Transcript Diapositive 1 - UJF) Grenoble

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
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