The timing of IFNß production affects early innate responses to Listeria monocytogenes and determines the overall outcome of lethal infection.

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a crucial role in the first phase of host defense against infections and tumors. Listeria monocytogenes (Lm) is an intracellular pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection. We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the molecular mechanisms underlying DCs interaction with Lm. Using a functional genomics approach, we found that Lm infection induced a cluster of late response genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I INFs were produced at the maximal level only at 24 h post infection indicating that the regulation of IFNs in the context of Lm infection is delayed compared to the rapid response observed with viral pathogens. We showed that during Lm infection, IFNγ production and cytotoxic activity were severely impaired in NK cells compared to E. coli infection. These defects were restored by providing an exogenous source of IFNß during the initial phase of bacterial challenge. Moreover, when treated with IFNß during early infection, NK cells were able to reduce bacterial titer in the spleen and significantly improve survival of infected mice. These findings show that the timing of IFNß production is fundamental to the efficient control of the bacterium during the early innate phase of Lm infection.

Synergism of NOD2 and NLRP3 activators promotes a unique transcriptional profile in murine dendritic cells.

NLRs are cytoplasmic proteins that sense cellular stress and intracellular damage resulting from pathogen uptake. To date, the role of NLRs has been studied using combinations of NLR and TLR agonists, but the interplay between two different NLRs remains uncharacterized. In this study, we employed microarrays to investigate in DCs the regulation of gene transcription mediated by activation of NOD2 and NLRP3 pathways using MDP and MSU. MDP and MSU co-stimulation of murine BMDCs up-regulated the expression of genes encoding molecules for antigen presentation and co-stimulation (MHC class II, CD80, CD86), integrins (ITGB3, ITGAV), cytokines (IL-1α, IL-1ß, IL-6, IL-2, IL-23p19, IL-12p40), and chemokines (CXCL1, CXCL2). Transcription of the cytokine genes induced by MDP and MSU partially depended on NOD2 but was independent of NLRP3. Finally, we showed that ERK1 and c-JUN activation increased upon MDP and MSU co-stimulation. As a whole, the results indicate that two different NLR activators synergize at the transcriptional level, leading to unique differential expression of genes involved in the innate immune response.

Generation of murine growth factor-dependent long-term dendritic cell lines to investigate host-parasite interactions.

Substantial progress has been made over the last several years in the development of protocols for the isolation of large numbers of dendritic cells (DCs) from different tissues and their short-term culture. Indeed, several stable DC lines and clones have been established from various tissues of mice and humans, providing useful experimental tools for studying the biology of DCs at both molecular and biochemical levels and for the establishment of new DC-based immunotherapies. In this chapter, we will describe the development of long-term DC lines that maintain the growth factor dependence and their immature functional state, thus providing a unique opportunity to study the mechanisms of the initiation of the immune response to infectious agents.

Synthesis and biological evaluation of novel lipid A antagonists.

A mimetic of Lipid A with a beta-N(OMe) glycosidic linkage, four linear C-14 hydrophobic chains and without phosphate groups has been prepared together with its beta-O-linked analogue. Both these molecules were active in inhibiting the inflammatory action of Escherichia coli lipid A on MT2 macrophages in a dose-dependent manner, while they were completely devoid of inflammatory activity.

A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana.

Recognition of pathogen-associated molecular patterns (PAMP) influences the response of dendritic cells (DC) and therefore development of innate and adaptive immunity. Different forms of Leishmania mexicana have distinct effects on DC, with promastigotes and amastigotes being activating and apparently neutral, respectively. We investigated whether stage-specific differences in surface composition might account for these distinct effects. Amastigotes and promastigotes lacking the lpg1 gene needed for lipophosphoglycan (LPG) biosynthesis could not activate DC in vitro. Genome-wide transcriptional profiling of DC infected with wild-type or mutant promastigotes or wild-type amastigotes revealed that wild-type promastigotes induce an inflammatory signature that is lacking in DC exposed to the other parasite forms. The proinflammatory response pattern was partly recovered by reconstitution of lpg1 expression in lpg1-/- parasites, and exposure to purified LPG increased the expression of MHC class II and CD86 on DC. Infection with wild-type but not lpg1-/- promastigotes increased the number of activated DC in draining lymph nodes, and this was correlated with lower early parasite burdens in wild-type-infected animals. These in vivo and in vitro results suggest an LPG-dependent activation of DC that contributes to host defense and agree with the notion that the parasites evolved under immune pressure to down-regulate PAMP expression in mammalian hosts.

