OBJECTIVECarbohydrate-responsive element-binding protein (ChREBP) is a transcription factor that has been shown to regulate carbohydrate metabolism in the liver and pancreatic β-cells in response to elevated glucose concentrations. Because few genes have been identified so far as bona fide ChREBP-target genes, we have performed a genome-wide analysis of the ChREBP transcriptome in pancreatic β-cells.RESEARCH DESIGN AND METHODSChromatin immunoprecipitation and high-density oligonucleotide tiling arrays (ChIP-chip; Agilent Technologies) using MIN6 pancreatic β-cell extracts were performed together with transcriptional and other analysis using standard techniques.RESULTSOne of the genes identified by ChIP-chip and linked to glucose sensing and insulin secretion was aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor-1β (HIF-1β), a transcription factor implicated in altered gene expression and pancreatic-islet dysfunction in type 2 diabetes. We first confirmed that elevated glucose concentrations decreased ARNT/HIF-1β levels in INS-1 (832/13) cells and primary mouse islets. Demonstrating a role for ChREBP in ARNT gene regulation, ChREBP silencing increased ARNT mRNA levels in INS-1 (832/13) cells, and ChREBP overexpression decreased ARNT mRNA in INS-1 (832/13) cells and primary mouse islets. We demonstrated that ChREBP and Max-like protein X (MLX) bind on the ARNT/HIF-1β promoter on the proximal region that also confers the negative glucose responsiveness.CONCLUSIONSThese results demonstrate that ChREBP acts as a novel repressor of the ARNT/HIF-1β gene and might contribute to β-cell dysfunction induced by glucotoxicity.

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Mesangial cell apoptosis occurs in experimental diabetic nephropathy, and this correlates with worsening albuminuria. This study examines the mechanism by which glucose modulates mesangial cell apoptosis. Apoptosis was induced in mesangial cells by serum deprivation in the presence of 5 or 25 mM D-glucose, and examined by expression of Annexin-V and disruption of mitochondrial transmembrane potential. Involvement of Bax, Bcl-2 and NF-jB were examined by RT-PCR and EMSA. Involvement of TGF-b1 was sought by determining the effect of recombinant TGF-b1on apoptosis and the mediators of the apoptotic pathway (Bcl2/ Bax and NF-jB). Culture of cells in the presence of 25 mM D-glucose (i) enhanced apoptosis stimulated by serum depletion, (ii) enhanced activation of caspase-3, (iii) inhibited NF-jB activation, and (iv) decreased Bcl-2:Bax ratio. Inhibition of NF-jB using SN50, also increased mesangial cell apoptosis, and decreased Bcl-2:Bax ratio. Addition of TGF-b1 to mesangial cells mimicked the effect of high glucose reducing NF-jB expression and Bcl-2:Bax ratio. Furthermore glucose-mediated enhanced apoptosis was inhibited by the addition of a blocking antibody to TGF-b1. Exposure of mesangial cells to 25 mM D-glucose stimulated the generation of both total and active TGF-b1 in the cell culture supernatant, this increase was only significant after 48-72 h, that is at a time point later than enhanced apoptosis. Addition of 25 mM D-glucose, however, increased sensitivity of mesangial cells to TGF-b1 as assessed by luciferase activity of a Smad sensitive reporter construct. The data suggest that elevated glucose concentration enhanced the pathway leading to apoptosis following serum deprivation. Furthermore, it is likely that this is dependent on glucose-mediated enhanced sensitivity to endogenous TGF-b1 rather than glucose stimulated de novo TGF-b1 synthesis.

Experimental autoimmune uveoretinitis is a model for noninfectious posterior segment intraocular inflammation in humans. Although this disease is CD4+ T cell dependent, in the persistent phase of disease CD8+ T cells accumulate. We show that these are effector memory CD8+ T cells that differ from their splenic counterparts with respect to surface expression of CD69, CD103, and Ly6C. These retinal effector memory CD8+ T cells have limited cytotoxic effector function, are impaired in their ability to proliferate in response to Ag-specific stimulation, and upregulate programmed death 1 receptor. Treatment with fingolimod (FTY720) during the late phase of disease revealed that retinal CD8+ T cells were tissue resident. Despite signs of exhaustion, these cells were functional, as their depletion resulted in an expansion of retinal CD4+ T cells and CD11b+ macrophages. These results demonstrate that, during chronic autoimmune inflammation, exhausted CD8+ T cells become established in the local tissue. They are phenotypically distinct from peripheral CD8+ T cells and provide local signals within the tissue by expression of inhibitory receptors such as programmed death 1 that limit persistent inflammation.

