Porcine Dendritic Cells Research Articles

Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells.

The purpose of this study was to develop a monoclonal antibody (mAb) that can identify porcine dendritic cells (DCs) that have differentiated from bone marrow progenitor cells. Hybridoma technology was used to obtain mAbs, and bone marrow-derived DCs (BMDCs) were employed as immunogens for producing antibodies. The generated PD9-9 mAbs exhibited considerable reactivity towards porcine BMDCs with applications in flow cytometry and immunostaining. The antibody was composed of heavy immunoglobulin gamma-1 chains and light kappa chains. The PD9-9 mAb recognized fully differentiated porcine BMDCs and cells undergoing DC differentiation. In contrast, bone marrow cells and macrophages were not recognized by PD9-9. In addition, the PD9-9 mAb promoted porcine DC proliferation. Consequently, the PD9-9 mAb may be a biomarker for porcine DCs and will be advantageous for investigating porcine DC biology.

Exopolysaccharides from Limosilactobacillus reuteri: their influence on in vitro activation of porcine monocyte-derived dendritic cells - brief report

The aim of this study was to evaluate the immunomodulatory potential of two α-D-glucans from Limosilactobacillus reuteri L26 Biocenol™ (EPS-L26) and L. reuteri DSM17938 (EPS-DSM17938), with respect to their influence on in vitro activation of porcine dendritic cells (DCs). We used immature DCs differentiated from porcine blood monocytes under in vitro conditions. Based on the surface expression of MHC II and costimulatory CD80/86 molecules, we showed that both used EPSs favour the maturation of monocyte-derived DCs (MoDCs) similarly to the commonly used stimulant tumour necrosis factor α (TNF-α). In contrast to TNF-α stimulation, MoDCs treated with both used EPSs significantly up-regulated the mRNA levels not only for interleukin (IL)-10 (P < 0.0001 for EPS-DSM17938; P = 0.0037 for EPS-L26), but also for IL-12 (P = 0.0176 for EPS-DSM17938; P = 0.0019 for EPS-L26). These cytokines are known to regulate T-cell kinetics and play a key role in maintaining immune homeostasis. Interestingly, only relatively linear α-D-glucan (EPS-DSM17938) significantly increased gene expression of the major pro-inflammatory cytokine IL-1β (P = 0.0011) and the “SOS” cytokine IL-6 (P = 0.0127). However, it is important to highlight the need for further studies aimed at cytokine kinetics in DCs, as well as a co-culture study with allogenic T-lymphocytes.

Ascaris suum excretory/secretory products differentially modulate porcine dendritic cell subsets.

Helminths produce excretory/secretory products (E/S) which can modulate the immune responses of their hosts. Dendritic cells (DC) are essential for initiating the host T cell response and are thus potential targets for modulation by helminth E/S. Here we study immunomodulation of porcine peripheral blood DC subsets following ex vivo stimulation with E/S from Ascaris suum, a common helminth of pigs with considerable public health and economic importance. Our data showed that the relative frequencies of DC subsets in porcine blood differ, with plasmacytoid DC (pDC) being the most prominent in healthy 6-month-old pigs. pDC are an important cytokine source, and we found that A. suum E/S suppressed production of the type 1 cytokines IL-12p40 and TNF-α by this subset following toll-like receptor (TLR) ligation. In contrast, conventional DC (cDC) are more efficient antigen presenters, and the expression of CD80/86, costimulatory molecules essential for efficient antigen presentation, were modulated differentially by A. suum E/S between cDC subsets. CD80/86 expression by type 1 cDC (cDC1) following TLR ligation was greatly suppressed by the addition of A. suum E/S, while CD80/86 expression by type 2 cDC (cDC2) was upregulated by A. suum E/S. Further, we found that IFN-γ production by natural killer (NK) cells following IL-12 and IL-18 stimulation was suppressed by A. suum E/S. Finally, in the presence of E/S, IFN-γ production by CD4+ T cells co-cultured with autologous blood-derived DC was significantly impaired. Together, these data provide a coherent picture regarding the regulation of type 1 responses by A. suum E/S. Responsiveness of pDC and cDC1 to microbial ligands is reduced in the presence of E/S, effector functions of Th1 cells are impaired, and cytokine-driven IFN-γ release by NK cells is limited.

