Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses.

The mechanisms regulating systemic and mucosal IgA responses in the respiratory tract are incompletely understood. Using virus-like particles loaded with single-stranded RNA as a ligand for TLR7, we found that systemic vs mucosal IgA responses in mice were differently regulated. Systemic IgA responses following s.c. immunization were T cell independent and did not require TACI or TGFbeta, whereas mucosal IgA production was dependent on Th cells, TACI, and TGFbeta. Strikingly, both responses required TLR7 signaling, but systemic IgA depended upon TLR7 signaling directly to B cells whereas mucosal IgA required TLR7 signaling to lung dendritic cells and alveolar macrophages. Our data show that IgA switching is controlled differently according to the cell type receiving TLR signals. This knowledge should facilitate the development of IgA-inducing vaccines.

Plasmacytoid dendritic cells: one-trick ponies or workhorses of the immune system?

Plasmacytoid dendritic cells (pDCs) were first described as interferon-producing cells and, for many years, their overlapping characteristics with both lymphocytes and classical dendritic cells (cDCs) created confusion over their exact ontogeny. In this Viewpoint article, Nature Reviews Immunology asks five leaders in the field to discuss their thoughts on the development and functions of pDCs--do these cells serve mainly as a major source of type I interferons or do they also make other important contributions to immune responses?

Immunology taught by lung dendritic cells.

Dendritic cells (DCs) are leukocytes specialised in the uptake, processing, and presentation of antigen and fundamental in regulating both innate and adaptive immune functions. They are mainly localised at the interface between body surfaces and the environment, continuously scrutinising incoming antigen for the potential threat it may represent to the organism. In the respiratory tract, DCs constitute a tightly enmeshed network, with the most prominent populations localised in the epithelium of the conducting airways and lung parenchyma. Their unique localisation enables them to continuously assess inhaled antigen, either inducing tolerance to inoffensive substances, or initiating immunity against a potentially harmful pathogen. This immunological homeostasis requires stringent control mechanisms to protect the vital and fragile gaseous exchange barrier from unrestrained and damaging inflammation, or an exaggerated immune response to an innocuous allergen, such as in allergic asthma. During DC activation, there is upregulation of co-stimulatory molecules and maturation markers, enabling DC to activate naïve T cells. This activation is accompanied by chemokine and cytokine release that not only serves to amplify innate immune response, but also determines the type of effector T cell population generated. An increasing body of recent literature provides evidence that different DC subpopulations, such as myeloid DC (mDC) and plasmacytoid DC (pDC) in the lungs occupy a key position at the crossroads between tolerance and immunity. This review aims to provide the clinician and researcher with a summary of the latest insights into DC-mediated pulmonary immune regulation and its relevance for developing novel therapeutic strategies for various disease conditions such as infection, asthma, COPD, and fibrotic lung disease.

Plasmacytoid Dendritic Cells Are Productively Infected and Activated through TLR-7 Early after Arenavirus Infection.

The antiviral response is largely mediated by dendritic cells (DCs), including conventional (c) DCs that function as antigen-presenting cells, and plasmacytoid (p) DCs that produce type I interferons, making them an attractive target for viruses. We find that the Old World arenaviruses lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) and Lassa virus bind pDCs to a greater extent than cDCs. Consistently, LCMV Cl13 targets pDCs early after in vivo infection of its natural murine host and establishes a productive and robust replication cycle. pDCs coproduce type I interferons and proinflammatory cytokines, with the former being induced in both infected and uninfected pDCs, demonstrating a dissociation from intrinsic virus replication. TLR7 globally mediates pDC responses, limits pDC viral load, and promotes rapid innate and adaptive immune cell activation. These early events likely help dictate the outcome of infections with arenaviruses and other DC-replicating viruses and shed light on potential therapeutic targets.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells.

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.

T Cell Priming by Activated Nlrc5-Deficient Dendritic Cells Is Unaffected despite Partially Reduced MHC Class I Levels.

