Fabrice Bureau

Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice

The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC-driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC-driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10-dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions.

Interleukin-6/STAT3 signaling regulates the ability of naive T cells to acquire B-cell help capacities.

The conditions leading to the activation/differentiation of T-helper (Th) cells dedicated for B-cell antibody production are still poorly characterized. We now demonstrate that interleukin-6 (IL-6) promotes the differentiation of naive T lymphocytes into helper cells able to promote B-cell activation and antibody secretion. IL-6-driven acquisition of B-cell help capacity requires expression of the signal transducer and activator of transcription 3 (STAT3), but not STAT4 or STAT6 transcription factors, suggesting that the ability to provide help to B cells is not restricted to a well-defined Th1 or Th2 effector population. T cell-specific STAT3-deficient mice displayed reduced humoral responses in vivo that could not be related to an altered expansion of CXCR5-expressing helper T cells. IL-6 was shown to promote IL-21 secretion, a cytokine that was similarly found to promote the differentiation of naive T cells into potent B-cell helper cells. Collectively, these data indicate that the ability to provide B-cell help is regulated by IL-6/IL-21 through STAT3 activation, independently of Th1, Th2, Th17, or follicular helper T cell (T(FH)) differentiation.

Selective Blockade of NF-κB Activity in Airway Immune Cells Inhibits the Effector Phase of Experimental Asthma

Knockout mice studies have revealed that NF-κB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-κB also plays a role in the effector phase of airway allergy and whether inhibiting NF-κB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-κB activity in the lungs during OVA challenge. Intratracheal administration of NF-κB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-κB activity in the airways upon OVA provocation. NF-κB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-κB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-κB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.

Nicotinamide Phosphoribosyl Transferase/Pre-B Cell Colony-Enhancing Factor/Visfatin Is Required for Lymphocyte Development and Cellular Resistance to Genotoxic Stress

Nicotinamide phosphoribosyl transferase (Nampt)/pre-B cell colony-enhancing factor (PBEF)/visfatin is a protein displaying multiple functional properties. Originally described as a cytokine-like protein able to regulate B cell development, apoptosis, and glucose metabolism, this protein also plays an important role in NAD biosynthesis. To gain insight into its physiological role, we have generated a mouse strain expressing a conditional Nampt allele. Lack of Nampt expression strongly affects development of both T and B lymphocytes. Analysis of hemizygous cells and in vitro cell lines expressing distinct levels of Nampt illustrates the critical role of this protein in regulating intracellular NAD levels. Consequently, a clear relationship was found between intracellular Nampt levels and cell death in response to the genotoxic agent MNNG (N-methyl-N′-nitro-N-nitrosoguanidine), confirming that this enzyme represents a key regulator of cell sensitivity to NAD-consuming stress secondary to poly(ADP-ribose) polymerases overactivation. By using mutant forms of this protein and a well-characterized pharmacological inhibitor (FK866), we unequivocally demonstrate that the ability of the Nampt to regulate cell viability during genotoxic stress requires its enzymatic activity. Collectively, these data demonstrate that Nampt participates in cellular resistance to genotoxic/oxidative stress, and it may confer to cells of the immune system the ability to survive during stressful situations such as inflammation.

Mechanisms of Persistent Nf-Kappa B Activity in the Bronchi of an Animal Model of Asthma

In most cells trans-activating NF-κB induces many inflammatory proteins as well as its own inhibitor, IκB-α, thus assuring a transient response upon stimulation. However, NF-κB-dependent inflammatory gene expression is persistent in asthmatic bronchi, even after allergen eviction. In the present report we used bronchial brushing samples (BBSs) from heaves-affected horses (a spontaneous model of asthma) to elucidate the mechanisms by which NF-κB activity is maintained in asthmatic airways. NF-κB activity was high in granulocytic and nongranulocytic BBS cells. However, NF-κB activity highly correlated to granulocyte percentage and was only abrogated after granulocytic death in cultured BBSs. Before granulocytic death, NF-κB activity was suppressed by simultaneous addition of neutralizing anti-IL-1β and anti-TNF-α Abs to the medium of cultured BBSs. Surprisingly, IκB-β, whose expression is not regulated by NF-κB, unlike IκB-α, was the most prominent NF-κB inhibitor found in BBSs. The amounts of IκB-β were low in BBSs obtained from diseased horses, but drastically increased after addition of the neutralizing anti-IL-1β and anti-TNF-α Abs. These results indicate that sustained NF-κB activation in asthmatic bronchi is driven by granulocytes and is mediated by IL-1β and TNF-α. Moreover, an imbalance between high levels of IL-1β- and TNF-α-mediated IκB-β degradation and low levels of IκB-β synthesis is likely to be the mechanism preventing NF-κB deactivation in asthmatic airways before granulocytic death.

Prostaglandin D2 Affects the Maturation of Human Monocyte-Derived Dendritic Cells: Consequence on the Polarization of Naive Th Cells

Among the factors produced at inflammatory sites and those capable of modulating dendritic cell (DC) functions, PGD2 may be important in the outcome of immune responses. The biological roles for PGD2 are in part effected through two plasma membrane G protein-coupled receptors: the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes (CRTH2). In this report, we studied the effects of PGD2 and of its major physiological metabolite, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), on the functions of human monocyte-derived DC. First, we show that PGD2 exerts in vitro chemotactic effects on monocytes via CRTH2 activation while it inhibits the chemokine-driven migration of monocyte-derived DC through DP. We also report that PGD2 and 15d-PGJ2 alter the LPS- and allergen-induced DC maturation and enhance the CD80/CD86 ratio on mature DC in a DP- and CRTH2-independent manner. Moreover, PGD2 and 15d-PGJ2 strongly reduce the secretion of the Th1 promoting cytokine IL-12 and affect the synthesis of chemokines involved in Th1 cell chemotaxis, particularly CXCL10. Inhibition of cytokine/chemokine secretion implicates at least in part DP, but not CRTH2. The effects exerted by PGD2 are associated with the phosphorylation of CREB, but do not parallel with the deactivation of the NF-κB and mitogen-activated protein kinase pathways. In contrast, 15d-PGJ2 seems to target other cellular proteins. Finally, in a model of Th CD45RA+ differentiation induced by allergen- and superantigen-pulsed DC, PGD2 impacts on the orientation of the immune response by favoring a Th2 response.

