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IMMUNODEPRESSION ACQUISE EN REANIMATION Outils diagnostiques et perspectives thérapeutiques DESC de Réanimation Médicale Juin 2009 Guillaume Monneret Laboratoire.

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Presentation on theme: "IMMUNODEPRESSION ACQUISE EN REANIMATION Outils diagnostiques et perspectives thérapeutiques DESC de Réanimation Médicale Juin 2009 Guillaume Monneret Laboratoire."— Presentation transcript:

1 IMMUNODEPRESSION ACQUISE EN REANIMATION Outils diagnostiques et perspectives thérapeutiques DESC de Réanimation Médicale Juin 2009 Guillaume Monneret Laboratoire d’Immunologie Cellulaire Hospices Civils de Lyon Hôpital E. Herriot guillaume.monneret@chu-lyon.fr Hôpitaux de Lyon

2 - Septic Syndromes : Leading cause of death in ICU - 3rd cause of death after cardiovascular diseases and cancers - 2005 US figures : 800 000 cases / year – 600 deaths / day - 2001 F figures : 70 000 cases / year Septic syndromes: a significant healthcare challenge NEJM 2003 Constant rise for many years 200,000 400,000 600,000 800,000 1,000,000 1,200,000 1,400,000 1,600,000 1,800,000 200120252050 Year 100,000 200,000 300,000 400,000 500,000 600,000 Severe Sepsis Cases US Population Sepsis Cases Total US Population/1,000 - Better care of co-morbidities - Increased longevity future

3 Hospitalization rate nearly doubled from 1993 to 2003 Population-based mortality rate rose by two thirds.

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6 Wenzel 2002 - N Engl J Med Increased severity Multiple Organ failure Definition Association of an infection and a systemic inflammatory response syndrome (SIRS)

7 Onset : microbiology 1 germ + site of infection + Inflammation Amplification ? Severe sepsis Septic shock Pathophysiology : uncontrolled inflammatory response organ systemic “The germ is nothing, the terrain is everything” Pasteur L. (1895) SEPSIS IS NOT CAUSED BY THE INFECTION ITSELF BUT BY THE HOST RESPONSE TO THIS INFECTION

8 Decreased arterial pressure emergency symptomatic treatment : agressive vascular resuscitation, vasoactive agents + Antibiotherapy Uncontrolled inflammatory response Multiple organ failure

9 Uncontrolled inflammatory response Anti-inflammatory drugs

10 Failure of clinical trials testing anti-inflammatory therapies 38 35 39 45 40 37 39 … 38 1203433Total 35 36 50 41 38 40 35 … 2010 755 870 4132 688 514 1267 … 4228239…4228239… Anti-endotoxine Anti-bradykinine Anti-PAF Anti-TNF R solubles TNF AINS Stéroïdes … Mortality (%) Placebo Drug Number of patients Number of studies Drug Zeni et al, Crit Care Med, 1997

11 Resultant immune response at the systemic level = immunosuppression Pro-inflammatory Response Anti-inflammatory Response Time Simplified description of systemic pro- and anti-inflammatory immune responses over time after septic shock - Accounts for more than 80 % of total mortality - Patients dying after the first 48 hours are all severly immunodepressed Similar mechanisms each time SIRS occurs: trauma, surgery, pancreatitis, burns……

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13 Summarized view of stress-induced immunosuppression immune functions Innate Immunity Monocyte anergy Adaptive Immunity Lymphocyte anergy IL-10 (and soluble mediators) Apoptosis (different mechanisms) Endotoxin tolerance Dendritic Cells DC anergy

14 Consequences - Decreased clearance of initial infection - Increased nosocomial infections - Viral reactivation => Directly contribute to mortality Diagnostic? No clinical sign => biological monitoring 1.Functional testing 2.Soluble mediators 3.Cellular approach 4.Genomics outcomes: - mortality - occurrence of nosocomial infections

15 - Because this directly measures ex vivo the capacity of a cell population to respond to an immune challenge, functional testing theoretically represents the method of reference - Monocyte capacity to release TNF in response to LPS challenge - Lymphocyte proliferation in response to recall antigens or mitogens - Phagocytosis, chemotaxis…. - Time consuming (days of incubation for lymphocyte proliferation), - Home-made protocols => difficult to standardize => Not suitable for routine monitoring - They remain essential to gain insights in the understanding of pathophysiology and to assess the validity of surrogate markers 1. Functional Testing

16 2. Soluble mediators - In septic shock, > 300 released mediators - Both pro- and anti-inflammatory mediators are elevated : not informative - A panel of markers is likely more desirable (or at least a ratio) => If a single one : IL-10 - Potent immunosuppressive cytokine - Many studies have identified it as the most informative - Standardized measurement

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18 20 40 60 80 100 120 140 160 180 200 IL-10 pg/ml 1-23-45-78-15 ** Non-survivors 20 40 60 80 100 120 140 TNF pg/ml 1-23-45-78-15 Non-survivors High IL-10 is associated with mortality – not TNF 2004

19 3. Cellular phenotyping and apoptosis (the power of flow cytometry) - monocytes - apoptosis - lymphocytes

20 Monocytes

21 mHLA-DR expression level = Integrated Σ of the effects of numerous mediators It is the true reflection of what force dominates at any given time-point Why using flow cytometry ? => example of mHLA-DR 100 %0 % Ting et al.

22 Low monocyte HLA-DR (measured in whole blood) is a reliable marker of monocyte anergy as demonstrated by functional testing - Monocytes from patients with low mHLA-DR are unable to produce TNF and IL-1 in response to LPS, SEB, PHA. (Astiz et al., J Lab Clin Med 1996) - Lymphocytes from patients with low mHLA-DR are unable to proliferate in response to TT.(Manjuck et al., J Lab Clin Med 2000) -Patients + GM-CSF : HLA-DR recovery accompagnied by increased release of TNF (Nierhaus et al., Intensive Care Med 2003) - Patients + G-CSF : HLA-DR recovery accompagnied by increased T proliferation and pro-inflammatory cytokines production (Schneider et al., Ann Surg 2004) Why focusing on HLA-DR ?

23 (n = 120 septic shock patients) 30 40 50 0 - 48 h48 – 96 h % HLA-DR + monocytes Survivors Non-survivors p : 0.2p < 0.001 Monneret et al., Intensive Care Med 2006 (Control values > 90-100 %) Low HLA-DR predicts mortality

24 Time from onset of shock (days) Proportion surviving (%) 51015202528 20 40 60 80 100 log rank test, p=0.0006 mHLA-DR >30 % mHLA-DR ≤30 % mHLA-DR >30 % 49454339354931 mHLA-DR ≤30 % 37302521183414 0 Number remaining at risk Monneret et al., Intensive Care Med 2006 Survival curves stratified on mHLA-DR at 30 %

25 Sex (F) Age > 64 years IGS II (onset) > 49 Type of admittance (surgery vs med.) Comorbidity (≥ 1) Type of infection (noso. vs commu.) Infection site (pulm., abdo., others) SOFA (≠ J1J2 vs J3J4) > 0 HLA-DR (J1-J2) < 30 % HLA-DR (J3-J4) < 30 % - 6.14 - 4.34 - 6.58 - 8.81 - 1.3 – 28.4 - 1.0 – 18.5 - 1.5 – 28.6 - 1.9 – 40.4 - 0.02 - 0.05 - 0.01 - 0.005 Odds ratio95 % CIp Multivariate analysis : mHLA-DR is an independent predictor of mortality (after adjustment for usual clinical confounders) 9-fold increased risk of death with mHLA-DR < 30 %

26 Flow chart n = 209 septic shock n = 153 with mHLA-DR at days 3-4 Missing samples (n = 33) Death (n = 23) n = 42 with NI n = 111 without NI Cox analysis n = 40 with NI n = 81 without NI n = 121 with mHLA-DR at days 6-9 Infected before days 6-9 (n = 2) ICU discharge (n = 14) Death (n =14) Missing samples (n = 2) Cox analysis

27 Day 1-2Day 3-4Day 6-9 48 IN+272835 161 IN-323949 p = 0.114p = 0.03p = 0.022 mHLA-DR as % of positive monocytes (median) => Secondary nosocomial infections Best threshold (that maximized sensitivity and specificity from ROC analysi) 25 %40 %

