IMMUNODEPRESSION ACQUISE EN REANIMATION Outils diagnostiques et perspectives thérapeutiques DESC de Réanimation 2011 Gui

1. Hôpitaux de Lyon IMMUNODEPRESSION ACQUISE EN REANIMATION Outils diagnostiques et perspectives thérapeutiques DESC de Réanimation 2011 Guillaume Monneret Laboratoire d’Immunologie Cellulaire Hospices Civils de Lyon Hôpital E. Herriot guillaume.monneret@chu-lyon.fr

2. Sepsis as a starting point : Septic syndromes an increasing and 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 • 2001 F figures : 70 000 cases / year Constant rise for many years future 1,800,000 600,000 Severe Sepsis Cases 1,600,000 500,000 1,400,000 1,200,000 400,000 Sepsis Cases Total US Population/1,000 1,000,000 300,000 US Population 800,000 200,000 600,000 400,000 100,000 200,000 2001 2025 2050 Year • Better care of co-morbidities • Increased longevity NEJM 2003

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

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

7. 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 Onset :germ(s) + site of infection + local inflammation organ systemic Amplification ? Lack of modulation ? Pathophysiology : uncontrolled inflammatory response (“The germ is nothing, the terrain is everything” - Pasteur L. - 1895)

8. Uncontrolled Inflammatory response Decreased arterial pressure Shock Multiple organ failure

9. Uncontrolled Inflammatory response Emergency symptomatic treatment : Antibiotherapy Agressive vascular resuscitation Vasoactive agents Decreased arterial pressure Shock Multiple organ failure

10. « Cytokine storm » => « Mediator storm » > 300 released mediators (Marshall, Nature Rev. 2003) Extensive description for many years….. Cytokines : TNF, IL-1, IL-6, IL-12 Activation, amplification Chemokines : IL-8, MIP-1, MCP-1 Mobilize and activate leukocytes Vasoactive hormones, Lipid mediators : PAF, PG’s, LK, TX, ADM, ANP… Multiple actions Oxygen radicals : superoxide, nitric oxide Antimicrobial properties regulation vascular tone

11. Uncontrolled inflammatory response Anti-inflammatory drugs

12. Drug Number of studies Number of patients Mortality (%) Placebo Drug Anti-endotoxine Anti-bradykinine Anti-PAF Anti-TNF R solubles TNF AINS Stéroïdes … 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 … Total 33 12034 38 38 Failure of clinical trials testing anti-inflammatory therapies Zeni et al, Crit Care Med, 1997

13. Reasons for such a perfect disaster • Inappropriate animal models of sepsis • Heterogeneity of septic population (germs, virulence factors, co-morbidities, inflammatory • response, site of infection….) • Pathophysiology largely unknown

14. > 70 % total mortality

15. Simplified description of systemic pro- and anti-inflammatory immune responses over time after septic shock 1. Inappropriate animal models of sepsis 2. Heterogeneity of septic population 3. Pathophysiology largely unknown Pro-inflammatory Response Onset of Anti-inflammatory-based RCT Resultant immune response at the systemic level = immunosuppression Time Anti-inflammatory Response Similar mechanisms each time SIRS occurs: trauma, surgery, pancreatitis, burns……

16. Summarized view of sepsis-induced immunosuppression immune functions IL-10 (and soluble mediators) Endotoxin tolerance Epigenetic regulation Apoptosis (different mechanisms) • PNN chemotaxis and phagocytosis Monocyte deactivation: • -  antigen presentation capacity • -  pro-inflammatory cytokine production • -  CX3CR1 •  dendritic cells nbr • Profound lymphopenia • Th2 shift • Lymphocyte anergy •  apoptosis •  % Treg Innate Immunity Monocyte / DC anergy Adaptive Immunity Lymphocyte anergy

17. Consequences • Decreased clearance of initial infection (Togersen 2009) • Increased nosocomial infections • Viral reactivation => May directly contribute to mortality Diagnostic? No clinical sign => biological monitoring • Functional testing • Soluble mediators • Cellular approach • Genomics outcomes: - mortality - occurrence of nosocomial infections

18. 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

19. 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

21. 2004 High IL-10 is associated with mortality – not TNF 140 TNF pg/ml 120 200 100 ** 180 IL-10 pg/ml 80 Non-survivors 160 60 140 40 Non-survivors 120 20 100 1-2 3-4 5-7 8-15 80 ** 60 40 ** ** 20 1-2 3-4 5-7 8-15

22. => Correlation with mortality but no data on nosocomial infections

23. 3. Cellular phenotyping • monocytes • lymphocytes

24. Monocytes Gold standard = Decreased mHLA-DR

25. 100 % 0 % Why using flow cytometry ? => example of mHLA-DR - 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 - Decreased mHLA-DR correlates with decreased functional testing (TNF release, Ag presentation) Ting et al.

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

28. Survival curves stratified on mHLA-DR at 30 % at days 3-4 (n = 120 patients)

29. 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 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 9-fold increased risk of death with mHLA-DR < 30 %

30. Multivariate analysis (including usual confonding factors): • SOFA, SAPSII, Intubation, catheterization • - Competitive risks

31. Does mHLA-DR predict nosocomial infections in other ICU contexts ?

32. 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

33. Normal values

34. AUC : 0.8 Recovery slope (> 1.2)

36. Low mHLA-DR predicts nosocomial infections after Day 15

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

38. Dendritic cells

40. 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)

41. Lymphocytes

42. Castelino et al. From lab computer in a university hospital over a 3 month-period Lymphopenia when Ly < 600 / microL => 1042 patients with lymphopenia

44. Lymphocytes from septic patients do not proliferate upon antigen / mitogen challenge Controls Septic patients Controls Septic patients Roth et al.,

45. An increased circulating percentage of Treg is associated with a decreased cell proliferation in septic shock patients FOXP3 inhibition restores lymphocyte proliferation

46. Lymphocytes: No recent data on correlation with motality and nosocomial infections

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

49. Cook CCM 2009

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