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Undifferentiated uveitis (intraocular inflammation, IOI) is an idiopathic sight-threatening, presumed autoimmune disease, accountable for ~ 10% of all blindness in the developed world. We have investigated the association of uveitis with inflammatory bowel disease (IBD) using a mouse model of spontaneous experimental autoimmune uveoretinitis (EAU). Mice expressing the transgene (Tg) hen egg lysozyme (HEL) in the retina were crossed with 3A9 mice expressing a transgenic HEL-specific TCR. Double transgenic (dTg TCR/HEL) mice with EAU also spontaneously develop clinical signs of colitis at ~ P30 (post-partum day) with diarrhoea, bowel shortening, oedema and lamina propria (LP) inflammatory cell infiltration. Single (s)Tg TCR (3A9) mice also show histological LP cell infiltration but no clinical signs. dTg TCR/HEL mice are profoundly lymphopenic at weaning. In addition, dTg TCR/HEL mice contain myeloid cells which express MHC Class II-HEL peptide (MHCII-HEL) complexes, not only in the inflamed retina but also in the colon. In this model the lymphopenia and reduction in the absolute Treg numbers in dTg TCR/HEL mice is sufficient to initiate eye disease. We suggest that cell-associated antigen released from the inflamed eye, activates colonic HEL-specific T cells which, in a microbial micro-environment, not only cause colitis but feed-back to amplify IOI.

Cell therapies for autoimmune diseases using tolerogenic dendritic cells (tolDC) have been promisingly explored. A major stumbling block has been generating stable tolDC, with low risk of converting to mature immunogenic DC (mDC), exacerbating disease. mDC induction involves a metabolic shift to lactate production from oxidative phosphorylation (OXPHOS) and β-oxidation, the homeostatic energy source for resting DC. Inhibition of glycolysis through the administration of 2-deoxy glucose (2-DG) has been shown to prevent autoimmune disease experimentally but is not clinically feasible. We show here that treatment of mouse bone marrow-derived tolDC ex vivo with low-dose 2-DG (2.5 mM) (2-DGtolDC) induces a stable tolerogenic phenotype demonstrated by their failure to engage lactate production when challenged with mycobacterial antigen (Mtb). ~ 15% of 2-DGtolDC express low levels of MHC class II and 30% express CD86, while they are negative for CD40. 2-DGtolDC also express increased immune checkpoint molecules PDL-1 and SIRP-1α. Antigen-specific T cell proliferation is reduced in response to 2-DGtolDC in vitro. Mtb-stimulated 2-DGtolDC do not engage aerobic glycolysis but respond to challenge via increased OXPHOS. They also have decreased levels of p65 phosphorylation, with increased phosphorylation of the non-canonical p100 pathway. A stable tolDC phenotype is associated with sustained SIRP-1α phosphorylation and p85-AKT and PI3K signalling inhibition. Further, 2-DGtolDC preferentially secrete IL-10 rather than IL-12 upon Mtb-stimulation. Importantly, a single subcutaneous administration of 2-DGtolDC prevented experimental autoimmune uveoretinitis (EAU) in vivo. Inhibiting glycolysis of autologous tolDC prior to transfer may be a useful approach to providing stable tolDC therapy for autoimmune/immune-mediated diseases. Electronic supplementary material The online version of this article (10.1007/s00018-020-03672-y) contains supplementary material, which is available to authorized users.

We showed previously that 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide hydrochloride (EDC) cross-linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4 years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers-bulky, aromatic N-cyclohexyl-N0-(2-morpholinoethyl)-carbodiimide (CMC), and nonbulky EDC-in a mouse corneal graft model. Murine corneas undergoing full-thickness implantation with these gels became opaque due to dense retro-corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c+ dendritic cells (DC) were found, suggesting a hydrogel-specific innate immune response. More DC infiltrated the stroma surrounding EDC-N-hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis-associated tenascin c expression. The opposite was true for CMC-NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor β1 (TGF-β1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF-β1 neutralisation. Furthermore, tenascin c staining was found in 40- to 50μm long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF-β1 alternatively activated (tolerising) DC regulate fibroblast-mediated tenascin c secretion, possibly via local production of TGF-β1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.

