Neutrophils in the Pathogenesis of Sepsis John C. Marshall, MD - PowerPoint PPT Presentation

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

Thank you!!