Delivery of antigen to porcine dendritic cells by fusing antigen with porcine dendritic cells targeting peptide.

Dendritic cells (DCs) are professional antigen-presenting cells that can recognize, capture, and process antigens. Fusing molecules targeting DCs with antigens can effectively improve the efficiency with which antigens are recognized and captured by DCs. This targeting strategy can be used for vaccine development to effectively improve the efficiency of antigen recognition and capture by DCs. The targeting sequence of porcine cytotoxic T-lymphocyte associated protein 4 (CTLA4), which binds porcine DCs, was identified in this study. Recombinant Lactobacillus reuteri (L. reuteri) expressing CTLA4-6aa (LYPPPY) and CTLA4-87aa fused to the porcine epidemic diarrhea virus (PEDV) protective antigen core neutralizing epitope (COE) were used to evaluate the ability of the two targeting motifs to bind the B7 molecule on DCs. Our results demonstrate that CTLA4-6aa could bind porcine DCs, and recombinant Lactobacillus expressing the CTLA4-6aa captured by porcine DCs was more efficient than those expressing CTLA4-87aa. In addition, the expression of DC markers, toll-like receptors, and cytokines was significantly higher in the 6aa-COE/L. reuteri-stimulated porcine DCs compared to DCs treated with 87aa-COE/L. reuteri (p<0.01) and recombinant Lactobacillus expressing CTLA4-6aa enhanced the ability of porcine DCs to activate T-cell proliferation. Our analysis of the protein structure revealed that CTLA4-87aa contains intramolecular hydrogen bonds, which may have weakened the intermolecular force between the residues on porcine CTLA4 and that on B7. In conclusion, recombinant Lactobacillus expressing CTLA4-6aa were more efficiently captured by porcine DCs and had a stronger ability to promote DC maturation and enhance T-cell proliferation. The LYPPPY motif is the optimal sequence for binding to porcine DCs. Piglets immunized with recombinant Lactobacillus showed that recombinant Lactobacillus expressing CTLA4-6aa induced significant levels of anti-PEDV-specific IgG and IgA antibody responses. Our study may promote research on DC-targeting strategies to enhance the effectiveness of porcine vaccines.

Mechanism of activation of porcine dendritic cells by an α-D-glucan nanoparticle adjuvant and a nanoparticle/poly(I:C) combination adjuvant.

Recent studies have shown that corn-derived cationic α-D-glucan nanoparticles, known as Nano-11, significantly increase the immune response when used as a vaccine adjuvant in mice and in pigs. Furthermore, the nanoparticles can be formulated with other immunostimulators such as poly(I:C), which further enhances the immune response. The current experiments were aimed at elucidating the mechanism of action of Nano-11 alone and in combination with poly(I:C). The effect of these adjuvants on porcine monocyte-derived dendritic cells (Mo-DCs) was determined by RNA-sequencing, supplemented with flow cytometry, cytokine analysis, and Western blots. Adsorption of poly(I:C) to Nano-11 reduced its cytotoxicity for Mo-DCs. Exposure of Mo-DCs to Nano-11 and Nano-11/poly(I:C) induced differential expression of 979 and 2016 genes, respectively. Gene Ontology enrichment and KEGG pathway analysis revealed many changes in gene expression related to inflammation, innate immunity, immune response to infections, and metabolism. Nano-11 and Nano-11/poly(I:C) induced maturation of the Mo-DCs as indicated by increased expression of costimulatory molecules and MHC II. Increased expression of genes downstream of p38 MAPK activation revealed a role for this signaling pathway in the activation of Mo-DCs by the adjuvants. This was confirmed by Western blot and inhibition of TNF-secretion upon incubation with the p38 inhibitor SB203580. These experiments provide insights into the mechanism of action of the novel adjuvants Nano-11 and Nano-11/poly(I:C).

Human dendritic cell targeting peptide can be targeted to porcine dendritic cells to improve antigen capture efficiency to stimulate stronger immune response.

Dendritic cells (DCs) play a key role in the natural recognition of pathogens and subsequent activation of adaptive immune responses due to their potent antigen-presenting ability. Dendritic cell-targeting peptide (DCpep) is strongly targeted to DCs, which often express antigens, to enhance the efficacy of vaccines. Our previous study showed that recombinant Lactobacillus expressing human DCpep could significantly induce stronger immune responses than recombinant Lactobacillus without DCpep, but the mechanism remains unclear. In this study, the mechanism by which DCpep enhances the immune response against recombinant Lactobacillus was explored. Fluorescence-labeled human DCpep was synthesized to evaluate the binding ability of human DCpep to porcine monocyte-derived dendritic cells (Mo-DCs) and DCs of the small intestine. The effects of Mo-DC function induced by recombinant Lactobacillus expressing human DCpep fused with the porcine epidemic diarrhea virus (PEDV) core neutralizing epitope (COE) antigen were also investigated. The results showed that human DCpep bind to porcine DCs, but not to porcine small intestinal epithelial cells. Human DCpep can also improve the capture efficiency of recombinant Lactobacillus by Mo-DCs, promote the maturation of dendritic cells, secrete more cytokines, and enhance the ability of porcine DCs to activate T-cell proliferation. Taken together, these results promote advanced understanding of the mechanism by which DCpep enhances immune responses. We found that some DCpeps are conserved between humans and pigs, which provides a theoretical basis for the development of a DC-targeted vaccine.

Effects of IFNγ and IL4 rich microenvironment on porcine monocyte-derived dendritic cell activation in vitro

Dendritic cells (DCs) represent a heterogeneous group of major antigen-presenting cells, responding to different stimuli in their microenvironment. They are able to activate naïve T-cells and drive their polarization towards effector types. However, the effect of different Th-polarizing cytokine microenvironment on porcine DCs remains poorly described. Therefore, the effect of IFNγ or IL4 rich microenvironment on porcine monocyte-derived dendritic cells and their activation, antigen recognition and cytokine secretion towards T-cell polarization were studied in vitro. IFNγ-rich microenvironment induced a higher proinflammatory response and release of Th1/Th17-polarizing cytokines (IL1β, IL12p35, IL23p19), while anti-inflammatory properties (IL10, TGFβ) was not affected by a cytokine microenvironment. From the achieved results, we propose that different cytokine microenvironment has the potential to modulate dendritic cells and their ability to activate T-cells.

Evaluation of the Effect of Inactivated Transmissible Gastroenteritis Virus Vaccine with Nano Silicon on the Phenotype and Function of Porcine Dendritic Cells.

A transmissible gastroenteritis virus (TGEV) is a porcine enteropathogenic coronavirus, causing acute swine enteric disease especially in suckling piglets. Mesoporous silica nanoparticles (MSNs) are safe vaccine adjuvant, which could enhance immune responses. Our previous research confirmed that nano silicon had immune-enhancing effects with inactivated TGEV vaccine. In this study, we further clarified the immune-enhancing mechanism of the inactivated TGEV vaccine with MSNs on porcine dendritic cells (DCs). Our results indicated that the inactivated TGEV vaccine with MSNs strongly enhanced the activation of the DCs. Expressions of TLR3, TLR5, TLR7, TLR9, and TLR10, cytokines IFN-α, IL-1β, IL-6, IL-12, and TNF-α, cytokine receptor CCR-7 of immature DCs were characterized and showed themselves to be significantly higher in the inactivated TGEV vaccine with the MSN group. In summary, the inactivated TGEV vaccine with MSNs has effects on the phenotype and function of porcine DCs, which helps to better understand the immune-enhancing mechanism.

Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation.

Containment of acute Toxoplasma gondii infection is dependent on an efficient interferon gamma response. However, the earliest steps of immune response initiation immediately following exposure to the parasite have not been previously characterized in pigs. Murine and human myeloid cells produce large quantities of interleukin (IL)-12 during early T. gondii infection. We therefore examined IL-12 expression by porcine peripheral blood monocytes and dendritic cell (DC) subsets following toll-like receptor (TLR) ligation and controlled T. gondii tachyzoite infection. We detected IL-12p40 expression by porcine plasmacytoid DC, but not conventional or monocyte-derived DC following TLR ligation. Unexpectedly, we also observed considerable IL-12p40 production by porcine CD3– NKp46+ cells—a classical natural killer cell phenotype—following TLR ligation. However, in response to T. gondii exposure, no IL-12 production was observed by either DC or CD3– NKp46+ cells. Despite this, IL-18 production by DC-enriched peripheral blood mononuclear cells was detected following live T. gondii tachyzoite exposure. Only combined stimulation of porcine peripheral blood mononuclear cells with recombinant IL-12p70 and IL-18 induced innate interferon gamma production by natural killer cells, while T cells and myeloid cells did not respond. Therefore, porcine CD3– NKp46+ cells serve as important IL-12 producers following TLR ligation, while IL-18 likely plays a prominent role in early immune response initiation in the pig following T. gondii infection.

