, Volume 29, Issue 3, pp 451–465 | Cite as

Lipocalin 2 alleviates iron toxicity by facilitating hypoferremia of inflammation and limiting catalytic iron generation

  • Xia Xiao
  • Beng San Yeoh
  • Piu Saha
  • Rodrigo Aguilera Olvera
  • Vishal Singh
  • Matam Vijay-Kumar


Iron is an essential transition metal ion for virtually all aerobic organisms, yet its dysregulation (iron overload or anemia) is a harbinger of many pathologic conditions. Hence, iron homeostasis is tightly regulated to prevent the generation of catalytic iron (CI) which can damage cellular biomolecules. In this study, we investigated the role of iron-binding/trafficking innate immune protein, lipocalin 2 (Lcn2, aka siderocalin) on iron and CI homeostasis using Lcn2 knockout (KO) mice and their WT littermates. Administration of iron either systemically or via dietary intake strikingly upregulated Lcn2 in the serum, urine, feces, and liver of WT mice. However, similarly-treated Lcn2KO mice displayed elevated CI, augmented lipid peroxidation and other indices of organ damage markers, implicating that Lcn2 responses may be protective against iron-induced toxicity. Herein, we also show a negative association between serum Lcn2 and CI in the murine model of dextran sodium sulfate (DSS)-induced colitis. The inability of DSS-treated Lcn2KO mice to elicit hypoferremic response to acute colitis, implicates the involvement of Lcn2 in iron homeostasis during inflammation. Using bone marrow chimeras, we further show that Lcn2 derived from both immune and non-immune cells participates in CI regulation. Remarkably, exogenous rec-Lcn2 supplementation suppressed CI levels in Lcn2KO serum and urine. Collectively, our results suggest that Lcn2 may facilitate hypoferremia, suppress CI generation and prevent iron-mediated adverse effects.


Lipocalin 2 Iron Catalytic iron Inflammation Anemia of inflammation Oxidative stress 



Lipocalin 2


Lipocalin 2 knockout




Neutrophil gelatinase-associated lipocalin




Lactate dehydrogenase


Alanine transaminase


Aspartate transaminase


Catalytic iron



We thank Dr. Gregory Shearer for his critical input.


This work was supported by grants from the National Institutes of Health (NIH) R01 (DK097865) and PSU Dean’s Schultz endowment, College of Health and Human Development Seed grant to M.V.-K. B.S.Y. is supported by NIH T32 (T32AI074551).

Compliance with ethical standards

Conflict of Interest

The authors have declared that no conflict of interest exists.

Supplementary material

10534_2016_9925_MOESM1_ESM.tif (594 kb)
Supplementary material 1 (TIFF 594 kb)
10534_2016_9925_MOESM2_ESM.tif (1.1 mb)
Supplementary material 2 (TIFF 1126 kb)
10534_2016_9925_MOESM3_ESM.docx (12 kb)
Supplementary material 3 (DOCX 11 kb)


