ACVP &ASVCP 2014, Atlanta, GA Mini-Symposium: Iron Biology and Anemia Iron Maldistribution and Infection Hal Drakesmith, PhD Weatherall Institute of Molecular Medicine John Radcliffe Hospital, University of Oxford, Oxford, UK hdrakes@hammer.imm.ox.ac.uk Introduction – (almost) everything needs iron Fundamental cellular physiological processes require iron. Oxygen carriage, generation of energy from oxygen, macromolecular synthesis and maintenance of genome fidelity through DNA repair are all iron-dependent activities. Iron is utilized because its valency states endow it with appropriate oxidoreductive properties and a capability to form bonds in multiple orientations, allowing it to contribute to catalysis and electron shuttling in iron-sulphur complexes, heme, and enzymes. Such basic biochemical machinations are so ancient that they are almost$ universally shared across the Kingdoms of life, with the result that not only do humans require iron to grow and thrive, so do the microorganisms that infect us(1). Indeed, of all the essential micronutrients, it does appear that iron has a very particular role in mediating host-pathogen interactions. The initial adoption of iron into newlyevolved life occurred at a (Hadean/Archean) time of high iron bioavailability, in conditions of a relatively reductive, oxygen-poor, acidic and sulphur-rich environment, in which iron is soluble. However since the Great Oxygenation Event of ~2.3 billion years ago, iron, although highly abundant, has become poorly bioavailable due to its negligible solubility at neutral pH. Therefore, because iron is both indispensable and difficult to obtain, it can be a key factor affecting the outcome of infectious diseases. Evidence supporting the latter statement comes from a large variety of sources. Multiple animal studies employing many different microoganisms have shown that increasing the amount of iron available to the invading pathogen can exacerbate infections. In humans too, correlations of high iron status and development of disease(2, 3), observations of infections developing after iron supplementation in both small groups and larger clinical trials(4-8), and case reports of genetic iron overload associating with infection(9) together support the concept that host iron matters. Examining the role of iron from the pathogen perspective corroborates this view; microbes have evolved a diverse array of mechanisms for scavenging iron from their hosts’ serum, hemoglobin and ferritin. For example there are more than 500 known bacterial siderophores (small, high- affinity iron-chelating compounds). Further confirmation of the centrality of iron for pathogen vigor is provided by the high level of genomic investment in iron-acquiring mechanisms and by the contribution of such genes to virulence. Highly pathogenic strains of Yersinia enterolytica, Y. pseudotuberculosis, and Y. pestis possess a common high-pathogenicity island that encodes proteins necessary for the synthesis, transport, and regulation of the siderophore yersiniabactin(10). Genetic detective work based on the full sequence of Y. pestis suggests that acquisition of these improved ironacquiring capabilities allowed the organism to make a niche transition from being an enteric to a (devastating) systemic pathogen(11). The host response to infection, and hepcidin In humans, iron and heme are usually tightly chaperoned, and free iron or heme in blood is an aberrant state. Under conditions of infection, serum iron is (generally) further ‘locked down’, becoming sequestered in reticuloendothelial macrophages and stored intracellularly in proteinaceous cages of ferritin. The depletion of iron from serum under conditions of $ rare exceptions include lactobacilli and the Lyme disease pathogen Borelia burgdorferi (Posey et al, Science 2000 vol288, p1651-3), which appear to make use of manganese in place of iron 1 ACVP &ASVCP 2014, Atlanta, GA Mini-Symposium: Iron Biology and Anemia invasion by pathogens is termed the hypoferremia of infection, and is thought to be a critical defence mechanism to protect against potentially fatal septicemia. The hypoferremia of infection appears to be a common mechanism in mammals and was first observed, and experimentally demonstrated, in the 1940s by pioneering work of Wintrobe and colleagues(12-14). Some iron