1. « Predisposition de la réponse à l’agression » D Payen, MD, Ph D dpayen1234@orange.fr Interface INSERM-SFAR-SRLF

3. Inflammation et métabolisme sont étroitement intriqués…

5. Argument phylogénique et « anatomique »

6. the Drosophila fat body incorporates the mammalian homologues of the liver and the haematopoietic and immune Systems. Function: sensing energy and nutrient availability, coordinates the appropriate metabolic and survival responses site of coordination of pathogen responses with metabolic status.

8. Therapeutic targets at the interface between metabolic and inflammatory pathways

9. Argument génétique constitutif

10. The constitutive view Topic: Studies on SNP’s associations with innate immune response. 3 examples of leading pathways: –TNF-  promoter variants –TLR4 haplotypes –HLA-DB variants Strategy: known prot  gene  SNPs association

11. Genetic Polymorphisms and Severe Sepsis Meningococcemia; Severe sepsis PAI-1 FactorV Leiden Severe Sepsis Viral Pneumonia IL-1 locus IL-4 Meningococcemia Septic Shock; Cerebral Malaria Severe Sepsis Severe Sepsis, Meningococcemia Severe sepsis TNF locus IL-18 IL-10 IL-6 Meningococcemia; Pneumococcemia FC  RII Receptor Meningococcemia, Pneumococcemia Severe sepsis Mannose Binding Lectin Gene Gram negative/positive Septic Shock Legionnaire’s Disease Septic Shock Toll-Like Receptor 4/2 Toll-Like Receptor 5 CD14 Susceptibility and/or Outcome

12. Limitations of gene by gene approach Hope in development of genome wide association methods (600 000 variants studied in one shot!) Clark et al. Intensive Care Med 2006 32:1706-1712

13. Genotyping of HLA-DRB (MHC class II) 4 HLA-DRB genes (B1, B3, B4, B5) B1 all humans Relation B1 – mHLA-DR or outcome N =183 pts; multicentric, multi focal infection mortality 38% Genotyping by high resolution DNA based typing (alleles) from leuco No relation with mortality nor HLA- DR expression B3 homozygous  less OF (p<0,007) B4 +  trend better survival (0.09) D Payen et al submitted

14. Argument génomique

15. Gene expression in whole blood leukocytes determined before and at 2, 4, 6, 9 and 24 h after the i.v LPS to 4 HV, compared to 4 additional subjects without LPS. Only 3% of the genes modified their expression Essentially under-expression  human blood leukocyte response to acute systemic inflammation  the transient dysregulation of leukocyte bioenergetics &modulation of translational machinery

16. Argument neuro-hormonal

19. Insulin-receptor signalling interfaces with inflammatory signalling at the level of insulin-receptor substrates through activation of serine kinases.

21. (b) ACTH responders P value for log rank test: 0.937 0 0.25 0.50 0.75 1.00 05101520 25 30 placebo steroid P value for log rank test: 0.850 0 0.25 0.50 0.75 1.00 05101520 25 30 (a) ACTH non-responders placebo steroid P value for log rank test: 0.813 0 0.25 0.50 0.75 1.00 05101520 25 30 day (c) All patients placebo steroid Figure 2 - Kaplan Meier Curves for 28 day all-cause mortality in (a) ACTH nonresponders (b) ACTH responders and (c) all patients

22. Figure 3 - Kaplan Meier Curves for time to reversal of shock in (a) ACTH nonresponders (b) ACTH responders and (c) all patients placebo steroid P value for log rank test: <0.001 0 0.25 0.50 0.75 1.00 05101520 25 30 day (c) All patients P value for log rank test: 0.038 0 0.25 0.50 0.75 1.00 05101520 25 3030 placebo steroid (a) ACTH non-responders(b) ACTH responders P value for log rank test: <0.001 0 0.25 0.50 0.75 1.00 05101520 25 30 placebo steroid

23. Sub-paper from Corticus data base Our article: shock reversal & outcome QS: Why success in shock reversal did not improve outcome?

24. Within the first five days Incidence of shock reversal according to treatment arm P<0.0001 0123456 0.0 0.2 0.4 0.6 0.8 1.0 Days after randomization Cumulative incidence Hydrocortisone Placebo

25. Within the first five days Incidence of death prior to shock reversal according to treatment arm P= 0.28

26. Argument biochimique

27. Free energy used by cells respiration or glycolysis Alternatively, reactions can be centred around ATP. ATP-producers include glycolysis & oxidative phosphorylation ATP-consumers: transport of cations & synthesis of macromolecules

28. energy consuming functions of immune cells Synthesis of macromolecule & ion transport; O 2 is mainly used by mitochondria, non-mitochondrial O 2 consumption is negligible

29. ADN/ARN synthesis O 2 is used for what type of functions? O2O2 ADP+Pi ATP H+ Actinomycine D ATP synthase Respiratoire Chain NADPH oxydase protein Synthesis Na +, K + ATPase Ca 2+ ATPase Antimycine A Cycloheximide Inhibition Buttgereit, Biochem J, 1995. Ouabaïne DPI Chlorure de lanthanum

