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  Director : Cavaillon Jean-Marc (jmcavail@pasteur.fr)


  abstract

 

We study the involvement of cytokines in severe inflammatory diseases in humans (sepsis, trauma, ischemia/reperfusion, and cystic fibrosis) and in murine models. These pathologies are associated with an exacerbated production of cytokines as well as an immune depression that we study at the intracellular signaling pathways level. We also study the effects of adherence on IL-10 properties and the regulation of the cytokine production by serotonin.



  report

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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 (Muñoz et al. J. Clin. Invest. 1991, 88, 1747), we have further characterized the immune-depression associated to this pathology. We have extended our observation to circulating neutrophils (Marie et al. Blood 1998, 91, 3439) and lymphocytes (Muret et al. Shock 2000, 13, 169) of sepsis patients and of patients suffering of non-infectious systemic inflammatory response syndrome (SIRS). In collaboration with Dr. C. Adrie (Hôp. de Saint-Denis), Prof. J-F. Dhainaut (Hôp. Cochin) and Drs P. Moine et K. Asehnoune (Hôp. du Kremlin Bicêtre), we study the intracellular molecular mechanisms responsible for the immune-depression observed in sepsis patients as well as in patients with SIRS (trauma, ischemia/reperfusion). A global decrease of nuclear factor-kB (NF-kB), an unbalance between its active (p65p50) and inactive (p50p50) forms and a weak cytoplasmic expression of its inhibitor (IkBa) 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-kB expression lasts for more than 10 days (Adib-Conquy et al. J. Leuk. Biol. 2001, 70, 30). We have shown that this hyporeactivity was restricted to Gram-negative bacteria, Escherichia coli lipopolysaccharide (LPS) and CpG oligonucleotide, whereas Leptospira interrogans LPS-induced TNF production was similar to that observed with healthy donors. TNF and IL-6 productions in response to Gram-positive bacteria were not altered as well. The expression of Toll-like receptor (TLR) 2 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 downregulation of TLR4. Indeed, unlike pro-inflammatory cytokines, the release of anti-inflammatory cytokines (IL-10 and IL-1ra) was increased as compared to healthy controls. The activation of the p38 MAPK and the Sp-1 transcription factor was increased in peripheral blood mononuclear cells 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). In conclusion, the immunodysregulation described for trauma patients is not a generalized phenomenon, but depends on the stimulus and the signaling pathway.

2- IMMUNOLOGICAL STATUS OF CARDIAC ARREST AND RECUSCITATED PATIENTS (M. Adib Conquy, C. Fitting)

In collaboration with Dr. C. Adrie (Hôp. de St Denis) 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 sequel, hemodynamic shock and possible multiple organ failure in 50% of the patients. We demonstrated the presence of circulating endotoxin in 46% of the patients within the first two days following admission in intensive care units. (n=35). We showed that at admission (around 3h 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-1ra was also a marker associated with prognosis. Endotoxin-induced TNF and IL-6 productions in in vitro whole blood culture were dramatically impaired in these patients as compared to healthy controls, while an unaltered production was observed with heat-killed staphylococcus aureus. In contrast, IL-1ra production was enhanced as compared to healthy controls. The productions of T-cell derived IL-10 and IFNg were 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 patients leukocytes, while an immunosuppressive activity was also present in their plasma. Altogether, these observations show that, despite a non-infectious stress, these patients harbor numerous parameters found in sepsis patients (Adrie et al. Circulation 2002, 106, 562).

3- CYTOKINES AND CYSTIC FIBROSIS (M. Adib-Conquy, A-F. Petit Bertron, C. Fitting)

In collaboration with Drs. H. Corvol and A. Clément (Hôp. Trousseau), we showed that neutrophils derived from sputum of young patients with cystic fibrosis had a high ex vivo spontaneous IL-8 production which cannot be up-regulated by the addition of LPS or downregulated by dexamethasone in contrast to what was observed in blood neutrophils. We are currently analyzing the expression of the TLR2 & TLR4, the level of apoptosis and the capacity of IL-10 to prevent the activation of these neutrophils. Other studies are undertaken to understand the interaction between neutrophils and epithelial cells from cystic fibrosis patients (collaboration with Drs J. Jacquot and O. Tabary).

4- PRODUCTION OF MIF (V. Maxime, C. Fitting)

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, which prevents glucocorticoid properties. In collaboration with Prof., D. Annane (Hôp. Poincaré, Garches) we have measured the plasma levels of MIF in sepsis patients, and showed that they correlate (r = 0.62, p = 0.001) with the glucocorticoid deficit (assessed as delta max of cortisol following ACTH test). We are currently studying the capacity of leukocytes of sepsis patients versus those of healthy controls to produce MIF in vitro in response to various activators and assessing the effect of a glucocorticoid treatment. An mRNA analysis is also performed by quantitative RT-PCR in collaboration with C. Behr and Ph. Bœuf.

5- CELL SIGNALING VIA TOLL-LIKE RECEPTORS-2 & 4 AND NOD1 (M. Adib-Conquy, C. Fitting)

In collaboration with Catherine Werts (in charge of the program) and Dana Philpott, 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 pathogens associated molecular patterns, PAMPs) and KO mice are used.