Heterogeneous populations from in vitro cultures of antigen presenting cells in pigs

Dendritic cells (DCs) are the most potent antigen presenting cells (APCs). Because of the difficulty in obtaining these cells directly from tissues, different sources of DCs are frequently used for in vitro experimentation and many of their biological and functional characteristics were studied using these systems. Until recently, it was assumed that specific culture conditions polarized the differentiation of either DCs or macrophages (Macs); however, it was shown that some DC culture systems in other species generate heterogeneous cell populations that can be identified according to their CD11c and MHC class II (MHC-II) expression. Following this approach, porcine DCs were directly isolated from peripheral blood or differentiated in vitro by culturing bone marrow (BM) progenitor cells or blood monocytes treated with growth factors. Mostly homogeneous monocyte-derived DCs (MoDCs) were obtained with similar phenotype and phagocytic characteristics to that of blood DCs. On the contrary, BM-derived DC (BMDC) cultures generated two distinct heterogeneous populations identified as MHC-II+ and MHC-II++ cells. BMDCs MHC-II+ had similar phenotypic and phagocytic characteristics to those of MoDCs and blood DCs. However, BMDCs MHC-II++ population expressed a higher amount of surface markers and transcribed genes associated with Macs-lineage exhibiting a higher phagocytic capacity than all the other cells. Noteworthy, every cell system expressed different genetic signatures. These results will help interpreting and re-interpreting data obtained using in vitro systems.

Development of Pig Conventional Dendritic Cells From Bone Marrow Hematopoietic Cells in vitro.

In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, studying the interaction of porcine DCs with pathogens is still difficult due to the scarcity of DCs in tissues. In the present work, the Flt3-ligand (Flt3L)-based in vitro derivation system was further characterized and compared with other cytokine derivation models using a combination of factors: stem cell factor (SCF), GM-CSF, and IL-4. The method using Flt3L alone or combined with SCF supported the development of pig bone marrow hematopoietic cells into in vivo equivalent conventional DCs (cDCs). The equivalent cDC1 (the minor population in the cultures) were characterized as CADM1+CD14–MHC-II+CD172a–/loCD1–CD163– DEC205+CD11R3loCD11R1+CD33+CD80/86+. They expressed high levels of FLT3, ZBTB46, XCR1, and IRF8 mRNA, were efficient in endocytosing dextran and in proliferating allogenic CD4+CD8+ T cells, but were deficient in phagocyting inactivated Staphylococcus aureus (S. aureus). Also, after poly I:C stimulation, they predominantly produced IL-12p40a and matured as indicated by the increase of MHC-I, MHC-II, and CD80/86. The equivalent cDC2 (the main population) were CADM1+CD14–MHC-II+C D172a+CD1+CD163–/loDEC205loCD11R3+CD11R1+CD33+CD80/86+; meanwhile, they overexpressed FcεR1α and IRF4 mRNA. They showed high efficiency in the endocytosis of dextran, but weak in phagocytosing bacteria. They supported allogenic CD4+CD8–/CD4+CD8+ T cell proliferation and were high producers of IL-12p40 (upon TLR7 stimulation) and IL-10 (upon TLR7 stimulation). TLR ligand stimulation also induced their maturation. In addition, a CD14+ population was identified with the phenotype CADM1+CD14+MHC-II+CD172a+ CD1+CD163+DEC205–CD11R3+CD11R1+CD33–/loCD80/86+. They shared some functional similarities with cDC2 and were distinguishable from macrophages. This CD14+ population was efficient in phagocyting S. aureus but showed less maturation upon TLR ligand stimulation than cDC1 or cDC2. The alternative methods of DC derivation including GM-CSF and/or IL-4 produced mostly CADM1– cells that did not fulfill the canonical phenotype of bona fide porcine DCs. Our study provides an exhaustive characterization of Flt3L-derived DCs with different methods that can help the in vitro study of the interaction of DCs with porcine-relevant pathogens.