Immune privilege (IP), a term introduced to explain the unpredicted acceptance of allogeneic grafts by the eye and the brain, is considered a unique property of these tissues. However, immune responses are modified by the tissue in which they occur, most of which possess IP to some degree. The eye therefore displays a spectrum of IP because it comprises several tissues. IP as originally conceived can only apply to the retina as it contains few tissue-resident bone-marrow derived myeloid cells and is immunologically shielded by a sophisticated barrier – an inner vascular and an outer epithelial barrier at the retinal pigment epithelium. The vascular barrier comprises the vascular endothelium and the glia limitans. Immune cells do not cross the blood-retinal barrier (BRB) despite two-way transport of interstitial fluid, governed by tissue oncotic pressure. The BRB, and the blood-brain barrier (BBB) mature in the neonatal period under signals from the expanding microbiome and by 18 months are fully established. However, the adult eye is susceptible to intraocular inflammation (uveitis; frequency ~200/100,000 population). Uveitis involving the retinal parenchyma (posterior uveitis, PU) breaches IP, while IP is essentially irrelevant in inflammation involving the ocular chambers, uveal tract and ocular coats (anterior/intermediate uveitis/sclerouveitis, AU). Infections cause ~50% cases of AU and PU but infection may also underlie the pathogenesis of immune-mediated “non-infectious” uveitis. Dysbiosis accompanies the commonest form, HLA-B27–associated AU, while latent infections underlie BRB breakdown in PU. This review considers the pathogenesis of uveitis in the context of IP, infection, environment, and the microbiome.

Histology (H&E) and transmission electron microscopy (TEM) data are provided showing age-related changes in the retinal structure of sTg-IRBP:HEL mice. These include substantial photoreceptor loss, atrophy of the retinal pigment epithelium, Bruch׳s membrane disruption and thickening, along with the presence of drusenoid deposits and changes in basal laminar infoldings. These features resemble some of those key characteristics found in the course of human dry (atrophic) age-related macular degeneration (AMD), particularly with regard to drusen. Hence, we believe the sTg-IRBP:HEL mouse model represents a useful and promising archetype for future study of the mechanism of drusen formation in AMD.

BACKGROUND: The protective role of mildly elevated bilirubin against CVD and diabetes mellitus type 2 (DMT2) is associated with a favorable lipid phenotype. As the mechanistic understanding of this protection in humans remains elusive, we aimed to assess the metabolomics profile of mildly hyperbilirubinaemic (Gilbert's syndrome; GS) individuals especially targeting lipid catabolism. METHODS AND RESULTS: Using NMR serum metabolomics of 56 GS individuals and 56 age and gender-matched healthy controls, GS individuals demonstrated significantly greater concentrations of acetylcarnitine (+20%, p

Heme catabolism exerts physiological functions that impact health through depressing inflammation. Upon reactive pathway progression, as in Gilbert’s Syndrome (GS; UGT1A1*28 polymorphism), aggravated health effects have been determined. Based on lower inflammation and improved metabolic health reported for GS, inter-group differences in heme catabolism were explored. Therefore, a case-control study including 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. Genetic expressions of HMOX-1 and BLVRA were measured. Additionally participants were genotyped for those polymorphisms that are known (UGT1A1*28) or likely (HMOX-1 microsatellites) to impact bilirubinemia. Intracellular interleukins (IL-6, IL-1β, TNFα), circulatory C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (Hpt) were analysed as inflammatory markers. To assess intracellular heme oxygenase 1 (HO-1) isolated PBMCs were used. In GS vs. C, inflammation markers were significantly decreased. This was supported by an altered heme catabolism, indirectly reflecting in elevated unconjugated bilirubin (UCB; main phenotypic feature of GS) and iron, decreased hemopexin (Hpx) and Hpt and in up-regulated biliverdin reductase (BLVRA) gene expressions. Moreover, HMOX (GT)n short alleles were non-significantly more prominent in female GS individuals. Herewith, we propose a concept to elucidate why GS individuals encounter lower inflammation, and are thus less prone to oxidative-stress mediated diseases.

