We study the early phase of innate immunity, namely, the mechanisms involved in cytokine induction by Gram-negative or Gram-positive bacteria and their pathogen-associated molecular patterns (PAMPs). While this response is a prerequisite to fight infection, its overwhelming activation may be associated with deleterious effects as those seen during septic shock or non-infectious systemic inflammatory response syndrome (SIRS). On another hand, the counteracting anti-inflammatory response may lead to an altered immune status (compensatory ant-inflammatory response syndrome, CARS). We study the production of cytokines and their involvement in different inflammatory disorders in humans (sepsis, trauma,
ischemia/reperfusion, cystic fibrosis). We investigate at the intracellular signaling pathways level, the reprogrammation of
circulatory cells, reminiscent of the endotoxin tolerance phenomenon, that could be linked to the increased susceptibility of these patients to nosocomial infections.
1- Endotoxin tolerance
Cellular signaling mechanisms associated with the inhibition of endotoxin tolerance by IFNgamma and GM-CSF
(Adib-Conquy & Cavaillon, J. Biol. Chem. 2002, 277; 27927).
Endotoxin tolerance was initially described as a failure of animals to develop fever in response to an injection of bacterial endotoxin after they had been pretreated with this molecule. The observation was extended to survival a lethal dose of lipopolysaccharide after a previous injection of a sub-lethal dose of LPS. In vitro, endotoxin tolerance is characterized by a decreased production of pro-inflammatory cytokines by cultured leukocytes in response to LPS following a first exposure to the same stimulus. Gamma interferon (IFNgamma) and granulocyte monocyte-colony stimulating factor (GM-CSF) are immunostimulatory cytokines that prime monocytes and prevent endotoxin tolerance. We have shown that the deactivating effects of LPS, as well as the priming effects of IFNgamma and GM-CSF or their capacity to restore tumor necrosis factor (TNF) production by LPS-tolerized human monocytes, are independent of the modulation of TLR4 or MD-2. In monocytes pretreated with IFNgamma or GM-CSF, IRAK expression was up-regulated. After LPS stimulation, an increased IRAK kinase activity, a higher MyD88/IRAK association and a stronger NF-kappaB activation were observed. In contrast, in LPS-tolerized monocytes, IRAK-1 expression and kinase activity, IRAK/MyD88 association and NF-kappaB activation were inhibited. Furthermore, we showed that the prevention of tolerance by IFNgamma and GM-CSF was independent of IRAK-1 kinase activity. Our results suggest that these cytokines prevent endotoxin tolerance, induced by low but not by high doses of LPS, by inhibiting IRAK-1 degradation and by promoting its association with MyD88 after a second LPS stimulation, which in turn leads to NF-kappaB activation and TNF production
Compartmentalization of endotoxin tolerance
(Fitting et al. J. Infect. Dis. 2004, 189, 1295).
Endotoxin tolerance has been defined and analyzed either entirely in vivo or entirely in vitro mostly with conventional LPS, extracted according to Westphal’s method, and purified according to conventional methods. In contrast, often referred to an endotoxin tolerance phenomenon, the hyporeactivity of circulating leukocytes reported in patients with sepsis is an ex vivo observation. Therefore, our objective was to set up an ex vivo model of endotoxin tolerance. Mice were injected intravenously with LPS and their leukocytes derived from different compartments were challenged in vitro with LPS or heat-killed Staphylococcus aureus (HKSA) and TNF production was measured. A rapid (1-3 h) and dramatic decrease in the production of TNF in response to LPS was observed with circulating leukocytes, splenocytes, peritoneal cells and bone-marrow cells after LPS injection. In contrast, LPS-induced TNF production by bronchoalveolar cells was reduced far less and only very briefly. The kinetics of acquisition of tolerance and recovery were different for the various compartments. “Cross-tolerance” with HKSA did not parallel the phenomenon of endotoxin tolerance as observed with LPS, in agreement with data suggesting that cross-tolerance between TLR2 and TLR4 agonists could not be generalized. These data show that endotoxin tolerance, as monitored by ex vivo analysis, is compartmentalized and that bronchoalveolar cells are less likely than peritoneal, splenic or marrow cells to develop endotoxin tolerance.
2- Cell signaling via toll-like receptors and Nod molecules
Contribution of phagocytosis and Nod2 to cytokine induction by Staphylococcus aureus: evidence for a regulatory role of Nod1.
