The molecular basis for the immunomodulatory activities of unconjugated bilirubin

The International Journal of Biochemistry & Cell Biology 45 (2013) 2843–2851

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Review

The molecular basis for the immunomodulatory activities of unconjugated bilirubin Sushrut Jangi a,b,∗ , Leo Otterbein a,b , Simon Robson a,b a b

Beth Israel Deaconess Medical Center, United States Harvard Medical School, United States

a r t i c l e

i n f o

Article history: Received 27 April 2013 Received in revised form 24 September 2013 Accepted 29 September 2013 Available online 19 October 2013 Keywords: Bilirubin Immunology Autoimmune disease Lymphocytes Immunomodulation

a b s t r a c t Nearly a century ago, jaundiced patients were observed to have surprising and spontaneous remissions from incurable immunologic diseases including rheumatoid arthritis, allergy, and asthma. The mystery of why this phenomenon occurred remains unresolved to this day. Bilirubin has traditionally been considered an excretory product resulting from heme metabolism with little benefit to human physiology. In the past few decades, however, the salutary role of this byproduct as a potent antioxidant has been repeatedly noted. Most recently, the molecule has been found to possess immunomodulatory properties that rival its redox capacity, possibly explaining its ability to suppress inflammation. In this review, we specifically examine unconjugated bilirubin (UCB) as an immunomodulator and explore the molecular basis for its immunosuppressive effects. © 2013 Elsevier Ltd. All rights reserved.

Contents 1.

2. 3.

4. 5.

6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2844 Historical anecdote: jaundice and relief of rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2844 1.1. 1.2. Bilirubin metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2844 Bilirubin as an antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2844 2.1. Bilirubin in coronary artery disease: a potent anti-oxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2844 Bilirubin as an immunomodulator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2845 3.1. Could bilirubin have an immunomodulatory role starting in infancy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2845 3.2. How might UCB modulate the immune response? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2845 3.3. Unconjugated bilirubin and the adaptive immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2847 A mechanistic model for unconjugated bilirubin as an immunomodulator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2847 Bilirubin in inflammatory disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2848 5.1. Rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2848 5.2. Inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2848 5.3. Multiple sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2849 5.4. Systemic lupus erythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2849 Modulating the immune response with bilirubin: recapitulating Hench’s observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2849 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2850 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2850

∗ Corresponding author at: Beth Israel Deaconess Medical Center, United States. Tel.: +1 5089040211. E-mail address: [email protected] (S. Jangi). 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.09.014

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1. Introduction Unconjugated Bilirubin – Why do we make it? For most of the past century, bilirubin has been considered to be a waste product of heme metabolism. Hyperbilirubinemia is also largely considered a bad sign: newborn infants with jaundice are susceptible to kernicterus, and the onset of jaundice in adults is often the harbinger of liver failure. Furthermore, the predominant form of bilirubin that circles in the serum – UCB – seems useless from multiple perspectives: it is water insoluble, it requires further metabolism for excretion, and it necessitates binding to albumin to move through the plasma. At high concentrations, UCB has cytotoxic effects on multiple tissues (Kapitulnik, 2004). Yet over the span of human evolution, nature has conserved the metabolic circuitry required to convert biliverdin, an already soluble and nontoxic heme breakdown product, into the potentially toxic and insoluble UCB, a step that furthermore costs the body a molecule of NADPH (Benaron and Bowen, 1991). The excretion of UCB is also dependent upon its conjugation by UDP-glucuronosyltransferase (UGT1A1); there are few other enzymes capable of bypassing this metabolic bottleneck (Gregory and Strickland, 1973). Given the lack of redundancy, the heme degradation pathway is under great selective pressure—yet this pathway leading to the production of UCB has been persistently conserved despite its costs. The teleological supposition is that UCB has a unique role with importance in physiology, and the molecule potentially confers fitness to the human organism. While UCB remains cytotoxic at high concentrations, UCB has been discovered to possess an astonishing potency in scavenging free radicals at physiologic concentrations. The same molecule also appears to have powerful immunosuppressive effects at physiologic or moderately elevated levels. In this review, we focus on cataloging the immunosuppressive potential of UCB, and bridge these insights into understanding the role of this molecule in autoimmune disease. We begin with a historical anecdote. 1.1. Historical anecdote: jaundice and relief of rheumatoid arthritis In 1929, Philip Hench, a rheumatologist at the Mayo Clinic, made a dramatic observation correlating relief of the otherwise incurable and relentless symptoms of rheumatoid arthritis with the onset of jaundice (Hench, 1938). Of these thirty patients with rheumatoid arthritis who had various types of spontaneous intercurrent jaundice twenty-five (83 percent) experienced marked or complete temporary remission of the arthritis irrespective of the concentrations of bilirubin in serum. When the level of serum bilirubin rose to more than 6 or 8 mg per 100 c.cm., the phenomenon of relief almost always occurred (in twenty-five, or 96 percent., of twenty-six such cases). When relief was induced, it was complete in 68 percent., marked but incomplete in the rest. Repeatedly, he noted that upon resolution of jaundice, the symptoms of rheumatoid arthritis returned. For years, he searched for the factor responsible for this phenomenon, investigating bile salts and bilirubin itself as the agent of relief. However, attempts to induce therapeutic hyperbilirubinemia with these agents were inconsistent. Later, his colleague would discover cortisone, and Hench surmised that during jaundice, elevations in cortisol may have been responsible for the effect (Hench et al., 1949). However, jaundice does not reproducibly cause cortisol elevations – the factor that Hench may have been searching for may have been the pigment of jaundice itself – bilirubin. In fact, between 1920 and 1950, several investigators reproducibly discovered that jaundice

