Université Paris Descartes École doctorale : Bio Sorbonne Paris Cité Institut Cochin, Hypoxia and iron homeostasis team The immunomodulatory role of epidermal hepcidin in infectious condition Par Mariangela MALERBA Thèse de doctorat de Physiopathologie Dirigée par Carole PEYSSONNAUX Présentée et soutenue publiquement le 25/09/2018 Devant un jury composé de : Dr. Carole PEYSSONNAUX Directeur de thèse Pr. Sylvie CHOLLET-MARTIN Rapporteur Dr. François CANONNE-HERGAUX Rapporteur Pr. Selim ARACTINGI Examinateur Dr. Agnès FOUET Membre invité 1 Contents ABSTRACT .......................................................................................................................................................... 6 INTRODUCTION ................................................................................................................................................. 8 HEPCIDIN ............................................................................................................................................... 9 1.1. Hepcidin: synthesis and structure ................................................................................................. 9 1.2. Hepcidin antimicrobial function .................................................................................................. 11 1.3. Hepcidin: iron regulatory hormone ............................................................................................. 12 1.3.1. Iron metabolism ...................................................................................................................... 12 1.3.2. Regulation of the iron homeostasis ......................................................................................... 16 1.4. Hepcidin/infection axis ................................................................................................................ 17 1.5. Hepcidin regulation ..................................................................................................................... 21 1.6. Extra-hepatic hepcidin and other roles. ...................................................................................... 25 2. SKIN ...................................................................................................................................................... 29 2.1. Mammalian skin architecture ...................................................................................................... 29 2.2. Skin Immunity .............................................................................................................................. 35 3. NEUTROPHIL ........................................................................................................................................ 47 3.1. Neutrophil production and bone marrow egress ........................................................................ 47 3.2. Neutrophils in tissue .................................................................................................................... 50 3.3. Chemoattractant molecules: the chemokine family ................................................................... 53 3.4. Role of neutrophils in fighting infection ...................................................................................... 55 4. GAS ...................................................................................................................................................... 63 4.1. Epidemiology of GAS infection .................................................................................................... 63 4.2. M1T1 : GAS invasive strain .......................................................................................................... 66 4.3. GAS-phagocyte fighting ............................................................................................................... 70 4.4. GAS-keratinocyte fighting ............................................................................................................ 73 4.5. GAS iron metabolism ................................................................................................................... 74 THE AIM OF THE STUDY ................................................................................................................................... 75 RESULTS ........................................................................................................................................................... 77 1. Hepcidin shares structural similarities with IL8 and displays neutrophil chemoattractant properties. 78 Hepcidin is expressed in the epidermis ............................................................................................... 85 Epidermal hepcidin has a protective role against GAS infection ........................................................ 88 Hepcidin has no antimicrobial effect against GAS in vitro and does not influence keratinocyte antimicrobial functions. ............................................................................................................................... 92 Hamp1 Δker mice do not display epidermal iron accumulation ......................................................... 93 Hepcidin modulates keratinocyte immune response ......................................................................... 96 Hepcidin- mediated CXCL1 production protects against GAS infection. ............................................. 99 2 Hepcidin is resistant to SpyCEP cleavage .......................................................................................... 101 Hepcidin subcutaneous injections have a therapeutic potential against GAS infection ................... 104 SUPPLEMENTARY FIGURES ............................................................................................................ 106 MATERIALS AND METHODS ........................................................................................................................... 110 DISCUSSION ................................................................................................................................................... 117 New chemoattractant role of hepcidin. ............................................................................................ 118 Hepcidin is expressed in the skin in pathophysiological conditions .................................................. 120 Keratinocyte- derived hepcidin has an immunomodulatory role during infection ........................... 122 Hepcidin as a therapeutic agent ........................................................................................................ 127 Conclusion and highlights .................................................................................................................. 130 BIBLIOGRAPHY ............................................................................................................................................... 131 ANNEXES ........................................................................................................................................................ 158 Epidermal hepcidin prevents systemic bacterial spreading in a Group A streptococcus-induced necrotizing soft tissue infection model ..................................................................................................... 159 Methods for treating gram positive bacterial infection .................................................................... 176 3 TABLE OF ABBREVIATIONS Aa amino acid IRP iron regulatory protein RLR RIG-like helicases AI anaemia of inflammation JAK Janus activated kinases ROS reactive oxygen species AMP antimicrobial peptide KRT keratin SC subcutaneous APC antigen presenting cell LF lactoferrin Scl1 streptococcus collagen-like ARF acute rheumatic fever LFA1 lymphocyte function- ScpA streptococcal C5a peptidase associated antigen1 A BM basement membrane LIP labile iron pool Sda1 Sterptococcal DNAse 1 BMP bone morphogenetic protein LPS lipopolysaccharide Shp streptococcal heme binding CLR C-type lectin receptor protein LTA lipoteicoic acid CXCL CXC-chemokine ligand Shr streptococcal heme protein MMP8/9 metrix metalloprotease receptor CXCR CXC-chemokine receptor 8/9 Ska1 Streptokinase 1 DAMP damage associated MPO myeloperoxidase molecular pattern SLO streptolysin O NE Neutrophil elastase DC dendritic cell SLS streptolysin S NET neutrophil extracellular trap dcytB duodenal cytochrome B sodA superoxide-detoxifying NLR NOD like receptor DMT1 Divalent metal transporter enzyme A 1 NoxA NADPH oxidase A SpeA/B Streptococcal Pyrogenic EPO erythropoiesis NRAMP1/2 natural resistance Exotoxin A/B associated macrophage protein Spy CEP streptococcus pyogenes fMLP N-Formylmethionyl- 1/2 leucyl-phenylalanine cell envelope protease NTBI non transferrin bound iron FPN ferroportin Sse Streptococcal esterase OH heme oxygenase GAS Group A streptococcus STAT signal transducers and PAMP pathogen associated activators of transcription GCPR G protein coupled receptor molecular pattern STSS streptococcal toxic shock G-CSF Granulocyte colony PDA 4 protein arginine deiminase syndrome stimulating factor type 4 Tf transferrin GM-CSF Granulocyte- PGN peptidoglycan macrophage colony-stimulating TfR1/2 transferrin receptor 1/2 PMN polymorphonuclear TJ tight junction HA hyaluronan capsule leukocyte TLR toll-like receptor HCP1 heme carrier protein PRR pattern recognition receptor TMPRSS6 matriptase HF hair follicle PSGL1 P-selectin glycoprotein HH hereditary hemochromatosis ligand 1 WT wild type HIFs hypoxia inducible factors