(Kapetanovic et al. Infect. Immun. 2007, 75, 830-837)
Toll-like receptors (TLR) are involved in the sensing of microbial-derived compounds. Nod1 and Nod2 are intracytoplasmic sensors that recognize distinct peptidoglycan-derived muropeptides. We analyzed the contribution of these receptors to cytokine production by macrophages following stimulation with whole bacteria. Using knockout mice, we determined that TLR4 and TLR2 contribution was predominant in the induction of TNF and IL-10 by Gram-negative bacteria. In contrast, the absence of TLR2 and/or TLR4 or TLR9 did not affect the response to Gram-positive bacteria. In the absence of TLR2, the phagocytic property of macrophages was essential for cytokine production in response to heat-killed Staphylococcus aureus (HKSA) and was markedly inhibited by cytochalasin D. By transfecting Raw 264.7 macrophages with dominant negative (DN) forms of Nod1 and Nod2, we showed that both molecules inhibited NF-?B activation in response to HKSA. The unexpected interference of DN Nod1 in the response of macrophages to Gram-positive bacteria was confirmed with a Nod2 agonist (muramyl dipeptide) in transfection experiments in HEK293T cell. Finally, HKSA up-regulated the expression of Nod1 but not Nod2 mRNA in mouse Raw 264.7 macrophages, Our study shows the contribution of phagocytosis for cytokine production and that of Nod2 for macrophage response to HKSA. It also identifies a cross-talk between Nod1 and Nod2.
Synergistic stimulation of monocytes and dendritic cells by Toll-like receptor 4, Nod1- and Nod2-activating agonists
(Fritz et al. Eur. J. Immunol 2005, 35, 2459-2470).
Muropeptides are degradation products of peptidoglycan (PGN). Interest in this family of molecules has increased recently with the discovery of Nod1 and Nod2 as sensors of PGN fragments (3, 4). One of these murapeptides, muramyl dipeptide (MDP) has been well characterized (13). MDP mediates signaling by Nod2, exerts adjuvant activity and synergizes with lipopolysaccharide to induce pro-inflammatory responses in vitro and in vivo. There are few and contradictory results on the stimulatory capacity of nod1 agonists toward myeloid cells. Thus, we examined the capacity of various nod1 (murnac-l-ala-?-d-glu-meso-diaminopimelic acid, m-tridap) and Nod2 (murnac-l-ala-d-isogln, MDP; murnac-l-ala-d-isogln-l-lys, m-trilys) agonists to stimulate primary human cells. We show that both cd14+ monocytes and cd1a+ immature dendritic cells express Nod1 and Nod2. Stimulation of primary human monocytes and dendritic cells with highly purified muropeptides (m-tridap, MDP and m-trilys) by itself induces release of pro-inflammatory mediators. Cooperatively with lps, Nod1 as well as Nod2 agonists stimulate release of both pro- and anti-inflammatory cytokines (IL-1beta, tnf, IL-6, IL-8, IL-10, IL-12p40) in these myeloid cell subsets. Nod1 and Nod2 agonists synergize with low amounts of lps to induce dendritic cell maturation, suggesting that nod protein agonists instruct the onset of adaptive immune responses, cooperatively with molecules sensed by toll-like receptors.
These studies have been carried out in collaboration with the group of Dana Philpott and were specifically funded by the Institut Pasteur.
3- Modulation of pro-inflammatory cytokine production
Modulation of the anti-inflammatory properties of interleukin-10 by adherence
(Adib-Conquy et al. Intern. Immunol. 1999, 11, 689-698; Petit-Bertron et al. J. Leuk. Biol. 2003, 73, 145-154; Petit-Bertron et al. Cytokine 2005, 29, 1-12).
Interleukin-10 (IL-10) is a well-recognized anti-inflammatory cytokine. IL-10 is also an immunostimulating cytokine, acting as a growth and differentiation factor for NK cells and B-lymphocytes. The dual role of IL-10 depends on its tissue levels, the nature of the target cell, the activating signal, the timing and sequence of cytokine exposure. We demonstrated that adherence is a parameter that markedly affects the properties of IL-10 on TNF production. Pre-incubation of whole blood samples with IL-10 before isolation of mononuclear leukocytes led to cells that were primed and, in response to LPS, produced more TNF than cells maintained in the absence of IL-10. We showed that prevention of adherence of monocytes during the pre-incubation step with IL-10 was responsible for this pro-inflammatory effect (figure).
We also showed that the pre-treatment of monocytes with IL-10 in the absence of adherence led to an enhanced TNF mRNA expression and NF-?B activation in monocytes / macrophages in the response to LPS. IL-10 is known to increase phagocytosis and we showed that this effect was significantly amplified when the pre-treatment occurred in the absence of adherence. Tyk2 and "signal transducer and activator of transcription" (STAT) 3 phosphorylation and suppressor of cytokine signaling (SOCS) 3 expression are induced by IL-10 in human monocytes. In the presence of adherence, a longer activation and/or expression of these molecules was observed than in the absence of adherence. Finally, heme oxygenase-1 (HO-1), an anti-inflammatory molecule, was induced by IL-10 in adherent monocytes, whereas its expression remained low in non-adherent cells.
We analyzed by macroarray the effect of IL-10 and adherence on the expression of 1050 genes coding for cytokines / chemokines and their receptors, integrins, cell surface markers, proteases, molecules of cell signaling, molecules involved in cell division and cell metabolism. Interestingly, IL-10 modulated differently on Teflon® and plastic the expression of 16 genes, whose products include SOCS2, SOCS3, coproporphyrinogen oxidase (an enzyme involved in heme biosynthesis), and several matrix metalloproteinases (see figure below). This was the first demonstration that IL-10 has a potent inhibitory effect on coproporphyrinogen oxidase expression (both at the mRNA and protein levels) in adherent cells. This could be one of the mechanisms by which IL-10 contributes to the anemia known to occur after injection of IL-10 in humans. These studies demonstrate that adherence has a profound modulatory effect on IL-10 properties and the signaling induced by IL-10.
The macroarray study was done in collaboration with Thierry Pedron (Molecular Microbial Pathogenesis Unit) using the DNA chips platform of the Institut Pasteur. The analysis of coproporphyrinogen oxidase was possible thanks to the collaboration with Ulrich Gross (Johann Wolfgang Goethe-Universität, Frankfurt-am-Main). The project, and Anne-France Petit-Bertron were funded by the Association Vaincre la Mucoviscidose.
Modulation of TNF and IL-1 production by serotonin
(Cloëz-Tayarani et al. Intern. Immunol. 2003, 15, 233-240 ; Cloëz-Tayarani et al. Life Sci. 2004, 76, 429-443)
Serotonin (or 5-hydroxytryptamine, 5-HT) is a mediator of the central nervous system. In addition to its role as a neuromediator, serotonin is present in the intestine and modulates intestinal physiology. Furthermore, serotonin is released by mast cells and by activated platelets and is present at micromolar concentrations at the inflammatory site. Several 7-fold transmembrane receptors of 5-HT have been characterized. In order to afford additional insight into the in vivo significance of 5-HT in inflammation, we examined its effects on the production of TNF, IL-1alpha, IL-1beta, IL-6, IL-10 and IL-1ra in LPS-stimulated peripheral blood mononuclear cells (PBMC). 5-HT inhibited TNF production and increased IL-1beta production in PBMC. The inhibitory effect of 5-HT on TNF production was antagonized by ketanserin, a selective 5-HT2A antagonist, and mimicked by DOI, a selective 5-HT2A/2C agonist. These findings suggest that the inhibition of TNF production by 5-HT involves the participation of the 5HT2A receptor subtype in PBMC. Accordingly, we detected the presence of 5-HT2A receptors in PBMC by Western blot. Our data support a role of 5-HT in inflammation through its effect on cytokine production by PBMC. The regulatory effect of 5-HT on cytokine production was recently confirmed with isolated monocytes (22). We also showed an activation of extracellular signal-regulated kinase (ERK) within cultured lymphocytes exposed to 5-HT. This phenomenon seems to be mediated via the 5-HT1A receptor since similar results were obtained with R-(+)-8-hydroxy-DPAT, a selective agonist of 5-HT1A receptor, and 5-HT-induced ERK phosphorylation was inhibited by WAY100635, a selective antagonist of 5-HT1A receptor. These results further illustrate that the properties and function of cells of the immune system can be modulated by numerous mediators, including neuromediators.
