Cytokines and inflammation - GDR CNRS 3048  

  HEADProf. Jean-Marc CAVAILLON /
  MEMBERSDr. Minou ADIB-CONQUY / Dr. Noëlle DOYEN / Catherine FITTING / Fernando GUIMARAES, Dr. Oumaïma IBRAHIM-GRANET / 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 aureusdiffer between peritoneal macrophages, alveolar macrophages and monocytes

Mononuclear phagocytes are among the first immune cells activated after pathogens invasion. Although they all derive from the same progenitor in the bone-marrow, their characteristics differ upon the compartment they are derived from. In this work, we investigated the contribution of phagocytosis for tumor-necrosis factor (TNF) production by murine mononuclear phagocytes (monocytes, peritoneal and alveolar macrophages) in response to heat-killed Staphylococcus aureus (HKSA). Mononuclear phagocytes behaved differently depending upon their compartment of residence. Indeed, when bacterial uptake or phagosome maturation was blocked, activation through membrane receptors, such asTLR2, was sufficient for a maximal production of TNF and IL-10 by peritoneal macrophages. In contrast, monocytes, and to a lesser extent alveolar macrophages, required phagocytosis for optimal cytokine production. While investigating on the different actors of cell signaling, we found that p38 and PI3K were playing an important role in HKSA phagocytosis and TNF production. Furthermore, blocking the α5β1 integrin on all three cell types significantly decreased TNF production in response toHKSA. Finally, using mononuclear phagocytes from TLR2 and/or NOD2 knockout mice, we observed a different role of these sensors in TNF production for each population. TNF production in response to HKSA was dependent on NOD2 for monocytes, on TLR2 for alveolar macrophages, and on both receptors for peritoneal macrophages. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to HKSA 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 andin vivo

Staphylococcus aureusis the most commonly found Gram-positive bacterium in patients admitted in intensive care units, causing septicaemia or pneumonia. In this work, we investigated the role of NOD2 in S. aureus-induced pneumonia. We found that the absence of NOD2 affected weight loss and recovery speed. Nod2-/- mice showed a reduced lung inflammation in comparison to wild-type animals, with lower presence of cytokines in broncho-alveolar lavage fluids and reduced recruitment of neutrophils. Furthermore, Nod2-/- alveolar macrophages displayed a deficient intracellular killing of S. aureus. Finally, histological analysis of the lungs revealed less severe lesions in Nod2-/- mice at day 2 and day 7 post-infection. In conclusion, we demonstrated that NOD2 is not a crucial receptor to fight S. aureus-induced pneumonia, but that it contributes to the inflammatory response in the lungs. Interestingly, the absence of NOD2 led to a lesser inflammation and was finally beneficial for the animal recovery. (In collaboration with Gregory Jouvion, Histotechnologie et Pathologie, Inst. Pasteur).

Biofilm-forming Pseudomonas aeruginosabacteria undergo LPS structuralmodifications and induce enhanced TNF production in human monocytes

Pseudomonas aeruginosaand 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 cytokines 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) – (Ciornei et al. Innate Immunity 2010, in press)

Aspergillus fumigatuspathophysiology

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 bioluminescentA. fumigatusstrain, which allows in vivomonitoring 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 found that experiments that perturb the number, recruitment, and function of neutrophils result in predictable patterns of invasive aspergillosis. Our study provides new insights into the innate immune response emphasizing an essential role for neutrophils as recruited phagocytes in the early innate response to A. fumigatus. The bioluminescent strain has been used to study the effectiveness of antifungals in vitroand in vivo. Our data shows the relationship between the effectiveness of antifungal and the site of infection.

We also initiated the study of the pathophysiology of Aspergillus terreusand Fusarium solani. Aspergillus terreusis an emerging human pathogen causing invasive aspergillosis in immunocompromised patients. Although A. terreus is less frequently isolated from patients with invasive aspergillosis than A. fumigatus, the resistance against the potent antifungal drug amphotericin B hampers treatment of A. terreus infections and leads to up to 100% mortality. Despite the severe outcome of A. terreus infections, pathogenesis has hardly been studied. To study the infection process, bioluminescent A. terreusstrains have been generated. Studies have shown that successful A. terreusinfections require extremely high conidia concentrations, implicating a reduced virulence in comparison to A. fumigatus. Further studies, including histopathologic analyses, will be needed to elucidate the reasons for the different aggressiveness of both strains during the infection process.

