Unit: Cytokines and inflammation

Director: Cavaillon Jean-Marc

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 (figure 1). 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- CONTRIBUTION OF PHAGOCYTOSIS, TLR2, NOD1 AND NOD2 FOR MACROPHAGE RESPONSE TO STAPHYLOCOCCUS AUREUS (R. Kapetanovic, C. Fitting, M. Adib-Conquy, in collaboration with D. Philpott, Innate Immunity & Signaling, Inst. Pasteur)

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, phagocytosis was essential for cytokine production in response to heat-killed Staphylococcus aureus (HKSA). By transfecting Raw 264.7 macrophages with dominant negative (DN) forms of Nod1 and Nod2, we showed that both molecules inhibited NF-kB 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, MDP) 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.

2- SYNERGISTIC STIMULATION OF MONOCYTES AND DENDRITIC CELLS BY TLR4, NOD1- AND NOD2-ACTIVATING AGONISTS (in collaboration with D. Philpott, Inst. Pasteur)

MDP mediates signaling by Nod2. It exerts adjuvant activity and synergizes with endotoxin (lipopolysaccharide, LPS) to induce pro-inflammatory responses in vitro and in vivo. Various Nod1 and Nod2 agonists stimulate CD14+ monocytes and CD1a+ immature dendritic cells to produce pro-inflammatory mediators. Cooperatively with LPS, Nod1 as well as Nod2 agonists stimulate release of both pro- and anti-inflammatory cytokines (IL-1β , 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 (Fritz et al. Eur. J. Immunol., 2005, 35, 2459-2470).

3- ENDOTOXIN TOLERANCE (C. Fitting)

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 (IFNg) and granulocyte monocyte-colony stimulating factor (GM-CSF) are immunostimulatory cytokines that prime monocytes and prevent endotoxin tolerance. We previously showed that in monocytes pretreated with IFNg or GM-CSF, IRAK-1 expression is up-regulated. IFNg and GM-CSF prevent endotoxin tolerance induced by low 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-k B activation and TNF production (Adib-Conquy & Cavaillon, J. Biol. Chem. 2002, 277: 27927). More recently we set up an ex vivo model of endotoxin tolerance. We showed that endotoxin tolerance is compartmentalized and that broncho-alveolar cells are less likely than peritoneal, splenic or marrow cells to develop endotoxin tolerance (Fitting et al. J. Infect. Dis. 2004, 189, 1295). We also showed that "cross-tolerance" with HKSA did not parallel the phenomenon of endotoxin tolerance as observed with LPS. We are now comparing the cross-tolerance phenomenon with conventional Escherichia coli LPS (Sigma) that contains trace amounts of lipoprotein contaminants, acting as TLR2 ligands, and a highly purified E. coli LPS. Conventional LPS tolerizes human monocytes for a further challenge by itself and also by highly purified LPS. In contrast, highly purified LPS has little or no capacity to tolerize monocytes to a challenge with conventional LPS. Pre-treatment with conventional LPS tolerizes cells to a challenge with a specific TLR2 ligand (Pam3CysSK4), whereas highly purified LPS primes the cells to a challenge by the TLR2 ligand.

4. MODULATION OF INTERLEUKIN-10 PROPERTIES BY ADHERENCE (A-F. Petit-Bertron, M. Adib-Conquy, in collaboration with T. Pedron and the plate forme 2 " puces à ADN " from Institut Pasteur)

We previously demonstrated that adherence is a parameter, which markedly affects the properties of IL-10 on TNF production, heme oxygenase-1 expression, and et by monocytes/macrophages (Adib-Conquy et al. Int. Immunol. 1999, 11, 689; Petit-Bertron et al. J. Leuk. Biol. 2003, 73, 145). We analyzed by macroarray the effect of IL-10 and adherence on the expression of 1050 genes. Interestingly, 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. The strong inhibition of coproporphyrinogen oxidase in adherent cells, could be one of the mechanisms by which IL-10 contributes to anemia. 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 (Petit-Bertron et al. Cytokine 2005, 29, 1).

5- LEUKOCYTE RESPONSE IN PATIENTS WITH SEVERE INFLAMMATORY DISORDERS

A/ Molecular basis of leukocyte reprogramming during systemic inflammation

(M. Adib-Conquy, in collaboration with Dr. Christophe Adrie, Hôpital Delafontaine, Saint-Denis, and Rudi Beyaert, Ghent University, Belgium).

