Arzu L. ARAL, Ayşe Başak ENGİN, Mehmet Ali ERGÜN, Alp Özgün BÖRCEK, Hayrunnisa BOLAY

Iron homeostasis is altered in response to hypoxia and hypothermic preconditioning in brain glial cells

Background/aim: Altered iron metabolism is one of the pathophysiological mechanisms occurring during hypoxic injuries in the central nervous system. Proper homeostasis of cellular iron is regulated by iron import, storage, and export proteins that prevent excess iron overload or iron starvation in cells. Therapeutic hypothermia is an approved treatment for hypoxic ischemia in newborns, but the underlying molecular mechanism is still unknown. We studied the effects of hypoxia, preceded with preconditioning, on the iron homeostasis of glial cells, known as a major actor in the inflammatory process during perinatal brain injury. Materials and methods: Primary microglia and astrocytes in culture were exposed to 12 h of hypoxia with or without mild hypothermic preconditioning. The mRNA expression was assessed using qPCR. Iron accumulation was visualized via modified Perl’s histochemistry. Cytokine levels in cell cultures were measured using ELISA. Results: Hypothermic preconditioning enhanced microglial viability, which previously was decreased in both cell types due to hypoxia. Hypoxia increased iron accumulation in the mixed glial cells and in ferritin expression in both microglia and astrocytes. Hypotermic preconditioning decreased the elevated ferritin-light chain expression significantly in microglia. Iron importer proteins, DMT1 and TfR1, both increased their mRNA expression after hypoxia, and hypothermic preconditioning continued to support the elevation of DMT1 in both glial cell types. Ferroportin expression increased as a survival factor of the glial cell following hypoxia. Hypothermic preconditioning supported this increase in both cell types and was especially significant in astrocytes. IL-10 levels were prominently increased in cell culture after hypothermic preconditioning. Conclusion: The data suggest that hypothermic preconditioning affects cellular iron homeostasis by regulating the storage and transfer proteins of iron. Regulation of the cellular iron traffic may prevent glial cells from experiencing the detrimental effects of hypoxia-related inflammation.

