Tissue injury, blood loss, hypoxia, transfusion, bacterial translocation, microbial infection, and cell activation by microbial products occur in patients with systemic inflammatory response syndrome (SIRS; e.g. trauma, hemorrhage, ischemia, burns, surgery or sepsis). These events contribute to the inflammatory response and affect the quality of the immune status. In addition, drugs (e.g. anesthetics, opioids…) also influence immune responses (figure 6). Depressed immune status including decreased blood cell counts, low expression of surface markers (e.g. MHC Class II antigen), altered natural killer (NK) cell activity, diminished cellular cytotoxicity, reduced antigen presentation, poor proliferation in response to mitogens and depressed cytokine production are seen in vitro, and illustrated in vivo by anergy to skin test antigens. These observations led Roger Bone to coin the concept of “compensatory anti-inflammatory response syndrome” or CARS. Bone postulated that when the SIRS response predominates, the syndrome is associated with organ dysfunction and cardiovascular compromise leading to shock. In contrast, when CARS predominates, it is characterized by anti-inflammatory responses associated with a suppressive effect on the immune system, also known as “immunoparalysis”. It is widely accepted that the SIRS response occurs first and is followed in some patients by the CARS response. However, as illustrated by our study on resuscitated patients after a cardiac arrest (see below), it is most probable that the two syndromes occur concomitantly. Although alterations in the immune response are probably associated with an enhanced sensitivity to nosocomial infections, there is no clear demonstration that they are directly responsible for poor outcome in sepsis. Furthermore, the mechanisms behind the maintenance of the sustained suppression of immune function remain incompletely understood.
Immunological status of cardiac arrest and resuscitated patients (Adrie et al. Circulation 2002, 106, 562).
We investigated the immuno-inflammatory profile of patients successfully resuscitated after cardiac arrest, which represents a model of whole-body ischemia-reperfusion syndrome. Prognosis of these patients is poor due to neurological sequelae, hemodynamic shock and possible multiple organ failure in 50% of them. We demonstrated the presence of circulating endotoxin in 46% of the patients (n=35) within the first two days following admission to intensive care units. We showed that at admission (around 3 h on average after cardiac arrest) the levels of plasma sTNFRII, IL-6, IL-8, and IL-10 were significantly higher among non-surviving patients than in survivors. On day 1, IL-1 receptor antagonist (IL-1Ra) was also a marker associated with prognosis. Endotoxin-induced TNF and IL-6 productions in ex vivo whole blood culture were dramatically impaired in these patients as compared to healthy controls, while an unaltered cytokine production was observed with heat-killed Staphylococcus aureus. In contrast, IL-1Ra production was enhanced as compared to healthy controls. The production of T-cell-derived IL-10 and IFNgamma was also impaired in these patients. Finally, we demonstrated using in vitro plasma exchange between healthy controls and patients that the endotoxin-dependent hyporeactivity was an intrinsic property of the patients’ leukocytes, while an immunosuppressive activity was also present in their plasma. Altogether, these observations show that, despite a non-infectious stress, these patients exhibit laboratory values reminiscent of those found in sepsis patients
Analysis of TLR- and Nod-induced signaling in sepsis and SIRS patients
Since our first report on the in vitro hyporeactivity of circulating monocytes in sepsis patients in terms of cytokine production (Muñoz et al. J. Clin. Invest. 1991, 88, 1747), we have further characterized the immune depression associated with this disease. In addition to the similar observation mentioned above with resuscitated patients after cardiac arrest, we have described an analogous but short-lasting alteration in patients after surgery (Cabié et al. Cytokine 1992, 4, 576). We extended our observations to circulating neutrophils (Marie et al. Blood 1998, 91, 3439) and T-lymphocytes (Muret et al. Shock 2000, 13, 169).