Introduction: Topical investigations have demonstrated that oxidative stress and inflammation play key roles in biological aging and determine incidence and course of age-related diseases. Lifestyle and environmental factors hugely impact epigenetic regulation and DNA stability with telomere attrition and epigenetic instability providing a potential record of the cumulative burden of endogenous and exogenous oxidative noxae. Certain physiologically active plant components exhibit antioxidative activities affecting epigenetic regulation of inflammation response and DNA repair. Methods: Against this background, the present study investigated green tea polyphenol epigallocatechin gallate (EGCG) in the context of telomere regulation in Caco-2 colorectal adenocarcinoma cells vs. ES-1 primary skin fibroblasts. Cell lines were treated with 20 and 200 µM EGCG for 36, 72 and 144 hours, respectively. Telomerase activity, relative telomere length as well as methylation status of hTERT and c-Myc from different culture conditions were assessed. Malondialdehyde (MDA) served as a surrogate marker of potential pro-oxidative effects of EGCG in a physiologically relevant tissue model. Results: EGCG incubation was associated with telomere shortening and decreased telomerase activity in Caco-2 cells, and relatively longer telomeres along with increased methylation of six 5'—C—phosphate—G—3' (CpG) sites in the promoter region of human Telomerase Reverse Transcriptase ( hTERT) in fibroblasts. At low concentrations, EGCG significantly decreased oxidative damage to lipids in Caco-2 cells and attenuated H 2 O 2 induced oxidation at higher concentrations. Conclusion: These results suggest differential EGCG-mediated telomeric modulation in cancer vs. primary cells and a specific antioxidant activity of EGCG against oxidative damage to lipids in abnormal cells. Keywords: Caco-2, epigallocatechin gallate, telomeres, hTERT, DNA methylation, telomerase, oxidative stress, malondialdehyde

Mild endogenous elevation of unconjugated bilirubin (UCB) as seen in Gilbert's syndrome (GS), might mitigate cardiovascular disease (CVD) risk factors including overweight/obesity. This study aimed to determine whether hyperbilirubinaemia is linked to improved anthropometric data and lipid profile.Our study considered GS and age-/gender-matched healthy controls (n = 248). Additionally, obese female type 2 diabetic patients (DM2) (n = 26) were included as a "disease control group".BMI, hip circumference (HC), and lipid profile were significantly lower in GS. UCB was inversely correlated with BMI (p 0 .001), HC as well as with fat mass (FM) and lipid variables (p 0.05). Moreover, DM2 patients had significantly lower UCB compared to GS and healthy controls. Older GS subjects (≥35 years) had significantly reduced anthropometric data and improved lipid profile.Our results propose that the health promoting potential of mild hyperbilirubinaemia may extend to protection from age-related weight gain and dyslipidaemia.

Bilirubin, the principal and biologically most relevant bile pigment was, until recently, considered a waste product of haem catabolism. However, current data suggest that bile pigments possess biological potential, related to their antioxidant and anti-mutagenic effects. In this context, it is now assumed that bile pigments and their derivatives exert these effects via multiple mechanisms, including discrete anti-oxidative and physico-chemical interactive effects. The major scientific focus so far has concentrated on the compounds' antioxidant action, and mechanistic investigations of possible mutagen-tetrapyrrole interaction are lacking. Therefore we tested structurally related bile pigments/derivatives (bilirubin/-ditaurate/-dimethyl ester, biliverdin/-dimethyl ester, urobilin, stercobilin and protoporphyrin) for anti-genotoxicity in the Salmonella reverse mutation assay (strains TA98, TA102), together with the synthetic mutagen 2,4,7-trinitro-9H-fluoren-9-one (TNFone). To explore possible structural interactions, molecular systems of chlorin e6 porphyrin/bilirubin/biliverdin with TNFone were assayed using circular dichroism. These data consistently revealed, at suprastoichiometric concentrations, that tetrapyrroles interact with TNFone. Addition of TNFone to chlorin e6 porphyrin, bilirubin-albumin and biliverdin-albumin led to a marked change in pigment spectra, providing evidence for tight tetrapyrrole-mutagen interaction. This conclusion was also supported by substantial, TNFone-induced decrease of bilirubin oxidation in the bilirubin-albumin system. This outcome was reflected in a bacterial model, in which most tetrapyrroles and especially protoporphyrin, significantly attenuated TNFone-induced mutagenesis. These data indicate that aromatic, tetrapyrrolic molecules interact with TNFone, providing a novel mechanism to suggest the anti-mutagenic effects of bile pigments in vivo are related to their physico-chemical interaction with genotoxins.