  1. Akrawinthawong K, Shaw MK, Kachner J et al (2013) Urine catalytic iron and neutrophil gelatinase-associated lipocalin as companion early markers of acute kidney injury after cardiac surgery: a prospective pilot study. Cardiorenal Med 3:7–16CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bao G, Clifton M, Hoette TM et al (2010) Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex. Nat Chem Biol 6:602–609CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bao GH, Xu J, Hu FL, Wan XC, Deng SX, Barasch J (2013) EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex. Biometals 26:1041–1050CrossRefPubMedPubMedCentralGoogle Scholar
  4. Barcia AM, Harris HW (2005) Triglyceride-rich lipoproteins as agents of innate immunity. Clin Infect Dis 41(Suppl 7):S498–S503CrossRefPubMedGoogle Scholar
  5. Boddaert N, Le Quan Sang KH, Rotig A et al (2007) Selective iron chelation in Friedreich ataxia: biologic and clinical implications. Blood 110:401–408CrossRefPubMedGoogle Scholar
  6. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302–310CrossRefPubMedGoogle Scholar
  7. Burkitt MJ, Milne L, Raafat A (2001) A simple, highly sensitive and improved method for the measurement of bleomycin-detectable iron: the ‘catalytic iron index’ and its value in the assessment of iron status in haemochromatosis. Clin Sci (Lond) 100(3):239–247CrossRefGoogle Scholar
  8. Cabantchik ZI (2014) Labile iron in cells and body fluids: physiology, pathology, and pharmacology. Front Pharmacol 5:45CrossRefPubMedPubMedCentralGoogle Scholar
  9. Carvalho FA, Aitken JD, Gewirtz AT, Vijay-Kumar M (2011) TLR5 activation induces secretory interleukin-1 receptor antagonist (sIL-1Ra) and reduces inflammasome-associated tissue damage. Mucosal Immunol 4:102–111CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chakraborty S, Kaur S, Guha S, Batra SK (2012) The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer. Biochim Biophys Acta 1826:129–169PubMedPubMedCentralGoogle Scholar
  11. Chassaing B, Aitken JD, Malleshappa M, Vijay-Kumar M (2014) Dextran sulfate sodium (DSS)-induced colitis in mice. Current protocols in immunology/edited by John E Coligan [et al] 104, Unit 15 25Google Scholar
  12. Chassaing B, Srinivasan G, Delgado MA, Young AN, Gewirtz AT, Vijay-Kumar M (2012) Fecal lipocalin 2, a sensitive and broadly dynamic non-invasive biomarker for intestinal inflammation. PLoS One 7:e44328CrossRefPubMedPubMedCentralGoogle Scholar
  13. Coudevylle N, Geist L, Hotzinger M et al (2010) The v-myc-induced Q83 lipocalin is a siderocalin. J Biol Chem 285:41646–41652CrossRefPubMedPubMedCentralGoogle Scholar
  14. Darshan D, Frazer DM, Wilkins SJ, Anderson GJ (2010) Severe iron deficiency blunts the response of the iron regulatory gene Hamp and pro-inflammatory cytokines to lipopolysaccharide. Haematologica 95:1660–1667CrossRefPubMedPubMedCentralGoogle Scholar
  15. Devireddy LR, Hart DO, Goetz DH, Green MR (2010) A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell 141:1006–1017CrossRefPubMedPubMedCentralGoogle Scholar
  16. Dixon SJ, Lemberg KM, Lamprecht MR et al (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149:1060–1072CrossRefPubMedPubMedCentralGoogle Scholar
  17. Duan LP, Wang HH, Wang DQ (2004) Cholesterol absorption is mainly regulated by the jejunal and ileal ATP-binding cassette sterol efflux transporters Abcg5 and Abcg8 in mice. J Lipid Res 45:1312–1323CrossRefPubMedGoogle Scholar
  18. Dupic F, Fruchon S, Bensaid M et al (2002) Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice. Gut 51:648–653CrossRefPubMedPubMedCentralGoogle Scholar
  19. Flo TH, Smith KD, Sato S et al (2004) Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432:917–921CrossRefPubMedGoogle Scholar
  20. Ganz T (2003) Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood 102:783–788CrossRefPubMedGoogle Scholar
  21. Ganz T (2011) Hepcidin and iron regulation, 10 years later. Blood 117:4425–4433CrossRefPubMedPubMedCentralGoogle Scholar
  22. Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, Strong RK (2002) The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10:1033–1043CrossRefPubMedGoogle Scholar
  23. Halliwell B, Gutteridge JM (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186:1–85CrossRefPubMedGoogle Scholar
  24. Handa P, Morgan-Stevenson V, Maliken BD et al (2016) Iron overload results in hepatic oxidative stress, immune cell activation, and hepatocellular ballooning injury, leading to nonalcoholic steatohepatitis in genetically obese mice. Am J Physiol Gastrointest Liver Physiol 310:G117–G127CrossRefPubMedGoogle Scholar
  25. Hentze MW, Muckenthaler MU, Andrews NC (2004) Balancing acts: molecular control of mammalian iron metabolism. Cell 117:285–297CrossRefPubMedGoogle Scholar
  26. Hoette TM, Abergel RJ, Xu J, Strong RK, Raymond KN (2008) The role of electrostatics in siderophore recognition by the immunoprotein Siderocalin. J Am Chem Soc 130:17584–17592CrossRefPubMedPubMedCentralGoogle Scholar
  27. Hu L, Hittelman W, Lu T et al (2009) NGAL decreases E-cadherin-mediated cell-cell adhesion and increases cell motility and invasion through Rac1 in colon carcinoma cells. Lab Investig 89:531–548CrossRefPubMedGoogle Scholar
  28. Huang H, Salahudeen AK (2002) Cold induces catalytic iron release of cytochrome P-450 origin: a critical step in cold storage-induced renal injury. Am J Transpl 2:631–639CrossRefGoogle Scholar
  29. Iwahashi H, Morishita H, Ishii T, Sugata R, Kido R (1989) Enhancement by catechols of hydroxyl-radical formation in the presence of ferric ions and hydrogen peroxide. J Biochem 105:429–434PubMedGoogle Scholar
  30. Jiang W, Constante M, Santos MM (2008) Anemia upregulates lipocalin 2 in the liver and serum. Blood Cells Mol Dis 41:169–174CrossRefPubMedPubMedCentralGoogle Scholar
  31. Kaitha S, Bashir M, Ali T (2015) Iron deficiency anemia in inflammatory bowel disease. World J Gastrointest Pathophysiol 6:62–72PubMedPubMedCentralGoogle Scholar
  32. Kakhlon O, Cabantchik ZI (2002) The labile iron pool: characterization, measurement, and participation in cellular processes(1). Free Radic Biol Med 33:1037–1046CrossRefPubMedGoogle Scholar
  33. Koskenkorva-Frank TS, Weiss G, Koppenol WH, Burckhardt S (2013) The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: insights into the potential of various iron therapies to induce oxidative and nitrosative stress. Free Radic Biol Med 65:1174–1194CrossRefPubMedGoogle Scholar
  34. Kruszewski M (2003) Labile iron pool: the main determinant of cellular response to oxidative stress. Mutat Res 531:81–92CrossRefPubMedGoogle Scholar
  35. Lele S, Shah S, McCullough PA, Rajapurkar M (2009) Serum catalytic iron as a novel biomarker of vascular injury in acute coronary syndromes. EuroIntervention 5:336–342CrossRefPubMedGoogle Scholar
  36. Maitra D, Shaeib F, Abdulhamid I et al (2013) Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway. Free Radic Biol Med 63:90–98CrossRefPubMedGoogle Scholar
  37. Martines AM, Masereeuw R, Tjalsma H, Hoenderop JG, Wetzels JF, Swinkels DW (2013) Iron metabolism in the pathogenesis of iron-induced kidney injury. Nat Rev Nephrol 9:385–398CrossRefPubMedGoogle Scholar
  38. Masaratana P, Patel N, Latunde-Dada GO, Vaulont S, Simpson RJ, McKie AT (2013) Regulation of iron metabolism in Hamp (−/−) mice in response to iron-deficient diet. Eur J Nutr 52:135–143CrossRefPubMedGoogle Scholar
  39. Mishra J, Mori K, Ma Q et al (2004) Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 15:3073–3082CrossRefPubMedGoogle Scholar
  40. Mitchell KM, Dotson AL, Cool KM, Chakrabarty A, Benedict SH, LeVine SM (2007) Deferiprone, an orally deliverable iron chelator, ameliorates experimental autoimmune encephalomyelitis. Mult Scler 13:1118–1126CrossRefPubMedGoogle Scholar
  41. Nairz M, Theurl I, Schroll A et al (2009) Absence of functional Hfe protects mice from invasive Salmonella enterica serovar Typhimurium infection via induction of lipocalin-2. Blood 114:3642–3651CrossRefPubMedPubMedCentralGoogle Scholar
  42. Nakamura K, Kawakami T, Yamamoto N et al. (2015) Activation of the NLRP3 inflammasome by cellular labile iron. Exp HematolGoogle Scholar
  43. Nemeth E, Rivera S, Gabayan V et al (2004) IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Investig 113:1271–1276CrossRefPubMedPubMedCentralGoogle Scholar
  44. Posey JE, Gherardini FC (2000) Lack of a role for iron in the Lyme disease pathogen. Science 288:1651–1653CrossRefPubMedGoogle Scholar
  45. Ratledge C (2007) Iron metabolism and infection. Food Nutr Bull 28:S515–S523CrossRefPubMedGoogle Scholar
  46. Roudkenar MH, Halabian R, Bahmani P, Roushandeh AM, Kuwahara Y, Fukumoto M (2011) Neutrophil gelatinase-associated lipocalin: a new antioxidant that exerts its cytoprotective effect independent on heme oxygenase-1. Free Radical Res 45:810–819CrossRefGoogle Scholar