High-Resolution Profiling of Innate Immune Responses by Porcine Dendritic Cell Subsets in vitro and in vivo.

The present study investigated the transcriptomic response of porcine dendritic cells (DC) to innate stimulation in vitro and in vivo. The aim was to identify DC subset-specialization, suitable Toll-like receptor (TLR) ligands targeting plasmacytoid DC (pDC), and the DC activation profile during highly and low virulent classical swine fever virus (CSFV, strain Eystrup and Pinar del Rio, respectively) infection, chosen as model for a virus causing a severe immunopathology. After identification of porcine conventional DC (cDC) 1, cDC2, pDC and a monocyte-derived subset in lymphoid tissues, we characterized DC activation using transcriptomics, and focused on chemokines, interferons, cytokines, as well as on co-stimulatory and inhibitory molecules. We demonstrate that porcine pDC provide important signals for Th1 and interferon responses, with CpG triggering the strongest responses in pDC. DC isolated early after infection of pigs with either of the two CSFV strains showed prominent upregulation of CCL5, CXCL9, CXCL10, CXCL11, and XCL1, as well as of the cytokines TNFSF13B, IL6, IL7, IL12B, IL15, IL27. Transcription of IL12B and many interferon genes were mostly restricted to pDC. Interestingly, the infection was associated with a prominent induction of inhibitory and cell death receptors. When comparing low and highly virulent CSFV strains, the latter induced a stronger inflammatory and antiviral response but a weaker cell cycle response, and reduced antigen presentation functions of DC. Taken together, we provide high-resolution information on DC activation in pigs, as well as information on how DC modulation could be linked to CSFV immunopathology.

Heterogeneity of porcine bone marrow-derived dendritic cells induced by GM-CSF

In vitro generation of dendritic cells (DCs) is advantageous for overcoming the low frequency of primary DCs and the difficulty of applying isolation techniques for studying DC immunobiology. The culture of bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used extensively to generate bone marrow-derived dendritic cells (BMDCs). Studies have reported the heterogeneity of cells grown in murine GM-CSF culture based on the levels of MHCII expression. Although porcine DCs are generated by this classical method, the exact characteristics of the BMDC population have not yet been defined. In this study, we discriminated GM-CSF-grown BMDCs from gnotobiotic miniature pigs according to several criteria including morphology, phenotype, gene expression pattern and function. We showed that porcine BMDCs were heterogeneous cells that differentially expressed MHCII. MHCIIhigh cells displayed more representative of DC-like morphology and phenotype, including costimulatory molecules, as well as they showed a superior T cell priming capacity as compared to MHCIIlow cell. Our data showed that the difference in MHCIIhigh and MHCIIlow cell populations involved distinct maturation states rather than the presence of different cell types. Overall, characterization of porcine BMDC cultures provides important information about this widely used cellular model.

Membrane-bound and soluble porcine CD83 functions antithetically in T cell activation and dendritic cell differentiation in vitro

Emerging evidence suggests that CD83, a dendritic cells (DCs) maturation marker in humans and mice, may prossess immunomodulatory capacities. Although porcine CD83 shares ∼75% sequence homology with its human counterpart, whether it functions as an immunoregulatory molecule remains unknown. To investigate porcine CD83 function, we deleted it in porcine DCs by RNA intereference. Results show that membrane-bound CD83 (mCD83) promotes DC-mediated T cell proliferation and cytokine production, thus confirming its immunoregulatory capacity. Intriguingly, porcine soluble CD83 (sCD83) treatment instead led to inhibition of DC-mediated T cell activation. Moreover, porcine sCD83 also inhibited differentiation of prepheral blood mononuclear cells (PBMCs) into DCs. These results collectively indicate that in addition to being a DC maturation maker, both membrane bound and souble porcine CD83 serve as immunoregulatory molecules with opposite effects on DC-mediated T cell activation and DC differentiation.

Porcine Dendritic Cells and Viruses: An Update.