28 p = 0.032 mHLA-DR < 25 % mHLA-DR > 25 % Kaplan Meier and multivariate analysis for nosocomial infection stratified on HLA-DR > 25 % at day 3-4 in 209 patients with septic shock Time from onset of septic shock (days) 30252015105 Probability of being free of NI 100 50 Parameter included in the model: age, SOFA, SAPSII, Intubation and HLA-DR Low HLA-DR was the sole parameter associated with nosocomial infections Hazard Ratio = 1.922 [IC95%:1.05 - 3.63] p = 0.038 Multivariate analysis Forward Stepwise (Likelihood Ratio)

29 p = 0.014 30252015105 mHLA-DR < 40 % mHLA-DR > 40 % Time from onset of septic shock (days) Probability of being free of NI 100 50 Kaplan Meier and multivariate analysis for nosocomial infection Stratified on HLA-DR > 40 % for day 6-9 in 209 patients with septic shock Parameter included in the model: age, SOFA, SAPSII, Intubation and HLA-DR Low HLA-DR was the sole parameter associated with nosocomial infections Hazard Ratio = 2.28 [IC95%:1.1-4.4] p = 0.013 Multivariate analysis Forward Stepwise (Likelihood Ratio)

30 HLA-DR expression and soluble HLA-DR levels in septic patients after trauma Ditschkowski et al. Ann. Surg. 1999 Minor injuries Severe injuries + secondary sepsis Severe injuries without secondary sepsis

31 Infected Not infected

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33 Low mHLA-DR predicts nosocomial infections after Day 15

34 ROC Curves Analysis for the prediction secondary infections (AUC = 0.9) According to secondary infection (n = 29 non infected n = 24 infected)

35 Dendritic cells

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37 Persisting low circulating myeloid dendritic cells number is associated with the development of nosocomial infections after septic shock Days after shock Pene, Chiche et al. (Société Réanimation Langue Française 2009)

38 Apoptosis

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40 (within 12 h) Non survivors controls 1 week before (within 12 h)

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42 Lymphocytes

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44 (in press 2009)

45 %Treg 1234 1 2 5 10 15 20 25 30 5000 10000 15000 20000 25000 30000 % of CD4+CD25+CD127- cells among CD4+ cpm Septic patientsHealthy individuals PHA (cpm) PWD (cpm) ConA (cpm) Normal values from the laboratory: PHA and ConA > 15 000 cpm / PWM > 5 000 cpm. An increased circulating percentage of Treg is associated with a decreased cell proliferation in septic shock patients

46 n = 1211 surgical ICU patients Skin testing (within 24 h after admission) Positive 30 % (n = 369) +/- 23 % (n = 272) Negative 47 % (n = 570) Mortality 9 % (n = 31) 25 % (n = 55) 32 % (n = 184) Nosocomial infection 27 % (n = 99) 33 % (n = 91) 42 % (n = 237) * p < 0.005 Skin testing with 5 antigens (positive if  2 reacted, +/- if solely 1 reacted, negative if none reacted) * *

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48 4. Transcriptomic approach (microarrays and qRT-PCR)

49 Genes coding for pro-inflammatory immune response are decreased while genes coding for apoptosis are increased

50 Immunol Lett 2006. 106 (1) :63-71 SurvivorsNon-survivors A cluster of 28 genes mostly linked with immunosuppression differentiated S from NS Septic shock patients > 48 h after the onset of shock 31 patients (10 NS) + 7 patients in a prospective control study (3 NS) HG-U133A oligonucleotide arrays (Affymetrix) – 14 500 genes Among them, the decreased expression of the fractalkine receptor CX3CR1 mRNA was the most interesting because of largest fold change between S and NS (  8-fold decreased in survivors)

51 CX3CR1 is mainly expressed on patroller monocytes that are the first to reach the site of secondary infections to initiate immune response. Due to decreased chemotaxis (and subsequent decreased inflammatory cytokines release), the loss of this receptor might have a role in the development of nosocomial infections