30. Global VO 2 : the different fractions % O2 75 80 85 90 95 100 050010001500200025003000 Sec Cellules DPI Cycloheximide Ouabaïne ActinomycineD Chlorure de lanthanum Antimycine A 78 ngatomes O 2

31. O 2 consumption of Peripheral Blood Mononuclear Cell (PBMC) ex vivo O 2 consumption rate, ngatoms O 2 /min/10 7 cells Stimulation by ADP 0 4 8 12 16 20 p=0.002 p=0.001 VoVo V stim VoVo CCM 2007

32. PBMC from volunteers + septic plasma O 2 consumption rate, ngatoms O 2 /min/10 7 cells Stimulation by ADP VoVo V stim VoVo 0 4 8 12 16 20 p<0.0001  Plasma induces immune cells bioenergy failure Belikova et al CCM 2007

33. Septic plasma  modifications similar to those observed in septic cells Plasmatic factors +++ Effects septic plasma septique on baseline (V0) & stimulated reponse

34. Mitochondrial VO 2 becomes more decoupled after incubation in septic plasma.

35. It cannot result from tissue hypoxia!!!!

36. M Integration of stress-signalling mechanisms.

38. Le sucre et inflammation…

39. J Appl Physiol 1997 Exogenous glucose (NUTRITION) Fed Fasted Fasted+iP G

40. J Appl Physiol 1997 In conclusion, glucose metabolism interferes with hemodynamic, metabolic, and inflammatory responses to LPS. MAJOR ROLE OF EXOGENOUS GLUCOSE Exogenous glucose (NUTRITION)

41. Fig. 4 O 2 consumption rate, ngatoms O 2 /min/10 7 cells p=0.003 A Delta increase in O 2 consumption, % p=0.004 B

42. Fig. 5 Vo, ngatoms O 2 /min/10 7 cells HLA-DR, site number Delta increase in O 2 consumption, % HLA-DR, site number Delta increase in O 2 consumption, % HLA-DR, site number r=0.44 p=0.009 r=0.57 p=0.0014 r=0.58 p=0.049 A B C Iono+PMA ADP

43. Un example of acute inflammation aigue in human beings: hepatic transplantation

44. Day 4 after OLT

45. Excès ou déficit de nutriments Stress, inflammation, infection, hypoxie Stress du téticumum endoplasmique (facteurs de transcriptions, molécules chaperons, enzymes Fonction des organelles mitochondries, RE Cytokines de l’inflammation et leurs récepteurs Chémokines et leurs récepteurs Métabolisme des ac gras Protéïnes chaperons Récepteurs nucléaires des hormones Activation des cascades de signalisation (JNK/IKK/ PKC) NF-  B, RLO PAMPs Bactéries: Flagelline, LPS, glycopeptide et peptidoglycane Virus: ssRNA, protéïnes de l’enveloppe Levures: zymozan, profiline DAMPs Cellules tumorales et nécrotiques, lésions tissulaires (trauma, ischémie): DNA, héparan sulfate, HMBH1, acide urique, matrice extracellulaire Récepteurs MRR et TLR Hypoxie/ischémie HIF Facteurs cytoprotecteurs Glycolyse anaérobie Fibrose Apoptose Angiogénèse NOS/HO Transcription de gènes Réponse immune innée Réponse immune adaptative

46. Argument inflammatoire

47. Immunity triggers inflammation Pathogen pathway  PAMPs or MAMPs  “stranger model”  Infection and sepsis Cell and tissue lesions  DAMPs  “danger model”  infection, trauma, postop, burns Kono et coll. Nat Rev Imm 2008

48. Cell death and inflammation. - Necrotic cell death  DAMPs  receptors on leukocytes  + prod of pro-inflam cytokines (IL ‑ 1). - Other molecules proteases; hydrolases act on EC components  + mediators (complement fragments) or DAMPs  prod of pro-inflam cytokines by host cells. - Pro-inflam mediators  local vascular endothelium  ‘leaky’, attracts neutrophils and monocytes/macrophages  soluble (antibody) and cellular defences in the tissue

49. Donc, les facteurs de prédisposition

50. Outcome Constitutive Polymorphisms SNPs… Acquired Polymorphisms? Epigenetic… Chronic disease Chronic treatment Poor or good Quality of feeding Pharmacogenomic Drug induced gene Expression changes Reversing shock ≠ Improve outcome Surviving shock increases the risks of disease Proteins released Are changed by Environment (nitrosylated…) Metabolic failure Not related to perfusion Need for Markers D Payen 2010

51. Difficult to reverse but good reserve Easy to reverse  But high co-morbidity in elderly… TISSUE RESERVE = Co-morbidity AGE Co-morbidity Severity Score INJURY Severe Injury + good tissue reserve Moderate injury + high Co-morbidity Similar severity score Shock severity is characterized by scores… Outcome may then depend on injury but also on acquired negative factors…

52. Survivors of hospitalization for community-acquired pneumonia are at increased risk of cardiovascular events, repeated infections, and death in the following months. But the cause is unknown…Early and long-lasting mechanisms?

53. Viral co-infections and tissue damage Crit Care Med 2009; 37:1850-1857 242 ICU patients, 39 CMV infections ICU mortality: 54% in CMV patients vs 37% in others (p=0.082) In-hospital mortality: 59% vs 41% (p=0.058) ICU LOS: 32d vs 12d (p<0.001) Length of MV: 27d vs 10d At least one bacterial nosoc inf: 69% vs 33% (p<0.001) 242 ICU patients, 39 CMV infections ICU mortality: 54% in CMV patients vs 37% in others (p=0.082) In-hospital mortality: 59% vs 41% (p=0.058) ICU LOS: 32d vs 12d (p<0.001) Length of MV: 27d vs 10d At least one bacterial nosoc inf: 69% vs 33% (p<0.001)

54. En conclusion Métabolisme et inflammation sont intriqués Les raisons et les preuves sont multiples Les facteurs enzymatiques, les substrats, les organelles, les facteurs nucléaires interférent pour assurer et moduler la réponse inflammatoire Le traitement et les biomarqueurs métaboliques et inflammatoires constituent un espoir diagnostique et thérapeutique

55. Attendons les données futures….

56. Mitochondrial oxygen consumption - oxidative phosphorilation (> 90 %). Electron transport chain Saraste M. Science, 1999. Migration Cytokinesis Phagocytosis Antigen processing Antigen presentation Activation Effector functions ATP Active transport of molecules and ions Synthesis of macromolecules (RNA/DNA/protein synthesis) Specific immune functions General housekeeping functions ENERGY CONSUMING FUNCTIONS OF IMMUNE CELLS Buttgereit F. Immunology Today, 2000.

57. Molecular pathways integrating stress and inflammatory responses with insulin action

58. LPS injection: an example of ACUTE INFLAMMATION …

59. Functional Gene expression Functional Gene expression constitutive factors constitutive factors Human genome sequence Variants (SNPs) What Predetermination means acute stress? It concerns both innate & acquired aspects Deleterious factors in Acute context? Deleterious factors in Acute context? Micro environment Micro environment Timing Co- infections Chronic context Recovery efficiency? Chronic context Recovery efficiency? Co-treatments diet Co-treatments diet Co-agression Cancer, diabetes, auto-immunity… Co-agression Cancer, diabetes, auto-immunity… Metabolic regulation ATP, co-factors, enz, … Metabolic regulation ATP, co-factors, enz, … ACQUIRED

61. La prédisposition de la réponse a l’agression... …. Implications potentielles

62. The future is predetermined in severe sepsis... ….what are the implications Didier Payen Anne Claire Lukaszewicz

63. Calgranulines: an example of DAMPs molecule May be a prognostic marker…

64. dead alive p<0,0001 0 5 10 15 20 25 S100A8/A9 pg/ml n=61n=50 Plasma S100 A8/A9 complex D0 SOFA D0 2 4 6 8 10 12 14 16 18 20 NS (p<0,09) Gobal population n = 111 Payen D, Lukazsewicz AC et al. (in review) (Patented in December 2008)

65. *p=<0.0001, test de Mann Whitney Cinétique des “alive”:D0-D28, p<0.0001, test de Friedman, n=34 D0-D14, p<0.0001, test de Friedman, n=41 D0-D7, p=0.0005, test de Friedman, n=57 Cinétique des “dead”: D0-D14,NS, test de Friedman, n=8 D0-D7, p=0.0010, test de Friedman, n=15 * * (41) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Plasma S100A8/A9 (  g/ml) D0D1D7D14D28 dead alive (15) (8) * * (43) (46) (59) (60) (50) Figure: trend over time of S100A8/A9 complex, according to outcome, in plasma. Results expressed in  g/ml. Effectives in brackets.

66. S100A8/A9 in patients without shock at D0 N=8 survivors N=8 non survivors Mann whitney test, p=0.0008 0 4 8 12 16 20 24 28 D0 dead alive Plasma S100A8/A9 (  g/ml) p=0.0008

67. Metabolic alterations: Singer et al. Lancet 2004, Belikova et al. CCM 2007 Impairment of capacity of repair? Exple: autophagy –to sustain metabolism during nutrient deprivation –to prevent the accumulation of damaged, toxic proteins and organelles during stress –Implicated in anti-microbial defense  inhibits cell death by necrosis Metabolic failure and cell recovery HOMEOSTASIS STRESS ADAPTATION Provision of nutrients during catabolism Generation of ATP in stressed cells Signals for heterophagic removal of apoptotic cells Degradation of misfolded proteins Removal of surplus or damage organelles Genomic stability Levine et al. Cell 2008 132:27-42

68. Autophagy is impaired in: degenerative disease, heart disease, aging, cancer, dysimmunity… Autophagy is impaired in: degenerative disease, heart disease, aging, cancer, dysimmunity… Levine et al. Cell 2008 132:27-42 Autophagy in innate and adaptive immunity