Unit: Cytokines and inflammation
Director: Cavaillon Jean-Marc
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 immune dysregulation observed in circulatory cells, reminiscent of the endotoxin tolerance phenomenon and that could be linked to the increased susceptibility of these patients to nosocomial infections. Finally, we analyzed the mechanisms involved in cytokine induction by pathogen-associated molecular patterns (PAMPs), the very early phase of innate immunity.
1- MOLECULAR BASIS OF IMMUNE-DEPRESSION DURING SYSTEMIC INFLAMMATION (M. Adib-Conquy)
Since we first reported 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 to this pathology. We have described a similar alteration in patients after surgery (Cabié et al. Cytokine 1992, 4, 576) and in patients resuscitated after cardiac arrest (Adrie et al. Circulation, 2002, 106, 562). We extended our observation to circulating neutrophils (Marie et al. Blood 1998, 91, 3439) and T-lymphocytes (Muret et al. Shock 2000, 13, 169). We study the intracellular molecular mechanisms responsible for the immune-depression observed in sepsis patients as well as in patients with non-infectious systemic inflammatory response syndrome (SIRS) (trauma, ischemia/reperfusion). A global decrease of nuclear factor-κB (NF-κB), an unbalance between its active (p65p50) and inactive (p50p50) forms and a weak cytoplasmic expression of its inhibitor (IκBα) have been observed within mononuclear cells of sepsis patients (Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2000, 162, 1877). This observation is a reminiscence of the events occurring in in vitro endotoxin-tolerized monocytes/macrophages. A similar study undertaken in trauma patients revealed that the defect in NF-κB expression lasts for more than 10 days (Adib-Conquy et al. J. Leuk. Biol. 2001, 70, 30).
The immune dysregulation is illustrated by a dramatic decrease in the capacity of peripheral blood mononuclear cells (PBMC) to release tumor necrosis factor (TNF) upon activation by Escherichia coli endotoxin (lipopolysaccharide, LPS) and CpG oligonucleotide, and to produce interleukin-6 (IL-6) in response to IL-1 and TNF. However, this is not a global defect, since cells remain fully reactive to other stimuli (Staphylococcus aureus, Streptococcus pyogenes, and LPS from Leptospira interrogans, a Toll-like receptor-2 (TLR2) agonist). 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, the hyporeactivity to Gram-negative bacteria and E. coli LPS cannot be fully explained by the down-regulation of TLR4. Indeed, PBMC produce enhanced levels of IL-10 and IL-1 receptor antagonist (IL-1ra) in response to E. coli LPS as compared to cells from healthy controls. The activation of the p38 MAPK and the Sp-1 transcription factor was increased in PBMC from trauma patients after E. coli LPS or heat-killed Staphylococci stimulation, and the addition of an inhibitor of p38 decreased IL-10 production. Furthermore, heterotrimeric Gi proteins and phosphatidylinositol-3'-kinase were involved in IL-10 production (figure 1). In conclusion, the immune-dysregulation described in SIRS patients is not a generalized phenomenon, but depends on the stimuli and the signaling pathways (Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2003, 168, 158).
2- PRODUCTION OF MIF (V. Maxime, C. Fitting)
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that prevents glucocorticoid activities. In collaboration with Prof., D. Annane (Hôp. Poincaré, Garches) we have 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 of the patients on the in vitro production of MIF by their leukocytes. PBMC from patients contained significantly higher amounts of MIF than cells from healthy controls. In culture, spontaneous release and release induced by LPS, heat-killed staphylococci 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 derived from animal studies, glucocorticoid treatment led to normalize the release of MIF by circulating PBMC from patients with septic shock.