Another project focuses on the development of an infection model for Fusarium solani. Although being generally a plant pathogen, this fungus is frequently isolated as a saprophyte from soil and is responsible for two thirds of all cases of human invasive fusariosis. Therefore, a bioluminescent F. solanistrain has been constructed. We established a fusariosis animal model suitable to study the study the pathophysiology of this fungus. Unlike A. fumigatus, our results have shown that after immunosuppression, intranasal administration of the macroconidia of F. solaniresults in dissemination to different organs with 100 % mortality in one week. Our tools will been used to study the effectiveness of antifungalsin vitro and against these opportunistic filamentous fungi.

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

Our research focuses on the role of innate immune mechanisms in the development of a protective adaptive T cell response to Leishmania majorinfection, with a particular emphasis on theToll-like receptors (TLR).We previously demonstrated that TLR9-deficient mice are more susceptible to infection withL. major.TLR9-deficient mice resolve their lesions and control parasite growth less effectively than wild-type mice. Dendritic cells (DCs) are generally activated byL. major, but DCs from TLR9-deficient mice are not. ThusL. majorDNA activates DCs in a TLR9-dependent manner.Remarkably, L.majorDNA-induced activation of DCs appears specific for parasite DNA, as purified DNA from mice, sheep, pigs, orfishes is non-reactive(J. Immunol. 2009,182 (3) 1386-1396).Our main objective will be to establish the origin of the specific activation caused by L. majorDNA. We will investigate if it is mediated by either particular DNA sequence/structure or its tropism to the TLR9-rich endocytic compartment.Also, the role of TLR9 in the curative immune response will be investigated by studying local recruitment and activation of immune cells.

Paradoxical effects of interleunkin-10 (IL-10)

IL-10 is a well-known anti-inflammatory cytokine that prevents the production of pro-inflammatory cytokines. 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 expressed (e.g. SOCS 2 & 3), and vice versa. Adherence modifies the regulation of gene expression induced by IL-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.

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 (IFNg) to prevent endotoxin tolerance in the alveolar environment and the cellular source of IFNg.

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

A program seeking to expand our knowledge about the effects of various endogenous plasma proteins on the response of macrophages to microbial products has been undertaken in collaboration with Dr. Shaw Warren,at the Massachusetts General Hospital in Boston. With the recent triumph of the laboratory mouse as the major experimental animal model for infectious diseases in humans, the striking difference between human and murine sensitivity to the toxicity of endotoxin of Gram-negative bacteria (lipopolysaccharide, LPS) seems to have become a "dirty little secret" of innate immunology, as highlighted in the editorial that accompanied our publication (Munford. J. Infect. Dis. 2010, 201, 175). This statement was referring to the words coined by Charles Janeway, a founder of modern immunology, underlying the importance of innate immunity (the requirement of adjuvants) to acquire an appropriate adaptive immune response. The possibility that the results of experiments using LPS and other stimulatory microbial molecules in mice might not apply to humans has been infrequently acknowledged. We pointed out a key factor or factors present in serum that account for the striking difference in sensitivity to endotoxin infusion. These results also raise the interesting possibility that these factors might be identified and harnessed to dampen human responses to bacterial diseases(Warren et al. J. Infect. Dis. 2010, 210, 223).

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). As most probably a "magic marker" does not exist, a program has been set up to define the best combination of markers that would allow the early diagnosis of an infectious process among intensive care patients.

Alteration of immune status of monocytes in sepsis and SIRS patients

The concept of 'Compensatory anti-inflammatory response syndrome' (CARS) was proposed in 1997 by Roger Bone (1941–1997) to qualify the consequences of the counter-regulatory mechanisms initiated to limit the overzealous inflammatory process in patients with infectious (sepsis) or non-infectious systemic inflammatory response syndrome (SIRS). One major consequence of CARS is the modification of the immune status that could favor the enhanced susceptibility of intensive care patients to nosocomial infections.

In collaboration with Dr. Rudi Beyaert (Ghent University), we showed that ABIN-3expression, an intracellular negative regulator of the TLR4-dependent pathway and NF-κB activation (Wullaert et al. J Biol Chem. 2007; 282: 81), was enhanced in monocytes of sepsis patients, and 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 evaluated the role of the inflammatory status and apoptosis activation in the development of organ dysfunction after brain death (85 patients : cardiac arrest, stroke, head injury) using plasma assays and macroarray analysis on skeletal muscle biopsies to look for evidence of remote tissue damage. Plasma endotoxin and cytokine levels indicated a marked systemic inflammatory response in brain-dead patients, which was strongest in the cardiac arrest group. Leukocyte dysfunction, as assessed by cytokines production in response to various stimuli, was noted in a subgroup of patients with brain death after stroke. Interestingly, skeletal muscle biopsies showed no increase in mRNAs for genes related to inflammation, whereas mRNAs for both antiapoptotic and proapoptotic genes were increased, the balance being in favor of apoptosis induction. The increased activation of the proapoptotic caspase 9 was further confirmed by Western-blot. In conclusion, 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 (Adrie et al. Shock, 2010, in press).

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 was 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 #61/064,633). We also measured levels of endotoxin bound to circulating leukocytes and in plasma. Patients undergoing carotid artery surgery (CAS) were included as a negative control group. Our detection system 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. (Kim et al. Critical Care 2009, 13:R124).

Decreased expression of HLA-DR on monocytes is a hallmark of altered immune status in patients with a systemic inflammatory response syndrome (SIRS). So far, the analyses were performed without taking into account monocytes subpopulations. We studied this modification on CD14HIGHand CD14LOWmonocytes of 20 SIRS patients undergoing abdominal aortic surgery (AAS), 20 patients undergoing carotid artery surgery (CAS), and 9 healthy controls, and we investigated mediators and intracellular molecules that may be involved in this process. HLA-DR on CD14HIGHmonocytes started to decrease during surgery, after blood reperfusion, and was further reduced post-surgery. In contrast, HLA-DR expression on CD14LOWcells only decreased after surgery, and to a lesser extent than on CD14HIGHmonocytes. Negative correlations were found between the reduction of HLA-DR expression and the change in cortisol levels for both subpopulations, whereas a negative correlation between IL-10 levels and HLA-DR modulation was only observed for CD14HIGH cells. In accordance with these ex vivoresults, HLA-DR on CD14HIGHand CD14LOWmonocytes of healthy donors was reduced following incubation with hydrocortisone, whereas IL-10 only acted on CD14HIGHsubpopulation. Furthermore, flow cytometry revealed that the expression of IL-10 receptor was higher on CD14HIGHversus CD14LOWmonocytes. In addition, hydrocortisone, and to a lesser extent IL-10, reversed the up-regulation of HLA-DR induced by bacterial products. Finally, MARCH-1 mRNA, a negative regulator of MHC class II, was up-regulated in monocytes of AAS patients on day one post-surgery, and in those of healthy subjects exposed to hydrocortisone. This study reveals that HLA-DR expression is modulated differently on CD14HIGH(“classical”) versus CD14LOW(“inflammatory”) monocytes after systemic inflammation.(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, Nod-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).


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

Abou Fakher FH, Rachinel N, Klimczak M, Louis J, Doyen N. TLR9-dependent activation of dendritic cells by DNA from Leishmania major favors Th1 cell development and the resolution of lesions. J Immunol. 2009 Feb 1;182(3):1386-96.

Warren HS, Fitting C,Hoff E, Adib-Conquy M, Beasley-Topliffe L, Tesini B, Liang X, Valentine C, Hellman J, Hayden D, Cavaillon J-M. Resilience to bacterial infection: difference between species could be due to proteins in serum. J.Infect. Dis. 2010; 201:223–32

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