BCR and TLR signalling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas.

The genetics and pathogenesis of splenic marginal zone lymphoma are poorly understood. The lymphoma lacks chromosome translocation, and ~30% of cases are featured by 7q deletion, but the gene targeted by the deletion is unknown. A recent study showed inactivation of A20, a 'global' NF-kB negative regulator, in 1 of 12 splenic marginal zone lymphoma. To investigate further whether deregulation of the NF-kB pathway plays a role in the pathogenesis of splenic marginal zone lymphoma, we screened several NF-kB regulators for genetic changes by PCR and sequencing. Somatic mutations were found in A20 (6/46=13%), MYD88 (6/46=13%), CARD11 (3/34=8.8%), but not in CD79A, CD79B and ABIN1. Interestingly, these genetic changes are largely mutually exclusive from each other and MYD88 mutation was also mutually exclusive from 7q deletion. These results strongly suggest that deregulation of the TLR (toll like receptor) and BCR (B-cell receptor) signalling pathway may play an important role in the pathogenesis of splenic marginal zone lymphoma.

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.

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

Monocyte differentiation toward regulatory dendritic cells is not affected by respiratory syncytial virus-induced inflammatory mediators.

Airway epithelial cells were shown to drive the differentiation of monocytes into dendritic cells (DCs) with a suppressive phenotype. In this study, we investigated the impact of virus-induced inflammatory mediator production on the development of DCs. Monocyte differentiation into functional DCs, as reflected by the expression of CD11c, CD123, BDCA-4, and DC-SIGN and the capacity to activate T cells, was similar for respiratory syncytial virus (RSV)-infected and mock-infected BEAS-2B and A549 cells. RSV-conditioned culture media resulted in a partially mature DC phenotype, but failed to up-regulate CD80, CD83, CD86, and CCR7, and failed to release proinflammatory mediators upon Toll-like receptor (TLR) triggering. Nevertheless, these DCs were able to maintain an antiviral response by the release of Type I IFN. Collectively, these data indicate that the airway epithelium maintains an important suppressive DC phenotype under the inflammatory conditions induced by infection with RSV.

Uric acid is a danger signal activating NALP3 inflammasome in lung injury inflammation and fibrosis.

RATIONALE: Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and the IL-1 receptor 1 (IL-1R1) signaling pathway. The molecular mechanisms by which lung injury triggers IL-1beta production, inflammation, and fibrosis remain poorly understood. OBJECTIVES: To determine if lung injury depends on the NALP3 inflammasome and if bleomycin (BLM)-induced lung injury triggers local production of uric acid, thereby activating the NALP3 inflammasome in the lung. Methods: Inflammation upon BLM administration was evaluated in vivo in inflammasome-deficient mice. Pulmonary uric acid accumulation, inflammation, and fibrosis were analyzed in mice treated with the inhibitor of uric acid synthesis or with uricase, which degrades uric acid. MEASUREMENTS AND MAIN RESULTS: Lung injury depends on the NALP3 inflammasome, which is triggered by uric acid locally produced in the lung upon BLM-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1beta production, lung inflammation, repair, and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88, and IL-1R1 pathways and Toll-like receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor. CONCLUSIONS: Uric acid released from injured cells constitutes a major endogenous danger signal that activates the NALP3 inflammasome, leading to IL-1beta production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1beta production and chronic inflammatory lung pathology.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

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.

Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor.

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.

The evolutionary history of the CD209 (DC-SIGN) family in humans and non-human primates

The CD209 gene family that encodes C-type lectins in primates includes CD209 (DC-SIGN), CD209L (L-SIGN) and CD209L2. Understanding the evolution of these genes can help understand the duplication events generating this family, the process leading to the repeated neck region and identify protein domains under selective pressure. We compiled sequences from 14 primates representing 40 million years of evolution and from three non-primate mammal species. Phylogenetic analyses used Bayesian inference, and nucleotide substitutional patterns were assessed by codon-based maximum likelihood. Analyses suggest that CD209 genes emerged from a first duplication event in the common ancestor of anthropoids, yielding CD209L2 and an ancestral CD209 gene, which, in turn, duplicated in the common Old World primate ancestor, giving rise to CD209L and CD209. K(A)/K(S) values averaged over the entire tree were 0.43 (CD209), 0.52 (CD209L) and 0.35 (CD209L2), consistent with overall signatures of purifying selection. We also assessed the Toll-like receptor (TLR) gene family, which shares with CD209 genes a common profile of evolutionary constraint. The general feature of purifying selection of CD209 genes, despite an apparent redundancy (gene absence and gene loss), may reflect the need to faithfully recognize a multiplicity of pathogen motifs, commensals and a number of self-antigens

Immunogenicity of dimorphic and C-terminal fragments of Plasmodium falciparum MSP2 formulated with different adjuvants in mice.

BACKGROUND: Plasmodium falciparum MSP2 is a blood stage protein that is associated with protection against malaria. It was shown that the MSP2 dimorphic (D) and constant (C) regions were well recognized by immune human antibodies, and were characterized by major conserved epitopes in different endemic areas and age groups. These Abs recognized merozoite-derived proteins in WB and IFA. Here, the goal was to determine in mice the immunogenicity of the two allelic MSP2 D and C domains formulated with different adjuvants, for their possible use in future clinical studies. METHOD: Female A/J, C3H, and ICR mice were immunized subcutaneously 3 times at 3-week interval with a mixture of allelic and conserved MSP2 long synthetic peptides formulated with different adjuvants. One week after the third injection, sera from each group were obtained and stored at -20°C for subsequent testing. RESULTS: Both domains of the two MSP2 families are immunogenic and the fine specificity and intensity of the Ab responses are dependent on mouse strains and adjuvants. The major epitopes were restricted to the 20-mer peptide sequences comprising the last 8aa of D and first 12aa of C of the two allelic families and the first 20aa of the C region, this for most strains and adjuvants. Strong immune responses were associated with GLA-SE adjuvant and its combination with other TLR agonists (CpG or GDQ) compared to alhydrogel and Montanide. Further, the elicited Abs were also capable of recognizing Plasmodium-derived MSP2 and inhibiting parasite growth in ADCI. CONCLUSION: The data provide a valuable opportunity to evaluate in mice different adjuvant and antigen formulations of a candidate vaccine containing both MSP2 D and C fragments. The formulations with GLA-SE seem to be a promising option to be compared with the alhydrogel one in human clinical trials.