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 SIRS. 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). 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 activation of the p38 MAPK and the Sp-1 transcription factor was increased in PBMC from trauma patients after ex vivo activation. This observation may explain the up-regulation of IL-10 production (Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2003, 168, 158).

More recently, we showed that the TNF production in response to Pam3CysSK4, a specific TLR2 ligand, was also reduced in sepsis as compared to healthy controls. However, responsiveness to HKSA, heat-killed E. coli, and MDP was similar to that obtained with cells from healthy donors. MDP is sensed by Nod2 intracellular receptor. We found that the expression of Nod2 mRNA was unchanged in sepsis, as well as that of Nod1, a sensor of peptidoglycan from Gram-negative bacteria. In contrast to TNF, the production of IL-10 by monocytes of septic patients in response to LPS and Pam3CysSK4 was enhanced, whereas that induced by heat-killed bacteria was unchanged. We investigated the expression of various molecules regulating negatively the TLR4-dependent signaling. 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 (figure 2). The later results were confirmed by quantitative real-time PCR. We also included a group of resuscitated patients after cardiac arrest (RCA), in order to compared sepsis with non-infectious SIRS. Their monocytes responsiveness displayed a reduced production of TNF only in response to LPS, but not Pam3CysSK4 nor heat-killed bacteria. In contrast to sepsis patients, there was no increase in IL-10 production after ex vivo stimulation. The expression of SIGIRR mRNA was increased in the monocytes from RCA patients, but not that of MyD88s. Thus, leukocytes hyporesponsiveness to microbial agonists is not a generalized phenomenon in sepsis and SIRS, and the terms "leukocyte reprogramming" better define this phenomenon, rather than the terms often employed such as anergy, immunodepression, or immunoparalysis (Adib-Conquy et al submitted).

In a mouse model of hemorrhage and resuscitation, we have demonstrate that α2-adrenoceptor ligands contribute to the ex vivo "blood deactivation" to LPS (Asehnoune et al. submitted).

B/ Production of MIF (V. Maxime, C. Fitting, In collaboration with Prof., D. Annane, Hôp. Poincaré, Garches)

"Macrophage migration inhibitory factor" (MIF) is an inflammatory cytokine known to couterregulate the effects of glucocorticoids. 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. PBMC from patients contained significantly higher amounts of MIF than cells from healthy controls. In culture, spontaneous release and release induced by LPS and HKSA 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 E. 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. Glucocorticoid treatment led to normalize the release of MIF by circulating PBMC from patients with septic shock (Maxime et al. J. Infect. Dis. 2005, 191, 138).

C/ Cytokines and cystic fibrosis (A-F. Petit-Bertron, M. Adib-Conquy, C. Fitting. This work is performed in collaboration with T. Pedron, Inst. Pasteur, and Drs J. Jacquot, O. Tabary, H. Corvol and A. Clément, Hôp. Trousseau, Paris; founded by Association Vaincre la Mucovosicidose)

We study neutrophils (PMN) derived from sputum of patients with cystic fibrosis (CF). CF PMN display a high ex vivo spontaneous IL-8 production which can neither be up-regulated by the addition of LPS nor 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 activated blood PMN, has no effect on sputum PMN. In CF patients, 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.

Comparative microArray analysis of PMN derived from sputum and blood from CF patients and from blood of healthy controls was performed. The expression of 42 genes in blood PMN of CF patients and 56 genes in sputum PMN (twenty common) was enhanced as compared to their respective expressions in PMN of healthy controls. The expression of 25 genes in blood PMN of CF patients and 16 genes in sputum PMN (twelve common) was reduced as compared to their respective expression in PMN of healthy controls. It is worth mentioning the up-regulation of numerous chemokine genes, growth factors and cytokine receptor genes. Most interesting is the up-regulation of 6 IFNγ -dependent genes in either CF blood or sputum PMN.

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 (Tabary et al. Am J Physiol. Lung Cell Mol. Physiol, in press).