52 Days after the onset of shock Survival distribution fraction CX3CR1 mRNA > 0.12 CX3CR1 mRNA < 0.12 p = 0.0002 Survival curves stratified on CX3CR1 mRNA level at day 1-2 (cut off: 0.12) Confirmation cohort (N = 160 septic shock patients) : qRT-PCR in whole blood

53 mRNA level for pro-apoptotic protein is increasedmRNA level for anti-apoptotic protein is decreased Apoptosis assessment by qRT-PCR in whole blood (Paxgen tubes)

54 Cytokines / HLA-DR assessment by qRT-PCR in whole blood (Paxgen tubes) Abe R et al.

55 0,2 0,4 0,6 0,8 1 1,2 HLA-DRB mRNA (ratio) p < 0.01 Pachot et al., Crit Care Med 2005 HLA-DRB1 mRNA in whole blood of septic patients Day 1-3Day 4-10 NS S S

56 5. Conclusion

57 Conclusions & perspectives - Tools and biomarkers for the monitoring of sepsis-induced immune alterations are currently under development - For now, the literature is very homogenous. For every biomarker/ immune dysfunction measured, the conclusion stays the same: => Septic patients who do not recover normal immune functions are those who die - We now need multicentric clinical studies to validate and reinforce these promising preliminary results - We need to establish standardized measurement protocols for each potential biomarker

58 Representative examples 0 10 20 30 40 50 60 70 80 1345672 HLA-DR (% + monocytes) Days post-shock Survivor Non-survivor 2 patients with septic shock – community acquired No comorbidity – first sample < 6 hours after the onset of shock

59 Pro-inflammatory Response Anti-inflammatory Response Time Simplified description of systemic pro- and anti-inflammatory immune responses over time after septic shock Anti-inflammatory drugs Pro-inflammatory drugs ? New Strategies: - Close Monitoring (PCT ?) to detect asap the beginning of infections - Preventive antibacterial therapy (when possible / risk of resistance) - Immunotherapy to restore immune functions

60 Harms et al. 2008

61 Summarized view of sepsis-induced immunosuppression Sepsis-induced immune dysfunctions Innate Immunity Monocyte anergy Adaptive Immunity Lymphocyte anergy IL-10 Apoptosis

62 Monocyte anergyLymphocyte anergy IL-10 Apoptosis  IL-7GM-CSF - - AS-101Ritonavir Sepsis-induced immune dysfunctions Immunotherapy to restore immune functions ?

63 IL-10 Apoptosis  IL-7GM-CSF - - AS-101Ritonavir Circulating IL-10mHLA-DR% TregAnnexin-V IMMUNOMONITORING Monocyte anergyLymphocyte anergy Sepsis-induced immune dysfunctions Ultimate objective: the concept of individualized/tailored and targeted immune therapy in sepsis-induced immunosuppression

64 Docke WD et al. Nat Med. 1997;3:678-81 Monocyte deactivation in septic patients: restoration by IFN-gamma treatment 9 patients sepsis severe HLA-DR < 30 % (2 jours de suite) Interferon-gamma Mortalité 33 % Mortalité 58 % 26 patients sepsis severe HLA-DR < 30 % (2 jours de suite) Non randomisée

65 Nakos G et al., Crit Care Med 2002;30:1488-1494 Immunoparalysis in patients with severe trauma and the effect of inhaled interferon-gamma 52 trauma patients HLA-DR at day 3 (BAL) HLA-DR > 30 % (n = 31) Usual monitoring HLA-DR < 30 % (n = 21) Placebo (n = 10) Infection II : 5 50 % Interferon-gamma (n = 11) Infection II : 1 9 % p < 0.05 Infection II : 3 10 % Pas d’impact sur mortalité

66 Antibiothé rapie Remplissage Vasculaire Agents Vaso- constricteurs Agents Inotropes Positifs Corticostéroïdes Protéine C Activée Insuline PROCEDURES THERAPEUTIQUES SYSTEME ENDOCRINIEN INFLAMMATION IMMUNITE SYSTEME CARDIOVASCULAIRE S. NERVEUX CENTRAL AGENT PATHOGENE SYSTEME HEMOSTATIQUE HOMEOSTASIEHOMEOSTASIE Place de la dépression immunitaire parmi les défaillances d’organes ?


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