3- CYTOKINES AND CYSTIC FIBROSIS (A-F. Petit-Bertron, M. Adib-Conquy, C. Fitting)
We study neutrophils (PMN) derived from sputum of young patients with cystic fibrosis (CF). CF PMN display a high ex vivo spontaneous IL-8 production which cannot be up-regulated by the addition of LPS or down-regulated by dexamethasone in contrast to what was observed in blood neutrophils (Corvol et al. Am. J. Physiol. 2003, 284, L997). As well, IL-10, that inhibits IL-8 production by blood PMN activated by LPS or peptidoglycan, has no effect on sputum PMN. In CF patients, TLR2 and TLR4 surface expression on PMN are modified as compared to blood PMN from healthy donors. TLR2 expression is significantly reduced on blood PMN from CF patients and TLR4 expression is significantly enhanced on sputum PMN. The expression of CD64 (Fcγ receptor type I), a marker of cell activation, is increased on blood PMN of CF patients. PMN from sputum of CF patients, kept in contact with respiratory epithelial cells, display an enhanced survival (reduced apoptosis) as compared to blood PMN from patients and healthy controls. A synergy in terms of IL-6 and IL-8 production is observed when PMN from CF patients are cultured in the presence of CF bronchial epithelial cells as compared to normal bronchial epithelial cells. This work is performed in collaboration with Drs J. Jacquot, O. Tabary, H. Corvol and A. Clément (Hôp. Trousseau, Paris) and founded by Association Vaincre la Mucovosicidose.
4- CELL SIGNALING VIA TOLL-LIKE RECEPTORS-2 & 4 AND NOD1 (M. Adib-Conquy, R.Kapetanovic, C. Fitting)
Within the Programme Transversal de Recherche (PTR) 94 and in collaboration with Dana Philpott and Stephen Girardin, we investigate the role of TLR2, TLR4, TLR9 and Nod1 molecules in cellular signaling which specifically lead to the production of TNF and IL-10. In this context, numerous well purified and characterized PAMPs and KO mice are used. In the absence of TLR2 and TLR4, TNF production by peritoneal macrophages is impaired in response to Gram-negative bacteria; in contrast, activation by Gram-positive bacteria is unchanged. This suggests that TLR2 and TLR4 are dispensable for macrophage activation by Gram-positive bacteria, while their phagocytosis appears as an important step involved in cytokine induction. Nod1-/- macrophages produce less IL-10 than control cells in response to heat-killed Staphylococci. Transfection experiments of a macrophage cell line with dominant negative forms of Nod1, Nod2 and MyD88 suggest that these molecules are important for cytokine induction by S. aureus.
5- ENDOTOXIN TOLERANCE (C. Fitting, M. Adib-Conquy)
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 (IFNγ) and granulocyte monocyte-colony stimulating factor (GM-CSF) are immunostimulatory cytokines that prime monocytes and prevent endotoxin tolerance. In monocytes pretreated with IFNγ or GM-CSF, IRAK-1 expression is upregulated. In LPS-tolerized monocytes, IRAK-1 expression and kinase activity, IRAK/MyD88 association and NF-κB activation are inhibited. The prevention of tolerance by IFNγ 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-κB activation and TNF production (Adib-Conquy & Cavaillon, J. Biol. Chem. 2002, 277: 27927).