6- CELLULAR SIGNALING MECHANISMS ASSOCIATED WITH THE INHIBITION OF ENDOTOXIN TOLERANCE BY IFNg and GM-CSF (M. Adib-Conquy)

Endotoxin tolerance is characterized by a decreased production of pro-inflammatory cytokines by cultured leukocytes in response to lipopolysaccharide (LPS) following a first exposure to the same stimulus. Gamma interferon (IFNg) 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 IFNg and GM-CSF or their capacity to restore TNF production by LPS-tolerized human monocytes are independent of the modulation of TLR2, TLR4 or MD-2. In monocytes pretreated with IFNg or GM-CSF, IRAK expression is upregulated. After LPS stimulation, an increased IRAK kinase activity, a higher MyD88/IRAK association and a stronger NF-kB activation are observed. In contrast, in LPS-tolerized monocytes, IRAK expression and kinase activity, IRAK/MyD88 association and NF-kB activation are inhibited. Furthermore, the prevention of tolerance by IFNg and GM-CSF was independent of IRAK kinase activity. Our results suggest that these cytokines prevent endotoxin tolerance, induced by low but not by high doses of LPS, by inhibiting IRAK degradation and by promoting its association with MyD88 after a second LPS stimulation, which in turn leads to NF-kB activation and TNF production (Adib-Conquy & Cavaillon, J. Biol. Chem. 2002, 277: 27927).

7- COMPARTMENTALIZATION DURING ENDOTOXIN TOLERANCE (C. Fitting)

The in vivo induction of endotoxin tolerance leads to diverse states of hyporeactivity (intensity, length) of the cells depending upon the compartment they derived from (spleen, bone marrow, peritoneal cavity, lungs, blood). This phenomenon is poorly influenced by the release of glucocorticoid in response to LPS as assessed by experiments performed with adrenalectomized mice and mice receiving dexamethasone or glucocorticoid antagonist (RU486).

8. MODULATION OF INTERLEUKIN-10 PROPERTIES BY ADHERENCE (M. Adib-Conquy, A-F. Petit-Bertron)

We have previously demonstrated that adherence is a parameter which markedly affects the properties of IL-10 on TNF production and NF-kB activation by monocytes/macrophages (Adib-Conquy et al. Intern. Immunol. 1999, 11, 689). We have further studied the effects of adherence on the properties of IL-10 on monocyte-enriched peripheral blood mononuclear cells. There is no significant difference in response to IL-10 with or without adherence in terms of cell surface marker involved in LPS signaling (CD14, TLR4, MD2), IL-10 responsiveness (IL-10R), antigen presentation (HLA-DR), co-activation (CD40) and adherence (CD11a, CD62L). Only CD11b expression was more markedly inhibited by IL-10 together with adherence as compared to non-adhering conditions. The absence of adherence prevented the inhibitory effects of IL-10 on LPS-induced TNF and G-CSF production, and increased IL-1b production and sTNFRII release in IL-10-pretreated cells. Similarly, the absence of adherence amplified the enhancement of phagocytosis induced by IL-10. Tyk2 and STAT3 phosphorylation and SOCS3 expression were induced by IL-10 in both conditions, 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. Altogether, these data illustrate that adherence modulates the properties and the anti-inflammatory effects of IL-10. We consider that our model can mimic some in vivo events when circulating monocytes encounter IL-10 before adhering to endothelium and marginating towards tissues (Petit-Bertron et al. J. Leuk. Biol. 2003, 73, 145).

5. SEROTONIN AND CYTOKINE PRODUCTION (I. Cloëz-Tayarani)

Serotonin (5-hydroxytryptamine = 5-HT) is released by activated platelets and can be present at the 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-1a, IL-1b, IL-6, IL-10 and IL-1ra in LPS-stimulated peripheral blood mononuclear cells (PBMC). 5-HT inhibited TNF production and increased IL-1b production in PBMC. The level of caspase-1 remained unchanged suggesting that the effect is not directly related to IL-1 b maturation process. TNF mRNA and IL-1b mRNA content remained unchanged in the presence of 5-HT. 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. Intern. Immunol. 2003, 15 Feb. Issue).

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-1b, 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 current and published works (Adib-Conquy M et al J. Leuk. Biol. 2001; 70:30) and other reports (Learn et al. J. Biol. Chem. 2001; 276:20234 ; Solomon et al. J. Clin. Invest. 1998; 102:2019)

Keywords: Toll-like receptor, sepsis, intra-cellular signaling, endotoxin, serotonin



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
    Minou Adib-Conquy, researcher IP, madib@pasteur.fr

Isabelle Cloëz-Tayarani, researcher IP, icloez@pasteur.fr

Anne-France Petit-Bertron PhD student ,afpetit@pasteur.fr

Virginie Maxime MD, Master student , virginie.maxime@libertysurf.fr

Shaw Warren Invited Professor , warren@helix.mgh.harvard.edu

Catherine Fitting Technician IP ,cfitting@pasteur.fr

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