Maria Foti

Coordinated events during bacteria-induced DC maturation.

Abstract The dendritic cell (DC) system represents a class of professional antigen-presenting cells whose primary function is to regulate the initiation of immune responses. Here, Maria Rescigno and colleagues describe the kinetics and molecular events of bacteria-induced DC maturation.

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation.

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor κB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca2+/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cγ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

Probing Host Pathogen Cross-Talk by Transcriptional Profiling of Both Mycobacterium tuberculosis and Infected Human Dendritic Cells and Macrophages.

Background Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells. Methodology/Principal Findings In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. Conclusions/Significance This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.

Mycobacterial P1-Type ATPases Mediate Resistance to Zinc Poisoning in Human Macrophages

Summary Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P1-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

Early events in dendritic cell maturation induced by LPS

Immature dendritic cells (Dcs) are characterised by high antigen uptake ability and poor T-cell stimulatory function. In contrast, mature DCs have a high stimulatory function and poor antigen uptake ability. Inflammatory stimuli induce DC maturation and migration from nonlymphoid tissues to lymphoid organs. We investigated the effect of lipopolysaccharide (LPS) on DC antigen uptake and migratory function at early and late stimulation time points. We observed that the transition from the immature to the mature state is not a progressive itinerary, but it is characterised by precise functional stages. At early time points after LPS stimulation DCs significantly decrease their intrinsic migratory ability and increase the antigen uptake function. Later, around 4 h after LPS activation, DCs show recovery of migratory ability and start to progressively lose their antigen uptake function until the mature stage in which they show poor antigen uptake and migratory activity.

Dendritic Cell Biology

Dendritic cells (DCs) are a special type of leukocytes able to alert the immune system to the presence of infections. They play a central role in the initiation of both innate and adaptive immune responses. This particular DC feature is regulated by the activation of specific receptors at the cell surface called Toll-like receptors (TLRs) that bind a number of microbial products collectively referred to as microbial-associated molecular patterns (MAMP). TLRs initiate a cascade of events, which together define the process of DC maturation. This phenomenon allows DCs to progressively acquire varying specific functions. DC maturation depends on the nature of the perturbation and permits unique and efficient immune responses for each pathogen. In this review the discussion is focused on DCs in the context of interactions with pathogens and DC-specific functions are highlighted.

TLR-Dependent Activation Stimuli Associated with Th1 Responses Confer NK Cell Stimulatory Capacity to Mouse Dendritic Cells

Dendritic cells (DCs) have an important role in the activation of NK cells that exert direct antitumor and antimicrobial effects and can influence the development of adaptive T cell responses. DCs acquire NK cell stimulatory capacity after exposure to various stimuli. In this study we investigated the nature of the stimuli that confer to DCs the NK cell-activating capacity. After exposure of DCs to TLR-dependent and -independent microbial stimuli and to nonmicrobial stimuli, we evaluated the ability of activated DCs to elicit IFN-γ production from NK cells in vitro and to promote NK cell activation in vivo. We show in this study that only TLR-dependent microbial stimuli typically associated with Th1 responses confer to DCs the ability to activate NK cells, whereas stimuli associated with Th2 responses do not have this property.

Both Treg cells and Tconv cells are defective in the Myasthenia gravis thymus: roles of IL-17 and TNF-α.

Myasthenia gravis (MG) is an autoimmune disease in which the thymus frequently presents follicular hyperplasia and signs of inflammation and T cells display a defect in suppressive regulation. Defects in a suppressive assay can indicate either the defective function of Treg cells or the resistance of Tconv cells to suppression by Treg cells. The aim of this study was to determine which cells were responsible for this defect and to address the mechanisms involved. We first performed cross-experiment studies using purified thymic Treg cells and Tconv cells from controls (CTRL) and MG patients. We confirmed that MG Treg cells were defective in suppressing CTRL Tconv proliferation, and we demonstrated for the first time that MG Tconv cells were resistant to Treg cell suppression. The activation of MG Tconv cells triggered a lower upregulation of FoxP3 and a higher upregulation of CD4 and CD25 than CTRL cells. To investigate the factors that could explain these differences, we analyzed the transcriptomes of purified thymic Treg and Tconv cells from MG patients in comparison to CTRL cells. Many of the pathways revealed by this analysis are involved in other autoimmune diseases, and T cells from MG patients exhibit a Th1/Th17/Tfh signature. An increase in IL-17-related genes was only observed in Treg cells, while increases in IFN-γ, IL-21, and TNF-α were observed in both Treg and Tconv cells. These results were confirmed by PCR studies. In addition, the role of TNF-α in the defect in Tconv cells from MG patients was further confirmed by functional studies. Altogether, our results indicate that the immunoregulatory defects observed in MG patients are caused by both Treg cell and Tconv cell impairment and involve several pro-inflammatory cytokines, with TNF-α playing a key role in this process. The chronic inflammation present in the thymus of MG patients could provide an explanation for the escape of thymic T cells from regulation in the MG thymus.

Retroviral immortalization of phagocytic and dendritic cell clones as a tool to investigate functional heterogeneity

We have developed a method to generate immortalized phagocytic and dendritic cell clones from various mouse tissues such as spleen, thymus, brain and bone marrow. The clones were phenotypically characterized and shown to retain the ability to respond to immune or inflammatory signals, e.g., IFN-gamma. Functional cytokine activity and nitric oxide production were maintained in activated macrophages, microglial and dendritic cell clones. Immune functions, such as antigen presentation was exhibited by all clones whereas tissue-specific properties such as the ability to respond to corticotropin-releasing hormone and produce beta-endorphin was shown in microglial cell clones but not in macrophage cell clones, indicating that heterogeneity of cells of the mononuclear-phagocytic lineage can be maintained in vitro after the immortalization procedure. Moreover, the continuous proliferation of the clones could be inhibited by various stimuli and further differentiation of the cells could be achieved in vitro.

Sox2 Is Required to Maintain Cancer Stem Cells in a Mouse Model of High-Grade Oligodendroglioma

The stem cell-determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor-induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells.