Francesca Granucci

Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria.

Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyers patches. However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration. This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells. We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs). DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria. In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved.

Inducible IL-2 production by dendritic cells revealed by global gene expression analysis

Dendritic cells (DCs) are strong activators of primary T cell responses. Their priming ability is acquired upon encounter with maturation stimuli. To identify the genes that are differentially expressed upon maturation induced by exposure to Gram-negative bacteria, a kinetic study of DC gene expression was done with microarrays representing 11,000 genes and ESTs (expressed sequence tags). Approximately 3000 differentially expressed transcripts were identified. We found that functional interleukin 2 (IL-2) mRNA, which gave rise to IL-2 production, was transiently up-regulated at early time-points after bacterial encounter. In contrast, macrophages did not produce IL-2 upon bacterial stimulation. Thus, IL-2 is an additional key cytokine that confers unique T cell stimulatory capacity to DCs.

CD14 Controls the LPS-Induced Endocytosis of Toll-like Receptor 4

The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.

Infection of dendritic cells by murine cytomegalovirus induces functional paralysis

Cytomegalovirus (CMV), measles and HIV are the main human pathogens known to induce immunosuppression. Unlike measles and HIV, and despite the availability of a well studied animal model, little is known about the mechanisms that control CMV-induced immunosuppression. We hypothesized that dendritic cells (DCs), which are crucial in generating and maintaining immune responses, represent a target for CMV and that the transient, but profound, immunosuppression that accompanies CMV infection results from viral interference with DC functions. Here we show that DCs were permissive to murine CMV infection. In addition, DC infection prevented delivery of the signals required for T cell activation. Thus, CMV-mediated impairment of DC function may be crucial for virally induced immunosuppression and interleukin 2 is implicated as a key factor.

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.

A Contribution of Mouse Dendritic Cell–Derived IL-2 for NK Cell Activation

Dendritic cells (DCs) play a predominant role in activation of natural killer (NK) cells that exert their functions against pathogen-infected and tumor cells. Here, we used a murine model to investigate the molecular mechanisms responsible for this process. Two soluble molecules produced by bacterially activated myeloid DCs are required for optimal priming of NK cells. Type I interferons (IFNs) promote the cytotoxic functions of NK cells. IL-2 is necessary both in vitro and in vivo for the efficient production of IFNγ, which has an important antimetastatic and antibacterial function. These findings provide new information about the mechanisms that mediate DC–NK cell interactions and define a novel and fundamental role for IL-2 in innate immunity.

The European dimension for the mouse genome mutagenesis program

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

Transcriptional reprogramming of dendritic cells by differentiation stimuli.

Immature and mature dendritic cells (DC) have been well characterized functionally and phenotypically. Microorganisms or bacterial products such as lipopolysaccharide (LPS) and inflammatory molecules, including tumor necrosis factor (TNF‐α), are both believed to activate the DC maturation program which allows DC to initiate and amplify innate and adaptive immune responses. However, there is increasing evidence that the functional state of DC, induced by different stimuli, may be relevant for the immune response outcome. Thus, we compared the transcriptional program of mature, transitional and immature DC, after either LPS or TNF‐α stimulation. GeneChip® oligonucleotide microarrays, representing approximately 6,500 murine genes and ESTs, were used for this analysis. A very diverse modulation of gene expression was observed with the two stimuli. Only LPS‐treated cells showed a pattern of expression of genes compatible with a definitive growth arrest and with a suitable activation and control of the immune response.

Early IL-2 Production by Mouse Dendritic Cells Is the Result of Microbial-Induced Priming

Dendritic cells (DCs) are professional APCs able to initiate innate and adaptive immune responses against invading pathogens. Different properties such as the efficient Ag processing machinery, the high levels of expression of costimulatory molecules and peptide-MHC complexes, and the production of cytokines contribute in making DCs potent stimulators of naive T cell responses. Recently we have observed that DCs are able to produce IL-2 following bacterial stimulation, and we have demonstrated that this particular cytokine is a key molecule conferring to early bacterial activated DCs unique T cell priming capacity. In the present study we show that many different microbial stimuli, but not inflammatory cytokines, are able to stimulate DCs to produce IL-2, indicating that DCs can distinguish a cytokine-mediated inflammatory process from the actual presence of an infection. The capacity to produce IL-2 following a microbial stimuli encounter is a feature shared by diverse DC subtypes in vivo, such as CD8α+ and CD8α− splenic DCs and epidermal Langerhans cells. When early activated DCs interact with T cells, IL-2 produced by DCs is enriched at the site of cell-cell contact, confirming the importance of DCs-derived IL-2 in T cell activation.

Glial TLR4 Receptor as New Target to Treat Neuropathic Pain: Efficacy of a New Receptor Antagonist in a Model of Peripheral Nerve Injury in Mice

Neuropathic pain remains a prevalent clinical problem because it is often poorly responsive to the currently used analgesics, thus it is crucial the identification of new potential targets and drugs. Recent evidence indicated that microglial cells in the spinal cord are critically involved in the development and maintenance of neuropathic pain, with a pivotal role of toll‐like receptor 4 (TLR4). Binding of an endogenous ligand to TLR4 might be considered an important step in the regulation of microglia activity in pain facilitation, suggesting that a mechanism aimed to inhibit such a binding could be effective against neuropathic pain. We have synthesized new ligands to TLR4 with antagonistic activity. In the present work we evaluated the efficacy of the most potent TLR4 antagonist synthesized by us (FP‐1), administered in mice with painful neuropathy. The repeated treatment of neuropathic mice with FP‐1 (5–10 mg/kg, i.p.) evoked a relief of both thermal hyperalgesia and mechanical allodynia, whereas the administration of the highest dose to TLR4 knockout neuropathic mice revealed that in the absence of TLR4 receptor, the compound lost its efficacy. As consequence of TLR4 binding, the repeated treatment with FP‐1 prevented the activation of the transcription factor NF‐kB and the TNFα overproduction in the spinal cord. Together, our findings support the previous evidence indicative for a contribution of glial TLR4 to the initiation of neuropathic pain, suggest it as potential innovative target to treat this debilitating disease, and propose FP‐1 as lead compound for the development of new effective drugs.