Olivier Vosters

Induction of FOXP3-expressing regulatory CD4pos T cells by human mature autologous dendritic cells

Current literature suggests that T cells recognizing antigen on mature dendritic cells (DC) differentiate into effector T cells whereas tolerance is induced when antigen is presented by immature DC. We investigated the consequences of the interactions between immature or lipopolysaccharide‐matured DC and CD4pos T lymphocytes in absence of foreign antigen. While immature DC did not induce significant CD4pos T cell activation, we observed that a significant fraction of CD4pos T cells cultured with mature autologous DC displayed phenotypic features of activation and produced IL‐2, IFN‐γ, IL‐10 and TGF‐β. Furthermore, CD4pos T lymphocytes primed by mature, but not immature, autologous DC acquired regulatory properties. Indeed, when added to an allogeneic mixed leukocyte reaction, they suppressed the response of alloreactive T lymphocytes to the priming DC while responses to third‐party stimulators were spared. The generation of CD4pos T cells with regulatory function by autologous stimulation did not require the presence of natural CD4posCD25pos regulatory T cells. In addition, the acquisition ofregulatory function by CD4posCD25neg T cells stimulated by autologous mature DC was accompanied by the induction of FOXP3 expression. Our data suggest that during inflammatory conditions, presentation of self antigens by mature DC to autologous T lymphocytes could contribute to the generation of regulatory mechanisms.

ChemR23 dampens lung inflammation and enhances anti-viral immunity in a mouse model of acute viral pneumonia.

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23−/− mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23−/− mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.

Dendritic cells exposed to nacystelyn are refractory to maturation and promote the emergence of alloreactive regulatory T cells

Background. Drugs blocking dendritic cell (DC) maturation might be useful in transplantation by inhibiting the induction of primary alloimmune responses and promoting the emergence of regulatory T lymphocytes (Treg). We investigated the effects of Nacystelyn (NAL), an N-acetyl-L-cysteine derivative, on human DCs, paying attention to the T-cell responses elicited by NAL-treated DCs in vitro. Methods. Lipopolysaccharide (LPS) was used to induce the maturation of DCs naturally present in blood or generated from human monocytes cultured in interleukin-4 and granulocyte-macrophage colony-stimulating activity. We first analyzed the consequences of NAL on cytokine production and expression of major histocompatibility complex class II and costimulatory molecules. Monocyte-derived DCs were then used as stimulators in mixed leukocyte cultures with naive CD4+ T cells. Cytokine levels were measured in culture supernatants; the phenotype of T cells and their capacity to inhibit the proliferation of third-party T-cell responders was determined at the end of the culture. Results. NAL proved to be a potent inhibitor of DC maturation in whole blood experiments and on monocyte-derived DCs. Alloreactive T cells stimulated with DCs pretreated with LPS in the presence of NAL produced much less interferon-&ggr; but similar levels of interleukin-13 compared with DCs treated with LPS alone. Immature DCs induced Treg, which was not observed with mature DCs. DCs cultured with LPS in the presence of NAL were as efficient as immature DCs to generate alloreactive T cells with regulatory activity. Conclusions. NAL is a potent inhibitor of DC maturation, which might be useful to promote allograft acceptance by inducing the differentiation of allospecific Treg.

CD40 expression on human pancreatic duct cells: role in nuclear factor-kappa B activation and production of pro-inflammatory cytokines.

