Porcine Monocyte-derived Dendritic Cells Research Articles

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.

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.

Epigenetic Modulation of TLR4 Expression by Sulforaphane Increases Anti-Inflammatory Capacity in Porcine Monocyte-Derived Dendritic Cells.

Simple SummaryEpigenetic modifications of the genes regulate the inflammation process that includes the DNA methylation and histone acetylation. Sulforaphane is well known for its immunomodulatory properties. Notably, the mechanism of its anti-inflammatory functions involving epigenetic modifications is unclear. This study highlighted the regulatory mechanism of sulforaphane in the innate immunity responses in an acute inflammatory state employ in vivo cell culture model. Porcine monocyte-derived dendritic cells were exposed to LPS with or without sulforaphane pre-treatment for these purposes. Epigenetics modulations of the important genes and regulatory factors were studies as well as the immune responses of the cells were vigorously studied over the period of time. This study deciphers the mechanism of SFN in restricting the excessive inflammatory reactions, thereby, exerting its protective and anti-inflammatory function though epigenetic mechanism.Inflammation is regulated by epigenetic modifications, including DNA methylation and histone acetylation. Sulforaphane (SFN), a histone deacetylase (HDAC) inhibitor, is also a potent immunomodulatory agent, but its anti-inflammatory functions through epigenetic modifications remain unclear. Therefore, this study aimed to investigate the epigenetic effects of SFN in maintaining the immunomodulatory homeostasis of innate immunity during acute inflammation. For this purpose, SFN-induced epigenetic changes and expression levels of immune-related genes in response to lipopolysaccharide (LPS) stimulation of monocyte-derived dendritic cells (moDCs) were analyzed. These results demonstrated that SFN inhibited HDAC activity and caused histone H3 and H4 acetylation. SFN treatment also induced DNA demethylation in the promoter region of the MHC-SLA1 gene, resulting in the upregulation of Toll-like receptor 4 (TLR4), MHC-SLA1, and inflammatory cytokines’ expression at 6 h of LPS stimulation. Moreover, the protein levels of cytokines in the cell culture supernatants were significantly inhibited by SFN pre-treatment followed by LPS stimulation in a time-dependent manner, suggesting that inhibition of HDAC activity and DNA methylation by SFN may restrict the excessive inflammatory cytokine availability in the extracellular environment. We postulate that SFN may exert a protective and anti-inflammatory function by epigenetically influencing signaling pathways in experimental conditions employing porcine moDCs.

Lactobacillus johnsonii activates porcine monocyte derived dendritic cells maturation to modulate Th cellular immune response

Lactobacilli are abundant in the intestinal tract where they constantly regulate immune system via interacting with a great diversity of immune cells, such as dendritic cells (DCs). Notably, DCs are powerful antigen-presenting cells and they are capable of initiating primary immune responses. In this study, we studied the effects of Lactobacillus johnsonii (L. johnsonii) and Lactobacillus johnsonii cell-free supernatant (L. johnsonii-CFS) on the activation of porcine monocyte-derived dendritic cells (MoDCs) and their regulation of Th cellular immune responses in vitro. The MoDCs generated from porcine peripheral blood monocytes were stimulated by L. johnsonii and L. johnsonii-CFS, respectively. Pre-incubation with L. johnsonii increased expression of CD172a, CD80, major histocompatibility complex class II (MHCII) in MoDCs, and enhanced the ability of MoDCs to induce the proliferation of CD4+ T cell, while pre-incubation with L. johnsonii-CFS merely upregulated the expression of MHCII. Analysis of the cytokines showed that L. johnsonii stimulated up-regulation of Th1-type cytokines (IL-12p40, IFN-γ, TNF-α), pro-inflammatory cytokine IL-1β, chemokine CCL20, and Treg-type / anti-inflammatory cytokines IL-10 in MoDCs. Notably, a high production of IL-10 was observed in the MoDCs treated with L. johnsonii-CFS, indicating L. johnsonii-CFS exerted anti-inflammatory effects. Furthermore, L. johnsonii induced up-regulation of TLR2 and TLR6, but L. johnsonii-CFS not. Moreover, MoDCs stimulated by L. johnsonii mainly promoted T cell differentiate into Th1/Th2/Treg cells and plays an important role in improving the balance between Th1/Th2/Treg-type cells, whereas MoDCs stimulated by L. johnsonii-CFS mainly directed T cell to Th2/Treg subset polarization. In conclusion, L. johnsonii and L. johnsonii-CFS exhibited the ability of modulating innate immunity by regulating immunological functions of MoDCs in vitro, suggesting their potential ability to use as microecological preparations and medicines.

