Display of native antigen on cDC1 that have spatial access to both T and B cells underlies efficient humoral vaccination

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination

A novel TLR3 inhibitor encoded by African swine fever virus (ASFV)

African swine fever virus (ASFV) encodes proteins that manipulate important host antiviral mechanisms. Bioinformatic analysis of the ASFV genome revealed ORF I329L, a gene without any previous functional characterization as a possible inhibitor of TLR signaling. We demonstrate that ORF I329L encodes a highly glycosylated protein expressed in the cell membrane and on its surface. I329L also inhibited dsRNA-stimulated activation of NFκB and IRF3, two key players in innate immunity. Consistent with this, expression of I329L protein also inhibited the activation of interferon-β and CCL5. Finally, overexpression of TRIF reversed I329L-mediated inhibition of both NFκB and IRF3 activation. Our results suggest that TRIF, a key MyD88-independent adaptor molecule, is a possible target of this viral host modulation gene. The demonstration of an ASFV host evasion molecule inhibiting TLR responses is consistent with the ability of this virus to infect vertebrate and invertebrate hosts, both of which deploy innate immunity controlled by conserved TLR systems

Synthetic Toll Like Receptor-4 (TLR-4) Agonist Peptides as a Novel Class of Adjuvants

Background: Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants. Methodology/Principal Findings: We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-kB nuclear translocation analyses in HEK-BLUE TM-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant

CCAAT/Enhancer-Binding Protein γ Is a Critical Regulator of IL-1β-Induced IL-6 Production in Alveolar Epithelial Cells

CCAAT/enhancer binding protein γ (C/EBPγ) is a member of the C/EBP family of transcription factors, which lacks known activation domains. C/EBPγ was originally described as an inhibitor of C/EBP transactivation potential. However, previous study demonstrates that C/EBPγ augments the C/EBPβ stimulatory activity in lipopolysaccharide induction of IL-6 promoter in a B lymphoblast cell line. These data indicate a complexing functional role for C/EBPγ in regulating gene expression. Furthermore, the expression and function of C/EBPγ during inflammation are still largely unknown. In this study, we demonstrate that C/EBPγ activation was induced by IL-1β treatment in lung epithelial cells. Importantly, we demonstrate for the first time that C/EBPγ plays a critical role in regulating IL-1β-induced IL-6 expression in both mouse primary alveolar type II epithelial cells and a lung epithelial cell line, MLE12. We further provide the evidence that C/EBPγ inhibits IL-6 expression by inhibiting C/EBPβ but not NF-κB stimulatory activity in MLE12 cells. These findings suggest that C/EBPγ is a key transcription factor that regulates the IL-6 expression in alveolar epithelial cells, and may play an important regulatory role in lung inflammatory responses

Crucial Role for BAFF-BAFF-R Signaling in the Survival and Maintenance of Mature B Cells

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools

Induction of IFN-β and the Innate Antiviral Response in Myeloid Cells Occurs through an IPS-1-Dependent Signal That Does Not Require IRF-3 and IRF-7

Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-α/β) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-α levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3−/−× IRF-7−/− double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-α response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-β response in DKO fibroblasts and cortical neurons. In contrast, the IFN-β response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-κB and ATF-2/c-Jun, the two other known components of the IFN-β enhanceosome, strongly reduced IFN-β gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-β response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-β gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-β enhanceosome by canonical constituent transcriptional factors

Evasion by Stealth: Inefficient Immune Activation Underlies Poor T Cell Response and Severe Disease in SARS-CoV-Infected Mice

Severe Acute Respiratory Syndrome caused substantial morbidity and mortality during the 2002–2003 epidemic. Many of the features of the human disease are duplicated in BALB/c mice infected with a mouse-adapted version of the virus (MA15), which develop respiratory disease with high morbidity and mortality. Here, we show that severe disease is correlated with slow kinetics of virus clearance and delayed activation and transit of respiratory dendritic cells (rDC) to the draining lymph nodes (DLN) with a consequent deficient virus-specific T cell response. All of these defects are corrected when mice are treated with liposomes containing clodronate, which deplete alveolar macrophages (AM). Inhibitory AMs are believed to prevent the development of immune responses to environmental antigens and allergic responses by interacting with lung dendritic cells and T cells. The inhibitory effects of AM can also be nullified if mice or AMs are pretreated with poly I:C, which directly activate AMs and rDCs through toll-like receptors 3 (TLR3). Further, adoptive transfer of activated but not resting bone marrow–derived dendritic cells (BMDC) protect mice from lethal MA15 infection. These results may be relevant for SARS in humans, which is also characterized by prolonged virus persistence and delayed development of a SARS-CoV-specific immune response in individuals with severe disease

