its subsequent activation of NFκB to protect ß-cells. ß-cell-specific Lgr4 -conditional knockout (cko) mice exhibit normal glucose homeostasis but increased ß-cell death in both sexes and decreased proliferation only in females. Male Lgr4 cko mice under stress display reduced ß-cell proliferation and a further increase in ß-cell death. Upon aging, both male and female Lgr4 cko mice display impaired ß-cell homeostasis, however, only female mice are glucose intolerant with decreased plasma insulin. We show that LGR4 is required for maintaining ß-cell health under basal and stress-induced conditions, through suppression of RANK. Teaser: LGR4 receptor is critical for maintaining ß-cell health under basal and stressed conditions, through suppression of RANK.

Cholesterol 25-Hydroxylase Protects Against Diabetic Kidney Disease by Regulating ADP Ribosylation Factor 4.

Cholesterol 25-hydroxylase (CH25H), an enzyme involved in cholesterol metabolism, regulates inflammatory responses and lipid metabolism. However, its role in kidney disease is not known.  The author found that CH25H transcript is expressed mostly in glomerular and peritubular endothelial cells and that its expression increased in human and mouse diabetic kidneys.  Global deletion of Ch25h in Leprdb/db mice aggravated diabetic kidney disease (DKD), which is associated with increased endothelial cell apoptosis. Treatment of 25-hydroxycholesterol (25-HC), the product of CH25H, alleviated kidney injury in Leprdb/db mice. Mechanistically, 25-HC binds to GTP-binding protein ADP-ribosylation factor 4 (ARF4), an essential protein required for maintaining protein transport in the Golgi apparatus. Interestingly, ARF4's GTPase-activating protein ASAP1 is also predominantly expressed in endothelial cells and its expression increased in DKD. Suppression of ARF4 activity by deleting ARF4 or overexpressing ASAP1 results in endothelial cell death. These results indicate that 25-HC binds ARF4 to inhibit its interaction with ASAP1, and thereby resulting in enhanced ARF4 activity to confer renoprotection. Therefore, treatment of 25-HC improves kidney injury in DKD in part by restoring ARF4 activity to maintain endothelial cell survival. This study provides a novel mechanism and a potential new therapy for DKD.

Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes ß-cell subpopulations with dynamic transcriptome profiles.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo. On the other hand, single-nucleus RNA sequencing (snRNA-seq) has compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and affords enhanced information from intronic sequences that can be leveraged to identify pre-mRNA transcripts. METHODS: We obtained nuclear preparations from fresh human islet cells and generated snRNA-seq datasets. We compared these datasets to scRNA-seq output obtained from human islet cells from the same donor. We employed snRNA-seq to obtain the transcriptomic profile of human islets engrafted in immunodeficient mice. In both analyses, we included the intronic reads in the snRNA-seq data with the GRCh38-2020-A library. RESULTS: First, snRNA-seq analysis shows that the top four differentially and selectively expressed genes in human islet endocrine cells in vitro and in vivo are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (ß-cells); PTPRT, FAP, PDK4, LOXL4 (α-cells); LRFN5, ADARB2, ERBB4, KCNT2 (δ-cells); and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ-cells). Second, by integrating information from scRNA-seq and snRNA-seq of human islet cells, we distinguish three ß-cell sub-clusters: an INS pre-mRNA cluster (ß3), an intermediate INS mRNA cluster (ß2), and an INS mRNA-rich cluster (ß1). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA-rich cluster (ß1) becomes the predominant sub-cluster in vivo. CONCLUSIONS: In summary, snRNA-seq and pre-mRNA analysis of human islet cells can accurately identify human islet cell populations, subpopulations, and their dynamic transcriptome profile in vivo.

Human Pancreatic α-Cell Heterogeneity and Trajectory Inference Analysis Using Integrated Single Cell- and Single Nucleus-RNA Sequencing Platforms.