Ir-LBP, an Ixodes ricinus Tick Salivary LTB4-Binding Lipocalin, Interferes with Host Neutrophil Function

Background During their blood meal, ticks secrete a wide variety of proteins that can interfere with their hosts defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. Methodology/Principal Findings We previously identified 14 new lipocalin genes in the tick Ixodes ricinus. One of them codes for a protein that specifically binds leukotriene B4 with a very high affinity (Kd: ±1 nM), similar to that of the neutrophil transmembrane receptor BLT1. By in silico approaches, we modeled the 3D structure of the protein and the binding of LTB4 into the ligand pocket. This protein, called Ir-LBP, inhibits neutrophil chemotaxis in vitro and delays LTB4-induced apoptosis. Ir-LBP also inhibits the host inflammatory response in vivo by decreasing the number and activation of neutrophils located at the tick bite site. Thus, Ir-LBP participates in the ticks ability to interfere with proper neutrophil function in inflammation. Conclusions/Significance These elements suggest that Ir-LBP is a “scavenger” of LTB4, which, in combination with other factors, such as histamine-binding proteins or proteins inhibiting the classical or alternative complement pathways, permits the tick to properly manage its blood meal. Moreover, with regard to its properties, Ir-LBP could possibly be used as a therapeutic tool for illnesses associated with an increased LTB4 production.

NF-κB activation in response to toxical and therapeutical agents: role in inflammation and cancer treatment

The NF-kappaB transcription factor is ubiquitously expressed and controls the expression of a large number of genes. Experimental data clearly indicate that NF-kappaB is a major regulator of the inflammatory reaction by controlling the expression of pro-inflammatory molecules in response to cytokines, oxidative stress and infectious agents. We demonstrated that NF-kappaB activation by IL-1beta follows three distinct cell-specific pathways. Moreover, our studies indicated that in one model of inflammatory diseases, horse recurrent airway obstruction (RAO), the extent of NF-kappaB basal activity correlates with pulmonary dysfunction. Another role of NF-kappaB activity protects cancer cells against apoptosis and could participate in the resistance to cancer treatment. However, we did not observe any increased cytotoxicity after treatment with anticancer drugs or TNF-alpha of cells expressing a NF-kappaB inhibitor. Therefore, we can conclude that the inhibition of apoptosis by NF-kappaB is likely to be cell type and stimulus-dependent. Further studies are required to determine whether NF-kappaB could be a target for anticancer treatments.

Dendritic Cells Genetically Engineered to Express IL-10 Induce Long-Lasting Antigen-Specific Tolerance in Experimental Asthma

Dendritic cells (DCs) are professional APCs that have a unique capacity to initiate primary immune responses, including tolerogenic responses. We have genetically engineered bone marrow-derived DCs to express the immunosuppressive cytokine IL-10 and tested the ability of these cells to control experimental asthma. A single intratracheal injection of OVA-pulsed IL-10-transduced DCs (OVA-IL-10-DCs) to naive mice before OVA sensitization and challenge prevented all of the cardinal features of airway allergy, namely, eosinophilic airway inflammation, airway hyperreactivity, and production of mucus, Ag-specific Igs, and IL-4. OVA-IL-10-DCs also reversed established experimental asthma and had long-lasting and Ag-specific effects. We furthermore showed, by using IL-10-deficient mice, that host IL-10 is required for mediating the immunomodulatory effects of OVA-IL-10-DCs and demonstrated a significant increase in the percentage of OVA-specific CD4+CD25+Foxp3+IL-10+ regulatory T cells in the mediastinal lymph nodes of OVA-IL-10-DC-injected mice. Finally, adoptive transfer of CD4+ mediastinal lymph node T cells from mice injected with OVA-IL-10-DCs protected OVA-sensitized recipients from airway eosinophilia upon OVA provocation. Our study describes a promising strategy to induce long-lasting Ag-specific tolerance in airway allergy.

Prostaglandin D2 inhibits the production of interleukin‐12 in murine dendritic cells through multiple signaling pathways

Prostaglandin (PG) D2, and its metabolites, are known to be important mediators during acute and chronic inflammation. However, their functions during the early phases of the immune response are poorly documented. In the present study, we show that PGD2 inhibits, in a dose‐dependent manner, the CD40‐ and LPS‐induced secretion of the Th1‐driving factor IL‐12 by murine splenic dendritic cells (DC), the most potent antigen‐presenting cells. The inhibition of IL‐12 production is mediated only in part by the cell surface Gαs protein‐coupled D prostanoid receptor (termed DP1) but not by the Gαi protein‐coupled DP receptor, DP2. We show that recruitment of DP1 in DC results in the activation of a cyclic AMP/protein kinase A pathway that is partially responsible for the inhibition of IL‐12 production. We also suggest that the DP1‐independent effects exerted by PGD2 on IL‐12 production may be due to the action of ist PGJ2, but not PGF2α, metabolites. Electrophoretic mobility shift assays demonstrated that PGD2 affects NF‐κB activation through (the) DP1‐independent pathway(s). Together these data suggest that PGD2, by interacting with DP1 and by binding to other target cellular proteins, may regulate immune responses by affecting IL‐12 production in DC.