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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 [email protected] Hôpitaux de Lyon Septic syndromes: a significant healthcare challenge - 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 Constant rise for many years Sepsis Cases 1,800,000 600,000 Severe Sepsis Cases 1,600,000 500,000 1,400,000 1,200,000 400,000 1,000,000 US Population 800,000 300,000 200,000 600,000 400,000 100,000 200,000 2001 2025 Year NEJM 2003 Total US Population/1,000 future 2050 - Better care of co-morbidities - Increased longevity Hospitalization rate nearly doubled from 1993 to 2003 Population-based mortality rate rose by two thirds. Wenzel 2002 - N Engl J Med Multiple Organ failure Increased severity Definition Association of an infection and a systemic inflammatory response syndrome (SIRS) SEPSIS IS NOT CAUSED BY THE INFECTION ITSELF BUT BY THE HOST RESPONSE TO THIS INFECTION “The germ is nothing, the terrain is everything” Pasteur L. (1895) Onset : microbiology 1 germ + site of infection + Inflammation organ systemic Amplification ? Pathophysiology : uncontrolled inflammatory response Severe sepsis Septic shock Uncontrolled inflammatory response emergency symptomatic treatment : agressive vascular resuscitation, vasoactive agents + Antibiotherapy Decreased arterial pressure Multiple organ failure Uncontrolled inflammatory response Anti-inflammatory drugs Failure of clinical trials testing anti-inflammatory therapies Drug Anti-endotoxine Anti-bradykinine Anti-PAF Anti-TNF R solubles TNF AINS Stéroïdes … Total Mortality (%) Placebo Drug Number of studies Number of patients 4 2 2 8 2 3 9 … 2010 755 870 4132 688 514 1267 … 35 36 50 41 38 40 35 … 35 39 45 40 40 37 39 … 33 12034 38 38 Zeni et al, Crit Care Med, 1997 Simplified description of systemic pro- and anti-inflammatory immune responses over time after septic shock Pro-inflammatory Response Resultant immune response at the systemic level = immunosuppression - Accounts for more than 80 % of total mortality - Patients dying after the first 48 hours are all severly immunodepressed Time Anti-inflammatory Response Similar mechanisms each time SIRS occurs: trauma, surgery, pancreatitis, burns…… Summarized view of stress-induced immunosuppression Endotoxin tolerance immune functions IL-10 (and soluble mediators) Apoptosis (different mechanisms) Innate Immunity Dendritic Cells Adaptive Immunity Monocyte anergy DC anergy Lymphocyte anergy 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 1. Functional Testing - 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 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 2004 High IL-10 is associated with mortality – not TNF 140 TNF pg/ml 120 200 IL-10 pg/ml 100 ** 180 80 160 Non-survivors 60 140 40 120 Non-survivors 20 100 1-2 80 3-4 5-7 8-15 ** 60 40 ** 20 1-2 3-4 5-7 ** 8-15 3. Cellular phenotyping and apoptosis (the power of flow cytometry) - monocytes - apoptosis - lymphocytes Monocytes Why using flow cytometry ? => example of mHLA-DR 100 % 0% 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 Ting et al. Why focusing on HLA-DR ? 