PDF

___

1. Van Steenwinckel J, Schang AL, Sigaut S, Chhor V, Degos V et al. Brain damage of the preterm infant: new insights into the role of inflammation. Biochemical Society Transactions 2014; 42 (2): 557-563.
2. Rezaie P, Dean A. Periventricular leucomalasia, inflammation and white matter lesions within the developing nervous system. Neuropathology 2002; 22: 106-132.
3. Gupta K, Sharma N, Sundaram V, Vasishta RK, Kakkar N. Periventricular leukomalacia-Fungal mimicker in newborn brain: A case report with review of literature. Neurology India 2010; 58 (6): 951-953. doi: 10.4103/0028-3886.73757
4. Mallard C, Davidson JO, Tan S, Green CR, Bennet L et al. Astrocytes and microglia in acute cerebral injury underlying cerebral palsy associated with preterm birth. Pediatric Research 2013; 75 (1-2): 234-240. doi: 10.1038/pr.2013.188
5. Fleiss B, Gressens P. Tertiary mechanisms of brain damage: a new hope for treatment of cerebral palsy? Lancet Neurology 2012; 11: 556-566.
6. Jeong SY, David S. Glycosylphosphatidylinositol-anchored ceruloplasmin is required for iron efflux from cells in the central nervous system. Journal of Biological Chemistry 2003; 278 (29): 27144-27148.
7. Shouman BO, Mesbah A, Aly H. Iron metabolism and lipid peroxidation products in infants with hypoxic ischemic encephalopathy. Journal of Perinatology 2008; 28 (7): 487-491.
8. Kaur C, Ling EA. Periventricular white matter damage in the hypoxic neonatal brain: role of microglial cells. Progress in Neurobiology 2009; 87: 264-280.
9. Rathnasamy G, Ling EA, Kaur C. Iron and iron regulatory proteins in ameboid microglial cells are linked to oligodendrocyte death in hypoxic neonatal rat periventricular white matter through production of proinflammatory cytokines and reactive oxygen/nitrogen species. Journal of Neuroscience 2011; 31 (49): 17982-17995.
10. Rathnasamy G, Ling EA, Kaur C. Hypoxia inducible factor-1 alpha mediates iron uptake which induces inflammatory response in ameboid microglial cells in developing periventricular white matter through MAP kinase pathway. Neuropharmacology 2014; 77: 428-440. doi: 10.1016/j. neuropharm.2013.10.024
11. Qian ZM, Wu XM, Fan M, Yang L, Du F et al. Divalent metil transporter 1 is a hypoxia inducible gene. Journal of Cellular Physiology 2011; 226: 1596-1603.
12. Yang L, Fan M, Du F, Gong Q, Bi ZG et al. Hypoxic preconditioning increases iron transport rate in astrocytes. Biochimica et Biophysica Acta 2012; 1822: 500-508.
13. Irace C, Scorziello A, Maffettone C, Pignataro G, Matrone C et al. Divergent modulation of iron regulatory proteins and ferritin biosynthesis by hypoxia-reoxygenation in neurons and glial cells. Journal of Neurochemistry 2005; 95: 1321-1331.
14. Wu LY, Ding AS, Zhao T, Ma ZM, Wang FZ et al. Underlying mechanism of hypoxic preconditioning decreasing apoptosis induced by anoxia in cultured hippocampal neurons. Neurosignals 2005; 14 (3): 109-116.
15. Edwards AD, Brocklehurst P, Gunn AJ, Halliday H, Juszczak E et al. Neurological outcomes at 18 months of age after moderate hypothermia for perinatal hypoxic ischaemic encephalopathy: synthesis and meta-analysis of trial data. British Medical Journal 2010; 340: c363. doi: 10.1136/bmj.c363
16. Seo JW, Kim JH, Kim JH, Seo M, Han HS et al. Time-dependent effects of hypothermia on microglial activation and migration. Journal of Neuroinflammation 2012; 9: 164. doi: 10.1186/1742- 2094-9-164
17. Diestel A, Troeller S, Billecke N, Sauer IM, Berger F et al. Mechanisms of hypothermia induced cell protection mediated by microglial cells in vitro. European Journal of Neuroscience 2010; 31 (5): 779-787. doi: 10.1111/j.1460-9568.2010.07128.x
18. Matsui T, Motoki Y, Yoshida Y. Hypothermia reduces TLR-3 activated microglial IFN-beta and NO production. Mediators of Inflammation 2013; 436263.
19. Jeong SY, David S. Age-related changes in iron homeostasis and cell death in the cerebellum of ceruloplasmin-deficient mice. Journal of Neuroscience 2006; 26 (38): 9810-9819.
20. Arslan N, Sayın O, Altun Z, Çavdar Z, Eğrilmez MY et al. İnsan beyni mikrovasküler endotel hücrelerinde in vitro iskemi reperfüzyon modelinin geliştirilmesi ve hasarın değerlendirilmesi. Turkish Journal of Biochemistry 2010; 35 (3): 195-202 (in Turkish).
21. Nishio S, Yunoki M, Chen ZF, Anzivino MJ, Lee KS. Ischemic tolerance in the rat neocortex following HT. Journal of Neurosurgery 2000; 93 (5): 845-851.
22. Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox generated free radicals. Proceedings of the National Academy of Sciences of the United States of America 1997; 94: 9866-9868.
23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using RT qPCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25 (4): 402-408.
24. Schulz K, Kroner A, David S. Iron efflux from astrocytes plays a role in remyelination. Journal of Neuroscience 2012; 32 (14): 4841-4847.
25. Ma H, Sinha B, Pandya RS, Lin N, Popp AJ et al. Therapeutic hypothermia as a neuroprotective strategy in neonatal hypoxicischemic brain injury and traumatic brain injury. Current Molecular Medicine 2012; 12 (10): 1282-1296.
26. Xiong M, Chen LX, Ma SM, Yang Y, Zhou WH. Short-term effects of hypothermia on axonal injury, preoligodendrocyte accumulation and oligodendrocyte myelination after hypoxia-ischemia in the hippocampus of immature rat brain. Developmental Neuroscience 2013; 35 (1): 17-27. doi: 10.1159/000346324
27. Rey-Funes M, Dorfman VB, Ibarra ME, Peña E, Contartese DS et al. Hypothermia prevents gliosis and angiogenesis development in an experimental model of ischemic proliferative retinopathy. Investigative Ophthalmology & Visual Science 2013; 54 (4): 2836-2846. doi: 10.1167/iovs.12-11198
