Cytokines and inflammation - GDR CNRS 3048  

  HEADProf. Jean-Marc CAVAILLON /
  MEMBERSDr. Minou ADIB-CONQUY / Dr. Noëlle DOYEN / Catherine FITTING / Dr. Oumaïma IBRAHIM-GRANET / Ronan KAPETANOVIC / Dr. Oh Yoen KIM / Dr. Tomohiro MATSUI / Véronique MÉRIAUX / Marianna PARLATO / Dr François PHILIPPART / Nora YAHIA

  Annual Report

Interaction host cells - microorganisms

Sensing and signalling in response to Staphylococcus aureus in monocytes and macrophages

We analyzed the contribution of Toll-like receptor 2 (TLR2) and phagocytosis for tumor-necrosis factor (TNF) production by murine mononuclear phagocytes in response to heat-killed Staphylococcus aureus. Mononuclear phagocytes behaved differently depending upon their compartment of residence. When bacterial uptake or phagosome maturation was blocked, activation through TLR2 was sufficient for a maximal production of TNF by peritoneal macrophages. In contrast, monocytes and alveolar macrophages required simultaneous stimulation through both TLR2 and phagocytosis for optimal cytokine production. Blocking the α5β1 integrin on all three cell-types significantly decreased TNF production in response to S. aureus. Furthermore, we found that the G-protein Rac1 and the MAPK ERK played a role in macrophage-signaling pathways but not in monocytes. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to S. aureus are specific for each mononuclear phagocyte population and involve different recognition processes and signaling pathways.

Contribution of NOD2 in the sensing of S. aureus in vitro and in vivo

Using mononuclear phagocytes from NOD2-/- mice together with anti-TLR2 blocking antibodies, we observed that TNF production in response to S. aureus following phagocytosis was independent of NOD2 in monocytes and macrophages. We also evaluated the role of NOD2 in vivoin a mouse model of S. aureusinduced pneumonia. The mortality was low and no difference was observed between wild-type and NOD2 -/- animals. However, the lost of weight was significantly higher for WT animals and more severe lesions were found in their lungs at day 7 post-infection. Thus, although NOD2 is dispensable for S. aureussensing in macrophages, it contributes to lung lesions induced by S. aureusinfection. (In collaboration with Gregory Jouvion, Histotechnologie et Pathologie, Inst. Pasteur).

Biofilm-forming Pseudomonas aeruginosa bacteria undergo LPS structural modifications and induce enhanced TNF production in human monocytes

Pseudomonas aeruginosa and Staphylococcus aureus are the two most common bacteria colonizing cystic fibrosis (CF) patients’ airways. In lungs, these bacteria can form a biofilm and express characteristic phenotypes. We compared the cytokine induction in human monocytes by planktonic and biofilm P. aeruginosaand S. aureus. Planktonic and biofilm S. aureus induced equivalent amounts of cytokine in human monocytes. In contrast, biofilm-forming P. aeruginosa isolates induced higher production of TNF and IL-6 than their planktonic counterpart. Thecell wall of Gram-negative bacteria contains lipopolysaccharide (LPS). Switch between the two life styles of P. aeruginosawas shown to cause several reversible LPS structure modifications affecting both lipid A and polysaccharide moieties. Modifications in secondary fatty acids hydroxylation at C2 and C2’ positions in lipid A were observed for both clinical isolates and laboratory strains. In addition, LPS isolated from biofilm-grown bacteria induced more inflammatory cytokines than that isolated from its planktonic counterpart. Our results therefore show that P. aeruginosabiofilm LPS undergoes structural modifications contributing to an increased inflammatory response from human mononuclear phagocytes. (In collaboration with Jean-Marc Ghigo, Unit Génétique des Biofilms, Inst. Pasteur and Martine Caroff, IBBM, Orsay)

Aspergillus fumigatus infection

Aspergillus fumigatuscauses invasive aspergillosis and is a serious problem for patients suffering from organ transplantation, leukemia, AIDS, and other diseases. The lethality is up to 90%, diagnosis is difficult and only a few antifungals are available. To bring light into the infection process, we constructed a bioluminescent A. fumigatusstrain, which allows in vivo monitoring of the infection process. Our studies showed that mice, immunosuppressed with corticosteroids, emit light from their lungs even 20 hours post infection, indicating a rapid outgrowth of the fungus. Therefore, early diagnosis of fungal infections is of tremendous importance. This strain was useful to study the recruitment kinetics of the innate immune cells following different immunosuppressive regimens. We also used this strain to study the effectiveness of antifungals in vitro and will apply this to in vivo systems, because only alive fungi emit light and biomass formation correlates well with light intensities. Due to highly reproducible results from small animal groups, this tool may save hundreds of animals in drug efficiency testings.