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) Low HLA-DR predicts mortality (n = 120 septic shock patients) % HLA-DR + monocytes (Control values > 90-100 %) 50 p : 0.2 p < 0.001 Survivors 40 30 Non-survivors 0 - 48 h 48 – 96 h Monneret et al., Intensive Care Med 2006 Survival curves stratified on mHLA-DR at 30 % Proportion surviving (%) 100 mHLA-DR >30 % 80 log rank test, p=0.0006 60 mHLA-DR ≤30 % 40 20 0 5 10 15 20 25 28 Time from onset of shock (days) Number remaining at risk mHLA-DR >30 % mHLA-DR ≤30 % 49 37 49 34 45 30 43 25 39 21 35 18 31 14 Monneret et al., Intensive Care Med 2006 Multivariate analysis : mHLA-DR is an independent predictor of mortality (after adjustment for usual clinical confounders) Odds ratio 95 % CI p Sex (F) - - - Age > 64 years - - - 6.14 1.3 – 28.4 0.02 - - - 4.34 1.0 – 18.5 0.05 Type of infection (noso. vs commu.) - - - Infection site (pulm., abdo., others) - - - 6.58 1.5 – 28.6 0.01 HLA-DR (J1-J2) < 30 % - - - HLA-DR (J3-J4) < 30 % 8.81 1.9 – 40.4 0.005 IGS II (onset) > 49 Type of admittance (surgery vs med.) Comorbidity (≥ 1) SOFA (≠ J1J2 vs J3J4) > 0 9-fold increased risk of death with mHLA-DR < 30 % Flow chart n = 209 septic shock Missing samples (n = 33) Death (n = 23) n = 42 with NI n = 153 with mHLA-DR at days 3-4 n = 111 without NI Cox analysis Infected before days 6-9 (n = 2) ICU discharge (n = 14) Death (n =14) Missing samples (n = 2) n = 40 with NI n = 121 with mHLA-DR at days 6-9 n = 81 without NI Cox analysis mHLA-DR as % of positive monocytes (median) => Secondary nosocomial infections Day 1-2 Day 3-4 Day 6-9 48 IN+ 27 28 35 161 IN- 32 39 49 p = 0.114 Best threshold (that maximized sensitivity and specificity from ROC analysi) p = 0.03 25 % p = 0.022 40 % Probability of being free of NI Kaplan Meier and multivariate analysis for nosocomial infection stratified on HLA-DR > 25 % at day 3-4 in 209 patients with septic shock 100 mHLA-DR > 25 % Multivariate analysis Forward Stepwise (Likelihood Ratio) Parameter included in the model: age, SOFA, SAPSII, Intubation and HLA-DR 50 p = 0.032 Low HLA-DR was the sole parameter associated with nosocomial infections Hazard Ratio = 1.922 [IC95%:1.05 - 3.63] p = 0.038 mHLA-DR < 25 % 5 10 15 20 25 30 Time from onset of septic shock (days) Probability of being free of NI Kaplan Meier and multivariate analysis for nosocomial infection Stratified on HLA-DR > 40 % for day 6-9 in 209 patients with septic shock 100 mHLA-DR > 40 % Multivariate analysis Forward Stepwise (Likelihood Ratio) Parameter included in the model: age, SOFA, SAPSII, Intubation and HLA-DR p = 0.014 50 Low HLA-DR was the sole parameter associated with nosocomial infections Hazard Ratio = 2.28 [IC95%:1.1-4.4] p = 0.013 mHLA-DR < 40 % 5 10 15 20 25 30 Time from onset of septic shock (days) HLA-DR expression and soluble HLA-DR levels in septic patients after trauma Ditschkowski et al. Ann. Surg. 1999 Minor injuries Severe injuries without secondary sepsis Severe injuries + secondary sepsis Infected Not infected Low mHLA-DR predicts nosocomial infections after Day 15 ROC Curves Analysis for the prediction secondary infections (AUC = 0.9) According to secondary infection (n = 29 non infected n = 24 infected) Dendritic cells 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) Apoptosis controls 1 week before (within 12 h) Non survivors (within 12 h) Lymphocytes (in press 2009) %Treg 30 30000 25 25000 20 20000 15 15000 10 10000 5 5000 1 2 3 4 Septic patients Normal values from the laboratory: PHA and ConA > 15 000 cpm / PWM > 5 000 cpm. 1 2 Healthy individuals cpm PHA (cpm) PWD (cpm) ConA (cpm) % of CD4+CD25+CD127- cells among CD4+ An increased circulating percentage of Treg is associated with a decreased cell proliferation in septic shock patients Skin testing Mortality Nosocomial infection 9 % (n = 31) 27 % (n = 99) +/23 % (n = 272) 25 % (n = 55) 33 % (n = 91) Negative 47 % (n = 570) 32 % (n = 184) (within 24 h after admission) n = 1211 surgical ICU patients Positive 30 % (n = 369) * 42 % (n = 237) * p < 0.005 Skin testing with 5 antigens (positive if 2 reacted, +/- if solely 1 reacted, negative if none reacted) * 4. Transcriptomic approach (microarrays and qRT-PCR) Genes coding for pro-inflammatory immune response are decreased while genes coding for apoptosis are increased Immunol Lett 2006. 