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

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

May 29, 2023 818 likes •1.12k views

Neutrophils in the Pathogenesis of Sepsis John C. Marshall, MD FRCSC Critical Care Canada Forum 2012 Toronto, Canada October 29, 2012 St. Michaels Hospital University of Toronto Thanks to Songhui Jia Maria Jimenez Yue Li Zeenat

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SLIDE 1

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

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SLIDE 2

Songhui Jia Maria Jimenez Yue Li Zeenat Malam Jean Parodo Ravi Taneja Jennifer Tsang Bill Watson

Thanks to …

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SLIDE 3
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SLIDE 4
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SLIDE 5

Neutrophils are a Dynamic Population

  • Constitutively apoptotic
  • 6 – 8 Hours In vivo
  • 24 – 36 Hours In vitro
  • 1011 PMN released and die by

apoptosis each day

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SLIDE 6

Neutrophils kill bacteria and phagocytose damaged tissue Activated neutrophils also damage adjacent normal tissues

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SLIDE 7

Neutrophil Antimicrobial Defenses

Production of reactive

  • xygen intermediates

Antimicrobial peptides

  • BPI
  • Defensins
  • Alkaline phosphatase
  • Lysozyme
  • Lactoferrin
  • Elastase
  • Cathepsin G
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SLIDE 8

Neutrophil extracellular traps (NETs)

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SLIDE 9

Time-dependent Effects of Neutrophil Depletion in CLP

  • Hoesel et al Shock

24:40, 2005

  • 24 Hours

+ 12 Hours

Log CFU/ml Blood

2 4 6 8

Non-specific IgG Anti-PMN

Time Relative to CLP

* **

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SLIDE 10
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SLIDE 11

Duration of In Vitro Culture

0 Hours 18 Hours 24 Hours

Percent Apoptosis

10 20 30 40 50 60 Control Sepsis

  • Taneja, Crit Care Med 32:1460,2004

PMN Apoptosis is Delayed in Sepsis

LPS

* **

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SLIDE 12

Microbial Products Host-derived Mediators

Endotoxin Interleukin (IL)-1β 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

Factors Inhibiting Neutrophil Apoptosis

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SLIDE 13

% Apoptosis

10 20 30 40

*

‡ ‡ ‡ ‡ ‡ ‡

*

Control Herb A PDTC CHX Control LPS (1mg/ml) GM-CSF (100U/ml)

Delayed Apoptosis Requires Cellular Activation and Protein Synthesis

  • Watson, J.Immunol. 161:957, 1998
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SLIDE 14

Control Anti-sense Sense Anti-sense + IL-1

Percent Apoptosis

10 20 30 40 50 Control LPS (1ug/ml) GM-CSF (100U/ml)

Effects of Anti-sense to IL-1 on LPS and GM-CSF Inhibition of PMN Apoptosis

  • Watson, J.Immunol. 161:957, 1998.
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SLIDE 15

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
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SLIDE 16

Con Lps

Sepsis Patients

1 2 3 4 5 6 7 8

PBEF mRNA is Highly Expressed in Sepsis PMN

PBEF G3PDH

  • Jia et al J Clin Invest 113:1318, 2004
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SLIDE 17

No Oligo Sense Non-sense Anti-sense

Percent Apoptosis

5 10 15 20 25 30

*

PBEF Anti-sense Partially Restores PMN Apoptosis in Sepsis

  • Jia et al J Clin Invest 113:1318:2004
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SLIDE 18

10 20 30 40 50 60 70 Control PBEF S199A S200A % apoptosis

Anti-apoptotic effect of Nampt active PBEF on trauma PMN is inhbited by prior addition of insulin (20 µU/mL)

N=6

no insulin add ins 2nd add ins 1st

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SLIDE 19

Hotchkiss et al Crit.Care Med. 28:3207, 2000

  • Imai et al, JAMA 289:2104, 2003

Trauma and Sepsis Result in Systemic Epithelial Cell Apoptosis

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SLIDE 20

Cytochrome C

CD95/TNFR

Caspase 8 Procaspase 9

APAF-1

Caspase 3

Bcl2 Mcl1

Inhibitors of Apoptosis: Survivin CIAP1,2 XIAP1, NAIP

DNA, Protein cleavage

SMAC/Diablo

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SLIDE 21

MDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIK DALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYL NTRKEEMERELQTPGRAQISAYRFHFCRMSWAEANSQ CQTQSVPFWRRVDHLLIRVMLYQISEEVSRSELRSFKF LLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKR VCAQINKSLLKIINDYEEFSKGEELCGVMTISDSPREQD SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKL HSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIY EILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPI YELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPV ETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNC VSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNY EVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD

Caspase-8

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SLIDE 22

A549 HEK293 Percent Hypodiploid DNA 10 20 30 40 50

* * ** **

DPI

  • +

+ -

  • Catalase
  • + -

GSH

  • +

No PMN + PMN Western: ~ Caspase-8 ~pY

Trauma Neutrophils Induce Caspase-8 Dephosphorylation and Apoptosis of A549 Lung Epithelial Cells

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SLIDE 23

pY pY

SHP-1 Cleavage of caspase-3 and progression of apoptosis

lyn PI3 Kinase Activation PBEF Transcription IAP Synthesis

CD95/TNFR FADD Pro-caspase-8 Caspase-8 Phosphorylation Caspase-8 Dephosphorylation

p10 p18

The Caspase-8 Survivalsome

IL-1R

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SLIDE 24

Candida : Neutrophil Ratio

Control 1 : 1 5 : 1 10 : 1 Percentage Apoptosis

10 20 30 50 60 70 80 90

Phagocytosis of Candida albicans Induces Neutrophil Apoptosis In Vitro

40

*

  • Rotstein, Shock 2000; 14:278
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SLIDE 25

Ischemia/reperfusion I/R + E. coli

Intratracheal E. coli Attenuates Pulmonary Neutrophilia

  • Sookhai, Ann Surg 2002; 235:285
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SLIDE 26

Time (Hours)

10 20 30 40 50

Percent Survival

20 40 60 80 10 Sham I/R + E.coli I/R + Saline

  • Sookhai Ann Surg 2002; 235:285

Intratracheal Heat-killed E. coli Attenuates Mortality Following Ischemia/Reperfusion

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SLIDE 27

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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SLIDE 28

Thank you!!