A Key Role of Dendritic Cells in Probiotic Functionality

BACKGROUND: Disruption of the intestinal homeostasis and tolerance towards the resident microbiota is a major mechanism involved in the development of inflammatory bowel disease. While some bacteria are inducers of disease, others, known as probiotics, are able to reduce inflammation. Because dendritic cells (DCs) play a central role in regulating immune responses and in inducing tolerance, we investigated their role in the anti-inflammatory potential of probiotic lactic acid bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Selected LAB strains, while efficiently taken up by DCs in vitro, induced a partial maturation of the cells. Transfer of probiotic-treated DCs conferred protection against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Protection was associated with a reduction of inflammatory scores and colonic expression of pro-inflammatory genes, while a high local expression of the immunoregulatory enzyme indolamine 2, 3 dioxgenase (IDO) was observed. The preventive effect of probiotic-pulsed DCs required not only MyD88-, TLR2- and NOD2-dependent signaling but also the induction of CD4+ CD25+ regulatory cells in an IL-10-independent pathway. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that selected probiotics can stimulate DC regulatory functions by targeting specific pattern-recognition receptors and pathways. The results not only emphasize the role of DCs in probiotic immune interactions, but indicate a possible role in immune-intervention therapy for IBD

The absence of MyD88 has no effect on the induction of alternatively activated macrophage during Fasciola hepatica infection

<p>Abstract</p> <p>Background</p> <p>Alternatively activated macrophages (AAMϕ) play important roles in allergies and responses to parasitic infections. However, whether signaling through toll-like receptors (TLRs) plays any role in AAMϕ induction when young <it>Fasciola hepatica </it>penetrates the liver capsule and migrates through the liver tissue is still unclear.</p> <p>Results</p> <p>The data show that the lack of myeloid differentiation factor 88 (MyD88) has no effect on the AAMϕ derived from the bone marrow (BMMϕ) <it>in vitro </it>and does not impair the mRNA expression of arginase-1, resistin-like molecule (RELMα), and Ym1 in BMMϕs. The Th2 cytokine production bias in splenocytes was not significantly altered in <it>F. hepatica</it>-infected mice in the absence of MyD88 <it>in vitro </it>and in the pleural cavity lavage <it>in vivo</it>. In addition, MyD88-deficiency has no effect on the arginase production of the <it>F. hepatica </it>elicited macrophages (Fe Mϕs), production of RELMα and Ym1 proteins and mRNA expression of Ym1 and RELMα of macrophages in the peritoneal cavity 6 weeks post <it>F. hepatica </it>infection.</p> <p>Conclusions</p> <p>The absence of MyD88 has no effect on presence of AAMϕ 6 weeks post <it>F. hepatica </it>infection.</p

Maturation of monocyte-derived dendritic cells with Toll-like receptor 3 and 7/8 ligands combined with prostaglandin E2 results in high interleukin-12 production and cell migration

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that play a key role in regulating T cell-based immunity. In vivo, the capacity of DC to activate T cells depends on their ability to migrate to the T cell areas of lymph nodes as well as on their maturation state. Depending on their cytokine-secreting profile, DC are able to skew the immune response in a specific direction. In particular, IL-12p70 producing DC drive T cells towards a T helper 1 type response. A serious disadvantage of current clinical grade ex vivo generated monocyte-derived DC is the poor IL-12p70 production. We have investigated the effects of Toll-like receptor (TLR)-mediated maturation on ex vivo generated human monocyte-derived DC. We demonstrate that in contrast to cytokine-matured DC, DC matured with poly(I:C) (TLR3 ligand) and/or R848 (TLR7/8 ligand) are able to produce vast amounts of IL-12p70, but exhibit a reduced migratory capacity. The addition of prostaglandin E2 (PGE2) improved the migratory capacity of TLR-ligand matured DC while maintaining their IL-12p70 production upon T cell encounter. We propose a novel clinical grade maturation protocol in which TLR ligands poly(I:C) and R848 are combined with PGE2 to generate DC with both high migratory capacity and IL-12p70 production upon T cell encounter