resulted in the improvement of several other inflammatory disorders, including psoriatic arthritis, hay fever, and asthma (Gorin, 1949; Hench, 1949). Others consistently reproduced Hench’s observations. Recently, authors describe a case of refractory asthma that spontaneously remitted following the onset of jaundice secondary to hepatitis B (Ohrui et al., 2003). The mechanisms underpinning the improvement in human inflammatory diseases secondary to hyperbilirubinemia have only recently been revisited nearly a century after Hench’s original observation. We review bilirubin metabolism, its known anti-oxidant effects, and then focus on the potential for UCB as an immunomodulator that may explain Hench’s original observations.

1.2. Bilirubin metabolism Bilirubin is a byproduct of heme degradation (Fig. 1). The enzyme heme-oxygenase (HO) catalyzes the degradation of heme to form three products: biliverdin, ferrous iron, and carbon monoxide (Tenhunen et al., 1968). There are two main forms of HO: HO-1, which is inducible by numerous factors, but particularly oxidative stress, and HO-2 which is constitutively active in specific cell types, such as neurons (Maines, 1997). Once heme degradation products are released into the serum, each has a separate fate. Biliverdin is reduced to UCB via biliverdin reductase. Because UCB is not water soluble, it largely binds to albumin and circulates in the blood. The circulation of UCB in the serum terminates after it encounters the hepatocyte. Once inside the liver, UCB moves to the endoplasmic reticulum and is conjugated with glucuronic acid by the enzyme UGT1A1. Subsequently, conjugated bilirubin is excreted into the bile. Following deconjugation by the intestinal flora, urobilinogens and urobilins are excreted in the stool; a proportion of unconjugated bilirubin may be reabsorbed in the gut (Fig. 2). Concentration of UCB can vary widely as the host’s physiology changes. Consequently, UCB can have different effects on the host based on its concentration. For example, in infancy, high turnover of red blood cells and inefficient conjugation and excretion of UCB leads to an unconjugated hyperbilirubinemia that usually peaks by day 3 of life at an average concentration of 5.5 mg/dL before resolving. But if the concentration exceeds 15 mg/dL at 1 week of life, the neonate is at risk for development of kernicterus, in which UCB accumulates and damages neurons and glial cells (Lauer and Spector, 2011). In vitro, UCB has been found to be toxic to numerous other cell lines, including fibroblasts, hepatocytes, erythrocytes, leukocytes, and platelets (Vetvicka et al., 1991). Consequently, such elevated UCB concentrations have been shown to cause oxidative stress and cytotoxicity. In the remainder of the review, we explore UCB’s beneficial effects. Such beneficial effects occur at physiologic or moderately elevated serum concentrations of UCB, between 2 and 12 mg/dL in adults. At and above 15–20 mg/dL, cytotoxic effects of UCB become apparent.

2. Bilirubin as an antioxidant 2.1. Bilirubin in coronary artery disease: a potent anti-oxidant UCB is a powerful antioxidant, possessing physiologic properties akin to the anti-oxidant effects of Vitamin C and Vitamin E. In particular, UCB has a major capacity to protect phospholipids against oxidative damage from free radicals generated from the peroxisome (Stocker et al., 1987). UCB effectively inhibited oxidation of LDL near physiologic serum levels. This effect was nearly twenty times more potent than that of Vitamin E, a known scavenger of free radicals (Wu et al., 1994).

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Fig. 1. Synthesis of bilirubin. Following destruction of red blood cells, heme oxygenase catalyzes the degradation of heme into carbon monoxide, iron, and biliverdin. Subsequently, biliverdin is reduced to unconjugated bilirubin, which primarily circulates in the serum bound to albumin.