4- Assessment of the inflammatory status of intensive care unit patients
Circulating cytokines and chemokines
(Cavaillon et al. Scand. J. Infect. Dis. 2003, 35, 535)
The presence within the blood stream of cytokines with pro- or anti-inflammatory properties is a hallmark of the systemic inflammation occurring in sepsis patients. Surprisingly, we showed that the levels of the chemokine RANTES (or CCL5) were inversely correlated with APACHE II severity score and outcome: in contrast to all cytokines reported so far, high levels of circulating RANTES correlated with a better prognosis. Most probably, these levels are linked to the number of platelets that are a major source of RANTES.
Enhanced plasma levels of soluble triggering expressed on myeloid cells-1 (sTREM-1) and procalcitonin (PCT) after cardiac surgery and cardiac arrest in the absence of infection
(Adib-Conquy et al. Shock 2007, 28, 406)
Soluble Triggering Expressed on Myeloid Cells-1 (sTREM-1) and Procalcitonin (PCT) have been claimed to be specific markers of infection. We decided to evaluate the plasma levels of sTREM-1 and PCT in non-septic etiologies of systemic inflammatory response syndrome. Plasma s-TREM-1 and PCT were determined in 76 non-infected patients undergoing an elective heart surgery with cardiopulmonary-bypass, 54 patients admitted after an out-of-hospital cardiac arrest, 55 patients with a severe sepsis and 31 healthy volunteers. We found a plasma sTREM value above the suggested threshold for sepsis in 58% cardiac surgery patients, 56% cardiac arrest patients, and 78 % septic patients. Plasma PCT values above the suggested threshold of 0.5 µg/l for sepsis were found in 33% cardiac surgery patients, 80% cardiac arrest patients, and 96% severe sepsis patients. Levels of sTREM-1 and PCT were significantly higher in cardiac arrest patients who died by a refractory shock than those dying from neurological damages or those surviving without major neurological sequalae. In the group of cardiac arrest patients with a refractory shock, levels of sTREM-1 and PCT were similar to that observed in severe sepsis patients. Thus, we established that sTREM-1 and PCT are not specific markers of infection, and are significantly increased in some acute inflammatory non-infectious situations.
5- Molecular basis of leukocyte reprogramming during systemic inflammation
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.
6- Status of circulating and airways-derived neutrophils in cystic fibrosis
Cystic fibrosis (CF) is a genetic multiorgan disease caused by mutation of the transmembrane conductance regulator (CFTR) gene. Obstructive lung disease is the predominant cause of morbidity and mortality. Lung inflammation and lung infection (primarily by S. aureus and Pseudomonas aeruginosa) are early events occurring in the course of the disease. We studied neutrophils (PMN) derived either from sputum or from blood of young patients with cystic fibrosis (CF). In contrast to what was observed in blood neutrophils, CF sputum PMN displayed a high ex vivo spontaneous IL-8 production which was neither up-regulated by the addition of LPS nor down-regulated by synthetic glucocorticoid such as dexamethasone (Corvol et al. Am. J. Physiol. 2003, 284, L997). Also, IL-10, which inhibits IL-8 production by blood PMN activated by LPS (or peptidoglycan), had no effect on sputum PMN. In CF patients, TLR2 expression was significantly reduced on blood PMN and TLR4 expression was significantly enhanced on sputum PMN as compared to blood PMN from healthy donors. The expression of CD64 (Fcgamma receptor type I), a marker of cell activation, was increased on blood PMN of CF patients (Petit-Bertron et al. Cytokine 2008, 41, 54).
Macroarray analysis of 1050 genes revealed that the expression of 25 genes in CF blood PMNs and 16 genes in CF airway PMNs was reduced as compared to healthy blood PMNs. By contrast, the expression of 42 genes in blood PMNs of CF patients and 56 genes in CF airway PMNs was enhanced as compared to healthy blood PMNs. When comparison was performed between blood and airway PMNs of CF patients, there was, unexpectedly, a very limited difference in terms of gene expression. Only the mRNA expression of amphiregulin and of tumor necrosis factor receptor was significantly higher in airway PMNs. The presence of amphiregulin was confirmed by ELISA in the sputum of CF patients. Altogether, this study clearly demonstrates that PMNs from CF patients display a profound modification of their gene expression profile associated with the disease, with a very limited difference between blood and airway PMNs (Adib-Conquy et al. Mol. Med. 2008, 14, 36-44).
