Network of dendritic cells within the muscular layer of the mouse intestine

Dendritic cells (DCs) are located at body surfaces such as the skin, respiratory and genital tracts, and intestine. To further analyze intestinal DCs, we adapted an epidermal sheet separation technique and obtained two intestinal layers, facing the lumen and serosa. Unexpectedly, immunolabeling of the layer toward the serosa revealed a regular, dense, planar network of cells with prominent dendritic morphology within the external muscular layer and with increasing frequency along the length of the intestine. Direct examination of the serosal-disposed layers showed a significant fraction of the DCs to express DEC-205/CD205, CD11c, Langerin/CD207, Fcγ receptor/CD16/32, CD14, and low levels of activation markers, CD25, CD80, CD86, and CD95. By more sensitive FACS analyses, cells from this layer contained two CD11c+ populations of CD45+ CD205+, CD19- leukocytes, MHC II + and MHC II-. When ovalbumin conjugated to an anti-DEC-205 antibody was injected into mice, the conjugate targeted to these DCs, which upon isolation were able to stimulate ovalbumin-specific, CD4 + and CD8+ T cell antigen receptor-transgenic T cells. In vivo, these DCs responded to two microbial stimuli, systemic LPS and oral live bacteria, by up-regulating CD80, CD86, DEC-205, and Langerin within 12 h. This network of DCs thus represents a previously unrecognized antigen-presenting cell system in the intestine

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

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

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent <i>Mycobacterium tuberculosis</i>

<div><p>Airways infection with <i>Mycobacterium tuberculosis</i> (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205⁺ APCs and to assess its <i>in vivo</i> effects on protection associated responses (IFN-γ production, <i>in vivo</i> CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4⁺ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ⁺ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. <i>In vivo</i> CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205⁺ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study <i>in vivo</i> the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.</p></div

α-DEC-ESAT treatment increases IFN-γ⁺ T cells in the lung during the acute phase of experimental tuberculosis.

<p>The production of IFN-γ by CD4⁺ and CD8⁺ T cells was assessed in different tissues at two time-points during the experimental Mtb infection. α-DEC-ESAT treatment increased the percentages of IFN-γ⁺ CD4⁺ (A) and CD8⁺ (B) T cells in the lungs, during the acute (day 14) but not during the chronic (day 60) phase of the disease. In the lymphoid organs analyzed such as mediastinal lymph nodes (MedLN; C-D), inguinal lymph nodes (IngLN; E-F), and spleen (G-H), we observed very low levels of IFN-γ production either by CD4⁺ (left side panels) or CD8⁺ (right side panels) T cells. Individual mice were analyzed and 3–5 mice were used per group. Data are presented as mean plus standard error. (*) indicates P < 0.05. All bars represent groups of infected mice with different treatments. Black bars: untreated mice (Inf/No-Tx); white bars: α-DEC-ESAT-treated mice (Inf/DE6-Tx); stripped bars: mice treated with isotype control antibody conjugated with ESAT-6 (Inf/IsoE6-Tx). Cell suspensions were cultured either with medium alone (M) or medium with ESAT-6 pool 1 of peptides (p1) as detailed in Materials and Methods. MedLN = Mediastinal lymph nodes, IngLN = Inguinal lymph nodes.</p

α-DEC-ESAT-treated mice show reduced bacterial burden and increased cellular infiltrate in the lungs.

<p>Pulmonary mycobacterial load (CFUs/lung) and cellular infiltrate (% pneumonic areas) were quantified at two time points during Mtb infection in the different experimental groups. Horizontally stripped bars indicate uninfected non-immunized mice (No-Inf). All other bars represent infected mice with different treatments. Black bars: untreated mice (Inf/No Tx); white bars: α-DEC-ESAT-treated mice (Inf/DE6-Tx); diagonally stripped bars: mice treated with isotype control antibody-ESAT (Inf/IsoE6-Tx). Quantification of the cellular infiltrate during the acute infection is shown in (A) while that of the chronic stage is shown in (C). The bacterial burden expressed as CFUs per lung is shown in (B) for the acute stage of the infection and in (D) for the chronic phase. (E) Representative picture of HE staining of lungs from infected mice which were α-DEC-ESAT-untreated (-) or α-DEC-ESAT-treated (+). Individual lungs were analyzed by duplicate and 3 mice were used per group. Data are presented as mean plus standard error. (*) Represents P<0.05.</p

Immunization with α-DEC-ESAT hybrid antibody induces Th1 responses to ESAT-6 in the lungs and mediastinal lymph nodes of non-infected mice.

<p>A) A fusion antibody was generated to murine DEC205, genetically coupled to ESAT-6 and produced by transfected 293T cells. This Ab (α-DE6) presented an electrophoretic delay (left and middle figures), while binding to surface DEC205 on DEC205-transfected CHO cells was not affected (FACS histograms in the figure to the right). B) The ESAT-6 peptide library is depicted here to illustrate the distribution of the three pools of peptides that were used to stimulate cell suspensions from the different tissues examined, as indicated. Control unstimulated cell suspensions treated with culture medium alone are indicated with (M) while those treated with ESAT-6 pool 1 of peptides are indicated with (p1). C) and D) The IFN-γ production by ESAT-6-specific CD4⁺ T cells is shown as dot plots in (C), and as integrated results of the experiments performed for the various tissues assessed in (D). Data are presented as mean plus standard error and percentage of IFN-γ producing T cells. (*) represents P<0.05; (**) indicates P<0.01; (***) represents P<0.001. All bars represent uninfected mice treated with α-DEC-ESAT (white bars) or untreated (gray bars). MedLN = Mediastinal lymph nodes, IngLN = Inguinal lymph nodes.</p