The powerful antioxidant effect of UCB translate clinically: asymptomatic males with low total bilirubin levels have more severe coronary artery disease on angiography. In fact, low total serum bilirubin is a strong, independent risk factor for coronary disease, with a significance approaching that of established risk factors such as smoking and hypertension (Schwertner et al., 1994). The inverse relationship between total bilirubin and coronary artery disease has been consistently confirmed (Hopkins et al., 1996; Hunt et al., 1996; Kronenberg et al., 2002; Endler et al., 2003; Ghem et al., 2010). Overall, a 40–50% reduction in coronary artery disease prevalence is noted in males with total bilirubin higher than 8 mg/L independent of other risk factors (Endler et al., 2003). Targeting HO-1, which induces bilirubin production, is under investigation as a therapy for coronary artery disease, as are attempts to regulate biliverdin reductase. Niacin is known to induce HO-1 which may partially explain its vascular protective properties (Wu et al., 2012a). Statins and fenofibrates may also upregulate HO-1 activity (Grosser et al., 2004a,b; Kronke et al., 2007). The antioxidant effects of total bilirubin have been investigated in numerous other diseases, including cerebrovascular disease, ischemia-reperfusion injury, and hypertension. 3. Bilirubin as an immunomodulator 3.1. Could bilirubin have an immunomodulatory role starting in infancy? Infants are particularly predisposed to developing neonatal jaundice, usually secondary to unconjugated hyperbilirubinemia. Neonates who develop jaundice have more difficulty producing antibodies in response to routine vaccination series against diphtheria, tetanus, and measles (Nejedla, 1970; Diaz del Casrillo, 1973; De Sanctis et al., 1968; Rola-Plezczynski et al., 1975). This effect is durable beyond the period of hyperbilirubinemia: even following resolution of jaundice, antibody titers against diphtheria and tetanus continued to be depressed even a year later. Hyperbilirubinemia curtails production of antibodies—total IgM and IgA levels

are lower in the jaundiced infant. Lymphocytes obtained from the serum of hyperbilirubinemic neonates have depressed proliferative ability. Elevations in UCB are thus associated with depressed antibody production in infants. Whether direct suppression of antibody production by UCB occurs or whether UCB helps modulate neonatal development of the immune system remains unexplored. 3.2. How might UCB modulate the immune response? The toxicity of significantly elevated concentrations of UCB has long overshadowed any potential benefits. The molecule uncouples oxidative phosphorylation and inhibits DNA and protein synthesis (Ernster and Zetterstrom, 1956). Consequently, glial cells, myocardium, renal tubules, and platelets can all be damaged by elevated levels of UCB (Ross and Fremland, 1968; Fernandes et al., 2006; Gordo et al., 2006; Vetvicka et al., 1991). The immune system is also impacted: neutrophils are unable to engage in phagocytosis (Thong et al., 1977). Furthermore, superoxide production by neutrophils is dismantled (Nakamura et al., 1987). The ability of the neutrophil to migrate and respond to chemotactic signals is compromised. Like colchicine, UCB appears to paralyze the neutrophil (Miler et al., 1981; Svejcar et al., 1984). However, UCB also appears to affect the immune system at physiologic concentrations, independent of cytotoxicity. UCB can affect complement physiology, an integral part of innate immunity, near physiologic serum concentrations of UCB. The thirty enzymes that participate in this reaction are triggered by the binding of the C1 complex to an antibody. UCB inhibits the complement cascade by interrupting binding of the C1 complex to antibody (Basiglio et al., 2007). This inhibitory effect has been replicated in vivo: UCB infusion into rats primed for intravascular hemolysis through complement fixation ameliorates the lysis of red blood cells (Sima et al., 1980). Therefore, UCB appears to directly impact innate immunity. UCB has also been observed to interact with macrophages. Their role as phagocytic cells depends upon the expression of Fc receptors on the surface. UCB alters the expression of Fc subsets

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Fig. 2. Uptake and excretion of bilirubin. Free UCB undergoes carrier-mediated diffusion into the hepatocyte via organic anion-transporting polypeptide 2 (OATP2). Following transport to the endoplasmic reticulum, UGT1A1 conjugates bilirubin with glucuronic acid. Conjugated bilirubin is then actively excreted through the biliary system into the small intestine, where flora deconjugate and degrade bilirubin into urobilinogens, some of which are reabsorbed via enterohepatic recycling. Brush-border ␤-glucuronidase also convert conjugated bilirubin to the unconjugated form for reabsorption Free UCB undergoes facilitated diffusion into the hepatocyte. Following transport to the endoplasmic reticulum, UGT1A1 conjugates bilirubin to glucuronic acid. Conjugated bilirubin is then actively excreted through the biliary system into the small intestine, where flora deconjugate and degrade bilirubin into urobilinogens, some of which are reabsorbed via enterohepatic recycling. Brush-border ≡-glucuronidase also convert conjugated bilirubin to the unconjugated form for reabsorption.