NLRC5, a member of the NOD-like receptor (NLR) protein family, has recently been characterized as the master transcriptional regulator of MHCI molecules in lymphocytes, in which it is highly expressed. However, its role in activated dendritic cells (DCs), which are instrumental to initiate T cell responses, remained elusive. We show in this study that, following stimulation of DCs with inflammatory stimuli, not only did NLRC5 level increase, but also its importance in directing MHCI transcription. Despite markedly reduced mRNA and intracellular H2-K levels, we unexpectedly observed nearly normal H2-K surface display in Nlrc5(-/-) DCs. Importantly, this discrepancy between a strong intracellular and a mild surface defect in H2-K levels was observed also in DCs with H2-K transcription defects independent of Nlrc5. Hence, alongside with demonstrating the importance of NLRC5 in MHCI transcription in activated DCs, we uncover a general mechanism counteracting low MHCI surface expression. In agreement with the decreased amount of neosynthesized MHCI, Nlrc5(-/-) DCs exhibited a defective capacity to display endogenous Ags. However, neither T cell priming by endogenous Ags nor cross-priming ability was substantially affected in activated Nlrc5(-/-) DCs. Altogether, these data show that Nlrc5 deficiency, despite significantly affecting MHCI transcription and Ag display, is not sufficient to hinder T cell activation, underlining the robustness of the T cell priming process by activated DCs.

Étude génétique et fonctionnelle des Interferon-producing Killer Dendritic Cells

L’idée qu’une cellule puisse effectuer la cytolyse de cellules transformées, comme une cellule Natural Killer (NK), tout en ayant la capacité de présenter des antigènes, comme une cellule dendritique (DC), peut sembler fantaisiste. Cependant, de telles cellules furent bel et bien identifiées chez la souris en 2006. Ces cellules, nommées Interferon-producing Killer Dendritic Cells (IKDC), furent l’objet d’une caractérisation extensive qui révéla leur énorme potentiel immunologique. La combinaison de fonctions associées à des cellules NK et à des DC a doté les IKDC d’un pouvoir antitumoral remarquable. D’ailleurs, il a été démontré que les IKDC sont plus efficaces que les cellules NK pour limiter la croissance tumorale. Ainsi, suite à leur découverte, les IKDC ont suscité beaucoup d’intérêt. Cependant, une controverse émergea sur la nature des IKDC. Plusieurs groupes indépendants tentèrent de reproduire les expériences attestant les fonctions de DC des IKDC, sans y parvenir. De plus, des études additionnelles révélèrent que les IKDC possèdent des similitudes très importantes avec les cellules NK. Ces observations ont mené la communauté scientifique à suggérer que les IKDC sont des cellules NK en état d’activation (aNK). Malgré cette controverse, les caractéristiques antitumorales des IKDC sont si uniques et considérables qu’il est primordial de poursuivre l’étude de ces cellules. Pour y arriver, il est essentiel de déterminer la nature des IKDC et de mettre fin à ce débat. Par la suite, il sera important d’identifier des façons de cibler spécifiquement les IKDC pour permettre leur usage dans le cadre de thérapies antitumorales. Ainsi, l’objectif de cette thèse est de définir l’identité des IKDC, puis de déterminer les facteurs génétiques responsables de la régulation de ces cellules. Nous avons démontré que les IKDC ne sont pas des cellules aNK, contrairement à ce qui avait été suggéré. Nous avons constaté que les IKDC prolifèrent activement et possèdent un phénotype unique, des caractéristiques associées à des cellules NK très immatures. Afin de déterminer si les IKDC peuvent acquérir un phénotype mature, nous avons effectué des expériences de transfert adoptif. Suite à leur injection in vivo, les IKDC acquièrent un phénotype de cellules matures, mais étonnamment, elles se différencient aussi en cellules NK. Ainsi, nous avons révélé que les IKDC sont un intermédiaire dans la différenciation des cellules NK. En parallèle, nous avons démontré que la proportion d’IKDC varie grandement entre des souris de fond génétique différent, indiquant que des facteurs génétiques sont impliqués dans la régulation de ces cellules. Nous avons alors effectué une analyse génétique qui a révélé que les IKDC sont régulées par des facteurs génétiques compris dans une région distale du chromosome 7. Les résultats présentés dans cette thèse constituent une avancée importante pour la recherche sur les IKDC. Ils ont permis de définir la nature des IKDC et d’identifier un intervalle génétique impliqué dans la régulation de ces cellules. Ces découvertes sont des connaissances précieuses pour l’identification des IKDC chez l’Homme et la création de nouvelles thérapies dans la lutte contre le cancer.

