Neutrophils in the Pathogenesis of Sepsis John C. Marshall, MD FRCSC Critical Care Canada Forum 2012 Toronto, Canada October 29, 2012 St. Michael’s Hospital University of Toronto
Thanks to … Songhui Jia Maria Jimenez Yue Li Zeenat Malam Jean Parodo Ravi Taneja Jennifer Tsang Bill Watson
Neutrophils are a Dynamic Population • Constitutively apoptotic • 6 – 8 Hours In vivo • 24 – 36 Hours In vitro • 10 11 PMN released and die by apoptosis each day
Neutrophils kill bacteria and phagocytose damaged tissue Activated neutrophils also damage adjacent normal tissues
Neutrophil Antimicrobial Defenses Antimicrobial peptides • BPI • Defensins • Alkaline phosphatase • Lysozyme • Lactoferrin • Elastase Production of reactive oxygen intermediates • Cathepsin G
Neutrophil extracellular traps (NETs)
8 * Non-specific IgG Log CFU/ml Blood Anti-PMN 6 Time-dependent 4 ** Effects of Neutrophil 2 Depletion in CLP 0 - 24 Hours + 12 Hours Time Relative to CLP - Hoesel et al Shock 24:40, 2005
PMN Apoptosis is Delayed in Sepsis 60 50 Percent Apoptosis Control LPS 40 Sepsis 30 * 20 10 ** 0 0 Hours 18 Hours 24 Hours Duration of In Vitro Culture - Taneja, Crit Care Med 32:1460,2004
Factors Inhibiting Neutrophil Apoptosis Microbial Products Host-derived Mediators Interleukin (IL)-1 β Endotoxin Lipoteichoic acid IL-2 Mannan IL-3 Modulins from CONS IL-4 E. coli verotoxin IL-6 H. pylori surface proteins IL-8 Butyric acid Tumor Necrosis Factor Propionic acid Interferons G-CSF, GM-CSF Respiratory syncytial virus Leptin PBEF C5a Cathelicidins Physiologic Processes Transendothelial migration
Delayed Apoptosis Requires Cellular Activation and Protein Synthesis - Watson, J.Immunol. 161:957, 1998 40 ‡ ‡ ‡ ‡ ‡ ‡ % Apoptosis 30 * Control Herb A 20 * PDTC CHX 10 0 LPS GM-CSF Control (1 m g/ml) (100U/ml)
Effects of Anti-sense to IL-1 on LPS and GM-CSF Inhibition of PMN Apoptosis 50 Percent Apoptosis 40 30 Control LPS (1ug/ml) 20 GM-CSF (100U/ml) 10 0 Control Anti-sense Sense Anti-sense + IL-1 - Watson, J.Immunol. 161:957, 1998.
Pre-B Cell Colony Enhancing Factor (PBEF) • 52 kDa secreted protein • Cytokine-like TATT motifs in mRNA • Synergizes with IL-7 to induce pre-B cell colony formation - Samal; Mol.Cell Biol. 14:1431, 1994
PBEF mRNA is Highly Expressed in Sepsis PMN Sepsis Patients Lps Con 1 2 3 4 5 6 7 8 PBEF G3PDH - Jia et al J Clin Invest 113:1318, 2004
PBEF Anti-sense Partially Restores PMN Apoptosis in Sepsis 30 * 25 Percent Apoptosis 20 15 10 5 0 No Oligo Sense Non-sense Anti-sense - Jia et al J Clin Invest 113:1318:2004
Anti-apoptotic effect of Nampt active PBEF on trauma PMN is inhbited by prior addition of insulin (20 µU/mL) N=6 70 no insulin 60 add ins 2nd 50 % apoptosis add ins 1st 40 30 20 10 0 Control PBEF S199A S200A
Trauma and Sepsis Result in Systemic Epithelial Cell Apoptosis Hotchkiss et al Crit.Care Med. 28:3207, 2000 - Imai et al, JAMA 289:2104, 2003
CD95/TNFR Caspase 8 APAF-1 Inhibitors of Apoptosis: Cytochrome C Survivin CIAP1,2 Procaspase 9 XIAP1, NAIP Caspase 3 Bcl2 SMAC/Diablo Mcl1 DNA, Protein cleavage
Caspase-8 MDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIK DALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYL NTRKEEMERELQTPGRAQISAYRFHFCRMSWAEANSQ CQTQSVPFWRRVDHLLIRVMLYQISEEVSRSELRSFKF LLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKR VCAQINKSLLKIINDYEEFSKGEELCGVMTISDSPREQD SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKL HSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQI Y EIL KIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPI YELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPV ETDSEEQP Y LEMDLSSPQTRYIPDEADFLLGMATVNNC VSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVN Y EVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD
Trauma Neutrophils Induce Caspase-8 Dephosphorylation and Apoptosis of A549 Lung Epithelial Cells ** ** Percent Hypodiploid DNA 50 40 No PMN + PMN Western: 30 ~ Caspase-8 * * 20 ~pY 10 - - + + - - DPI - - - + - Catalase 0 - - - - + GSH A549 HEK293
The Caspase-8 Survivalsome IL-1R CD95/TNFR FADD Pro-caspase-8 SHP-1 lyn pY Caspase-8 pY Dephosphorylation Caspase-8 Phosphorylation p18 p10 PI3 Kinase Activation PBEF Transcription IAP Synthesis Cleavage of caspase-3 and progression of apoptosis
Phagocytosis of Candida albicans Induces Neutrophil Apoptosis In Vitro * 90 Percentage Apoptosis 80 70 60 50 40 30 20 10 0 Control 1 : 1 5 : 1 10 : 1 Candida : Neutrophil Ratio - Rotstein, Shock 2000; 14:278
Intratracheal E. coli Attenuates Pulmonary Neutrophilia Ischemia/reperfusion I/R + E. coli - Sookhai, Ann Surg 2002; 235:285
Intratracheal Heat-killed E. coli Attenuates Mortality Following Ischemia/Reperfusion 10 0 Percent Survival 80 Sham 60 I/R + E.coli I/R + Saline 40 20 0 0 10 20 30 40 50 Time (Hours) - Sookhai Ann Surg 2002; 235:285
Conclusions • Functional neutrophils are critical to the early response against infection • Their prolonged activity contributes to inadvertent organ injury • Accelerating their involution through apoptosis may limit this delayed harm
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