Cd40–Cd40 Ligand Interactions in Vivo Regulate Migration of Antigen-Bearing Dendritic Cells from the Skin to Draining Lymph Nodes

Whereas CD40–CD40 ligand interactions are important for various dendritic cell (DC) functions in vitro, their in vivo relevance is unknown. We analyzed the DC status of CD40 ligand −/− mice using a contact hypersensitivity (CHS) model system that enables multiple functions of DCs to be assessed in vivo. Immunohistochemistry of skin sections revealed no differences in terms of numbers and morphology of dendritic epidermal Langerhans cells (LCs) in unsensitized CD40 ligand −/− mice as compared with wild-type C57BL/6 mice. However, after contact sensitization of CD40 ligand −/− mice, LCs failed to migrate out of the skin and substantially fewer DCs accumulated in draining lymph nodes (DLNs). Furthermore, very few antigen-bearing DCs could be detected in the paracortical region of lymph nodes draining sensitized skin. This defect in DC migration after hapten sensitization was associated with defective CHS responses and decreased cutaneous tumor necrosis factor (TNF)-α production and was corrected by injecting recombinant TNF-α or an agonistic anti-CD40 monoclonal antibody. Thus, CD40–CD40 ligand interactions in vivo regulate the migration of antigen-bearing DCs from the skin to DLNs via TNF-α production and play a vital role in the initiation of acquired T cell–mediated immunity

Travelling with Dengue: From the Skin to the Nodes

Dengue virus (DENV) infects humans through the skin. The early infection and encounters between DENV and cutaneous immune and non‐immune cells only recently are under investigation. We have reported DENV‐infected cutaneous dendritic cells (DCs), also keratinocytes and dermal fibroblasts permissive to DENV infection. Now, upon cutaneously inoculating fluorescently labeled DENV into immune‐competent mice, we found DENV mostly in dermis from 1 h post‐inoculation. Afterwards, DENV rapidly localized in the subcapsular sinus of draining lymph nodes (DLNs) associated with CD169+ macrophages, suggesting virus travelling through lymph flow. However, DENV association with CD11c+ DCs in the paracortex and T:B border suggests DENV being ferried from the skin to DLNs by DCs too. DENV was not associated with F4/80+ macrophages nor with DEC205+ DCs, but it was inside B cell follicles early after cutaneous inoculation. DENV inside B follicles likely affects the development of humoral responses. Antibody responses deserve very careful scrutiny as neutralizing memory antibodies are crucial to counteract homotypic reinfections whereas non‐neutralizing ones might facilitate heterotypic DENV infection or even Zika infection, another flavivirus. Unravelling the DENV journey from skin to lymph into regional nodes and the cellular compartments will aid to understand the disease, its pathology and how to counteract it

Soluble flagellin coimmunization attenuates Th1 priming to Salmonella and clearance by modulating dendritic cell activation and cytokine production

Soluble flagellin (sFliC) from Salmonella Typhimurium (STm) can induce a Th2 response to itself and coadministered antigens through ligation of TLR5. These properties suggest that sFliC could potentially modulate responses to Th1 antigens like live STm if both antigens are given concurrently. After coimmunization of mice with sFliC and STm there was a reduction in Th1 T\ua0cells (T-bet+IFN-γ+ CD4 T\ua0cells) compared to STm alone and there was impaired clearance of STm. In contrast, there was no significant defect in the early extrafollicular B-cell response to STm. These effects are dependent upon TLR5 and flagellin expression by STm. The mechanism for these effects is not related to IL-4 induced to sFliC but rather to the effects of sFliC coimmunization on DCs. After coimmunization with STm and sFliC, splenic DCs had a lower expression of costimulatory molecules and profoundly altered kinetics of IL-12 and TNFα expression. Ex vivo experiments using in vivo conditioned DCs confirmed the effects of sFliC were due to altered DC function during a critical window in the coordinated interplay between DCs and naïve T\ua0cells. This has marked implications for understanding how limits in Th1 priming can be achieved during infection-induced, Th1-mediated inflammation

Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

In this work, we demonstrate that that both human whole skin and freshly isolated skin fibroblasts are productively infected with Dengue virus (DENV). In addition, primary skin fibroblast cultures were established and subsequently infected with DENV-2; we showed in these cells the presence of the viral antigen NS3, and we found productive viral infection by a conventional plaque assay. Of note, the infectivity rate was almost the same in all the primary cultures analyzed from different donors. The skin fibroblasts infected with DENV-2 underwent signaling through both TLR3 and RIG-1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 IRF7, when compared with mock-infected fibroblasts. Our data suggest that fibroblasts might even participate producing mediators involved in innate immunity that activate and contribute to the orchestration of the local innate responses. This work is the first evaluating primary skin fibroblast cultures obtained from different humans, assessing both their susceptibility to DENV infection as well as their ability to produce molecules crucial for innate immunity

The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment. OMVs have been studied extensively in bacterial pathogens, however, information related with the composition of Aeromonas hydrophila OMVs is missing. In this study we analyzed the composition of purified OMVs from A. hydrophila ATCC® 7966TM by proteomics. Also we studied the effect of OMVs on human peripheral blood mononuclear cells (PBMCs). Vesicles were grown in agar plates and then purified through ultracentrifugation steps. Purified vesicles showed an average diameter of 90–170 nm. Moreover, 211 unique proteins were found in OMVs from A. hydrophila; some of them are well-known as virulence factors such as: haemolysin Ahh1, RtxA toxin, extracellular lipase, HcpA protein, among others. OMVs from A. hydrophila ATCC® 7966TM induced lymphocyte activation and apoptosis in monocytes, as well as over-expression of pro-inflammatory cytokines. This work contributed to the knowledge of the composition of the vesicles of A. hydrophila ATCC® 7966TM and their interaction with the host cell

Jak3 Is Involved in Dendritic Cell Maturation and CCR7-Dependent Migration

BACKGROUND: CCR7-mediated signalling is important for dendritic cell maturation and homing to the lymph nodes. We have previously demonstrated that Jak3 participates in the signalling pathway of CCR7 in T lymphocytes. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we used Jak3(-/-) mice to analyze the role of Jak3 in CCR7-mediated dendritic cells migration and function. First, we found no differences in the generation of DCs from Jak3(-/-) bone marrow progenitors, when compared to wild type cells. However, phenotypic analysis of the bone marrow derived DCs obtained from Jak3(-/-) mice showed reduced expression of co-stimulatory molecules compared to wild type (Jak3(+/+)). In addition, when we analyzed the migration of Jak3(-/-) and Jak3(+/+) mature DCs in response to CCL19 and CCL21 chemokines, we found that the absence of Jak3 results in impaired chemotactic responses both in vitro and in vivo. Moreover, lymphocyte proliferation and contact hypersensitivity experiments showed that DC-mediated T lymphocyte activation is reduced in the absence of Jak3. CONCLUSION/SIGNIFICANCE: Altogether, our data provide strong evidence that Jak3 is important for DC maturation, migration and function, through a CCR7-mediated signalling pathway