A Nanoparticle-Poly(I:C) Combination Adjuvant Enhances the Breadth of the Immune Response to Inactivated Influenza Virus Vaccine in Pigs.

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-α and IL-1ß cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFNγ secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response.

Corrigendum to “Effects of inactivated porcine epidemic diarrhea virus on porcine monocyte-derived dendritic cells and intestinal dendritic cells” [Research in Veterinary Science 106 (2016) 149–158

Corrigendum to “Effects of inactivated porcine epidemic diarrhea virus on porcine monocyte-derived dendritic cells and intestinal dendritic cells” [Research in Veterinary Science 106 (2016) 149–158

The Probiotic BB12 Induces MicroRNAs Involved in Antigen Processing and Presentation in Porcine Monocyte-Derived Dendritic Cells.

MicroRNAs (miRNAs) mediate the regulation of gene expression. Several reports indicate that probiotics induce miRNA-mediated immunomodulation at different levels, such as cytokine production and the up-regulation of several markers related to antigen presentation in antigen-presenting cells. The objective of this work was to identify target genes of miRNAs that are involved in the processing and presentation of antigens in monocyte-derived dendritic cells (moDCs) stimulated with the probiotic Bifidobacterium animalis ssp. lactis BB12 (BB12). First, an in silico prediction analysis for a putative miRNA binding site within a given mRNA target was performed using RNAHybrid software with mature sequences of differentially expressed miRNAs retrieved from a Genbank data set that included BB12-stimulated and unstimulated porcine monocytes. From them, 23 genes resulted in targets of 19 miRNAs, highlighting miR-30b-3p, miR-671-5p, and miR-9858-5p, whose targets were costimulatory molecules, and were overexpressed (p < 0.05) in BB12-stimulated moDCs. The analysis of moDCs showed that the percentage of cells expressing SLA-DR+CD80+ decreased significantly (p = 0.0081) in BB12-stimulated moDCs; interleukin (IL)-10 production was unchanged at 6 h but increased after 24 h of culture in the presence of BB12 (p < 0.001). In summary, our results suggest that SLA-DR and CD80 can be down-regulated by miRNAs miR-30b-3p, miR-671-5p, and miR-9858-5p, while miR-671-5p targets IL-10.

Inflammatory Responses of Porcine MoDC and Intestinal Epithelial Cells in a Direct-Contact Co-culture System Following a Bacterial Challenge.

Intestinal epithelial cells (IEC) and immune cells, such as dendritic cells (DC), jointly control the immune response towards luminal pathogens in the intestinal mucosa. Crosstalk between IEC and DC is crucial for coordinating immune responses and occurs via soluble factors and direct cell-cell contacts. The present study aimed at establishing a direct-contact co-culture model of porcine IEC and DC to mimic these interactions. The effects of (1) co-cultivation of the two cell types and (2) bacterial infection on the inflammatory response patterns of each of the cell types were determined with a special focus on the canonical and non-canonical inflammasome signaling pathways. In infection experiments, in vitro cultures were exposed to either the probiotic Enterococcus (E.) faecium NCIMB 10415 or enterotoxigenic Escherichia coli (ETEC). In porcine IEC (IPEC-J2), co-cultivation with porcine monocyte-derived DC (MoDC) resulted in reduced basal NLRP3 (nucleotide oligomerization domain [NOD]-like receptor [NLR] family, pyrin domain containing 3) inflammasome mRNA levels in unstimulated conditions. In porcine MoDC, the presence of IPEC-J2 cells evoked a noticeable decrease of interleukin (IL)-8 and transforming growth factor-β (TGF-β) mRNA and protein expression. ETEC, in contrast to E. faecium, modulated the inflammasome pathway in IPEC-J2 cells and porcine MoDC. Co-cultured IPEC-J2 cells showed an augmented inflammasome response to ETEC infection. By contrast, MoDC revealed a weakened ETEC response under such co-culture conditions as indicated by a reduction of inflammasome-related IL-1β protein release. Our data indicate that the close contact between IEC and resident immune cells has a major effect on their immunological behavior.