Background Moderately elevated unconjugated bilirubin concentrations protect against inflammatory diseases and are present in individuals with Gilbert's syndrome. This study examined the relationship between circulating haem oxygenase catabolites, unconjugated bilirubin, carboxy haemoglobin, iron and inflammatory parameters. Materials and methods Seventy-six matched individuals were allocated to Gilbert's syndrome (GS) or control group (unconjugated bilirubin ≥ or

In vitro anti-genotoxic properties of bile pigments have been explored and confirmed recently. Despite these reports mechanisms to explain DNA protection by endogenous bile pigments remain unclear. Surprisingly, the quantification of cellular pigment absorption which could represent a fundamental prerequisite for intracellular (e.g., anti-mutagenic) effects, has not been explored. Therefore, we aimed to measure the amounts of un-/conjugated bilirubin as well as biliverdin absorbed into colonies of Salmonella typhimurium, utilising HPLC analyses, and to observe whether intracellular compound concentrations could predict anti-genotoxic effects. HPLC analyses confirmed that bacterial bile pigment absorption was concentration-dependent. Plate bile pigment concentrations were inversely associated with genotoxicity of all tested mutagens, irrespective of strain and test conditions. However, protection against frame-shift mutation in strain TA98 most strongly depended on the bacterial absorption of bilirubin and biliverdin, which indicates that bile pigments can protect by intercepting mutations extracellularly and specifically inhibit frame-shift mutations intracellularly.

The antioxidant properties of protoporphyrin IX and related tetrapyrroles are poorly characterized. Therefore, eight tetrapyrroles, five of which are produced in vivo, were tested to assess their antioxidant capacities in the Salmonella reverse mutation, TEAC, FRAP and ORAC assays. Tertiary-butyl hydroperoxide (tert-BOOH) in the presence or absence of metabolic activation (±S9) was added to Salmonella strain TA102 together with the test compounds. In the absence of metabolic activation, the order of effectiveness was protoporphyrinbiliverdinbilirubin ditauratebilirubinbiliverdin dimethyl esterstercobilinbilirubin dimethyl esterurobilin. In the presence of S9, the effectiveness was reversed, with urobilinbiliverdin dimethyl esterbilirubin dimethyl esterstercobilinbiliverdinbilirubinbilirubin ditaurateprotoporphyrin. In the antioxidant capacity assays FRAP, TEAC and ORAC, mainly bilirubin, bilirubin ditaurate, biliverdin and protoporphyrin showed antioxidant activity. This study reports that previously untested tetrapyrroles of related structure prevent oxidatively induced genotoxicity, and for some, novel underlying mechanisms of antioxidant action were revealed. These results support the physiological importance and biological relevance of tetrapyrroles including protoporphyrin that might act as antioxidants, protecting from oxidatively induced DNA damage, particularly in the tissues/organs where they accumulate.

Energy metabolism, involving the ATP-dependent AMPK-PgC-Ppar pathway impacts metabolic health immensely, in that its impairment can lead to obesity, giving rise to disease. Based on observations that individuals with Gilbert’s syndrome (GS; UGT1A1*28 promoter mutation) are generally lighter, leaner and healthier than controls, specific inter-group differences in the AMPK pathway regulation were explored. Therefore, a case-control study involving 120 fasted, healthy, age- and gender matched subjects with/without GS, was conducted. By utilising intra-cellular flow cytometry (next to assessing AMPKα1 gene expression), levels of functioning proteins (phospho-AMPK α1/α2, PgC 1 α, Ppar α and γ) were measured in PBMCs (peripheral blood mononucleated cells). In GS individuals, rates of phospho-AMPK α1/α2, -Ppar α/γ and of PgC 1α were significantly higher, attesting to a boosted fasting response in this condition. In line with this finding, AMPKα1 gene expression was equal between the groups, possibly stressing the post-translational importance of boosted fasting effects in GS. In reflection of an apparently improved health status, GS individuals had significantly lower BMI, glucose, insulin, C-peptide and triglyceride levels. Herewith, we propose a new theory to explain why individuals having GS are leaner and healthier, and are therefore less likely to contract metabolic diseases or die prematurely thereof.

