mechanisms Kieran ODea PhD Critical Care Research Group Anaesthetics Pain Medicine and Intensive Care Areas covered Innate immune response in sepsis Targeting inflammation in sepsis New concepts in sepsis pathogenesis ID: 1047880
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1. SepsisCellular and humoural mechanismsKieran O’Dea PhDCritical Care Research GroupAnaesthetics, Pain Medicine and Intensive Care
2. Areas coveredInnate immune response in sepsisTargeting inflammation in sepsisNew concepts in sepsis pathogenesis
3. Innate immunity and sepsisInnate immunityEarly detection of invasive organismsInflammation microbial killingAntigen presenting cell adaptive immunityEffector cells: macrophages, neutrophils, dendritic cellsSepsisSystemic activation of innate responseExcessive response ‘Cytokine Storm’
4. Innate recognition of microbes PAMPS: Pathogen associated molecule patterns Conserved structural motifs in molecules common to different microorganisms and not present in higher eukaryotes Essential for structure and/or functionGram -ve: lipopolysaccharide (LPS), peptidoglycanGram +ve: lipoteichoic acid, peptidoglycan
5. Pattern Recognition ReceptorsSecretedComplementKilling,OpsonisationExtracellular/membraneMannose,ScavengerPhagocytosisToll-like(TLR)Gene expressionIntracellular/cytoplasmNOD-like(NLR)Gene expression,protein maturationRIG-like(RLR)Gene expression
6. TLR 2/1TLR 2/6TLR4Lipoteichoic acidLipopeptidesLPSNLROrganisation of PAMP recognition and gene expressionEndosomeTLR3,7,8,9Caspase 1(ICE)Pro-IL1,IL18IL1, IL18AP1IBp65p50NF-BCytokines etcFlagellinsensorsNucleic acidsensorsMicrobial membranesensorsTLR5
7. The LPS as a primary mediator of sepsisGram-ve bacterial endotoxinAbundant: ~70% of outer bacterial wallSystemic injection reproduces symptoms of sepsisExtremely potent: 1ug/kg induces shock in humans
8. Lipopolysaccharide (LPS) structure-activityO-antigen polysaccharide: Exposed and structurally diverse region = antigenLipid A:Highly conserved anchoring region = PAMPBacterial wallGlycolipid
9. [LPS]nLPS binding cofactorsMonocyteLBP+TLR4-MD2CD14LPSLBP+CD14Endothelial cellLPSLBPLPSLBPLBP: LPS Binding ProteinMD-2: myeloid differentiation protein
10. (Lu et al. Cytokine 2008;42;145)LPS mediated activation of gene transcription by TLR4 signallingLPS binding to the TLR4/MD-2 complex causes oligomerisation of TLR4 Toll-interleukin 1 receptor (TIR) domain recruits adaptor proteinsMyD88: myeloid differentiation primary response geneTIRAP: TIR adaptor proteinTRIF: TIR domain-containing adaptor inducing IFN-TRAM: TRIF-related adaptor molecule The MyD88 pathway induces pro-inflammatory genes The MyD88-independent pathway induces Type I interferon genes (, , IP-10)TIR
11. MyD88-dependent NF-B activation triggers a coordinated inflammatory responseResponse amplification/transmission:Tumour necrosis factor Interleukin-1βIL-6, IL-12Anti-inflammatory mediators:IL-10, IL-11, IL-4, TGF-bSoluble cytokine antagonists: soluble TNF receptors, IL-1 receptor antagonistLeukocyte mobilisation and recruitmentIL-8MIP-, MCP-1, MCP-3Leukocyte adherenceICAM-1VCAM-1E-selectinActivation of endothelium, permeability,vascular tone:Inducible nitric oxide synthaseInducible cyclooxygenase-25-LipoxygenaseCytosolic phospholipase A2Increased leukocyte productionGM-CSFM-CSFG-CSFCoagulation:Tissue factor
12. Edwin Deitch: Multiple Organ Failure: Pathophysiology and Potential Future Therapy. 1992. Ann. Surg. 216 (2) 117-134 ‘.. the 1990s will see the transition of molecular biology from the laboratory to the clinic… …it is almost certain that molecular biologic therapies will become part of the therapeutic armamentarium of most practising surgeons’
13. Clinical trials: anti-inflammatory therapies “Graveyard” for pharmaceutical companiesAnti-endotoxin therapyAnti-endotoxin monoclonal antibodiesNo significant effect on mortalityrBPI (bactericidal/permeability increasing protein)No significant effect on mortalityTLR4Eritoran: no significant effect on mortality (2011)Anti-cytokine therapyAnti-TNF monoclonal AbNo significant effect on mortalityTNF receptor p75 fusion proteinNo effect, possible increase in mortality at high doseTNF receptor p55 fusion proteinNo significant effect on mortalityIL-1 receptor antagonistNo significant effect on mortality
14. Anti-coagulants treatments could have dual effects:Anti-inflammatory: Protease Activated Receptor (PAR)-1 and PAR-2 signallingActivated protein C:Modulates thrombin-induced PAR-1 signalling to anti-inflammatory, anti-apoptotic effect.Anti-coagulation therapyAnti-thrombin IIINo significant effect on mortalityIncreased risk of haemorrhage when administered with heparinTFPI (tissue factor pathway inhibitor)No significant effect on mortalityActivated Protein CSignificant effect on mortality (19% decrease)
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16. Why has translation from the lab to the clinics failed? Pre-clinical animal models oversimplistic:Animals too young, no co-morbidities, no antibioticsNo supportive care during organ dysfunctionImmunity: Inflammatory response is required to control infectionAnti-endotoxin/LPS: wrong molecule! Gram-positive>Gram-negative>fungal infectionsComplexity: Redundancy, multiple overlapping inflammatory pathways, no magic bullet!