Monocyte-Derived Dendritic Cells as Model to Evaluate Species Tropism of Mosquito-Borne Flaviviruses.

Several mosquito-borne Flaviviruses such as Japanese encephalitis virus (JEV), West Nile virus (WNV), Dengue Virus (DENV), and Zika virus (ZIKV) can cause severe clinical disease. Being zoonotic, Flaviviruses infect a wide variety of terrestrial vertebrates, which dependent of the virus-host interactions, can enhance ongoing epidemics and maintain the virus in the environment for prolonged periods. Targeted species can vary from amphibians, birds to various mammals, dependent on the virus. For many mosquito-borne flaviviruses the spectrum of targeted species is incompletely understood, in particular with respect to their contribution to the maintenance of virus in certain geographical regions. Furthermore, little is known about virus and host factors contributing to species tropism. The present study utilized human and porcine monocyte-derived dendritic cells (MoDC) as a cell culture model to better understand Flavivirus species tropism and innate immune responses. MoDC were selected based on their presence in the skin and their role as an early target cell for several Flaviviruses and their role as immune sentinels. While differences in viral infectivity and replication were minor when comparing porcine with human MoDC for some of the tested Flaviviruses, a particularly strong replication in human MoDC was found with USUV, while JEV appeared to have a stronger tropism for porcine MoDC. With respect to innate immune responses we found high induction of TNF and IFN-β in both human and porcine MoDC after infection with JEV, WNV, and USUV, but not with DENV, ZIKV, and Wesselsbron virus. Spondweni virus induced these cytokine responses only in porcine MoDC. Overall, innate immune responses correlated with early infectivity and cytokine production. In conclusion, we demonstrate Flavivirus-dependent differences in the interaction with MoDC. These may play a role in pathogenesis but appear to only partially reflect the expected species tropism.

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.

Oral administration of inactivated porcine epidemic diarrhea virus activate DCs in porcine Peyer\u2019s patches

BackgroundPeyer’s patches (PPs) can be considered as the immune site of the intestine. Within PPs, Dendritic cells (DCs) can uptake antigens from the gut lumen by extending dendrites into epithelium, and process it and then present to lymphocytes, which effectively antigen produces an immune response. Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), an acute and highly contagious enteric viral disease. The interaction between inactivated porcine epidemic diarrhea virus and porcine monocyte-derived dendritic cells (Mo-DCs) has been reported. However, little is known about the interaction between inactivated PEDV and DCs in porcine PPs.ResultsIn this study, for the first time we investigated the role of DCs in porcine PPs after oral administration inactivated PEDV. Firstly, a method to isolate DCs from porcine PPs was established, in which the purity of SWC3a+/MHC-II+ DCs was more than 90%. Our findings clearly indicate that DCs in porcine PPs after oral administration of inactivated PEDV not only stimulated the proliferation of allogeneic lymphocytes, but also secreted cytokines (IL-1, IL-4). Furthermore, the number of DCs and IgA+ cells in porcine intestinal mucosal significantly increased and the levels of anti-PEDV specific IgG antibody in the serum and SIgA antibody in the feces increased after oral administration inactivated PEDV.ConclusionsOur findings indicate that oral administration of inactivated PEDV activate DCs in porcine Peyer’s patches and inactivated PEDV may be a useful and safe vaccine to trigger adaptive immunity.