  47. Roudkenar MH, Halabian R, Ghasemipour Z et al (2008) Neutrophil gelatinase-associated lipocalin acts as a protective factor against H(2)O(2) toxicity. Arch Med Res 39:560–566CrossRefPubMedGoogle Scholar
  48. Sanders CJ, Moore DA 3rd, Williams IR, Gewirtz AT (2008) Both radioresistant and hemopoietic cells promote innate and adaptive immune responses to flagellin. J Immunol 180:7184–7192CrossRefPubMedGoogle Scholar
  49. Schiefner A, Skerra A (2015) The menagerie of human lipocalins: a natural protein scaffold for molecular recognition of physiological compounds. Acc Chem Res 48:976–985CrossRefPubMedGoogle Scholar
  50. Shah SV, Rajapurkar MM, Baliga R (2011) The role of catalytic iron in acute kidney injury. Clin J Am Soc Nephrol 6:2329–2331CrossRefPubMedGoogle Scholar
  51. Shi H, Bencze KZ, Stemmler TL, Philpott CC (2008) A cytosolic iron chaperone that delivers iron to ferritin. Science 320:1207–1210CrossRefPubMedPubMedCentralGoogle Scholar
  52. Singh V, Yeoh BS, Carvalho F, Gewirtz AT, Vijay-Kumar M (2015) Proneness of TLR5 deficient mice to develop colitis is microbiota dependent. Gut Microbes 6:279–283CrossRefPubMedGoogle Scholar
  53. Srinivasan G, Aitken JD, Zhang B et al (2012) Lipocalin 2 deficiency dysregulates iron homeostasis and exacerbates endotoxin-induced sepsis. J Immunol 189:1911–1919CrossRefPubMedPubMedCentralGoogle Scholar
  54. Stanley ER (1985) The macrophage colony-stimulating factor, CSF-1. Methods Enzymol 116:564–587CrossRefPubMedGoogle Scholar
  55. Stein J, Hartmann F, Dignass AU (2010) Diagnosis and management of iron deficiency anemia in patients with IBD. Nat Rev Gastroenterol Hepatol 7:599–610CrossRefPubMedGoogle Scholar
  56. Sulieman M, Asleh R, Cabantchik ZI et al (2004) Serum chelatable redox-active iron is an independent predictor of mortality after myocardial infarction in individuals with diabetes. Diabetes Care 27:2730–2732CrossRefPubMedGoogle Scholar
  57. Sullivan JL (2009) Iron in arterial plaque: modifiable risk factor for atherosclerosis. Biochimica et biophysica acta 1790:718–723CrossRefPubMedGoogle Scholar
  58. Thethi TK, Parsha K, Rajapurkar M et al (2011) Urinary catalytic iron in obesity. Clin Chem 57:272–278CrossRefPubMedGoogle Scholar
  59. Torrance JD, Bothwell TH (1968) A simple technique for measuring storage iron concentrations in formalinised liver samples. S Afr J Med Sci 33:9–11PubMedGoogle Scholar
  60. Walmsley TA, George PM, Fowler RT (1992) Colorimetric measurement of iron in plasma samples anticoagulated with EDTA. J Clin Pathol 45:151–154CrossRefPubMedPubMedCentralGoogle Scholar
  61. Wang J, Pantopoulos K (2011) Regulation of cellular iron metabolism. Biochem J 434:365–381CrossRefPubMedPubMedCentralGoogle Scholar
  62. Wang L, Harrington L, Trebicka E et al (2009) Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis in mice. J Clin Investig 119:3322–3328PubMedPubMedCentralGoogle Scholar
  63. Wang L, Johnson EE, Shi HN, Walker WA, Wessling-Resnick M, Cherayil BJ (2008) Attenuated inflammatory responses in hemochromatosis reveal a role for iron in the regulation of macrophage cytokine translation. J Immunol 181:2723–2731CrossRefPubMedPubMedCentralGoogle Scholar
  64. Weinberg ED (1997) The Lactobacillus anomaly: total iron abstinence. Perspect Biol Med 40:578–583CrossRefPubMedGoogle Scholar
  65. Weiss G (2009) Iron metabolism in the anemia of chronic disease. Biochimica et biophysica acta 1790:682–693CrossRefPubMedGoogle Scholar
  66. Yamada Y, Miyamoto T, Kashima H et al. (2016) Lipocalin 2 attenuates iron-related oxidative stress and prolongs the survival of ovarian clear cell carcinoma cells by up-regulating the CD44 variant. Free Radic Res, 1–36Google Scholar
  67. Yang J, Goetz D, Li JY et al (2002) An iron delivery pathway mediated by a lipocalin. Mol Cell 10:1045–1056CrossRefPubMedGoogle Scholar
  68. Zhang X, Goncalves R, Mosser DM (2008) The isolation and characterization of murine macrophages. Current protocols in immunology/edited by John E Coligan [et al] Chapter 14, Unit 14 11Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Xia Xiao
    • 1
  • Beng San Yeoh
    • 1
  • Piu Saha
    • 1
  • Rodrigo Aguilera Olvera
    • 1
  • Vishal Singh
    • 1
  • Matam Vijay-Kumar
    • 1
    • 2
  1. 1.Department of Nutritional SciencesThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Department of MedicineThe Pennsylvania State University Medical CenterHersheyUSA

Personalised recommendations