sequestering proteins, for example ferritin, lactoferrin (which bind iron), haptoglobin and hemopexin (which bind heme) are acute-phase response proteins that contribute to iron withholding from pathogens as an innate immune response to infection gets underway. The molecular mechanism of the hypoferremia of infection has been further elucidated in recent years since the identification of the key molecules that regulate systemic iron homeostasis, hepcidin and its target ferroportin. In brief, ferroportin mediates export of iron from macrophages and from enterocytes (and to a lesser extent, from hepatocytes), and hepcidin is a small peptide hormone that binds and inhibits ferroportin(15-18). Relevant to the present context, hepcidin is ancestrally related to anti-microbial peptides called defensins, although hepcidin itself has only limited direct microbicidal activity. However hepcidin may be indirectly beneficial to the host in terms of protecting against infection, because inflammatory cytokines such as IL-6, IL-22 and Type I interferon, released by the host following sensing of infection, strongly induce transcription of hepcidin(19-21) and so decrease iron availability to blood-borne pathogens via the blockade and degradation of ferroportin. In addition, transcription of the ferroportin gene is itself suppressed by inflammation, mediated at least in part by Type II interferon and TNF-alpha. The net effect is that inflammation following infection inhibits active ferroportin and prevents synthesis of new ferroportin; clearly these effects would be expected to synergize and result in acute macrophage iron deposition and a ‘scorched earth’ of serum depleted of iron. Indeed, in acute (experimental) infections in humans, transferrin saturation can rapidly fall to <5%. Iron redistribution in specific infections and pathological sequelae The redistribution of iron into macrophages and out of serum due to inflammation in an infectious context is not without consequences. If an infection persists and becomes chronic, continually raised hepcidin and hypoferremia decreases iron availability to tissues. The major demand for iron is from the erythron where iron is incorporated into heme, and eventually, erythropoiesis is limited by iron availability, and anemia results(22, 23). The anemia of chronic disease, also known as the anemia of inflammation is highly prevalent in hospitalized patients in the UK and is a substantial burden on the National Health Service. Increased hepcidin levels may help to distinguish anemia due to inflammation and anemia due to other causes(24). In animal models of the disease, anemia can be rescued by prevention of hepcidin activity(25, 26), and treatments for humans based on this concept are under development. A second consequence of iron redistribution is the effect on predisposition to secondary infections. Iron loading in macrophages may inhibit immunological macrophage functions(27) and favor growth of macrophage-tropic infections (e.g. Mycobacterium tuberculosis)(28). Iron redistribution at enrolment into a Gambian HIV cohort was predictive of incident tuberculosis(29), and common secondary infections (Mycobacterium, Candida, Pneumocystis) were more common in HIV-1-infected individuals with a high degree of macrophage iron loading(30). Iron redistribution is particularly common in malaria, with increased levels of hepcidin(31-33) and enhanced erythrophagocytosis contributing to an increase in macrophage iron, a relative paucity of iron in hepatocytes and decreased dietary iron absorption(34), which together lead to a lack of availability for erythropoiesis and an increase in prevalence of anemia (for example, at the end of a malaria season(35)). This iron redistribution has effects on both superinfection and on co-infection in the context of malaria. Hepcidin has been shown to play a crucial role in determining the multiplicity of malaria infections within a single host. The obligate liver stage of the malaria parasite 2 ACVP &ASVCP 2014, Atlanta, GA Mini-Symposium: Iron Biology and Anemia requires iron: hepcidin peptide injection or hepcidin overexpression by transgene or viral vector can reduce parasite survival at the crucial hepatic bottleneck (36). This same effect was observed when mice already harbouring a hepcidin-inducing blood-stage