ObjectivesRandomized controlled trials (RCTs) deliver robust internally valid evidence but generalizability is often neglected. Design features built into the Prostate testing for cancer and Treatment (ProtecT) RCT of treatments for localized prostate cancer (PCa) provided insights into its generalizability.Study Design and SettingPopulation-based cluster randomization created a prospective study of prostate-specific antigen (PSA) testing and a comprehensive-cohort study including groups choosing treatment or excluded from the RCT, as well as those randomized. Baseline information assessed selection and response during RCT conduct.ResultsThe prospective study (82,430 PSA-tested men) represented healthy men likely to respond to a screening invitation. The extended comprehensive cohort comprised 1,643 randomized, 997 choosing treatment, and 557 excluded with advanced cancer/comorbidities. Men choosing treatment were very similar to randomized men except for having more professional/managerial occupations. Excluded men were similar to the randomized socio-demographically but different clinically, representing less healthy men with more advanced PCa.ConclusionThe design features of the ProtecT RCT provided data to assess the representativeness of the prospective cohort and generalizability of the findings of the RCT. Greater attention to collecting data at the design stage of pragmatic trials would better support later judgments by clinicians/policy-makers about the generalizability of RCT findings in clinical practice.

Tumour necrosis factor‐α (TNF‐α) is a key mediator of inflammation in host defence against infection and in autoimmune disease. Its production is controlled post‐transcriptionally by multiple RNA‐binding proteins that interact with the TNF‐α AU‐rich element and regulate its expression; one of these is Fragile X mental retardation‐related protein 1 (FXR1). The anti‐inflammatory cytokine transforming growth factor‐β1 (TGF‐β1), which is involved in the homeostatic regulation of TNF‐α, causes post‐transcriptional suppression of lipopolysaccharide (LPS)‐induced TNF‐α production. We report here that this depends on FXR1. Using RAW 264.7 cells and bone marrow‐derived macrophages (BMDMϕ) stimulated with LPS and TGF‐β1, we show that TGF‐β1 inhibits TNF‐α protein secretion, whereas TNF‐α mRNA expression remains unchanged. This response is recapitulated by the 3′‐UTR of TNF‐α, which is known to bind FXR1. TGF‐β1 induces FXR1 with a pattern of expression distinct from that of tristetraprolin, T‐cell intracellular antigen 1, or human antigen R. When FXR1 is knocked down, TGF‐β1 is no longer able to inhibit LPS‐induced TNF‐α protein production, and overexpression of FXR1 suppresses LPS‐induced TNF‐α protein production. Targeting the p38 mitogen‐activated protein kinase pathway of LPS‐treated cells with small molecule inhibitors can induce FXR1 protein and mRNA expression. In summary, TGF‐β1 opposes LPS‐induced stabilization of TNF‐α mRNA and reduces the amount of TNF‐α protein, through induction of expression of the mRNA‐binding protein FXR1.

SummaryClinically available anti-tumour necrosis factor (TNF) biologics, which inhibit both soluble (sTNF) and transmembrane forms (tmTNF) of TNF, eliminating all TNF signalling, have successfully treated autoimmune diseases including uveitis. These have potentially serious side effects such as reactivation of latent Mycobacterium tuberculosis and, therefore, more specific inhibition of TNF signalling pathways may maintain clinical efficacy while reducing adverse effects. To determine the effects of specific pharmacological inhibition of sTNF on macrophage activation and migration, we used a mouse model of uveitis (experimental autoimmune uveoretinitis; EAU). We show that selective inhibition of sTNF is sufficient to suppress EAU by limiting inflammatory CD11b+ macrophages and CD4 + T cell migration into the eye. However, inhibition of both sTNF and tmTNF is required to inhibit interferon-g-induced chemokine receptor 2, CD40, major histocompatibility complex class II and nitric oxide (NO) up-regulation, and signalling via tmTNF is sufficient to mediate tissue damage. In confirmation, intravitreal inhibition of sTNF alone did not suppress disease, and inflammatory cells that migrated into the eye were activated, generating NO, thus causing structural damage to the retina. In contrast, intravitreal inhibition of both sTNF and tmTNF suppressed macrophage activation and therefore disease. We conclude that sTNF is required for inflammatory cell infiltration into target tissue, but at the tissue site inhibition of both sTNF and tmTNF is required to inhibit macrophage activation and to protect from tissue damage.

Ocular function depends on a high level of anatomical integrity. This is threatened by inflammation, which alters the local tissue over short and long time-scales. Uveitis due to autoimmune disease, especially when it involves the retina, leads to persistent changes in how the eye interacts with the immune system. The normal pattern of immune surveillance, which for immune privileged tissues is limited, is re-programmed. Many cell types, that are not usually present in the eye, become detectable. There are changes in the tissue homeostasis and integrity. In both human disease and mouse models, in the most extreme cases, immunopathological findings consistent with development of ectopic lymphoid-like structures and disrupted angiogenesis accompany severely impaired eye function. Understanding how the ocular environment is shaped by persistent inflammation is crucial to developing novel approaches to treatment.