Bilirubin prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing upregulation of inducible nitric oxide synthase.

Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Male C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving intraperitoneal injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels were determined, and intestinal specimens were analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration but did not alter IL-5 production. In conclusion, bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression.

Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase.

It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide ( [Formula: see text] ) production, respectively. The generation of both nitrate and [Formula: see text] in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated [Formula: see text] production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-ß, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-ß release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin.

Sodium taurocholate inhibits intestinal adenoma formation in APCMin/+ mice, potentially through activation of the farnesoid X receptor.

In light of clinical and biological evidence that bile constituents exert preventive effects against colorectal cancer, we evaluated the influence of oral bilirubin and sodium taurocholate (NaTC) on intestinal tumor formation in APC(Min/+) mice. Mice received bilirubin and/or bovine serum albumin (BSA) and NaTC in the drinking water for 8 weeks, after which the number, size and location of intestinal adenomas were determined. Tissue specimens were analyzed by light microscopy, TUNEL staining, immunohistochemistry for beta-catenin and Ki-67 and quantitative polymerase chain reaction for farnesoid X receptor (FXR)-dependent gene expression. Colon tumor formation also was assessed in azoxymethane (AOM)-treated hyperbilirubinemic Gunn (j/j) and wild-type (+/+) rats. Compared with untreated APC(Min/+) mice, the mean number of intestinal adenomas was markedly lower in both bilirubin (10.5 +/- 0.9 versus 37.0 +/- 5.2; +/-SEM; P < 0.001) and NaTC plus BSA (14.3 +/- 5.4; P = 0.01)-treated animals. Both treatment groups exhibited reduced levels of cellular proliferation in the ileum (by Ki-67 staining), but no differences in TUNEL staining or the percentage of beta-catenin-positive crypts. Bilirubin feeding reduced intestinal inducible nitric oxide synthase expression, but did not alter adenoma multiplicity in APC(Min/+) mice or in AOM-treated j/j versus +/+ rats. Mice receiving NaTC manifested increased intestinal expression of the FXR-regulated genes, Shp, FGF15 and IBABP, and a concomitant decrease in cyclin D1 message. Administering NaTC to APC(Min/+) mice causes a marked reduction in intestinal adenomas. We postulate that this effect is mediated through activation of FXR, leading to increased Shp expression and consequent downregulation of cyclin D1.

Alternative splicing of an rnp-4f mRNA isoform retaining an evolutionarily-conserved 5'-UTR intronic element is developmentally regulated and shown via RNAi to be essential for normal central nervous system development in Drosophila melanogaster.

Two major mRNA isoforms arise via alternative splicing in the 5'-UTR of Drosophila splicing assembly factor rnp-4f pre-mRNA, designated "long" (unspliced) and "short" (alternatively spliced). The coding potential for the two isoforms is identical, raising interesting questions as to the control mechanism and functional significance of this 5'-UTR intronic splicing decision. Developmental Northerns show that two temporally distinct rnp-4f mRNA degradation episodes occur during embryogenesis. The first occurs at the midblastula transition (MBT) stage and involves degradation of both maternally-derived transcripts, while the second involves only the long mRNA isoform and occurs during late embryo stages. Immunostaining of ovaries and staged embryos combined with results from developmental Westerns shows that maternal RNP-4F protein persists into fertilized eggs at high levels, associated with a burst of long isoform-specific transcription which begins just after the MBT and peaks in mid-embryo stages. These observations are discussed in support of a putative negative feedback control model for modulation of RNP-4F translation. In situ hybridization shows that the long isoform is relatively abundant throughout the developing embryonic germ band and central nervous system (CNS), especially along the dorsal roof of the ventral nerve cord. Long rnp-4f mRNA knockdown via RNAi reveals a variety of CNS abnormalities, which leads us to postulate that this isoform acts upstream of other genes which have been shown to be important for normal CNS development.

Hyperbilirubinemia is not a major contributing factor to altered bone mineral density in patients with chronic liver disease.

Reduced bone density is commonly encountered in patients with chronic liver disease. Prior studies have shown that unconjugated bilirubin inhibits osteoblast activity and function in vitro and in animal models of bone mineralization. To determine whether hyperbilirubinemia promotes the development of hepatic osteodystrophy, bone mineral density (BMD) was measured by dual energy X-ray absorptiometry in a cohort of 86 consecutive patients with chronic liver disease referred for liver transplant evaluation. The mean age of the study population was 52 years (range, 22-73), in which 52% were female and 90% were white. Average bone density values were significantly lower than expected for age, race, and sex, with Z-scores for the femoral neck and spine of -0.50 (95% confidence interval [CI] -0.63 to -0.37; p=0.0003) and -0.69 (95% CI -0.85 to -0.52; p=0.0001), respectively. Sixty-one subjects (71%) exhibited reduced BMD (T-score of femoral neck or spine

Bilirubin inhibits iNOS expression and NO production in response to endotoxin in rats.