Dynamic epigenetic changes in immune responses to infection in human dendritic cells

La méthylation de l'ADN est une marque épigénétique importante chez les mammifères. Malgré le fait que la méthylation de la cytosine en 5' (5mC) soit reconnue comme une modification épigénétique stable, il devient de plus en plus reconnu qu'elle soit un processus plus dynamique impliquant des voies de méthylation et de déméthylation actives. La dynamique de la méthylation de l'ADN est désormais bien caractérisée dans le développement et dans le fonctionnement cellulaire des mammifères. Très peu est cependant connu concernant les implications régulatrices dans les réponses immunitaires. Pour se faire, nous avons effectué des analyses du niveau de transcription des gènes ainsi que du profilage épigénétique de cellules dendritiques (DCs) humaines. Ceux-ci ont été faits avant et après infection par le pathogène Mycobacterium tuberculosis (MTB). Nos résultats fournissent le premier portrait génomique du remodelage épigénétique survenant dans les DCs en réponse à une infection bactérienne. Nous avons constaté que les changements dans la méthylation de l'ADN sont omniprésents, identifiant 3,926 régions différentiellement méthylées lors des infections par MTB (MTB-RDMs). Les MTB-RDMs montrent un chevauchement frappant avec les régions génomiques marquées par les histones associées avec des régions amplificatrices. De plus, nos analyses ont révélées que les MTB-RDMs sont activement liées par des facteurs de transcription associés à l'immunité avant même d'être infecté par MTB, suggérant ces domaines comme étant des éléments d'activation dans un état de dormance. Nos données suggèrent que les changements actifs dans la méthylation jouent un rôle essentiel pour contrôler la réponse cellulaire des DCs à l'infection bactérienne.

CD40 Triggering of Heterodimeric IL-12 p70 Production by Dendritic Cells In Vivo Requires a Microbial Priming Signal

CD40 ligation triggers IL-12 production by dendritic cells (DC) in vitro. Here, we demonstrate that CD40 cross-linking alone is not sufficient to induce IL-12 production by DC in vivo. Indeed, resting DC make neither the IL-12 p35 nor IL-12 p40 subunits and express only low levels of CD40. Nevertheless, after DC activation by microbial stimuli that primarily upregulate IL-12 p40 and augment CD40 expression, CD40 ligation induces a significant increase in IL-12 p35 and IL-12 p70 heterodimer production. Similarly, IL-12 p70 is produced during T cell activation in the presence but not in the absence of microbial stimuli. Thus, production of bioactive IL-12 by DC can be amplified by T cell–derived signals but must be initiated by innate signals.

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

Effects of oxidised low density lipoprotein on dendritic cells: a possible immunoregulatory component of the atherogenic micro-environment?

Objective: The objective of this study was to explore the relationship between low density lipoprotein (LDL) and dendritic cell (DC) activation, based upon the hypothesis that reactive oxygen species (ROS)-mediated modification of proteins that may be present in local DC microenvironments could be important as mediators of this activation. Although LDL are known to be oxidised in vivo, and taken up by macrophages during atherogenesis; their effect on DC has not been explored previously. Methods: Human DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4. Plasma LDLs were isolated by sequential gradient centrifugation, oxidised in CuSO4, and oxidation arrested to yield mild, moderate and highly oxidised LDL forms. DCs exposed to these LDLs were investigated using combined phenotypic, functional (autologous T cell activation), morphological and viability assays. Results: Highly-oxidised LDL increased DC HLA-DR, CD40 and CD86 expression, corroborated by increased DC-induced T cell proliferation. Both native and oxidised LDL induced prominent DC clustering. However, high concentrations of highly-oxidised LDL inhibited DC function, due to increased DC apoptosis. Conclusions: This study supports the hypothesis that oxidised LDL are capable of triggering the transition from sentinel to messenger DC. Furthermore, the DC clustering–activation–apoptosis sequence in the presence of different LDL forms is consistent with a regulatory DC role in immunopathogenesis of atheroma. A sequence of initial accumulation of DC, increasing LDL oxidation, and DC-induced T cell activation, may explain why local breach of tolerance can occur. Above a threshold level, however, supervening DC apoptosis limits this, contributing instead to the central plaque core.

What have dendritic cells ever done for adjuvant design? Cellular and molecular methods for the rational development of vaccine adjuvants

Our new molecular understanding of immune priming states that dendritic cell activation is absolutely pivotal for expansion and differentiation of naïve T lymphocytes, and it follows that understanding DC activation is essential to understand and design vaccine adjuvants. This chapter describes how dendritic cells can be used as a core tool to provide detailed quantitative and predictive immunomics information about how adjuvants function. The role of distinct antigen, costimulation, and differentiation signals from activated DC in priming is explained. Four categories of input signals which control DC activation – direct pathogen detection, sensing of injury or cell death, indirect activation via endogenous proinflammatory mediators, and feedback from activated T cells – are compared and contrasted. Practical methods for studying adjuvants using DC are summarised and the importance of DC subset choice, simulating T cell feedback, and use of knockout cells is highlighted. Finally, five case studies are examined that illustrate the benefit of DC activation analysis for understanding vaccine adjuvant function.