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netosis [Autosaved].pptx

  1. NETOSIS Ritasman Baisya
  2. Points of discussion • Definition of NET & NETosis • Brief history • Types • Mechanism of NET formation • Action of NETs • NETs and autoimmunity • Therapy related to NET • COVID & NETosis
  3. NET – Definition • NETs are large, extracellular, web-like structures composed of cytosolic and granule proteins that are assembled on a scaffold of decondensed chromatin • Majority of DNA from nucleus , also from mitochondria • NETs trap, neutralize and kill bacteria, fungi, viruses and parasites and are thought to prevent bacterial and fungal dissemination
  4. Components of NETs
  5. History • In 2004 , Brinkmann et al described NET • Stimulation with IL8, PMA , LPS & visualization with EM and IF - NETs were observed • Under EM - NETs were composed of linear elements about 15–17 (nm) in diameter and are studded with globules • The backbone of these NETs was composed of DNA and histones as demonstrated by IF
  6. Electron microscopic view
  7. Immunochemistry
  8. NETOSIS -definition • NET release occurs primarily through a cell death process termed NETosis • 2007 - it is declared as unique cell death pathway of neutrophil • During NETosis, neutrophils release NETs, which can capture and kill bacteria and other pathogens to prevent them from spreading.
  9. Different types cell death
  10. Different forms of neutrophil death
  11. Types of NETosis
  12. lytic NETosis – suicidal • Slow cell death • Neutrophils arrest their actin dynamics and depolarize • Nuclear envelope disassembles • Delobulation of nuclei • Nuclear chromatin decondenses into the cytoplasm mixing with cytoplasmic and granule components • Plasma membrane then permeabilizes • NETs expand into the extracellular space 3–8 hours after neutrophil activation
  13. Basic types of NETosis
  14. Non-lytic NETosis – vital • It leads to the rapid release of NETs within minutes of exposure to Staphylococcus aureus via the secretion of chromatin and granule contents • No cell death – rapid response • Seen in small fraction of neutrophils during systemic S. aureus infection • Generates NETs and anucleated cytoplasts that crawl and phagocytose bacteria
  15. Suicidal versus Vital NETosis
  16. Other classification types
  17. Mechanism of NET formation
  18. Basic steps 1. Activation of ROS by NOX or NOX independent pathway 2. Release of NE and translocation to nucleus 3. MPO- NE mediated chromatin decondensation 4. Hyper-citrullination of chromatin 5. Nuclear and cell membrane permeabilization 6. Release of NET
  19. ROS activation • ROS generated by NADPH oxidase stimulate MPO to trigger the activation and translocation of NE to the nucleus • ROS-inducing receptors & kinases - MEK (MAPK/ERK kinase), ERK, IRAK, PKC, PI3K and AKT – activate ROS in response to PMA, microorganisms, parasites and immobilized immune complexes • Requirement for PI3K in NETosis has also implicated a role for autophagy
  20. MPO- NE pathway • NE binds to F-actin filaments in the cytoplasm and degrade them & enter the nucleus • NE proteolytically processes histones to disrupt chromatin packaging • MPO binds chromatin and synergizes with NE in decondensing chromatin independently of its enzymatic activity • MPO–NE pathway is induced by many NET stimuli, such as fungi and crystals
  21. Other biochemical events • In resting neutrophils - fraction of MPO is bound to NE as part of a complex called the azurosome • H2O2 - selectively releases NE into the cytosol in an MPO- dependent manner at physiological condition • Chlorinated polyamines - crosslink NET proteins, increasing NET stability and integrity and potentiating the capture of microorganisms
  22. NOX – independent pathway • Immune complexes, ionomycin and nicotine, trigger NETosis independently of NOX , relying instead on mitochondrial ROS • Non-lytic NETosis is also thought to occur independently of ROS • Glycans in saliva induce NETs via an unknown mechanism
  23. Chromatin decondensation • Histone deamination or hyper citrullination • Driven by peptidylarginine deiminase 4 (PAD4) • H202 - is sufficient to activate PAD4 which requires calcium and is activated by PKC • PAD4 inhibition blocks NET release in mouse models of sepsis and cancer • Histone citrullination and MPO-NE activity lead to chromatin decondensation
  24. Membrane permeabilization • Plasma membrane permeabilization occurs in a programmed manner & not as a consequence of physical disruption by the expanding chromatin • Monosodium urate (MSU) crystals promote necroptosis with receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3,