17. The compensatory anti-inflammatoryresponse (CARS)Systemic inflammationSystemicInfection(LungsAbdomenUrinary)EarlyMOD/shockCytokine stormLateMOD/shockSIRSPro-inflammatoryAnti-inflammatoryNosocomialinfectionriskTraumaBurnsCARS
18. HLA-DRCD8CD4ControlControlSepsisSepsisChanges in splenocytes T cell activation Inhibitory co-receptors Stimulatory co-receptors T-regulatory (suppressor cells)Extensive depletion of CD4 and CD8 T cells
19. Immunorestoration: IL-7IL-7 stimulates T cell proliferation and repertoire diversityUndergoing Phase I and II clinical trials in HIV to restore lymphocyte function
20. Innate response to danger/damageSIRSCelldeathPAMPDAMPDamaged cells release molecules during injury and traumaTrigger innate immune responseClose off the site and initiate tissue repairAlarmins or DAMPs: ‘Danger’ or ‘Damage’ Associated Molecular Pattern. CARSInfectionPAMPDAMPEarly MODLate MODInfectionTraumaImmuno-suppression
21. Eukaryotic RNA stimulate TLR3 signallingTLR3 deficient mice survived peritoneal sepsisNecrotic neutrophils activate macrophages through TLR3
22. Nuclear protein in eukaryotic cellsReleased from stimulated and necrotic cells (DAMP) Found in septic patient plasmaSignals through TLR2, TLR4 and RAGENeutralisation protective in later stage of sepsis = Wide therapeutic windowHigh-mobility group box 1(HMGB1)
23. Microvesicles: a new regulator of inflammation in sepsisSub-cellular particles released from activated or dying cellsAct as signals between cells over long distances. Correlations found in sepsis with clinical statusMicrovesicles from septic patients induceorgan inflammation when administered to mice
24. Fundamental role in vascular homeostasis:Biomarker of endothelial dysfunction in sepsisTherapeutic target
25. SummarySepsis represents a complex interaction between the innate and adaptive arms of the immune responseDriven by reaction to pathogen and damaged host cellsTherapies should aim to restore system homeostasis
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28. Sepsis produces a pro-coagulant stateTissue factor expression induced in monocytes, enhancing activation of the coagulation cascade.Activated protein C (anticoagulant) levels decreased due reduced hepatic synthesis and systemic consumption.Thrombomodulin expression decreased (TNF, IL-1) and inactivated by neutrophil elastase.Tissue factor pathway inhibitor administration reduces intravascular coagulation and severity in experimental models of sepsis
29. The Toll-Like Receptor family The Toll-Like-Receptor family has principle role in innate recognition 13 TLR indentified in mammals. Single TLR types recognise different molecules All pathogens: Bacteria, viruses, fungi, protozoan and helminth parasites.+Recognition domain:Leucine-rich repeatsToll/IL-1R homology(TIR) domainConformationalchangesActivation signalTLRdimerLigand
30. Circulating mitochondrial PAMPS/DAMPS cause inflammatory responses to injury.Nature 2010. Zhang et al. 464, 104-108Mitochondria contain bacterial-like DNA (CpG repeats) and formylated peptidesMitochondrial DNA levels elevated in trauma patient plasma, 1000x normal levelsMitochondrial DAMPS activate signaling (p38 MAPK, Ca2+) and IL-8/MMP8 expression in neutrophils.Mitochondrial DAMPS induce systemic inflammation and acute lung injury (TNF, IL6, PMN influx, oedema) Lung: eosin/haematoxylinOxidant stain
31. Improvements cited: Low-dose corticosteroids; intensive glucose control;‘early goal directed resuscitation’Dombrovskiy et al. Critical Care Medicine 2007, 35; 1244-1250Reduction in case fatality rates