Several viral infections of swine are responsible for major economic losses and represent a threat to the swine industry worldwide. New tools are needed to prevent and control endemic, emerging, and re-emerging viral diseases. Dendritic cells (DC) play a central role in linking the innate and adaptive arms of the immune system, so knowledge regarding their interaction with pathogens is necessary to understand the mechanisms underlying diseases pathogenesis and protection. In the first part of this review, we provide an update on the heterogeneous cell subsets that comprise the porcine DC family. In the second part of this review, we provide an overview of how three viruses, affecting pork production at a global level, African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine circovirus 2 (PCV2), modulate DC function.

The Inflammatory Response to Enterotoxigenic E. coli and Probiotic E. faecium in a Coculture Model of Porcine Intestinal Epithelial and Dendritic Cells.

The gut epithelium constitutes an interface between the intestinal contents and the underlying gut-associated lymphoid tissue (GALT) including dendritic cells (DC). Interactions of intestinal epithelial cells (IEC) and resident DC are characterized by bidirectional crosstalk mediated by various factors, such as transforming growth factor-β (TGF-β) and thymic stromal lymphopoietin (TSLP). In the present study, we aimed (1) to model the interplay of both cell types in a porcine in vitro coculture consisting of IEC (cell line IPEC-J2) and monocyte-derived DC (MoDC) and (2) to assess whether immune responses to bacteria are altered because of the interplay between IPEC-J2 cells and MoDC. With regard to the latter, we focused on the inflammasome pathway. Here, we propose caspase-13 as a promising candidate for the noncanonical inflammasome activation in pigs. We conducted challenge experiments with enterotoxigenic Escherichia coli (ETEC) and probiotic Enterococcus faecium (E. faecium) NCIMB 10415. As potential mediators of IEC/DC interactions, TGF-β and TSLP were selected for analyses. Cocultured MoDC showed attenuated ETEC-induced inflammasome-related and proinflammatory interleukin (IL)-8 reactions compared with MoDC monocultures. Caspase-13 was more strongly expressed in IPEC-J2 cells cocultured with MoDC and upon ETEC incubation. We found that IPEC-J2 cells and MoDC were capable of releasing TSLP. The latter cells secreted greater amounts of TSLP when cocultured with IPEC-J2 cells. TGF-β was not modulated under the present experimental conditions in either cell types. We conclude that, in the presence of IPEC-J2 cells, porcine MoDC exhibited a more tolerogenic phenotype, which might be partially regulated by autocrine TSLP production. Noncanonical inflammasome signaling appeared to be modulated in IPEC-J2 cells. Our results indicate that the reciprocal interplay of the intestinal epithelium and GALT is essential for promoting balanced immune responses.

Effects of a pathogenic ETEC strain and a probiotic Enterococcus faecium strain on the inflammasome response in porcine dendritic cells

Dendritic cells (DC) are crucial for maintaining intestinal homeostasis and generating proper immune responses to bacteria occurring in the gut. Microbial stimuli can be recognized by intracellular receptors called inflammasomes, e.g., nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3). The aim of the present study was to unravel the inflammasome response of porcine monocyte-derived DC (MoDC). We investigated the capacity of probiotic Enterococcus faecium NCIMB 10415 (E. faecium) and enterotoxigenic Escherichia coli (ETEC) to elicit inflammasome activation. Since inflammasome activation normally requires a two-step process, MoDC were initially incubated with lipopolysaccharide (LPS) in order to prime cells. Primed and unprimed cells were then stimulated with the aforementioned bacterial strains. We also assessed whether preincubation with the probiotic prior to ETEC infection modified the immune response via the inflammasome pathway.Phenotypical analysis by flow cytometry showed that monocytes and MoDC expressed the surface markers CD14, CD16, and CD1 continuously, whereas swine leucocyte antigen (SLA) II was upregulated during differentiation.Following LPS priming, NLRP3, interleukin (IL)-1β and IL-18 mRNA expression, and IL-1β protein release increased. In unprimed cells, ETEC upregulated the expression of inflammasome components at later time points than in LPS-primed MoDC. Preincubation with the probiotic did not influence NLRP3 inflammasome activation in comparison with cells infected with ETEC alone.We conclude that ETEC, but not E. faecium, was able to stimulate inflammasome components in porcine MoDC. The present experimental conditions revealed no NLRP3 inflammasome-dependent protective effects of E. faecium during a pathogenic ETEC challenge.