106 (1) :63-71 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 A cluster of 28 genes mostly linked with immunosuppression differentiated S from NS Non-survivors Survivors 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) 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 Confirmation cohort (N = 160 septic shock patients) : qRT-PCR in whole blood Survival distribution fraction CX3CR1 mRNA > 0.12 Survival curves stratified on CX3CR1 mRNA level at day 1-2 (cut off: 0.12) p = 0.0002 CX3CR1 mRNA < 0.12 Days after the onset of shock Apoptosis assessment by qRT-PCR in whole blood (Paxgen tubes) mRNA level for pro-apoptotic protein is increased mRNA level for anti-apoptotic protein is decreased Abe R et al. Cytokines / HLA-DR assessment by qRT-PCR in whole blood (Paxgen tubes) HLA-DRB1 mRNA in whole blood of septic patients HLA-DRB mRNA (ratio) 1,2 p < 0.01 1 0,8 S 0,6 0,4 NS S NS 0,2 Day 1-3 Day 4-10 Pachot et al., Crit Care Med 2005 5. Conclusion 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 Representative examples HLA-DR (% + monocytes) 2 patients with septic shock – community acquired No comorbidity – first sample < 6 hours after the onset of shock 80 70 Survivor 60 50 40 30 20 Non-survivor 10 0 1 2 3 4 Days post-shock 5 6 7 Simplified description of systemic pro- and anti-inflammatory immune responses over time after septic shock Pro-inflammatory Response Anti-inflammatory drugs Pro-inflammatory drugs ? Anti-inflammatory Response Time 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 Harms et al. 2008 Summarized view of sepsis-induced immunosuppression Sepsis-induced immune dysfunctions IL-10 Apoptosis Innate Immunity Adaptive Immunity Monocyte anergy Lymphocyte anergy Immunotherapy to restore immune functions ? Sepsis-induced immune dysfunctions IL-10 Apoptosis - Monocyte anergy AS-101 GM-CSF Lymphocyte anergy IL-7 Ritonavir Ultimate objective: the concept of individualized/tailored and targeted immune therapy in sepsis-induced immunosuppression Sepsis-induced immune dysfunctions IL-10 Apoptosis - Monocyte anergy AS-101 Circulating IL-10 GM-CSF mHLA-DR Lymphocyte anergy IL-7 % Treg IMMUNOMONITORING Ritonavir Annexin-V Docke WD et al. Nat Med. 1997;3:678-81 Monocyte deactivation in septic patients: restoration by IFN-gamma treatment 9 patients sepsis severe 26 patients sepsis severe HLA-DR < 30 % (2 jours de suite) HLA-DR < 30 % (2 jours de suite) Non randomisée Interferon-gamma Mortalité 33 % Mortalité 58 % 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 = 21) HLA-DR > 30 % (n = 31) Usual monitoring Placebo (n = 10) Interferon-gamma (n = 11) Infection II : 5 50 % Infection II : 1 9% Infection II : 3 10 % p < 0.05 Pas d’impact sur mortalité Place de la dépression immunitaire parmi les défaillances d’organes ? PROCEDURES THERAPEUTIQUES Antibiothé rapie SYSTEME HEMOSTATIQUE Remplissage Vasculaire SYSTEME ENDOCRINIEN Agents Vasoconstricteurs Agents Inotropes Positifs INFLAMMATION IMMUNITE Corticostéroïdes Protéine C Activée Insuline SYSTEME CARDIOVASCULAIRE S. NERVEUX CENTRAL AGENT PATHOGENE H O M E O S T A S I E