modulating the macrophage’s phagocytic and antigen-presenting function, by upregulating Fc␥1, Fc␮, Fc␧, and Fc␣, and downregulating Fc␥2B. Moreover, this effect occurs almost immediately and surface expression does not return to baseline until at least 24 h after the initial exposure of UCB (Vetvicka et al., 1985a,b).

UCB also inhibits cell-surface expression of MHC II class molecules on antigen-presenting cells (Liu et al., 2008; Wu et al., 2005). B7 receptor expression is also inhibited on the surface of antigen-presenting cells; analogously, CD28 expression is also inhibited on CD4 cells. Since MHC II, B7, and C28 expression are

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reduced by UCB, there is potential for UCB to impair antigen presentation to lymphocytes. Interception of the complement system, modulation of Fc receptors, and modification of MHC II expression at near physiologic serum concentrations of UCB suggests the molecule has the ability to modulate innate immunity independent of its cytotoxic effects. 3.3. Unconjugated bilirubin and the adaptive immune response Blood from jaundiced patients who have liver disease secondary to a variety of etiologies inhibits T cell proliferation. When patients with biliary obstruction have resolution of jaundice, the same sera no longer exerts an inhibitory effect on lymphocyte proliferation (Newberry et al., 1973). Following direct incubation of lymphocytes with UCB, lymphocytes demonstrate impaired proliferation. UCB enters cells as a consequence of incubation, and the degree of intracellular accumulation of UCB is directly related to the depth of lymphocyte inhibition. The concentrations of UCB to achieve this effect was as low as 6 mg/dL, a modest elevation (Rola-Plezczynski et al., 1975; Haga et al., 1996a,b; Roughneen et al., 1986; Rubaltelli et al., 1982). This effect is independent of cytotoxicity, as the T cells remained viable; the effect is also independent of UCB’s antioxidant function since other potent antioxidants did not have any effects of lymphocyte suppression. Clinically, UCB’s effect on the effector T cell may result in anergy, however this was only observed at moderately elevated bilirubin concentrations (12 mg/dL). Patients with jaundice due to various causes are more likely to test anergic in response to antigenic skin tests against tetanus, diphtheria, streptococcus, tuberculin, candida, tricophyton, and proteus mirabilis. This anergic response vanishes when serum UCB levels return to normal (Cainzos et al., 1992). How might UCB induce lymphocyte hypo-responsiveness—and does this occur near physiologic concentrations? Pro-inflammatory cytokines, including interleukin-2 (IL-2), are suppressed upon exposure to UCB. Concentrations of IL-2 were measured in cancer patients with obstructive jaundice before and after biliary stenting and resolution of hyperbilirubinemia. Following intervention, IL-2 and interleukin-1 (IL-1) concentrations significantly increased; the production of both of these interleukins negatively correlated with total serum bilirubin levels. Similarly, when infants with hyperbilirubinemia are treated with phototherapy, as UCB levels drop, IL-2 levels increase (Haga et al., 1989; Vane et al., 1988; Sirota et al., 1999; Kimura et al., 2001). These epidemiologic associations suggest that UCB suppresses IL-2 production and this effect has been recapitulated through in vitro studies: intracellular accumulation of UCB suppresses IL2 production by lymphocytes at concentrations of 8–12 mg/dL in a dose-dependent manner. Higher concentrations of UCB also suppress other pro-inflammatory cytokines, including IFN-␥ and TNF-␣ (Liu et al., 2008). The secretion of IL-2 and pro-inflammatory cytokines is under control of nuclear factor kappa B (NF-␬B). Normally, Nf-␬B is bound to I␬B, which locks the transcription factor in the cytosol. Upon phosphorylation of I␬B, Nf-␬B is free to move to the nucleus. UCB, a known inhibitor of several cytosolic protein kinases, prevents Nf-␬B translocation to the nucleus, potentially through inhibition of I␬B phosphorylation (Liu et al., 2008; Hansen et al., 1996; Mazzone et al., 2009). Furthermore, UCB may also directly bind Nf-␬B interrupting its translocation. A similar process may interfere with CIITA–the regulator of MHC II receptor expression (Wu et al., 2005). These mechanisms have only been preliminary explored, however they provide initial evidence supporting the role of UCB in suppression of the effector Th1 response. In these experiments, this immunosuppression requires moderate elevations of UCB, to near 8 mg/dL.