TLR9-dependent activation of dendritic cells by DNA from Leishmania major favors Th1 cell development and the resolution of lesions

In its vertebrate host, Leishmaniaencounter cells that express TLRs. Using genetically resistant C57BL/6 mice deficient in either TLR2, 4 or 9 we show here that only TLR9-deficient mice are more susceptible to infection with L. major. TLR9-deficient 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. Weshowed that dendritic cells DCs in the draining lymph nodes are activated following infection with L. major. Furthermorebone 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. majorin vitro. In sharp contrast L. majorfailed to activate DCs from TLR9-/-mice. Noteworthy, activation of DCs was abolished either following treatment of the parasites with DNase or after acidification of the endosomal compartment of DCs by chloroquine, pinpointing the DNA of L. majoras the possible ligand of TLR9 leading to the activation of DCs. Results showed that DNA purified from L. majorwas indeed capable of activating DCs in a strictly TLR9-dependent manner. Moreover we showed that the L. majorDNA-induced TLR9 signaling in DCs condition these cells to promote IFN-γproduction by CD4+ T cells. (Hkima Abou Fakher et al. J. Immunol. 2009,182 (3) in press)

Endotoxin- and TLR-tolerance

The TLR-tolerance phenomenon is a complex orchestrated counter regulatory response to inflammation. We studied the cross-talk between different TLR agonists to induce tolerance and compare the phenomenon with different mononuclear phagocytes. In contrast to other mouse mononuclear phagocytes, alveolar macrophages neither display endotoxin tolerance, nor cross-tolerance. Indeed, in the later case the cells are primed rather than tolerized. We currently study the relative contribution of GM-CSF and gamma-interferon to prevent endotoxin tolerance in the alveolar environment.

Paradoxical effects of IL-10

IL-10 is a well-known anti-inflammatory cytokine that prevents the production of pro-inflammatory cytokine. Inin vitro experiments, we previously showed that the adherence of monocytes is a key parameter that allows the observation of the well-characterized property of IL-10 (Adib-Conquy et al. Int Immunol. 1999; 11: 689). Surprisingly, in the absence of adherence, IL-10 can enhance LPS-induced TNF production and turn-off genes that are otherwise turned-on (e.g. SOCS 2 & 3), and vice versa. Adherence modifies the regulation of gene expression induced by interleukin-10. (Petit-Bertron et al. Cytokine. 2005; 29: 1-12). We have now developed a mouse model that allows us to study in vivothis paradoxical property of IL-10.

The high resitance of mice to endotoxin renders them poorly suitable to mimick human sepsis.

We have undertaken studies to understand why mice are so resistant to endotoxin in comparison with humans (10,000 fold). We have shown that the induction of pro-inflammatory cytokines, such as tumor necrosis factor (TNF), from macrophages in response to inflammatory stimuli is down regulated by proteins present in mouse serum. This finding suggests that the mechanism for the difference in species sensitivity to pathogen associated molecular patterns (PAMPs) may be due to differences in a serum protein or proteins rather than intrinsic cellular differences between species. Localization of the mechanism to circulating compounds in blood raises hope that these proteins can be manipulated to create better animal models and potentially new drug targets. (In collaboration with Dr. Shaw Warren, MGH, Boston, USA)

Translational research

Markers of infection

Many inflammatory markers are found in plasma of sepsis and SIRS patients (Adib-Conquy & Cavaillon J-M. FEBS Lett. 2007, 581: 3723). Some of them are claimed to be specific markers of infection (e.g. procalcitonin, soluble TREM-1). However, we showedthat their plasma levels could also be very high in the absence of infection, such as in patients who died after after cardiac arrest and resuscitation (Adib-Conquy et al. Shock 2007, 28: 406).

Alteration of immune status of monocytes in sepsis and SIRS patients

We study the alteration of the immune status of patients with sepsis and systemic inflammatory response syndrome (SIRS; e.g. resuscitated patients after cardiac arrest; major trauma; vascular surgery) (for a review see: Adib-Conquy M, Cavaillon JM. Compensatory anti-inflammatory response syndrome. Thromb Haemost. 2009; 101: 36-47).In collaboration with Dr. Rudi Beyaert (Ghent University) we studied the involvement of ABIN-3, an intracellular negative regulator of the TLR4-dependent pathway and NF-κB activation (Wullaert et al. J Biol Chem. 2007; 282: 81), in the hyporeactivity of septic patients monocytes. A significantly enhanced expression of ABIN-3 was found in monocytes of sepsis patients, which was restored to control levels by corticotherapy (Verstrepen et al.J Cell Mol Med., 2008, 12: 316-29).