We studied the intracellular molecular mechanisms responsible for the immune depression observed in sepsis patients as well as in patients with non-infectious SIRS. We showed a global decrease of nuclear factor-?B (NF-kappaB), an imbalance between its active (p65p50) and inactive (p50p50) forms, and a weak cytoplasmic expression of its inhibitor (I?B?) within mononuclear cells of sepsis patients (Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2000, 162, 1877). A similar study undertaken in trauma patients revealed that the defect in NF-kappaB expression was also present and that it lasted for more than 10 days (Adib-Conquy et al. J. Leuk. Biol. 2001, 70, 30).The immune dysregulation in these patients is illustrated by a dramatic decrease in the capacity of PBMC to release TNF upon activation by Escherichia coli lipopolysaccharide (a TLR4 agonist) and an oligonucleotide carrying CpG motifs (a TLR9 agonist). More recently, we showed that TNF production in response to Pam3CysSK4, a specific TLR2 agonist, was also reduced in sepsis as compared to healthy controls. In contrast, in resuscitated patients after cardiac arrest (RCA), a group of non-infectious SIRS, monocyte responsiveness displayed a reduced production of TNF only in response to LPS, but not to Pam3CysSK4. We have shown that the surface expression of TLR2 was not reduced on patients’ monocytes as compared to healthy controls, whereas that of TLR4 was reduced. However, lower TNF production in response to E. coli LPS cannot be fully explained by the down-regulation of TLR4 expression because simultaneously the LPS-induced production of IL-10 was enhanced. The reduced production was also found for interleukin-6 release in response to IL-1 and TNF. While IL-1 shares with TLR ligands several signaling adaptor molecules, this is not the case for TNF. This means that the alteration observed in leukocytes of SIRS patients affects different intracellular cell signaling pathways.
Nevertheless, this hyporeactivity is not a global defect, since cells from SIRS patients remain fully reactive to heat-killed bacteria (Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli) and muramyl dipeptide (MDP). MDP is sensed by Nod2, an intracellular receptor. We found that the expression of Nod2 mRNA was unchanged in sepsis, as was that of Nod1, a sensor of peptidoglycan from Gram-negative bacteria. In contrast to TNF, the production of anti-inflammatory cytokines by monocytes of SIRS patients in response to LPS and Pam3CysSK4 was enhanced, whereas that induced by heat-killed bacteria was unchanged. The activation of the p38 mitogen-activated-kinase (MAPK) and the Sp-1 transcription factor (both involved in IL-10 production) was increased in PBMC from trauma patients after E. coli LPS or heat-killed Staphylococcus stimulation, and the addition of an inhibitor of p38 decreased IL-10 production(Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2003, 168, 158).
More recently, we investigated the expression of various molecules negatively regulating TLR4 signaling. Several molecules are known to regulate LPS-activated signaling pathways negatively in animal or in vitro models. Toll interacting protein (Tollip) is an adaptor protein which potently suppresses the activity of IL-1 receptor-associated kinase (IRAK) after TLR activation. Suppressor of cytokine signaling-1 (SOCS1) is one of eight members of a family involved in the negative regulation of cytokine signal transduction pathways, particularly the JAK/STAT pathway. An LPS-inducible splicing variant of myeloid differentiation 88 (MyD88), termed MyD88 short (MyD88s), is defective in its ability to induce IRAK phosphorylation and behaves as a dominant-negative inhibitor of LPS-induced NF-kappaB activation. Single immunoglobulin IL-1R-related molecule (SIGIRR), a member of the TLR/IL-1R superfamily, is a negative modulator of the signaling induced by IL-1 or LPS. RP105, another surface molecule, is a negative regulator of TLR4.With the exception of IRAK-M, another negative regulator of TLR signaling, the contribution of these molecules has yet to be fully established in human sepsis. We showed by RT-PCR that in monocytes of sepsis patients the expression of mRNA for Tollip and SOCS1 was similar to that of healthy controls, while MyD88s and SIGIRR expression was significantly enhanced. The expression of SIGIRR mRNA was increased in the monocytes from RCA patients, but not that of MyD88s. The results were confirmed by real-time PCR. We showed by transfection experiments that SIGIRR negatively regulates TLR4-dependent activation while MyD88s regulates both TLR2- and TLR4-dependent NF-kappaB activation. This would explain why TLR2-dependent TNF production was not inhibited in non-infectious SIRS (Adib-Conquy et al., Crit. Care Med. 2006, 34, 2377-2385).