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At very high levels of UCB concentration, pro-inflammatory, rather than anti-inflammatory responses have been noted in the brain. Astrocytes incubated with high UCB concentrations release TNF-␣ and IL-1␤ via activation of MAP kinases and Nf-␬B. Microglia may demonstrate an increased phagocytic response. Therefore, while UCB may have an immunosuppressive effect at physiologic or moderate elevations of UCB, severe UCB elevations may precipitate a pro-inflammatory response in the brain and at other sites. The varying inflammatory or immunosuppressive effects at high and moderate concentrations of UCB respectively, may underlie the dual toxic and beneficial properties of the molecule to the host (Silva et al., 2010). Regulatory T cells are implicated in the downregulation of the effector lymphocyte response and the induction of tolerance. Some lines of evidence suggest that UCB does not promote generation of Foxp3 positive regulatory T cells (Biburger et al., 2010). However, multiple investigations have shown that UCB induces Treg expansion (Rocuts et al., 2010; Lee et al., 2007). In an islet transplantation mouse model, recipient mice were treated with UCB with intraperitoneal injections of UCB at a dose of 20 ␮g/kg twice per day peri-operatively and for a duration of two weeks after surgery. A week after transplantation, recipient mice treated with UCB had a more than 50% increase in regulatory T cell populations compared to controls. Without UCB treatment, rejection of the graft occurred within 20 days. The administration of UCB prolonged graft survival to 40 days. How might UCB induce regulatory T cell function or expansion? Treg function and development is controlled by the master regulatory gene Foxp3. Transfection of Foxp3 into undifferentiated T cells leads to suppression of IL-2 and hypoproliferation of lymphocytes. Surprisingly, Foxp3 transfection into T cells also leads to heme-oxygenase 1 induction. Consequently, blocking HO-1 induction prevents the anti-proliferative effect of Foxp3 on lymphocytes (Choi et al., 2005). Foxp3 may thereby shape regulatory T cell development through HO-1 induction–indeed, HO-1 is constitutively expressed in regulatory T cells. Recently, it was shown that in place of HO-1 induction, administration of total bilirubin or carbon monoxide to donor mice induced prolonged islet survival following transplant into the recipient via expansion of Treg subsets (Rocuts et al., 2010; Lee et al., 2007). Therefore, Treg expansion may be directly linked to HO-1 induction and the products of heme degradation, including total bilirubin and UCB. Recently the aryl hydrocarbon receptor, also linked to Tr1 production, has also been linked to HO-1 induction, suggesting that AHR is part of the same network that links heme oxygenase to the induction of tolerance (Lee et al., 2007; Bock and Kohle, 2010). The various immunomodulatory effects of UCB on the different branches of the immune system are summarized in Fig. 3.

4. A mechanistic model for unconjugated bilirubin as an immunomodulator Mechanistically, how can we explain UCB’s numerous and varied effects on the immune system? UCB has minimal aqueous solubility when it is not bound to albumin (a free species) due to the formation of extensive intramolecular hydrogen bonds. In such condition it is able to pass through plasma membranes without difficulty (Zucker et al., 1999). Conjugated bilirubin does not show this facility, which may render it ineffective as an immunomodulator. Following the entry of UCB into a target cell, UCB might engage in widespread inhibition of protein kinases (Hansen et al., 1996). In vitro studies have demonstrated that UCB interacts with catalytic domains of various kinases, including PKC and I␬B kinase.

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Fig. 3. Bilirubin as an immunomodulator. Free bilirubin diffuses through plasma membranes and acts on the immune system at various arms, including disruption of antigen presentation, suppression of effector T cell responses, interception of the complement cascade, and promotion of regulatory T cell expansion. Mechanistically, UCB may achieve these effects via widespread protein kinase inhibition, apoptosis, and interference with membrane receptors at or near physiologic levels.