Prior studies have shown that pancreatic α-cells can transdifferentiate into ß-cells, and that ß-cells de-differentiate and are prone to acquire an α-cell phenotype in type 2 diabetes (T2D). However, the specific human α-cell and ß-cell subtypes that are involved in α-to-ß-cell and ß-to-α-cell transitions are unknown. Here, we have integrated single cell RNA sequencing (scRNA-seq) and single nucleus RNA-seq (snRNA-seq) of isolated human islets and human islet grafts and provide additional insight into α-ß cell fate switching. Using this approach, we make seven novel observations. 1) There are five different GCG -expressing human α-cell subclusters [α1, α2, α-ß-transition 1 (AB-Tr1), α-ß-transition 2 (AB-Tr2), and α-ß (AB) cluster] with different transcriptome profiles in human islets from non-diabetic donors. 2) The AB subcluster displays multihormonal gene expression, inferred mostly from snRNA-seq data suggesting identification by pre-mRNA expression. 3) The α1, α2, AB-Tr1, and AB-Tr2 subclusters are enriched in genes specific for α-cell function while AB cells are enriched in genes related to pancreatic progenitor and ß-cell pathways; 4) Trajectory inference analysis of extracted α- and ß-cell clusters and RNA velocity/PAGA analysis suggests a bifurcate transition potential for AB towards both α- and ß-cells. 5) Gene commonality analysis identifies ZNF385D, TRPM3, CASR, MEG3 and HDAC9 as signature for trajectories moving towards ß-cells and SMOC1, PLCE1, PAPPA2, ZNF331, ALDH1A1, SLC30A8, BTG2, TM4SF4, NR4A1 and PSCK2 as signature for trajectories moving towards α-cells. 6) Remarkably, in contrast to the events in vitro , the AB subcluster is not identified in vivo in human islet grafts and trajectory inference analysis suggests only unidirectional transition from α-to-ß-cells in vivo . 7) Analysis of scRNA-seq datasets from adult human T2D donor islets reveals a clear unidirectional transition from ß-to-α-cells compatible with dedifferentiation or conversion into α-cells. Collectively, these studies show that snRNA-seq and scRNA-seq can be leveraged to identify transitions in the transcriptional status among human islet endocrine cell subpopulations in vitro , in vivo , in non-diabetes and in T2D. They reveal the potential gene signatures for common trajectories involved in interconversion between α- and ß-cells and highlight the utility and power of studying single nuclear transcriptomes of human islets in vivo . Most importantly, they illustrate the importance of studying human islets in their natural in vivo setting.

FDA-Approved Excipient N, N-Dimethylacetamide Attenuates Inflammatory Bowel Disease in In Vitro and In Vivo Models.

Inflammatory bowel disease (IBD) affects almost 7 million people worldwide and is increasing in incidence. While the precise pathogenesis of IBD remains unknown, the production of inflammatory cytokines and chemokines play a central role. We have previously found that N, N-dimethylacetamide (DMA), a widely used non-toxic drug excipient, suppresses cytokine and chemokine secretion in vitro and prevents inflammation-induced preterm birth in vivo. Using sandwich enzyme-linked immunosorbent assays (ELISAs), we tested whether DMA attenuates cytokine and chemokine secretion from LPS- or TNFα-stimulated human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. To test our hypothesis that the mechanism of DMA's effects in in vitro and in vivo models of IBD is inhibition of the NF-κB pathway, we used western blotting to track levels of the nuclear factor kappa B (NF-κB) inhibitory molecule I kappa B alpha (IκBα) in THP-1 human monocytes in the absence or presence of DMA. Finally, we induced colitis in C57Bl/6 mice with dextran sodium sulfate (DSS) and then tested whether i.p injections of DMA at 2.1 g/kg/day attenuates clinical and histopathologic signs of colitis. DMA attenuated cytokine and chemokine release from human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. Importantly, DMA prevented degradation of IκBα in THP-1 cells, thereby suggesting one mechanism for DMA's effects. Finally, we show here, for the first time, that DMA attenuates clinical and histologic features of DSS-induced colitis. Based on these data, DMA should be further explored in preclinical and clinical trials for its potential as novel drug therapy for IBD.

Potentiation of Th17 cytokines in aging process contributes to the development of colitis.

Th17 cells, which produce IL-17 and IL-22, promote autoimmunity in mice and have been implicated in the pathogenesis of autoimmune/inflammatory diseases in humans. However, the Th17 immune response in the aging process is still not clear. In the present study, we found that the induction of IL-17-producing CD4(+) T cells was significantly increased in aged individuals compared with young healthy ones. The mRNA expression of IL-17, IL-17F, IL-22, and RORC2 was also significantly increased in aged people. Similar to humans, Th17 cells as well as mRNAs encoding IL-17, IL-22 and RORγt were dramatically elevated in naïve T cells from aged mouse compared to young ones. In addition, CD44 positive IL-17-producing CD4(+) T cells were significantly higher in aged mice, suggesting that memory T cells are an important source of IL-17 production. Furthermore, the percentage of IL-17-producing CD4(+) T cells generated in co-culture with dendritic cells from either aged or young mice did not show significant differences, suggesting that dendritic cells do not play a primary role in the elevation of Th17 cytokines in aged mouse cells. Importantly, transfer of CD4(+)CD45Rb(hi) cells from aged mice induced more severe colitis in RAG(-/-) mice compared to cells from young mice, Taken together, these results suggest that Th17 immune responses are elevated in aging humans and mice and may contribute to the increased development of inflammatory disorders in the elderly.

Dextran Sulfate Protects Pancreatic ß-Cells, Reduces Autoimmunity, and Ameliorates Type 1 Diabetes.