The persistent presence of PMN in airways is the hallmark of cystic fibrosis disease. We assessed whether there is a CF-related anomaly of PMN adherence, rate of apoptosis and production of inflammatory mediators (IL-6, IL-8 and lipoxin A4) when PMN are in contact with airway epithelial cells. We examined co-cultures of PMN isolated from airways and peripheral blood of CF patients and non-CF subjects with human bronchial epithelial cells bearing mutated CFTR, compared to CFTR-corrected and normal epithelial bronchial cells. Our data provided causal evidence that CFTR-deficient bronchial epithelial cells markedly increased sputum PMN adherence, prolonged their survival and induced altered inflammatory response in CF airways. We speculate that the prolonged lifespan of an elevated number of PMN adhering to CF airway epithelium may contribute to the exaggerated inflammatory response in lungs of CF patients (Tabary et al. Am J Physiol. Lung Cell Mol. Physiol, 2006; 290: L588-96)
7. Innate immunity against Aspergillus fumigatus
Aspergillus fumigatus (A.f.) is an ubiquitous thermophilic, saprophytic filamentous fungus. The inhalation of conidia of A.f. may results in different pulmonary diseases. Allergic bronchopulmonary aspergillosis, aspergilloma and invasive pulmonary aspergillosis (are among the most common and threatening pathologies due to A. fumigatus. Invasive pulmonary aspergillosis is most often fatal among immunocompromised patients. Indeed, A.f. has become today the major fungal air-borne pathogen in terms of morbidity and mortality in developed countries. The purpose of our studies is to better understand the innate immunity against this fungus in the immunocompetent and immunocompromised host. We have previously demonstrated that human, mouse or cell-line alveolar macrophages are able to efficiently phagocytize the conidia of A.f. Internalisation is mediated by the actin network and PI-3 kinases. Once internalized, the conidium is directed toward the phagolysosome. The intracellular phagocytosis of conidia triggers the phosphorylation of ERK and p38 MAP-kinases. Few hours after phagocytosis, the conidium grows isodiametrically. This first step in the process of conidial germination is not inhibited in the cells of an immunocompetent individual. The swelling of conidia occurs concomitantly with the translocation and activation of the transcription factor of NF-kB and the production of proinflammatory cytokines such as TNF? and IL-6. Most importantly, swollen conidia are sensitive to phagocytic killing and an immunocompetent mouse is able to clear all inhaled conidia within 3 days. Under immunosuppression, the inflammatory response is altered and the alveolar macrophages fail to clear the conidia. The germination of inhaled conidia results in a massive hyphal growth invading all the lungs alveoli and vessels. Although a massive infiltration of polymorphonuclear neutrophils is observed at the site of infection, the mycelium overgrowth results in the death of animals within 3 to 4 days.
8. Innate immunity against Leishmania major
In its vertebrate host, Leishmania major encounters cells that express Toll-Like Receptors (TLRs) The importance of the MyD88 adaptor molecule for the development of protective immunity against L. major has been documented. Some TLR (2, 3, 4, and 9) have been reported to be the target of some PAMPs from Leishmania. Using genetically resistant C57Bl/6 mice deficient for either TLR2, 4 or 9, we showed that only TLR9-deficient (TLR 9-/-) mice are more susceptible to infection with L. major. TLR9-/- mice resolved their lesions and controlled parasites growth with much lower efficiency than wild type C57Bl/6 mice. The absence of TLR9 also transiently inhibited the development of curative Th1 response. In an attempt to analyze the possible basis for such aberrant response in TLR9-/- mice, we have studied the importance of TLR9 for activation of dendritic cells (DCs) by L. major. Our results showed that bone marrow derived DCs as well as DCs freshly isolated from the spleen of C57Bl/6 mice can be activated by either heat-killed or live L. major in vitro. In sharp contrast, L. major failed to activate DCs from TLR9-/- mice. Interestingly, DNA purified from L. major was capable of activating DCs in a strictly TLR9-dependent manner. Similarly purified vertebrate DNA had no effect, suggesting that this TLR9-dependent response of DCs was restricted to L. major DNA. The importance of TLR9 signaling in the capacity of DCs to recruit and activate T cells is currently assessed in vitro. We will assess the capacity of the different subtypes of DCs (alone or in combination) to activate specific T cells. To this end we will use easily traceable T cells, i.e. T cells from mice transgenic for a TCR specific for OVA. The specific activation by L. major DNA will also be investigated.