Characterization of the attachment and infection by Porcine reproductive and respiratory syndrome virus 1 isolates in bone marrow-derived dendritic cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect porcine dendritic cells (DC). Previous studies indicated that different PRRSV1 isolates regulated differently the cytokine profiles and expression of surface molecules of DC. However, the characterisation of the infection is lacking. The current study aimed to characterise the replication and attachment of different PRRSV1 isolates in bone marrow-derived DC (BMDC). For this purpose, immature (i) and mature (m) BMDC were infected with three PRRSV1 isolates. The replication kinetics showed that titres in iBMDC were significantly (p < 0.05) higher than in mBMDC by 24 hpi, and for two isolates titres peaked earlier in iBMDC, suggesting that iBMDC were more efficient in supporting PRRSV1 replication than mBMDC. The attachment was revealed by a three-colour confocal microscopy staining. All three isolates were seen attached to iBMDC even in cells lacking CD163 -the essential receptor for PRRSV- or porcine sialoadhesin (PoSn). The attachment was not fully avoided after removal of heparan sulphate by heparinase I. Furthermore, the infection was examined with regards to CD163 expression. By flow cytometry and confocal microscopy, positive signals of PRRSV1 nucleocapsid could be observed in CD163− iBMDC. Additional sorting experiment demonstrated that CD163− iBMDC were infected only when CD163lo/hi cells were present. This can be interpreted in different ways: susceptible CD163− cells arose as result of milieu created by CD163+ infected BMDC; CD163− cells were infected by receptor-independent mechanisms (i.e. exosomes) or, some cells expressed CD163 at levels beyond the technical sensitivity.

Porcine monocyte-derived dendritic cells can be differentially activated by vesicular stomatitis virus and its matrix protein mutants

Vesicular stomatitis virus (VSV) can cause serious vesicular lesions in pigs, and the matrix (M) protein is its predominant virulence factor. Dendritic cells (DCs) act as the bridge between innate and adaptive immune responses. However, the susceptibility of porcine DCs to VSV infection and the role of M protein in modulating the function of infected DCs are still poorly defined. Thus, this study aimed to determine the ability of virulent wild-type VSV(wtVSV) and two attenuated M protein variants (VSVΔM51 and VSVMT) to induce maturation of porcine monocyte-derived DCs (MoDCs) in vitro. It was found that both wtVSV and the M protein mutant VSVs could productively replicate in porcine MoDCs. Infection with wtVSV resulted in weak proinflammatory cytokine responses and interfered with DC maturation via downregulation of the costimulatory molecule complex CD80/86. Whilst VSVΔM51 could activate porcine MoDCs, VSVMT, a highly attenuated recombinant VSV with triple mutations in the M protein, induced a potent maturation of MoDCs, as evidenced by efficient cytokine induction, and upregulation of CD80/86 and MHC class II. Overall, our findings reveal that porcine MoDCs are differentially activated by VSV, dependent on the presence of a functional M protein. M protein plays a crucial role in modulating porcine DC-VSV interactions. The data further support the potential use of VSVMT as a vaccine vector for pigs.

Characterization and expression of DEC205 in the cDC1 and cDC2 subsets of porcine dendritic cells from spleen, tonsil, and submaxillary and mesenteric lymph nodes

Conventional dendritic cells (cDCs) are divided into the following different subtypes: cDC1, which promotes a Th1 response, and cDC2, which stimulates a Th2 and Th17 response. These cells have not been characterized in porcine lymphoid tissues. DEC205 is a receptor that increases antigen presentation and allows DCs to cross-present antigens. The objectives of this work were to characterize cDCs subsets in the tonsil, submaxillary and mesenteric lymph nodes and spleen lymphoid tissues and to determine their expression of DEC205 by flow cytometry. The cDC1 (MHCIIhighCADM1highCD172a−/low) and cDC2 (MHCIIhighCADM1highCD172a+) phenotypes were confirmed by the expression of characteristic cDC1 and cDC2 transcripts (FLT3, XCR1 and FCER1α). Among all lymphoid tissues, the spleen had the highest frequency of total cDCs. The cDC1:cDC2 ratio showed that all lymph tissues had higher levels of cDC1 than levels of cDC2. DEC205+ cDCs were found in all analyzed tissues, albeit with different frequencies. Our research will facilitate the study on the function of these cells and the investigation of the strategies for DEC205 targeting and functional studies.