Inhibition of kinase activity interrupts downstream signaling cascades, including Nf-␬B and STAT-1, both of which prevent synthesis of proinflammatory cytokines, the appearance of costimulatory molecules on the surface of antigen-presenting cells, and expression of MHC II receptors. Thus, through widespread kinase inhibition, proinflammatory signaling is intercepted. While kinase inhibition by UCB was observed in vitro at high UCB concentrations, the cumulative effects of UCB at lower, physiologic levels likely still cause kinase inhibition (Hansen et al., 1996). Furthermore, enrichment of UCB in subcellular compartments in the cytosol may also locally raise UCB concentrations and result in inhibition of kinase catalytic domains, even when serum UCB concentrations are near physiologic levels. In a separate pathway, UCB has also been shown to lead to apoptosis of cells through an association of UCB with mitochondrial membranes, leading to cytochrome c release into the cytosol and activation of caspase-9; apoptosis of lymphocytes has been through to contribute to UCB’s immunosuppressive effect (Keshavan et al., 2004). The affinity of UCB for membrane lipids also suggests that UCB interacts with the membranes of cells involved in innate immunity, including macrophages, which demonstrate profound morphologic change following UCB treatment (Leipe et al., 1983). In particular, these morphologic changes are accompanied by modulation of Fc receptor expression, including upregulation of Fc␮R, Fc␣R, Fc␧R, and downregulation of Fc␥2␤R. Similar UCB-membrane interactions have been observed between UCB and the C1 q membrane receptor of complement. Therefore, UCB’s affinity for membranes allow for the molecule to gain access to the cytosol and inhibit proinflammatory signaling cascades, potentially through inhibition of catalytic kinase domains. Association with plasma cell membranes of target cells can also modulate expression and insertion of receptors, including Fc and complement receptors. Finally, increasing serum levels of UCB can lead to apoptosis of immune cells through association with mitochondrial membranes. Potently elevated levels of UCB may actually have toxic effects on various cell types,

potentially eliciting a pro-inflammatory rather than an immunosuppressive effect (Fernandes et al., 2011). 5. Bilirubin in inflammatory disorders 5.1. Rheumatoid arthritis HO-1 is induced in the inflamed joint (Kitamura et al., 2011; Takahashi et al., 2009). The inflamed joints of rheumatoid arthritis patients are metabolically active causing relative hypoxia, inducing transcription of (HIF-1) alpha, or hypoxia inducible factor. Expression of this factor correlates with induction of heme-oxygenase. When HO-1 expression is induced, pro-inflammatory cytokines in the synovium decrease. Following induction of HO-1, inflammation is consequently dampened. In a murine collagen induced arthitis model, animals with inflamed joints were treated with biliverdin and carbon monoxide (Bonelli et al., 2012). Pathologic analysis 60 days after treatment revealed that cartilage degradation was inhibited by biliverdin administration. The results of carbon monoxide administration were even more significant, with significantly reduced inflammation, erosions and osteoclast activity. Both biliverdin and carbon monoxide appear to be protective against inflammatory arthritis; their production is a consequence of HO-1 transcription. A large epidemiologic study using the NHANES database concludes that higher total serum bilirubin levels were associated with a reduced risk of rheumatoid arthritis in humans even after adjustment for age, sex, and socioeconomic factors (Fischman et al., 2010). UCB concentrations were not available in this study, but total bilirubin concentrations remained near physiologic levels. 5.2. Inflammatory bowel disease Ulcerative colitis gives us a unique vantage point to study the effects of hyperbilirubinemia because a subset of these patients have primary sclerosing cholangitis (PSC), which raises serum UCB

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concentrations. Patients with both PSC and UC have milder or asymptomatic colitis compared to patients with low UCB concentrations (Papatheodoridis et al., 1998). But when patients with PSC and UC undergo liver transplantation, UC often becomes suddenly symptomatic, and often times severe, prompting colectomy. Why liver transplantation should worsen the UC course is mysterious, but one hypothesis is that once normal total bilirubin levels are restored, inflammation in the colon becomes disinhibited (Papatheodoridis et al., 1998). Similarly, in Gilbert’s syndrome, the affected patient has a deficiency in UGT1A1. These patients have chronically elevated levels of UCB in their serum. Patients with Gilbert’s syndrome are significantly less likely to have Crohn’s disease—suggesting that the bilirubinemia of Gilbert’s syndrome may be protective against gut inflammation (de Vries et al., 2011). UCB concentrations in Gilbert’s rarely exceed 4 mg/dL, suggesting these immunosuppressive effects occur near physiologic levels.

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Table 1 Inducing unconjugated hyperbilirubinemia. Mechanism

Medication

Heme oxygenase induction

Hemin Niacin Isodon serra Curcumin (turmeric) Ferulic acid (component of asafoetida) BG-12 (dimethyl fumarate)

UGT1A1 inhibition

Chrysin (from passion-flower) Silibinin (from milk thistle) Tangeretin (from citrus) Itraconazole, ketoconazole

Inducing a Gilbert’s syndrome-like phenotype may be a useful strategy as an adjunct to established therapies for autoimmune diseases. Medicines that induce heme oxygenase or inhibit UGT1A1 can raise unconjugated serum bilirubin levels to a range of 1.0–3.5 mg/dL (Wu et al., 2012b; Fromke and Miller, 1972; Capuzzi et al., 1998; Nash et al., 2011; Wang et al., 2012; Motterlini et al., 2000; Ma et al., 2011; Walsky et al., 2012).