Endotoxin tolerance has been defined and analyzed either entirely in vivo or entirely in vitro. In contrast, the hyporeactivity of circulating leukocytes reported in patients with sepsis and often referred to an endotoxin tolerance phenomenon, 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 (SAC) for production of TNF. Cells acquired LPS tolerance within 1 to 3 hours after in vivo injection of LPS. A dramatic decrease in the production of TNF in response to LPS was observed with circulating leukocytes, splenocytes, peritoneal cells and bone-marrow cells 24hrs post-LPS injection. In contrast, LPS-induced TNF production by bronchoalveolar cells was far less reduced and only very briefly. The kinetics of acquisition of tolerance and recovery were different for the various compartments. "Cross-tolerance" with SAC did not parallel the phenomenon of endotoxin tolerance as observed with LPS. 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 (Fitting et al. J. Infect. Dis. 2004, in press)
6. MODULATION OF INTERLEUKIN-10 PROPERTIES BY ADHERENCE (A-F. Petit-Bertron, M. Adib-Conquy)
We previously demonstrated that adherence is a parameter, which markedly affects the properties of IL-10 on TNF production, TNF mRNA expression and NF-κB activation by monocytes/macrophages (Adib-Conquy et al. Int. Immunol. 1999, 11, 689). Tyk2 and STAT3 phosphorylation and suppressor of cytokine signaling (SOCS) 3 expression were induced by IL-10 in human monocytes both in the presence and in the absence of adherence, but a longer activation and/or expression was observed in adherent monocytes. 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 (Petit-Bertron et al. J. Leuk. Biol. 2003, 73, 145). Recently, we analyzed by macroarray the effect of IL-10 and adherence on the expression of 1050 genes. On adherent monocytes, IL-10 induced the expression of 21 genes and repressed the expression of 50 genes. On non-adherent monocytes, IL-10 induced the expression of 45 genes and repressed that of 67. Only 3 common genes were induced while 35 common genes were repressed by IL-10 in the two culture conditions. Interestingly, we showed that IL-10 modulated conversely on Teflon® and plastic the expression of 16 genes, of which SOCS2, SOCS3, coproporphyrinogen oxidase (an enzyme involved in heme biosynthesis)), and several matrix metalloproteinases. We confirmed by RT-PCR the modulation of the SOCS molecules and showed that the expression of the other proteins (except for CD35) was modulated similarly to that of their mRNA. The strong inhibition of coproporphyrinogen oxidase in adherent cells, could be one of the mechanisms by which IL-10 contributes to anemia. In the absence of adherence, IL-10 inhibited the expression of SOCS1, 2 and 3 molecules, which were strongly induced by this cytokine in adherent cells. The inhibition of SOCS could contribute to the pro-inflammatory effects of IL-10 in the absence of adherence, since these molecules have been shown to negatively regulate LPS signaling via TLR4. In addition, IL-10 induced in non-adherent monocytes the expression of various matrix metalloproteinases, molecules found in the blood stream during inflammatory states. This study demonstrates that adherence has a profound modulatory effect on the properties and the signaling induced by IL-10. This observation may partially explain the dual role of this cytokine. We consider that our model can mimic some in vivo events when circulating monocytes encounter IL-10 before adhering to endothelium and marginating within tissues towards inflammatory foci (fig. 2). The macroarray study was done in collaboration with T. Pedron and the plate forme 2 " puces à ADN " from Institut Pasteur.
7. SEROTONIN AND CYTOKINE PRODUCTION (I. Cloëz-Tayarani)
Serotonin (5-hydroxytryptamine, 5-HT) is released by activated platelets and can be present at micromolar concentration within the inflammatory site. In order to provide additional insight into the in vivo significance of 5-HT in inflammation, we examined its effects on the production of TNF, IL-1α, IL-1β, IL-6, IL-10 and IL-1ra in LPS-stimulated PBMC. 5-HT inhibited TNF production and increased IL-1β 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 subtypes in PBMC. Accordingly, we detected the presence of 5-HT2A receptors in PBMC by Western-blots. Our data support a role of 5-HT in inflammation through its effect on cytokine production in PBMC (Cloëz-Tayarani et al. Int. Immunol. 2003, 15 233). More recently we showed an activation of extracellular signal-regulated kinase (ERK) within 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.
Figure 1. Schematic cartoon of cellular signaling pathways, altered (grey background), or enhanced (thick lines) in leukocytes of trauma patients leading to the production of TNF, IL-1β, IL-1ra and IL-10 in response to TLR2 and TLR4 ligands derived from Gram negative (BG-) or Gram positive (BG+) bacteria. This cartoon illustrates our published works (Adib-Conquy M et al J. Leuk. Biol. 2001; 70:30; Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2003, 168, 158) and other reports (Learn et al. J. Biol. Chem. 2001; 276:20234 ; Solomon et al. J. Clin. Invest. 1998; 102:2019)
Figure 2. Sequence of signaling, local levels, nature of target cells, role of co-signals, and nature of environmental cells can modify the properties of IL-10 (see Cavaillon J-M. Pro- versus anti-inflammatory cytokines : myth or reality, Cell Mol. Biol. 2001, 47, 695)
Keywords: Toll-like receptor, sepsis, intracellular signaling, endotoxin, innate immunity