  25. Regulation of NETosis • Larger microorganisms- NET • Larger particle size -NET • Small virulent microorganisms that interfere with phagosomal killing • Large aggregates or abscess – favors NET • Microbial interference with phagosome maturation ( N . gonorrhea ) • Presence of motile flagella ( pseudomonas ) • Expression of enzyme ( invasin in yersinia ) • Lacking phagocyte promoting protease – cause NET
  26. Regulation of pH
  27. Intercellular regulation
  29. Microbiocidal action • Kill or suppress bacterial ,fungal, viral proliferation • Underlying mechanisms poorly understood. • Histones, defensins & cathelicidins - potent antimicrobials in NETs • NETosis is also likely to be the major route for the release of calprotectin • Physical sequestration of microorganisms by NETs is also thought to prevent systemic dissemination
  30. Mechanisms of NET-mediated pathology
  31. NETs damage tissues • NETs directly kill epithelial , endothelial cells • Excessive NETosis damages the epithelium in pulmonary fungal infection & endothelium in transfusion-related acute lung injury • NET-bound histones - central role in NET-mediated cytotoxicity • Defensins , permeabilize cells and NE targets extracellular matrix proteins that disrupt cell junctions
  32. NET causing organ injury
  33. NETs promote vaso-occlusion • Provide a scaffold that promotes DVT • Hypoxia-induced release of VWF & P-selectin from the endothelium initiate NET formation • Thromboxane A2 induces endothelial cell expression of ICAM1 to strengthen neutrophil interactions • Platelet- derived high mobility group protein B1 (HMGB1), ROS and integrins trigger NETosis
  34. Vaso-occlusion • NETs recruit Factor XIIa • Mobilizes endothelial cell granules (Weibel–Palade bodies) that contain VWF, P-selectin , Factor XIIa • Extracellular NET histones bind VWF and fibrin to recruit platelets and red blood cells • NET-bound NE cleaves tissue factor pathway inhibitor (TFPI) and proteolytically activates platelet receptors to increase platelet accumulation
  35. NET in vascular inflammation
  36. Other vascular events • NETs form in response to the build-up of bicarbonate salts and occlude pancreatic ducts to drive pancreatitis • NETosis in response to free haem may contribute to vaso- occlusion in sickle cell disease
  37. NETs modulate sterile inflammation • NETs regulate inflammatory cytokines directly or indirectly by modulating immune cells • Early inflammatory stages induce NETs by IL-6 & pro-IL- 1β in macrophages via TLR2 & 4 • Th 17 cell increased • Increased myeloid cell recruitment to site of inflamed lesions like atherosclerotic plaque
  38. Atherosclerosis and NET
  39. Sterile inflammation ( contd ) • Mouse model of ischaemia– reperfusion injury - NETs amplify inflammation and liver damage • NETs and neutrophil-derived IL-17 – role in Alzheimer disease
  40. NETosis in Alzheimer Disease
  41. • NETs promote inflammation and tissue destruction - delay wound healing in diabetes • Glucose is required for NETosis & neutrophils from patients with diabetes release NETs more readily • NETs - detected in adipose tissues of obese mice Sterile inflammation ( contd )
  42. Adiposity releases NET
  43. Anti-inflammatory response • NETs were suggested to have an anti-inflammatory role in mouse models of gout induced by MSU crystals • It is possible that NETs initiate inflammation and, as they build up over time, potentiate its resolution.
  44. NET as anti-inflammatory
  45. Neutrophils shield necrotic tissue by the formation of NETs building an anti-inflammatory barrier
  46. NETS in autoimmunity • NETs - source of self-antigen in autoimmune diseases • Autoantibodies against neutrophil- derived proteins • NET - first reported in kidney biopsy in ANCA- associated vasculitis - antibodies against NET components , MPO & proteinase 3 • Nuclear material released from NETs more immunogenic • Oxidised DNA in NET – promote IFN 1 signalling
  47. SLE • NETs activate plasmacytoid dendritic cells (pDCs) via TLR9 and TLR7 signalling & promote type I IFN expression • Mitochondrial ROS oxidize NET DNA to increase its ability to activate the stimulator of interferon genes (STING) pathway and trigger IFN production by pDCs • small population of circulating low-density granulocytes (LDGs) releases NETs spontaneously
  48. Basic steps in SLE
  49. Roles of mitochondria in NETosis in SLE
  50. Low density granulocyte in SLE
  51. Rheumatoid arthritis • Enhanced NETosis - detected in circulating & synovial-fluid neutrophils, synovial tissue, rheumatoid nodules, skin of affected patients • NETs are a source of extracellular citrullinated autoantigens • Release of active PAD isoforms through NETosis, citrullinate extracellular histones and fibrinogen in RA • It stimulate production of proinflammatory cytokines, chemokines & adhesion molecules in synovial fibroblasts