The inducible isoform of heme oxygenase (HO), HO-1, has been shown to play an important role in attenuating tissue injury. Because HO-1 catalyzes the rate-limiting step in bilirubin synthesis, we examined the hypothesis that bilirubin is a key mediator of HO-1 cytoprotection, employing a rat model of endotoxemia. Bilirubin treatment resulted in improved survival and attenuated liver injury in response to lipopolysaccharide infusion. Serum levels of NO and tumor necrosis factor alpha, key mediators of endotoxemia, and hepatic inducible nitric oxide synthase (iNOS) expression were significantly lower in bilirubin-treated rodents versus control animals. Both intraperitoneal and local administration of bilirubin also was found to ameliorate hindpaw inflammation induced by the injection of lambda-carrageenan. Consistent with in vivo results, bilirubin significantly inhibited iNOS expression and suppressed NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. In contrast, bilirubin treatment induced a threefold increase in LPS-mediated prostaglandin synthesis in the absence of significant changes in cyclooxygenase expression or activity, suggesting that bilirubin enhances substrate availability for eicosanoid synthesis. Bilirubin had no effect on LPS-mediated activation of nuclear factor kappaB or p38 mitogen-activated protein kinase, consistent with a nuclear factor kappaB-independent mechanism of action. Taken together, these data support a cytoprotective role for bilirubin that is mediated, at least in part, through the inhibition of iNOS expression and, potentially, through stimulation of local prostaglandin E2 production. In conclusion, our findings suggest a role for bilirubin in mollifying tissue injury in response to inflammatory stimuli and support the possibility that the phenomenon of "jaundice of sepsis" represents an adaptive physiological response to endotoxemia. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization.

Bilirubin is the principal end product of heme degradation. Prompted by epidemiologic analyses demonstrating an inverse correlation between serum bilirubin levels and cancer mortality, we examined the effect(s) of bilirubin on the growth and survival of colon adenocarcinoma cells. Adenocarcinoma cell monolayers were treated with bilirubin over a range of bilirubin:BSA molar ratios (0-0.6), and viability was assessed colorimetrically. Apoptosis was characterized by TUNEL assay, annexin V staining and caspase-3 activation. The mechanism(s) by which bilirubin induces apoptosis was investigated by Western blotting for cytochrome c release, assaying for caspase-8 and caspase-9 activation and for mitochondrial depolarization by JC-1 staining. The direct effect of bilirubin on the membrane potential of isolated mitochondria was evaluated using light-scattering and fluorescence techniques. Bilirubin decreased the viability of all colon cancer cell lines tested in a dose-dependent manner. Cells exhibited substantial apoptosis when exposed to bilirubin concentrations ranging 0-50 microM, as demonstrated by an 8- to 10-fold increase in TUNEL and annexin V staining and in caspase-3 activity. Bilirubin treatment evokes specific activation of caspase-9, enhances cytochrome c release into the cytoplasm and triggers the mitochondrial permeability transition in colon cancer monolayers. Additionally, bilirubin directly induces the depolarization of isolated rat liver mitochondria, an effect that is not inhibited by cyclosporin A. Bilirubin stimulates apoptosis of colon adenocarcinoma cells in vitro through activation of the mitochondrial pathway, apparently by directly dissipating mitochondrial membrane potential. As this effect is triggered at concentrations normally present in the intestinal lumen, we postulate a physiologic role for bilirubin in modulating colon tumorigenesis.

Deletion of the Aspergillus fumigatus gene encoding the Ras-related protein RhbA reduces virulence in a model of Invasive pulmonary aspergillosis.

Aspergillus fumigatus is the predominant mold pathogen in patients who lack functional innate immunity. The A. fumigatus rhbA gene was first identified as a transcript that was upregulated when the organism was grown in the presence of mammalian cells. To gain insight into the function of rhbA in the growth and pathogenesis of A. fumigatus, we constructed a strain that lacks a functional rhbA gene. The Delta rhbA mutant showed a significant reduction in virulence compared to the virulence of the wild type in a mouse model of invasive aspergillosis. Complementation of the deletion with the wild-type gene restored full virulence. Although the Delta rhbA mutant grew as well as the wild type on solid medium containing the rich nitrogen source ammonium, the growth of the mutant was impaired on medium containing poor nitrogen sources. Like the Saccharomyces cerevisiae rhb1 mutant, the Delta rhbA mutant exhibited increased uptake of arginine. In addition, the Delta rhbA strain underwent asexual development in submerged cultures, even under ammonium-excess conditions. Growth of the mutant with poor nitrogen sources eliminated both the arginine uptake and submerged asexual development phenotypes. The mutant showed enhanced sensitivity to the TOR kinase inhibitor rapamycin. These findings establish the importance of rhbA for A. fumigatus virulence and suggest a role for rhbA in nutrient sensing.