infection were exposed to liver-tropic sporozoites; the superinfection failed to thrive and did not emerge from the liver into the blood. Therefore it appears that the hepcidin upregulation initiated by one blood-stage malaria infection blocks the establishment of a second, potentially competing, Plasmodium infection (36). The physiological redistribution of iron as a consequence of malaria may also have a significant effect on host susceptibility to other bacterial, viral, or protozoa parasites. The increase in macrophage iron that occurs in blood-stage malaria may benefit pathogens that exploit the macrophage niche (37). In particular, hepcidin upregulation may help to explain the association between malaria infections and susceptibility to non-typhoid salmonella (NTS). The epidemiological link between malaria and NTS is well-established (38). Iron has been implicated in the contribution of malaria to NTS susceptibility through increases in both free heme and heme-oxygenase expression (39). By routing iron to accumulate in macrophages, the hepcidin response to malaria may also render the host more vulnerable to NTS directly (37). Finally mention must be made of an important exception to the rule of the hypoferremia of infection; we recently found that in the acute phases of both Hepatitis C virus and Hepatitis B virus infections, during the first 10 days when detectable virus emerges into the circulation, there was no evidence of hepcidin increase or of hypoferremia, and for HBV there was an indication of an increase in serum iron. This is despite (or potentially because?) the infection is localised in the organ that produces hepcidin. Chronic HCV and to a lesser extent HBV are characterised by iron accumulation in the liver, and chronic HCV is also associated with decreased hepcidin, even in the presence of ongoing inflammation(40, 41). Hepatic iron accumulation can contribute to organ damage in these chronic viral diseases, and more generally is a significant health problem both in humans (where overload may be caused due to aberrantly low levels of hepcidin and/or blood transfusion) and in some species of animals in zoo conditions - the best characterised appears to be browsing Rhinos (42) (where the cause is at least in part nutritional). Conclusion Iron is a critical nutrient required for growth and optimal function both by humans / mammals and by the microbial organisms that can pathogenically infect us. A part of the innate immune defence against most pathogens is the hypoferremic response, caused largely by increased hepcidin and decreased ferroportin activity, and which leads to iron redistribution out of serum and into macrophages. This iron relocalisation is likely protective against fatal septicemia, but may have deleterious consequences in terms of predisposition to develop inflammatory anemia and susceptibility to macrophage-tropic secondary infections. Hepatotropic viral infections represent interesting and important exceptions to the hyoperremia rule, as these conditions are associated with normal or decreased hepcidin and toxic accumulation of iron in the liver. The ability to therapeutically manipulate the hepcidin/iron axis, as well as offering possible new treatment options for disorders of iron metabolism, may eventually contribute to therapy for infectious diseases – because while pathogens can and do evade antibody and T-cell immunity, and develop antibiotic resistance, they cannot avoid their requirement for iron. References 1. Drakesmith, H., and Prentice, A.M. 2012. Hepcidin and the iron-infection axis. Science 338:768-772. 3 ACVP &ASVCP 2014, Atlanta, GA 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Mini-Symposium: Iron Biology and Anemia McDermid, J.M., Jaye, A., van der Loeff, M.F., Todd, J., Bates, C., Austin, S., Jeffries, D., Awasana, A.A., Whittle, H.C., and Prentice, A.M. 2007. Elevated Iron Status Strongly Predicts Mortality in West African Adults With HIV Infection. J Acquir Immune Defic Syndr. McDermid, J.M., van der Loeff, M.F., Jaye, A., Hennig, B.J., Bates, C., Todd, J., Sirugo, G., Hill, A.V., Whittle, H.C., and Prentice, A.M. 2009. Mortality in HIV infection is independently predicted by host iron status and SLC11A1 and HP genotypes, with new evidence of a gene-nutrient interaction. Am J Clin Nutr 90:225-233. Murray, M.J., Murray, A.B., Murray, M.B., and Murray, C.J. 1978. The adverse effect of iron repletion on the course of certain infections. Br Med J 2:1113-1115. Murray, M.J., Murray, N.J., Murray, A.B., and Murray, M.B. 1975. Refeeding-malaria and hyperferraemia. Lancet 1:653-654. Sazawal, S., Black, R.E., Ramsan, M., Chwaya, H.M., Stoltzfus, R.J., Dutta, A., Dhingra, U., Kabole, I., Deb, S., Othman, M.K., et al. 2006. Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: communitybased, randomised, placebo-controlled trial. Lancet 367:133-143. Soofi, S., Cousens, S., Iqbal, S.P., Akhund, T., Khan, J., Ahmed, I., Zaidi, A.K., and Bhutta, Z.A. 2013. Effect of provision of daily zinc and iron with several micronutrients on growth and morbidity among young children in Pakistan: a clusterrandomised trial. Lancet. Trousseau, A. 1872. True and false chlorosis. In Lectures on clinical medicine: Lindsay & Blakiston, Philadelphia. 95-117. Frank, K.M., Schneewind, O., and Shieh, W.J. 2011. Investigation of a researcher's death due to septicemic plague. The New England journal of medicine 364:25632564. Carniel, E. 1999. The Yersinia high-pathogenicity island. International microbiology : the official journal of the Spanish Society for Microbiology 2:161-167. Parkhill, J., Wren, B.W., Thomson, N.R., Titball, R.W., Holden, M.T., Prentice, M.B., Sebaihia, M., James, K.D., Churcher, C., Mungall, K.L., et al. 2001. Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413:523-527. Cartwright, G.E., Lauritsen, M.A., Humphreys, S., Jones, P.J., Merrill, I.M., and Wintrobe, M.M. 1946. The Anemia Associated With Chronic Infection. Science 103:72-73. Cartwright, G.E., Lauritsen, M.A., Humphreys, S., Jones, P.J., Merrill, I.M., and Wintrobe, M.M. 1946. The Anemia of Infection. Ii. The Experimental Production of Hypoferremia and Anemia in Dogs. The Journal of clinical investigation 25:81-86. Cartwright, G.E., Lauritsen, M.A., Jones, P.J., Merrill, I.M., and Wintrobe, M.M. 1946. The Anemia of Infection. I. Hypoferremia, Hypercupremia, and Alterations in Porphyrin Metabolism in Patients. The Journal of clinical investigation 25:65-80. Abboud, S., and Haile, D.J. 2000. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem 275:19906-19912. Donovan, A., Brownlie, A., Zhou, Y., Shepard, J., Pratt, S.J., Moynihan, J., Paw, B.H., Drejer, A., Barut, B., Zapata, A., et al. 2000. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 403:776-781. McKie, A.T., Marciani, P., Rolfs, A., Brennan, K., Wehr, K., Barrow, D., Miret, S., Bomford, A., Peters, T.J., Farzaneh, F., et al. 2000. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 5:299-309. Nemeth, E., Tuttle, M.S., Powelson, J., Vaughn, M.B., Donovan, A., Ward, D.M., Ganz, T., and Kaplan, J. 2004. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 306:2090-2093. 4 ACVP &ASVCP 2014, Atlanta, GA 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. Mini-Symposium: Iron Biology and Anemia Armitage, A.E., Eddowes, L.A., Gileadi, U., Cole, S., Spottiswoode, N., Selvakumar, T.A., Ho, L.P., Townsend, A.R., and Drakesmith, H. 2011. Hepcidin regulation by innate immune and infectious stimuli. Blood 118:4129-4139. Nemeth, E., Rivera, S., Gabayan, V., Keller, C., Taudorf, S., Pedersen, B.K., and Ganz, T. 2004. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 113:1271-1276. Ryan, J.D., Altamura, S., Devitt, E., Mullins, S., Lawless, M.W., Muckenthaler, M.U., and Crowe, J. 2012. Pegylated interferon-alpha induced hypoferremia is associated with the immediate response to treatment in hepatitis C. Hepatology 56:492-500. Theurl, I., Aigner, E., Theurl, M., Nairz, M., Seifert, M., Schroll, A., Sonnweber, T., Eberwein, L., Witcher, D.R., Murphy, A.T., et al. 2009. Regulation of iron homeostasis in anemia of chronic disease and iron deficiency anemia: diagnostic and therapeutic implications. Blood 113:5277-5286. Weiss, G., and Goodnough, L.T. 2005. Anemia of chronic disease. N Engl J Med 352:1011-1023. Pasricha, S.R., Atkinson, S.H., Armitage, A.E., Khandwala, S., Veenemans, J., Cox, S.E., Eddowes, L.A., Hayes, T., Doherty, C.P., Demir, A.Y., et al. 2014. Expression of the iron hormone hepcidin distinguishes different types of anemia in African children. Science translational medicine 