Bilirubin, the principal tetrapyrrole, bile pigment and catabolite of haem, is an emerging biomarker of disease resistance, which may be related to several recently documented biological functions. Initially believed to be toxic in infants, the perception of bilirubin has undergone a transformation: it is now considered to be a molecule that may promote health in adults. Data from the last decade demonstrate that mildly elevated serum bilirubin levels are strongly associated with reduced prevalence of chronic diseases, particularly cardiovascular diseases (CVDs), as well as CVD-related mortality and risk factors. Recent data also link bilirubin to other chronic diseases, including cancer and Type 2 diabetes mellitus, and to all-cause mortality. Therefore, there is evidence to suggest that bilirubin is a biomarker for reduced chronic disease prevalence and a predictor of all-cause mortality, which is of important clinical significance. In the present review, detailed information on the association between bilirubin and all-cause mortality, as well as the pathological conditions of CVD, cancer, diabetes and neurodegenerative diseases, is provided. The mechanistic background concerning how bilirubin and its metabolism may influence disease prevention and its clinical relevance is also discussed. Given that the search for novel biomarkers of these diseases, as well as for novel therapeutic modalities, is a key research objective for the near future, bilirubin represents a promising candidate, meeting the criteria of a biomarker, and should be considered more carefully in clinical practice as a molecule that might provide insights into disease resistance. Clearly, however, greater molecular insight is warranted to support and strengthen the conclusion that bilirubin can prevent disease, with future research directions also proposed.

The bile pigment bilirubin is a known antioxidant and is associated with protection from cancer and cardiovascular disease (CVD) when present in too strong concentrations. Unconjugated bilirubin (UCB) might also possess anti-genotoxic potential by preventing oxidative damage to DNA. Moderately elevated bilirubin levels are found in individuals with Gilbert syndrome and more severe in the hyperbilirubinemic Gunn rat model. This study was therefore aimed to assess the levels of oxidative damage to DNA in Gilbert syndrome subjects and Gunn rats compared to matched controls. Seventy-six individuals (age- and sex-matched) were allocated into Gilbert syndrome (UCB ≥17.1 μmol/L; n = 38) or control groups (UCB < 17.1 μmol/L; n = 38). In addition, 40 Gunn rats were used to support the results of the human trial. Single-cell gel electrophoresis (SCGE) assay measuring standard conditions (strand breaks, apurinic/apyrimidinic sites) and formamidopyrimidine glycosylase (FPG)-sensitive sites was conducted in human peripheral blood mononuclear cells (PBMC) and rat PBMCs, colon, and hepatocytes. Furthermore, urinary 8-oxo-2′-deoxyguanosine (8oxodGuo, DNA oxidation) and 8-oxo-guanosine (8oxoGuo, RNA oxidation) were measured in humans. The Gilbert syndrome and Gunn rat groups had significantly higher UCB levels (P < 0.001) than the corresponding controls. No further differences in damage to DNA or RNA were detected between the two groups, except higher strand breaks (PBMCs) in Gunn rats when compared with controls. However, when demographic effects were analyzed, lower 8oxodGuo concentrations were detected in the human group with a BMI ≥25 kg/m2 (1.70 ± 0.67 vs. 1.38 ± 0.43 nmol/mmol creatinine, P < 0.05), although this group showed lower UCB levels than normal weight subjects. This study suggests that the disease preventative effect of UCB is unrelated to DNA oxidation/strand breaks in human and animal models of hyperbilirubinaemia. Cancer Prev Res; 6(10); 1056–63. ©2013 AACR.

Circulating unconjugated bilirubin (UCB) has been reported to protect against lung and colorectal cancer. The present study aimed to explore, for the first time, whether mildly elevated circulating UCB, as found in Gilbert`s syndrome (GS), is associated with changes of DNA damage. A random 76 individuals, matched for age and gender, were recruited from the general population and allocated into the GS group (UCB ≥ 17.1 µM; n = 38) or control group (UCB