  52. NET in RA
  53. ANCA associated vasculitis • Antibodies to MPO & PR3 – pathogenic • They activate neutrophils primed by a proinflammatory stimuli, leading to respiratory burst • Enhanced NET formation in vitro • Lvels of NET remnants (MPO–DNA complexes), and neutrophil granular proteins, such as calprotectin , were increased in sera of AAV • NET formation is involved in vascular damage and immune system activation in AAV
  54. AAV – NET • ANCA induced NETs generated by C5a-primed neutrophils cause enhanced thrombosis & inflammation in AAV by promoting the expression of tissue factor • NETs can also present PR3 and MPO to dendritic cells • NETs - important sources of modified autoantigens in the kidney. • In necrotizing crescentic glomerulonephritis, neutrophil serine proteases like cathepsin G, neutrophil elastase and PR3 promoted IL-1β generation and kidney injury
  55. Front. Immunol., 30 June 2016
  56. APS • NETs - important activators of the coagulation cascade & integral components of arterial and venous thrombin • Serum of APS displays - decreased ability to degrade NETs • Elevated levels of both cell-free DNA and NET remnants • aPLs promote the release of NETs in a ROS and TLR4- dependent manner • A LDG population has also been described in primary AP
  57. NET in APS
  58. NETosis in APS
  59. A brief of NETs in autoimmune diseases
  61. Targeting NET
  62. Therapeutic strategy in autoimmunity • Inhibition of ROS production by targeting NADPH or mitochondria • ROS scavenger - N-acetyl cysteine (NAC) reduce NET release • MPO inhibitors - 4-aminobenzoic acid hydrazide or PF-1355- reduce NETosis, neutrophil recruitment and levels of circulating cytokines • TLR inhibitor - TAK-242 • PAD inhibitors - Cl-amidine , BB-Cl-amidine
  63. • Targeting B cells & plasma cells - reduction of autoantibody- induced NET formation • Inhibitors of calcineurin or GPCR phospholipase C ( staurosporine ) - suppress or modulate NETosis • DNase 1 - enhance their clearance • Targeting CXCL5 - decrease TH17-mediated autoimmunity - crescentic glomerulonephritis • Inhibition of TNF and IL-17 decreases NET in RA • Anti-C5 mAb therapy Therapeutic strategy
  64. Targeting NET in lupus
  65. Targeting NETs in other autoimmune disease
  66. Pictorial presentation of targets
  67. A Role for NETosis in COVID-19 Infection? • Cytokine storm seen with COVID-19 elaborates role of NET • NETs can induce macrophages to secrete IL1β & form a loop between macrophages and neutrophils, leading to progressive inflammatory damage. • Virus -induced NETs can circulate in an uncontrolled way, leading to an extreme systemic response of the body like ARDS . • NETs can be detected in tissues by immunohistochemistry & in blood by sandwich ELISA
  68. NET – lung damage – therapeutic aspect in COVID 19
  69. Take home message 1. NETosis- programmed cell death , unique in neutrophil 2. NETs are extracellular web like structure on scaffold of decondensed chromatin with granules 3. Either suicidal causing cell death or vital where no lysis happens 4. Mecahnism - activation of ROS , MPO-NE pathway , chromatin decondensation and increased permeability 5. It has multiple actions – microbiocidal , damage host tissue , sterile inflammation ( atherosclerosis , diabetes ),vaso –occlusion(DVT) and autoimmunity, malignancy 6. SLE , AAV ,gout ,RA , cresecentic GN – NET has important role 7. Therapeutic target of NETs are emerging 8. COVID 19 – NET – association is highly possible
  70. References 1. Papayannopoulos, V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 18, 134–147 (2018) 2. Gupta, S., Kaplan, M. The role of neutrophils and NETosis in autoimmune and renal diseases. Nat Rev Nephrol 12, 402–413 (2016). 3. De Bont, C.M., Boelens, W.C. & Pruijn, G.J.M. NETosis, complement, and coagulation: a triangular relationship. Cell Mol Immunol 16, 19–27 (2019). 4. Miguel Antonio Mesa1 and Gloria Vasque . NETosis . Autoimmune Diseases / 2013 5. Elsherif L, Sciaky N, Metts CA, et al. Machine Learning to Quantitate Neutrophil NETosis. Sci Rep. 2019;9(1):16891.
  71. 6.. Bryan G. Yipp, Paul Kubes; NETosis: how vital is it?. Blood 2013; 122 (16): 2784–2794 7. Boilard,E., Fortin, P. Mitochondria drive NETosis and inflammation in SLE. Nat Rev Rheumatol 12, 195–196 (2016 8. Xavier Bosch. Systemic Lupus Erythematosus and the Neutrophil. N Engl J Med 2011; 365:758-760 9. Nicoletta Sorvillo. Circulation Research. Extracellular DNA NET-Works With Dire Consequences for Health, Volume: 125, Issue: 4, References