5.3. Multiple sclerosis HO-1 is strongly expressed in the CNS lesions of the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Induction of HO-1 leads to reduction in the clinical signs of EAE, delayed onset, and reduced duration of disease (Liu et al., 2001). Carbon monoxide and bilirubin administration results in profound immunosuppression and EAE amelioration (Liu et al., 2008; Chora et al., 2007). In particular, administration of total bilirubin inhibits T cell proliferation, induces T cell apoptosis or anergy, inhibits CD28 expression on the T cell surface and B7 on the antigen-presenting cell, inhibits Nf-␬B translocation to the nucleus, and prevents MHC II receptor class presentation. Total bilirubin does not seem to skew the T cell axis toward a Th2 or immunoregulatory response in EAE—rather, all T cell responses, including Th2 and Tregs are mitigated (Liu et al., 2008). This contradicts other authors who suggest that bilirubin leads to regulatory T cell expansion. Patients with MS are found to have significantly lower levels of total bilirubin, conjugated and UCB (Peng et al., 2011). In a variant of multiple sclerosis that predominantly affects the optic nerve and the spinal cord, neuromyelitis optica (NMO), serum total bilirubin levels were not only lower in patients with NMO compared to healthy controls, but also comparable to typical multiple sclerosis (Peng et al., 2012). Among the medications currently being used in clinical trials to treat relapsing-remitting multiple sclerosis, the drug BG-12, a fumaric acid ester, is thought to act through induction of Nrf-2 which strongly regulates HO-1 induction. BG-12 has recently been shown in a phase III trial to decrease the annual relapse rate by 51% (Gold et al., 2012). Whether HO-1 induction is the primary cause for decreasing the relapse rate is unclear, however the effect of inducing this enzyme and the formation of heme degradation products on ameliorating human MS remains a promising objective. 5.4. Systemic lupus erythematosus Complement system aberrations, defects in antigen presentation and an abnormal adaptive immune response have been implicated in SLE. Patients with SLE had serum total bilirubin levels that were almost 50% lower than healthy controls, even in the absence of underlying liver disease (Vitek et al., 2010). These effects were seen in both men and women. Those patients with the lowest concentrations of serum UCB were more likely to have multi-organ disease secondary to lupus, such as nephritis. Conversely, patients with relatively higher total bilirubin levels have lower autoantibody titers directed against nuclear antigens and dsDNA, recapitulating the observation that hyperbilirubinemia may suppress antibody production (Yang et al., 2011). Biliverdin

has also recently been shown to inhibit the expression of cellsurface Toll-like receptors, which are receptors implicated in the chronic inflammation associated with lupus (Wegiel et al., 2011). 6. Modulating the immune response with bilirubin: recapitulating Hench’s observations As Hench discovered, the induction of hyperbilirubinemia in patients proves challenging. He tried transfusing patients with icteric serum, directly infusing bilirubin and bile salts into the blood, and prescribing hepatotoxic oral agents such as toluenediamine. His methods had an inconsistent and incomplete immunomodulatory effect. There are no current studies exploring the induction of hyperbilirubinemia in humans. But the most practical way to hijack the potential immunomodulatory effect of UCB is through induction of HO. The promoter region of HO contains multiple copies of an antioxidant response element that responds to oxidant triggers including lipopolysaccharide A, cytokines, and heat shock. However, some medications can also induce HO-1 transcription. Hemin, a drug used in the management of acute porphyria attacks, leads to expression of HO-1 and in experimental studies has been shown to suppress production of tumor necrosis factor-␣ (TNF-␣) and interleukin6 (Wu et al., 2012b). Niacin, historically used to change lipid profiles, probably confers cardioprotection independent of its influence on lipid metabolism. Niacin upregulates HO-1. In animal models and humans, the drug protects against inflammation, oxidation, and atherosclerosis; it can also counter the immunologic storm of reperfusion injury, and arteriosclerosis in cardiac transplantation (Fromke and Miller, 1972). The intravenous or long term oral administration of niacin elevates UCB levels, suggesting that niacin’s immunomodulatory effects may raise UCB via HO-1 induction (Capuzzi et al., 1998; Nash et al., 2011). Niacin is thus an attractive candidate to investigate the role of HO-1 and UCB in immune disease. BG-12, or dimethyl fumarate, can induce HO-1 transcription via Nrf-2. Finally, several herbal medications induce HO-1, including the Chinese preparation nodosin (Isodon serra) (Wang et al., 2012). Curcumin and ferulic acid have similar effects (Motterlini et al., 2000; Ma et al., 2011). An alternative strategy includes preventing excretion of UCB, through inhibition of UGT1A1, akin to inducing a state similar to that present in patients with Gilbert’s syndrome. Several agents significantly inhibit UGT1A1, including chrysin, silibinin (found in milk thistle), and tangeretin (found in tangerines) (Walsky et al., 2012). Itraconazole, ketoconazole, and ritonavir also inhibit UGT1A1. The medications that raise levels of UCB are summarized in Table 1.