Aims/hypothesisHuman pancreatic duct cells are closely associated with islet beta cells, and contaminate islet suspensions transplanted in Type 1 diabetes mellitus patients. Activated duct cells produce cytotoxic mediators and possibly contribute to the pathogenesis of Type 1 diabetes mellitus or islet graft rejection. As CD40 transduces activation signals involved in inflammatory and immune disorders, we investigated CD40 expression on duct cells and their response to CD40 engagement.MethodsCD40 expression on human pancreatic duct cells was analysed by flow cytometry and immunohistochemical analyses. To assess the function of CD40 expression on duct cells, activation of the transcription factor nuclear factor-kappa B was determined using electrophoretic mobility shift assay and ELISA. Cytokine mRNA levels were quantified by real-time RT-PCR, and protein levels by Luminex technology.ResultsIsolated human pancreatic duct cells and Capan-2 cell lines were found to express constitutively CD40. The expression of CD40 on duct cells was confirmed in vivo on human normal and pathological pancreatic specimens. CD40 ligation on Capan-2 cells induced rapid nuclear factor-kappa B activation, and supershift assays demonstrated that p50/p65 heterodimers and p50/p50 homodimers were present in the activated complexes in the nucleus. This activation was accompanied by tumour necrosis factor-α and interleukin-1β mRNA accumulation. Tumour necrosis factor-α protein secretion was confirmed in CD40-activated Capan-2 cells and in isolated human pancreatic duct cells.Conclusions/interpretationInteraction between activated T lymphocytes expressing CD40 ligand and duct cells expressing CD40 may contribute to the immune responses involved in Type 1 diabetes mellitus and islet graft rejection. Interfering with CD40-mediated duct cell activation could alleviate beta cell damage of immune origin.

N-Acetylcysteine derivative inhibits procoagulant activity of human islet cells

Aims/hypothesisThe early loss of beta cells after islet cell transplantation has been attributed in part to blood coagulation at the implant site. Tissue factor expressed by beta cells and contaminating duct cells is considered to activate this process. Here, we investigated the ability of N-acetyl-l-cysteine to suppress the in vitro procoagulant activity of duct cells and human islet cell preparations.Materials and methodsThe effects of Nacystelyn, a salt derivative of N-acetyl-l-cysteine, were first assessed on procoagulant activity induced in human plasma by recombinant tissue factor, human primary duct cells or human islet cell preparations. The influence of Nacystelyn on clot formation, platelet counts and d-dimers were measured in a whole blood tubing loop model. Human beta cell viability and insulin synthesis after Nacystelyn treatment were assessed to exclude cytotoxicity of Nacystelyn.ResultsNacystelyn efficiently inhibited the procoagulant activity of human recombinant tissue factor, primary duct cells and human islet cell preparations at clinically relevant concentrations without cellular toxicity.Conclusions/interpretationNacystelyn is a pharmaceutical candidate to reduce early beta cell loss related to tissue factor-dependent coagulation after islet transplantation.

Adult human hepatocytes promote CD4(+) T-cell hyporesponsiveness via interleukin-10-producing allogeneic dendritic cells.

The success of liver cell therapy remains closely dependent on how well the infused cells can be accepted after transplantation and is directly related to their degree of immunogenicity. In this study, we investigated the in vitro immunogenic properties of isolated human hepatocytes (hHeps) and adult-derived human liver progenitor cells (ADHLPCs), an alternative cell candidate for liver cell transplantation (LCT). The constitutive expression of immune markers was first analyzed on these liver-derived cells by flow cytometry. Human liver-derived cells were then cocultured with allogeneic human adult peripheral blood mononuclear cells (PBMCs), and the resulting activation and proliferation of PBMCs was evaluated, as well as the cytokine levels in the coculture supernatant. The effect of liver-derived cells on monocyte-derived dendritic cell (MoDC) properties was further analyzed in a secondary coculture with naive CD4+ T-cells. We report that hHeps and ADHLPCs expressed human leukocyte antigen (HLA) class I and Fas but did not express HLA-DR, Fas ligand, and costimulatory molecules. hHeps and ADHLPCs did not induce T-cell activation or proliferation. Moreover, hHeps induced a cell contact-dependent production of interleukin (IL)-10 that was not observed with ADHLPCs. The IL-10 was produced by a myeloid DC subset characterized by an incomplete mature state. Furthermore, hHep-primed MoDCs induced an antigen-independent hyporesponsiveness of naive CD4+ T lymphocytes that was partially reversed by blocking IL-10, whereas nonprimed MoDCs (i.e., those cultured alone) did not. hHeps and ADHLPCs present a low immunogenic phenotype in vitro. Allogeneic hHeps, but not ADHLPCs, promote a cell contact-dependent production of IL-10 by myeloid DCs, which induces naive CD4+ T-cells antigen-independent hyporesponsiveness.