In collaboration with Dr Christophe Adrie, (Intensive Care Unit, Delafontaine hospital, Saint-Denis) we investigated the inflammatory status, apoptosis and endotoxinemia in brain dead patients. The presence of inflammation and apoptosis induction may explain the rapid organ dysfunction seen after brain death. Both abnormalities may play a role in organ dysfunction associated with brain death. However, the level of systemic inflammation, or the presence of circulating endotoxin was not associated with lower graft survival.

Cystic Fibrosis and neutrophils

We have studied blood and sputum neutrophils of cystic fibrosis (CF) patients. We have shown by macroarray technology that numerous genes are turned-on even before bacterial colonization of the lungs. When comparison was performed between blood and airway neutrophils of CF patients, there was a limited difference in terms of gene expression. Only the mRNA expression of amphiregulin and TNF receptor p55 were significantly higher in airway neutrophils. We also showed that circulating neutrophils from CF patients display a distinct pattern of surface markers, including TLRs, as compared to neutrophils from healthy donors, and that airways neutrophils from CF patients are primed and resistant to anti-inflammatory signals delivered by IL-10. (Petit-Bertron et al. Cytokine 2008, 41, 54; Adib-Conquy et al. Mol Med. 2008, 14, 36).

Vascular surgery, inflammatory response and bacterial translocation

Abdominal aortic surgery (AAS) is thought to be associated with bacterial translocation following manipulation of the gut and aortic clamping. This translocation from the gut may amplify the inflammatory response and alter the immune status. To investigate translocation in AAS patients we developed a new tool to detect circulating peptidoglycanusing a NOD2-transfected cell line (US Patent 2008 circulating leukocytes and in plasma. Patients undergoing carotid artery surgery (CAS) were included as a negative control group. Our tool efficiently detected peptidoglycan from aerobic/anaerobic Gram-positive and Gram-negative bacteria. In 90.5% of the AAS patients, a peptidoglycan peak was detected in plasma before aortic clamping and persisted after blood reperfusion. As expected, no peak was detected in plasma from CAS patients. Regarding leukocyte-bound endotoxin levels, a peak appeared after blood reperfusion in AAS patients, but not in CAS patients (P=0.006). Regarding cytokines and inflammatory markers, levelsof interleukin (IL)-6, IL-10, C-reactive protein, and procalcitonin were maximal at postoperative day 1 in both groups, but the levels were much higher in the AAS group. The levels of circulating peptidoglycan after reperfusion positively correlated with those of CRP on day 1 post-AAS (P<0.001). The measurement of circulating peptidoglycan gives a highly sensitive tool for early and sensitive detection of bacterial translocation. Circulating peptidoglycan may contribute to further enhance the post-surgery inflammatory process.

(In collaboration with Prof. P. Coriat, Department of Anesthesiology and Critical Care,Hôpital Pitié-Salpétrière).

Keywords: Acute inflammation, Cytokines, Endotoxin, Innate immunity, Infection, Macrophages, Pathogen associated molecular patterns (PAMPs), Sepsis, Toll-like receptors


From bacteria to infectious diseases. Interaction of microbial derived molecules with host's cells leads to the production of cytokines, a prerequisite for innate immunity. Overwhelming production is associated with severe inflammation while the concomitant anti-inflammatory response may be associated with increased susceptibility to nosocomial infection (Annane D, Bellissant E, Cavaillon J-M (2005)Septic Shock. The Lancet 365: 63-78).


Maxime V, Fitting C, Annane D, Cavaillon J-M (2005) Corticoids normalize leukocyte production of macrophage migration inhibitory factor in septic shock. J. Infect. Dis. 191: 138-144

Fritz H.J., Girardin S.E., Fitting C., Werts C., Mengin-Lecreulx D., Caroff M., Cavaillon J-M., Philpott D.J., Adib-Conquy M. (2005) Synergistic stimulation of monocytes and dendritic cells by Toll-like receptor 4, Nod1- and Nod2-activating agonists. Eur. J. Immunol. 2005, 35, 2459-2470

Adib-Conquy M, Adrie C, Fitting C, Gattolliat O, Beyaert R, Cavaillon J-M (2006) Up-regulation of MyD88s and SIGIRR, molecules inhibiting Toll-like receptor signaling, in monocytes from septic patients. Crit Care Med. 34: 2377-2385

Kapetanovic R., Nahori M-A., Balloy V, Fitting C., Philpott D.J., Cavaillon J-M., Adib-Conquy M (2007) Contribution of phagocytosis and intracellular sensing for cytokine production by Staphylococcus aureus-activated macrophages. Infect. Immun. 75: 830-837

Brock M, Jouvion G, Droin-Bergère S, Dussurget O, Nicola MA, Ibrahim-Granet O. (2008) Bioluminescent Aspergillus fumigatus, a new tool for drug efficiency testing and in vivo monitoring of invasive aspergillosis. Appl. Environ. Microbiol. 74: 7023-35

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