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7. Conclusions Despite the metabolic costs of producing, transporting, and excreting bilirubin, its role in physiology has been evolutionarily conserved. UCB has a transient lifespan in the serum. The generation and elimination of UCB is regulated at either end by heme oxygenase and a UDP-glucuronosyltransferase. During its life, UCB serves as a (1) reducing agent and (2) an immunomodulator. Its potency as an antioxidant is likely derived from its two pyrrole rings which can donate electrons to free radicals. Numerous studies have supported a mechanistic role for UCB as a powerful antioxidant; clinically UCB has a protective effect in diseases thought to result from oxidant damage, such as coronary artery disease. More recently, UCB has been shown to possess potent immunomodulatory properties through interception of the innate and adaptive immune systems. While UCB has cytotoxic effects at high concentrations, moderate elevations of UCB are associated with immunosuppressive effects. At 6–8 mg/dL, UCB may interfere with MHC II, B7, and CD28 presentation, along with suppressing effector T cell function and expansion, while promoting regulatory T cell function and expansion. Closer to physiologic concentrations, UCB can also interrupt complement binding, thereby affecting the innate arms of immunity. The mechanism for how UCB may exert such varied effects likely rests on the ease of UCB entry through plasma membranes of target cells. Once inside the cell, UCB may interrupt proinflammatory signaling cascades, potentially through inhibition of kinases, although further investigation of this mechanism is warranted. The affinity of UCB for membranes of innate immune cells can modulate Fc receptor and C1 q receptor expression. Consequently, immunosuppression is observed in animal models of multiple inflammatory disorders; epidemiologic studies in analogous human autoimmune disorders also supports UCB leading to milder disease courses when UCB concentrations are mildly elevated. It may be possible to therapeutically harness these benefits through induction of heme oxygenase or suppression of UDP-glucuronosyltransferases to potentiate the lifespan of circulating UCB. As such, clinical trials of these agents as adjuncts to established therapy may prove useful in treating autoimmune disorders. References Basiglio CL. Protective role of unconjugated bilirubin on complement-mediated hepatocytolysis. Biochimica et Biophysica Acta 2007;1770(7):1003–10. Benaron DA, Bowen FW. Variation of initial serum bilirubin rise in newborn infants with type of illness. Lancet 1991;338(8759):78–81. Biburger M. Pivotal advance: heme oxygenase 1 expression by human CD4+ T cells is not sufficient for their development of immunoregulatory capacity. Journal of Leukocyte Biology 2010;87(2):193–202. Bock KW, Kohle C. Contributions of the Ah receptor to bilirubin homeostasis and its antioxidative and atheroprotective functions. Biological Chemistry 2010;391(6):645–53. Bonelli M. Heme oxygenase-1 end-products carbon monoxide and biliverdin ameliorate murine collagen induced arthritis. Clinical and Experimental Rheumatology 2012;30(1):73–8. Cainzos M. Hyperbilirubinemia: jaundice and anergy. Hepato-gastroenterology 1992;39(4):330–2. Capuzzi DM, Guyton JR, Morgan JM, Goldberg AC, Kreisberg RA, Brusco OA. Efficacy and safety of an extended-release niacin (niaspan): a long-term study. American Journal of Cardiology 1998;82:74–81. Choi BM, Pae HO. Critical role of heme oxygenase-1 in Foxp3-mediated immune suppression. Biochemical and Biophysical Research Communications 2005;327(4):1066–71. Chora AA. Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinflammation. The Journal of Clinical Investigation 2007;117(2):438–47. De Sanctis C. Neonatal hyperbilirubinemia and response of lymphocytes to phytohemagglutinin. Minerva Pediatrica 1968;20(38):2010–3. de Vries HS. A functional polymorphism in UGT1A1 related to hyperbilirubinemia is associated with a decreased risk for Crohn’s disease. Journal of Crohn’s & Colitis 2011;6(5):597–602. Diaz del Casrillo E. Neonatal hyperbilirubinemia. Its relation to the immunological response capacity of the newborn infant. Gaceta Medica de Mexico 1973;105(2):185–207.

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