28. Imai F, Suzuki H, Oda J, Ninomiya T, Ono K et al. Neuroprotective effect of exogenous microglia in global brain ischemia. Journal of Cerebral Blood Flow and Metabolism 2007; 27: 488-500.
29. Huang J, Liu G, Shi B, Shi G, He X et al. Inhibition of microglial activation by minocycline reduced preoligodendrocyte injury in a neonatal rat brain slice model. Journal of Thoracic Cardiovascular Surgery 2018; 156 (6): 2271-2280.
30. Alva N, Bardallo RG, Basanta D, Palomeque J, Carbonell T. Preconditioning-like properties of short-term hypothermia in isolated perfused rat liver (IPRL) system. International Journal of Molecular Sciences 2018; 19 (4): 1023. doi: 10.3390/ ijms19041023
31. Jiang S, Wu Y, Fang D, Chen Y. Hypothermic preconditioning but not ketamine reduces oxygen and glucose deprivation induced neuronal injury correlated with downregulation of COX-2 expression in mouse hippocampal slices. Journal of Pharmacological Sciences 2018; 137 (1): 30-37. doi: 10.1016/j. jphs.2018.04.001
32. Kaur C, Sivakumar V, Ang LS, Sundaresan A. Hypoxic damage to the periventricular white matter in neonatal brain: role of vascular endothelial growth factor, nitric oxide and excitotoxicity. Journal of Neurochemistry 2006; 98 (4): 1200- 1216.
33. Greenhalgh AD, Zarruk JG, Healy LM, Baskar Jesudasan SJ, Jhelum P et al. Peripherally derived macrophages modulate microglial function to reduce inflammation after CNS injury. PLoS Biology 2018; 16 (10): e2005264.
34. Peyssonnaux C, Zinkernagel AS, Schuepbach RA, Rankin E, Vaulont S et al. Regulation of iron homeostasis by the hypoxiainducible transcription factors (HIFs). Journal of Clinical Investigation 2007; 117: 1926-1932.
35. Zarruk JG, Berard JL, Passos dos Santos R, Kroner A, Lee J et al. Expression of iron homeostasis proteins in the spinal cord in experimental autoimmune encephalomyelitis and their implications for iron accumulation. Neurobiology of Disease 2015; 81: 93-107. doi: 10.1016/j.nbd.2015.02.001
36. Ryan F, Zarruk JG, Lößlein L, David, S. Ceruloplasmin plays a neuroprotective role in cerebral ischemia. Frontiers in Neuroscience 2019; 8 (12): 988.
37. Kroner A, Greenhalgh AD, Zarruk JG, Passos Dos Santos R, Gaestel M et al. TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord. Neuron 2014; 83 (5):1098-1116.
38. McCarthy RC, Kosman DJ. Mechanisms and regulation of iron trafficking across the capillary endothelial cells of the BBB. Frontiers in Molecular Neuroscience 2015; 8: 31.
39. Rouault TA, Cooperman S. Brain iron metabolism. Seminars in Pediatric Neurology 2006; 13: 142-148.
40. Aguirre P, Mena N, Tapia V, Arredondo M, Núñez MT. Iron homeostasis in neuronal cells: a role for IREG1. BMC Neuroscience 2005; 6: 3.
41. Arosio P, Levi S. Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage. Biochimica et Biophysica Acta 2010; 1800: 783-792. doi: 10.1016/j.bbagen.2010.02.005
42. Vercellotti GM, Khan FB, Nguyen J, Chen C, Bruzzone CM et al. H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice. Frontiers in Pharmacology 2014; 5: 79.
43. Harrison PM, Arosio P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochimica et Biophysica Acta 1996: 1275: 161-203.
44. Yang L, Wang D, Wang X, Lu Y, Zhu L. The roles of hypoxiainducible Factor-1 and iron regulatory protein 1 in iron uptake induced by acute hypoxia. Biochemical and Biophysical Research Communications 2018; 507 (1-4): 128-135.
45. Duck KA, Simpson IA, Connor JR. Regulatory mechanisms for iron transport across the blood-brain barrier. Biochemical and Biophysical Research Communications 2017; 494: 70-75.
46. Lee JH, Wei ZZ, Cao W, Won S, Gu X et al. Regulation of therapeutic hypothermia on inflammatory cytokines, microglia polarization, migration and functional recovery after ischemic stroke in mice. Neurobiology of Disease 2016; 96: 248-260. doi: 10.1016/j.nbd.2016.09.013
47. Matsui T, Yoshida Y, Yanagihara M, Suenaga H. Hypothermia at 35°C reduces the time dependent microglial production of pro-inflammatory and anti-inflammatory factors that mediate neuronal cell death. Neurocritical Care 2014; 20 (2): 301-310.
48. Chiou B, Connor JR. Emerging and Dynamic Biomedical Uses of Ferritin. Pharmaceuticals (Basel) 2018; 11 (4). doi: 10.3390/ ph11040124
49. Dignass A, Farrag K, Stein J. Limitations of Serum Ferritin in Diagnosing Iron Deficiency in Inflammatory Conditions. International Journal of Chronic Diseases 2018, 9394060.
50. Weis S, Carlos AR, Moita MR, Singh S, Blankenhaus B et al. Metabolic Adaptation Establishes Disease Tolerance to Sepsis. Cell 2017; 169: 1263-1275.
51. Gozzelino R, Andrade BB, Larsen R, Luz NF, Vanoaica L et al. Metabolic adaptation to tissue iron overload confers tolerance to malaria. Cell Host and Microbe 2012; 12: 693-704. doi: 10.1016/j.chom.2012.10.011
52. Fan Y, Zhang J, Cai L, Wang S, Liu C et al. The effect of anti-inflammatory properties of ferritin light chain on lipopolysaccharide-induced inflammatory response in murine macrophages. Biochimica et Biophysica Acta 2014; 1843: 2775- 2783. doi: 10.1016/j.bbamcr.2014.06.015
53. Parthasarathy N, Torti SV, Torti FM. Ferritin binds to light chain of human H-kininogen and inhibits kallikrein-mediated bradykinin release. Biochemical Journal 2002; 365: 279-286.
54. Aral LA, Pinar L, Göktaş G, Deveden EY, Erdoğan D. Comparison of hippocampal interleukin-6 immunoreactivity after exhaustive exercise in both exercise-trained and untrained rats. Turkish Journal of Medical Sciences 2014; 44 (4): 560-568.
55. Bobbo VCD, Jara CP, Mendes NF, Morari J, Velloso LA et al. Interleukin-6 expression by hypothalamic microglia in multiple inflammatory contexts: a systematic review. BioMed Research International 2019; 22: 1365210.