6- WHY MICE ARE SO RESISTANT TO ENDOTOXIN ? (C. Fitting, M. Adib-Conquy, in collaboration with Prof. Shaw Warren, Boston Mass. Gen. Hosp., and founded by NIH)

Mice are often used as animal model for sepsis and endotoxin-induced shock. However, mice are 105 fold more resistant to endotoxin than humans. We decided to investigated what was the basis for this difference, and showed that the difference may be due to a soluble factor present in mouse serum. We showed that, in contrast to human serum or fetal calf serum, mouse serum inhibits TNF production by human monocytes, and by mouse peritoneal or bone marrow-derived macrophages in response to numerous PAMPs (endotoxin, peptidoglycan, peptidoglycan-associated lipoprotein, Pam3CysSK4, bacterial DNA, toxic shock syndrome toxin), to zymosan or whole heat-killed bacteria. Mouse serum inhibits NF-κ B activation by LPS in human cells. Exposure of mouse serum to trypsin removes all inhibitory activity, proving that the inhibitory substance is a protein. Pre-exposure of human peripheral mononuclear cells to media containing mouse (but not human serum) downregulates the cells, indicating that the inhibitory of the mouse serum factor(s) does not need concomitant action with the activating signal. Furthermore, mouse serum inhibits the activation of human umbilical vein endothelial cells in the presence of human serum or recombinant sCD14. Biochemical characterization of the factor(s) responsible of the effect is underway in Prof. Warren's laboratory. Our unit is in charge of the study of the mechanisms of action of this factor and its capacity to modulate cell signaling in monocytes.

Pictures :

Figure 1. From bacteria to disease. During infection, host cells sense bacterial derived products (lipopolysaccharide, LPS; peptidoglycan, PGN; DNA…] via specific receptors (Toll-like receptors, TLR; peptidoglycan recognition proteins, PGRP; nucleotide-binding oligomerization domain, Nod). The activation of leukocyte results in the production of numerous mediators of innate immunity and inflammation (tumor necrosis factor, TNF; interleukin-1, IL-1; nitric oxide, NO…). Other events triggered by bacterial derived products (complement activation, coagulation) amplify the inflammatory process. An exacerbated production of inflammatory mediators contribute to organ dysfunction and eventually death. Concomitantly, the inflammation process is counter-regulated by the induction of anti-inflammatory mediators (interleukin-10, IL-10; IL-1 receptor antagonist, IL-1ra; soluble TNF receptors, sTNFR…), acute phase proteins (including LPS binding protein, LBP; soluble CD14, sCD14…), neuropeptides and hormones. These mediators together with the occurrence of apoptosis of leukocytes contribute to an altered immune status (Annane D., Bellissant E., Cavaillon J-M. Septic Shock, The Lancet, 2005, 365, 63).

Figure 2. LPS-induced intracellular signaling cascade. Following its binding to CD14, LPS interacts with MD2 / TLR4 signaling complex. Two main pathways are activated either MyD88-dependent leading to NF-κ B activation and IL-1 and TNF production, or TRAM/TICAM-2 and TRIF/TICAM-1-dependent leading to the activation IRF3 and interferon-β (IFNβ ) production. Other down-stream pathways lead to the activation of Sp1, a prerequisite for IL-10 production, while pI-3 kinase is involved in IL-1ra induction. Numerous intracellular molecules (MyD88s, Tollip, IRAK-M, SOCS-1, pI-3-kinase) and cell surface molecules (ST2, SIGIRR) down-regulate the activating cascade. During sepsis, leukocyte reprogramming is associated with some altered (grey background), or enhanced (yellow background) cellular signaling pathways that lead to the modified production of TNF, IL-1β  IL-1ra and IL-10 in response to TLR4 ligands. This cartoon illustrates our published works (Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2000, 162, 1877, Adib-Conquy et al J. Leuk. Biol. 2001; 70:30; Adib-Conquy et al. Am. J. Respir. Crit Care Med. 2003, 168, 158), unpublished work and other reports (Learn et al. J. Biol. Chem. 2001; 276:20234 ; Solomon et al. J. Clin. Invest. 1998; 102:2019). (adapted from Annane D., Bellissant E., Cavaillon J-M. Septic Shock, The Lancet, 2005, 365, 63).

Keywords: Toll-like receptor, sepsis, intracellular signaling, endotoxin, innate immunity


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