A failure in self-tolerance leads to autoimmune destruction of pancreatic ß-cells and type 1 diabetes (T1D). Low-molecular-weight dextran sulfate (DS) is a sulfated semisynthetic polysaccharide with demonstrated cytoprotective and immunomodulatory properties in vitro. However, whether DS can protect pancreatic ß-cells, reduce autoimmunity, and ameliorate T1D is unknown. In this study, we report that DS, but not dextran, protects human ß-cells against cytokine-mediated cytotoxicity in vitro. DS also protects mitochondrial function and glucose-stimulated insulin secretion and reduces chemokine expression in human islets in a proinflammatory environment. Interestingly, daily treatment with DS significantly reduces diabetes incidence in prediabetic NOD mice and, most importantly, reverses diabetes in early-onset diabetic NOD mice. DS decreases ß-cell death, enhances islet heparan sulfate (HS)/HS proteoglycan expression, and preserves ß-cell mass and plasma insulin in these mice. DS administration also increases the expression of the inhibitory costimulatory molecule programmed death-1 (PD-1) in T cells, reduces interferon-γ+CD4+ and CD8+ T cells, and enhances the number of FoxP3+ cells. Collectively, these studies demonstrate that the action of one single molecule, DS, on ß-cell protection, extracellular matrix preservation, and immunomodulation can reverse diabetes in NOD mice, highlighting its therapeutic potential for the treatment of T1D.

Myc Is Required for Adaptive ß-Cell Replication in Young Mice but Is Not Sufficient in One-Year-Old Mice Fed With a High-Fat Diet.

Failure to expand pancreatic ß-cells in response to metabolic stress leads to excessive workload resulting in ß-cell dysfunction, dedifferentiation, death, and development of type 2 diabetes. In this study, we demonstrate that induction of Myc is required for increased pancreatic ß-cell replication and expansion during metabolic stress-induced insulin resistance with short-term high-fat diet (HFD) in young mice. ß-Cell-specific Myc knockout mice fail to expand adaptively and show impaired glucose tolerance and ß-cell dysfunction. Mechanistically, PKCζ, ERK1/2, mTOR, and PP2A are key regulators of the Myc response in this setting. DNA methylation analysis shows hypomethylation of cell cycle genes that are Myc targets in islets from young mice fed with a short-term HFD. Importantly, DNA hypomethylation of Myc response elements does not occur in islets from 1-year-old mice fed with a short-term HFD, impairing both Myc recruitment to cell cycle regulatory genes and ß-cell replication. We conclude that Myc is required for metabolic stress-mediated ß-cell expansion in young mice, but with aging, Myc upregulation is not sufficient to induce ß-cell replication by, at least partially, an epigenetically mediated resistance to Myc action.

Dendritic cell-derived nitric oxide inhibits the differentiation of effector dendritic cells.

Dendritic cells (DCs) play a pivotal role in the development of effective immune defense while avoiding detrimental inflammation and autoimmunity by regulating the balance of adaptive immunity and immune tolerance. However, the mechanisms that govern the effector and regulatory functions of DCs are incompletely understood. Here, we show that DC-derived nitric oxide (NO) controls the balance of effector and regulatory DC differentiation. Mice deficient in the NO-producing enzyme inducible nitric oxide synthase (iNOS) harbored increased effector DCs that produced interleukin-12, tumor necrosis factor (TNF) and IL-6 but normal numbers of regulatory DCs that expressed IL-10 and programmed cell death-1 (PD-1). Furthermore, an iNOS-specific inhibitor selectively enhanced effector DC differentiation, mimicking the effect of iNOS deficiency in mice. Conversely, an NO donor significantly suppressed effector DC development. Furthermore, iNOS-/- DCs supported enhanced T cell activation and proliferation. Finally iNOS-/- mice infected with the enteric pathogen Citrobacter rodentium suffered more severe intestinal inflammation with concomitant expansion of effector DCs in colon and spleen. Collectively, our results demonstrate that DC-derived iNOS restrains effector DC development, and offer the basis of therapeutic targeting of iNOS in DCs to treat autoimmune and inflammatory diseases.

Reprogramming macrophage orientation by microRNA 146b targeting transcription factor IRF5.

The regulation of macrophage orientation pathological conditions is important but still incompletely understood. Here, we show that IL-10 and Rag1 double knockout mice spontaneously develop colitis with dominant M1 macrophage phenotype, suggesting that IL-10 regulates macrophage orientation in inflammation. We demonstrate that IL-10 stimulation induced miR-146b expression, and that the expression of miR-146b was impaired in IL-10 deficient macrophages. Our data show that miR-146b targets IRF5, resulting in the regulation of macrophage activation. Furthermore, miR-146b deficient mice developed intestinal inflammation with enhanced M1 macrophage polarization. Finally, miR-146b mimic treatment significantly suppresses M1 macrophage activation and ameliorates colitis development in vivo. Collectively, the results suggest that IL-10 dependent miR-146b plays an important role in the modulation of M1 macrophage orientation.