6:235re233. Sasu, B.J., Cooke, K.S., Arvedson, T.L., Plewa, C., Ellison, A.R., Sheng, J., Winters, A., Juan, T., Li, H., Begley, C.G., et al. 2010. Anti-hepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammationinduced anemia. Blood:blood-2009-2009-245977. Theurl, I., Schroll, A., Sonnweber, T., Nairz, M., Theurl, M., Willenbacher, W., Eller, K., Wolf, D., Seifert, M., Sun, C.C., et al. 2011. Pharmacologic inhibition of hepcidin expression reverses anemia of chronic inflammation in rats. Blood 118:4977-4984. Theurl, I., Fritsche, G., Ludwiczek, S., Garimorth, K., Bellmann-Weiler, R., and Weiss, G. 2005. The macrophage: a cellular factory at the interphase between iron and immunity for the control of infections. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 18:359-367. Boelaert, J.R., Vandecasteele, S.J., Appelberg, R., and Gordeuk, V.R. 2007. The effect of the host's iron status on tuberculosis. J Infect Dis 195:1745-1753. McDermid, J.M., Hennig, B.J., van der Sande, M., Hill, A.V., Whittle, H.C., Jaye, A., and Prentice, A.M. 2013. Host iron redistribution as a risk factor for incident tuberculosis in HIV infection: an 11-year retrospective cohort study. BMC Infect Dis 13:48. de Monye, C., Karcher, D.S., Boelaert, J.R., and Gordeuk, V.R. 1999. Bone marrow macrophage iron grade and survival of HIV-seropositive patients. AIDS 13:375-380. de Mast, Q., Nadjm, B., Reyburn, H., Kemna, E.H., Amos, B., Laarakkers, C.M., Silalye, S., Verhoef, H., Sauerwein, R.W., Swinkels, D.W., et al. 2009. Assessment of urinary concentrations of hepcidin provides novel insight into disturbances in iron homeostasis during malarial infection. J Infect Dis 199:253-262. de Mast, Q., Syafruddin, D., Keijmel, S., Riekerink, T.O., Deky, O., Asih, P.B., Swinkels, D.W., and van der Ven, A.J. 2010. Increased serum hepcidin and alterations in blood iron parameters associated with asymptomatic P. falciparum and P. vivax malaria. Hemeatologica 95:1068-1074. de Mast, Q., van Dongen-Lases, E.C., Swinkels, D.W., Nieman, A.E., Roestenberg, M., Druilhe, P., Arens, T.A., Luty, A.J., Hermsen, C.C., Sauerwein, R.W., et al. 2009. Mild increases in serum hepcidin and interleukin-6 concentrations impair iron incorporation in hemeoglobin during an experimental human malaria infection. Br J Hemeatol 145:657-664. 5 ACVP &ASVCP 2014, Atlanta, GA 34. 35. 36. 37. 38. 39. 40. 41. 42. Mini-Symposium: Iron Biology and Anemia Prentice, A.M., Doherty, C.P., Abrams, S.A., Cox, S.E., Atkinson, S.H., Verhoef, H., Armitage, A.E., and Drakesmith, H. 2012. Hepcidin is the major predictor of erythrocyte iron incorporation in anemic African children. Blood 119:1922-1928. Atkinson, S.H., Armitage, A.E., Khandwala, S., Mwangi, T.W., Uyoga, S., Bejon, P.A., Williams, T.N., Prentice, A.M., and Drakesmith, H. 2014. Combinatorial effects of malaria season, iron deficiency, and inflammation determine plasma hepcidin concentration in African children. Blood 123:3221-3229. Portugal, S., Carret, C., Recker, M., Armitage, A.E., Goncalves, L.A., Epiphanio, S., Sullivan, D., Roy, C., Newbold, C.I., Drakesmith, H., et al. 2011. Host-mediated regulation of superinfection in malaria. Nature medicine 17:732-737. van Santen, S., de Mast, Q., Swinkels, D.W., and van der Ven, A.J. 2013. The iron link between malaria and invasive non-typhoid Salmonella infections. Trends in parasitology 29:220-227. Mabey, D.C., Brown, A., and Greenwood, B.M. 1987. Plasmodium falciparum malaria and Salmonella infections in Gambian children. J Infect Dis 155:1319-1321. Cunnington, A.J., de Souza, J.B., Walther, M., and Riley, E.M. 2012. Malaria impairs resistance to Salmonella through heme- and heme oxygenase-dependent dysfunctional granulocyte mobilization. Nat Med 18:120-127. Fujita, N., Sugimoto, R., Takeo, M., Urawa, N., Mifuji, R., Tanaka, H., Kobayashi, Y., Iwasa, M., Watanabe, S., Adachi, Y., et al. 2007. Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C. Mol Med 13:97-104. Girelli, D., Pasino, M., Goodnough, J.B., Nemeth, E., Guido, M., Castagna, A., Busti, F., Campostrini, N., Martinelli, N., Vantini, I., et al. 2009. Reduced serum hepcidin levels in patients with chronic hepatitis C. J Hepatol 51:845-852. Beutler, E., West, C., Speir, J.A., Wilson, I.A., and Worley, M. 2001. The hHFE gene of browsing and grazing rhinoceroses: a possible site of adaptation to a low-iron diet. Blood Cells Mol Dis 27:342-350. 6