In conclusion, the immune dysregulation described in SIRS patients is not a generalized phenomenon but depends on the stimuli and the signaling pathways. The term “cellular reprogramming,” previously proposed by Zhang and Morisson to characterize endotoxin tolerance better defines the phenomenon than the terms anergy, immunodepression, or immunoparalysis commonly used to characterize the immune status of septic patients.
Ex vivo production of MIF (Maxime et al. J. Infect. Dis. 2005, 191, 138)
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that prevents glucocorticoid activities. In collaboration with Prof. D. Annane (Hôp. Poincaré, Garches) we studied the capacity of leukocytes of sepsis patients versus those of healthy controls to produce MIF in vitro in response to various activators, and assessed the effect of a glucocorticoid treatment on the in vitro production of MIF by the patients’ leukocytes. PBMC from patients contained significantly higher amounts of MIF than cells from healthy controls. In culture, the spontaneous release and that induced by LPS, HKSA and red blood cell lysates were significantly higher in patients than in controls. PBMC from patients treated with glucocorticoids showed a lower release of MIF in response to LPS, heat-killed Escherichia coli and peptidoglycan than PBMC from untreated patients, and had levels similar to those obtained with PBMC from healthy controls. Thus, MIF is the first pro-inflammatory cytokine of which ex vivo release by circulating cells is shown to be enhanced in sepsis. In contrast to the knowledge obtained from animal studies, glucocorticoid in vivo treatment normalized the release of MIF by circulating PBMC from patients with septic shock.
All clinical studies have been conducted in collaboration with different colleagues in charge of intensive care units (Prof. D. Payen, Hôp. Lariboisière; Prof. D. Annane, Hôp. Raymond Poincaré; Dr. Adrie, Hôp. Delafontaine) or working in such ICU (Drs. P. Moine & K. Asehnoune, Hôp. Kremlin Bicêtre)Selective beta 2-adrenoceptor modulation of ex vivo cytokine production following hemorrhage in mice.(Asehnoune et al. Cytokine 2006, 34, 212-218)
Sub-lethal hemorrhagic shock is associated with a reduction of ex vivo TNF production in response to LPS by cells from different compartments (33). In order to investigate the role of catecholamine in this process, we studied TNF and IL-10 cytokine production by blood cells stimulated by E. coli LPS or by HKSA in a hemorrhagic shock model in mice, in the presence of non-selective (?1-?2-, propranolol) or selective (?2-, ICI 118,551) ?-adrenoceptor antagonists. Hemorrhagic shock was obtained in BALB/c mice by a controlled bleeding through a cardiac puncture. Shed blood volume (SBV) was restored 60 min after hemorrhage. Animals were sacrificed 60 min later by exsanguination. Animals were assigned to nine groups (n = 8-10): Control: effect of anesthesia with and without pretreatment (beta1-beta2- or ?2-,); Sham: effect of cardiac puncture with and without pretreatment, and hemorrhage: SBV was restored with and without pretreatment.Similar to what was found with septic and non-infectious SIRS patients, hemorrhage resulted in a major decrease in LPS-induced TNF production, while the response to HKSA was unaltered. In contrast, IL-10 production was significantly enhanced. Blockade of beta1beta2 and ?2-adrenoceptor attenuated the decrease in TNF production but ?2-adrenoceptor blockade enhanced IL-10 production. Hemorrhage did not modify HKSA-induced TNF production levels, whereas IL-10 production was increased. ?2-adrenoceptor blockade further increased the production of both TNF and IL-10 upon HKSA stimulation. We showed that leukocyte responsiveness, following hemorrhage, varies with the microbial activators that trigger different signaling pathways, and we demonstrate that ?2-adrenoceptor ligands play a central role in the ex vivo “blood deactivation” to LPS after hemorrhage and resuscitation.
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