A type I IFN-dependent pathway induced by Schistosoma mansoni eggs in mouse myeloid dendritic cells generates an inflammatory signature.

Schistosomes are helminth parasites that display a dual impact on the immune system of their hosts. Although the larval stage, also known as schistosomulum, appears to subvert the host defenses, the egg stage induces strong inflammatory reactions. Given the pivotal role of dendritic cells (DC) in initiating and regulating immune responses, we compared the distinct transcriptional programs induced in immature mouse DC by S. mansoni eggs or schistosomula. Although SLA abrogated the transcription of many genes implicated in DC functions, eggs caused myeloid DC to produce IFN-beta. Autocrine/paracrine signaling through the type I IFN receptor in response to eggs was necessary for the induction of known IFN-responsive genes and enhanced the synthesis of key inflammatory products. Taken as a whole, our data provide molecular insights into the immune evasion mechanism of schistosomula and suggest an unexpected role for type I IFN in the innate response to helminth eggs.

The scavenger receptor MARCO mediates cytoskeleton rearrangements in dendritic cells and microglia.

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor expressed in peritoneal macrophages and in a subpopulation of macrophages in the marginal zone of the spleen and in the medullary cord of lymph nodes. By global gene expression analysis, it has been found that the MARCO mRNA was one of the most up-regulated in splenic dendritic cells (DCs) following lipopolysaccharide or bacterial activation and in granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated microglial cells. Here we show that MARCO is expressed on splenic DCs at late time points after activation and that its expression correlates with profound changes in actin cytoskeleton organization in DCs and microglia. During maturation, DCs undergo profound rearrangements of actin cytoskeleton. Immature DCs are adherent with visible actin cables, while fully mature, MARCO-expressing, splenic DCs are nonadherent, round in shape, and have an actin cytoskeleton with a punctate distribution. The simple expression of MARCO was sufficient to induce these cytoskeleton modifications in DCs. MARCO-transfected immature DCs acquired a typical morphology of mature DCs and did not rearrange the actin cytoskeleton following activation. Moreover, DCs in which MARCO was knocked down did not reach the mature phenotype and maintained the typical morphology of transitional DCs. MARCO expression in DCs and microglial cells was also associated with a decrease of antigen internalization capacity. Thus, the MARCO receptor is important for actin cytoskeleton rearrangements and the down-regulation of antigen uptake function during DC and microglial cell maturation.

Toll-like receptor 4 is not required for the full maturation of dendritic cells or for the degradation of Gram-negative bacteria.

Toll-like receptor 4 (TLR4) has been recently associated with cellular responses to lipopolysaccharide (LPS), and mice mutated in tlr4, such as C57BL/10ScCr or C3H/HeJ mice, become hyporesponsive to LPS. In this study, we have analyzed the capacity of bone marrow-derived dendritic cells (BMDC) from C57BL/10ScCr (ScCr-BMDC) or C3H/HeJ (HeJ-BMDC) mice to respond to LPS or to Gram-negative bacteria. We show that ScCr- or HeJ-BMDC are insensitive to LPS, but can mature in response to live and killed Gram-negative bacteria. Interestingly, only ScCr-BMDC but not HeJ-BMDC, stimulated with bacteria, have reduced capacity to produce pro- and anti-inflammatory cytokines as compared to BMDC from control mice, probably due to genetic defects unrelated to the tlr4 mutation. Nevertheless, ScCr-BMDC and ScCr BM-macrophages (BM-Mphi) phagocytose Salmonella typhimurium similarly to control cells, indicating that TLR4 is not compulsory for bacterial uptake. Moreover, BM-Mphi, but not BM-DC from B10ScCr or C3H/HeJ mice, are impaired in their capacity to kill intracellular bacteria and to produce NO as compared to wild type controls. However, the bacteria killing property of BM-Mphi is completely restored by pretreating the cells with IFN-gamma. Hence, TLR4 plays different roles in DC versus Mphi.