The Chemerin/ChemR23 System Does Not Affect the Pro-Inflammatory Response of Mouse and Human Macrophages Ex Vivo

Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23−/− mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1β and IL-6) and anti-inflammatory (IL-10) cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells.

PET/CT with 18F-FDG– and 18F-FBEM–Labeled Leukocytes for Metabolic Activity and Leukocyte Recruitment Monitoring in a Mouse Model of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. Methods: C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with 18F-FDG– or 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM)–labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. Results: In bleomycin-treated mice, a higher metabolic activity was measured on 18F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by 18F-FBEM–labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. Conclusion: 18F-FDG– and 18F-FBEM–labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected.

Clinical Parameters vs Cytokine Profiles as Predictive Markers of IgE-Mediated Allergy in Young Children.

Background Allergy afflicts one third of children, negatively impacting their quality of life and generating a significant socio-economic burden. To this day, this disorder remains difficult to diagnose early in young patients, with no predictive test available. Objective This study was designed to correlate cytokine profiles with clinical phenotypes of allergy development. Methods Three hundred patients were recruited and followed from birth to 18 months of age. They were given a clinical exam at birth and at 2, 6, 12, and 18 months of age, with skin prick tests at 6, and 18 months, in order to have a record of their medical history and determine their allergic status. In addition, mononuclear cells from 131 patients were isolated from cord blood and from peripheral blood samples at 2, 6 and 18 months of age, to analyse their cytokine and chemokine production. Results Cord blood mononuclear cells (CBMCs) from future Immunoglobulin (Ig) E-mediated allergic children produced significantly less Interleukin (IL)-12p70 and IL-15 than cells from the rest of the cohort. Multivariate analyses revealed that the best predictive model of allergy was built on cytokine data, whereas the best predictive model of IgE-mediated allergy was built on clinical parameters. Conclusions and clinical relevance Although univariate analyses can yield interesting information regarding the immune responses of allergic children, finding predictive markers of the disorder will likely rely on monitoring multiple parameters. Nonetheless these analyses suggest a potential key role for IL-15 in the development of atopic disease. In addition, the study highlights the importance of clinical parameters in predicting the development of IgE-mediated allergy.

N-acetylcysteine derivative inhibits CD40-dependent proinflammatory properties of human pancreatic duct cells.

Objectives: We recently observed that duct cells constitutively express CD40, a membrane molecule whose engagement results in duct cell activation and proinflammatory cytokine secretion. This observation suggests a potential role of this pathway in the pathogenesis of type 1 diabetes, islet graft rejection, or acute pancreatitis. In this article, we investigated whether a salt derivative of N-acetyl-l-cysteine, Nacystelyn, could modulate CD40 expression on duct cells and the response of activated duct cells to CD40 engagement. Methods: We assessed the effects of Nacystelyn on CD40 expression and function in human caucasian pancreatic adenocarcinoma, ATCC n° THB-80 (CAPAN-2) cells, a human pancreatic duct cell line. CD40 expression was analyzed by flow cytometry. To assess CAPAN-2 cell responses to CD40 engagement, we looked at nuclear factor-&kgr;B transcription factor activation using enzyme-linked immunosorbent assay and electrophoretic mobility shift assay and cytokine mRNA levels by quantitative real-time reverse transcriptase polymerase chain reaction. Results: We observed that Nacystelyn dose-dependently inhibited CD40 expression on CAPAN-2 cells as well as CD40-induced nuclear factor &kgr;B activation and proinflammatory cytokines up-regulation. Conclusions: Our data suggest that Nacystelyn could be considered as a useful tool to prevent immune and inflammatory responses in pancreatic disorders by interfering with the CD40 pathway in pancreatic duct cells.Abbreviations: NAL - Nacystelyn, NF-&kgr;B - nuclear factor &kgr;B, CD40L - CD40 ligand, EMSA - electrophoretic mobility shift assay, mAb - monoclonal antibody