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.

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.

Interaction of porcine monocyte-derived dendritic cells with African swine fever viruses of diverse virulence

African swine fever (ASF) is a devastating disease for which there is no vaccine. The ASF virus (ASFV) can infect dendritic cell (DC), but despite the critical role these cells play in induction of adaptive immunity, few studies investigated their response to ASFV infection. We characterized the in vitro interactions of porcine monocyte-derived DCs (moDC) with a virulent (22653/14), a low virulent (NH/P68) and an avirulent (BA71V) ASFV strain. At a high multiplicity of infection (MOI = 1), all three strains infected immature moDC. Maturation of moDC, with IFN-α/TNF-α, increased susceptibility to infection with 22653/14 and other virulent strains, but reduced susceptibility to NH/P68 and BA71V. The reduced moDC susceptibility to BA71V/NH/P68 was IFN-α mediated, whereas increased susceptibility to 22653/14 was induced by TNF-α. Using an MOI of 0.01, we observed that BA71V replicated less efficiently in moDC compared to the other isolates and we detected increased replication of NH/P68 compared to 22653/14. We observed that BA71V and NH/P68, but not 22653/14, downregulated expression of MHC class I on infected cells. All three strains decreased CD16 expression on moDC, whereas ASFV infection resulted in CD80/86 down-regulation and MHC class II DR up-regulation on mature moDC. None of the tested strains induced a strong cytokine response to ASFV and only modest IL-1α was released after BA71V infection. Overall our results revealed differences between strains and suggest that ASFV has evolved mechanisms to replicate covertly in inflammatory DC, which likely impairs the induction of an effective immune response.

Porcine epidemic diarrhea virus does not replicate in porcine monocyte-derived dendritic cells, but activates the transcription of type I interferon and chemokine

Porcine epidemic diarrhea virus (PEDV) belongs to the alphacoronavirus of the Coronaviridae. It is the major etiological agent of the recent outbreaks of piglet diarrhea and death in the US. Limited knowledge is currently available regarding the role of dendritic cells in PEDV infection. Here, we observed that PEDV did not replicate in monocyte-derived dendritic cells as evidenced by the decrease of viral gene transcript copies in infected cells by qRT-PCR and the absence of viral proteins by immunofluorescence staining as well as the absence of virus particles in infected cells by transmission electron microscopy. In addition, PEDV did not compromise cell viability at 48, 72, and 96h after infection at either a MOI of 2.5 or 5. Interestingly, an increased transcription of type I interferon including interferon-α and β was observed in infected cells compared to mock infected cells. Surprisingly, we did not detect any interferon-β in the supernatants of infected cells. A slight increase in interferon-α protein production in the supernatants of PEDV-infected cells was observed compared to mock infected cells. We also observed a markedly increased transcription of interferon inducible protein −10 (IP-10). Overall, PEDV does not replicate in porcine Mo-DC, but activates the transcription of type I interferon and chemokine IP-10.

Species-specific immunity to Helicobacter suis.

Helicobacter (H.) suis is mainly associated with pigs, but is also the most prevalent gastric non-H.pylori Helicobacter species found in humans. Both H.pylori and H.suis may cause persistent infection of the stomach. Several immune evasion mechanisms have been proposed for H.pylori, which focus to a great extent on its major virulence factors, which are absent in H.suis. The aim of this study was to gain more knowledge on immune evasion by H.suis. Cytokine expression kinetics were monitored in the stomach of BALB/c mice experimentally infected with H.suis. The cytokine expression profile in the stomach of naturally H.suis-infected pigs was also determined. Subsequently, the effect of H.suis on murine and porcine dendritic cell (DC) maturation and their ability to elicit T-cell effector responses was analyzed. Despite a Th17/Th2 response in the murine stomach, the inflammatory cell influx was unable to clear H.suis infection. H.suis-stimulated murine bone marrow-derived dendritic cells induced IL-17 secretion by CD4+ cells in vitro. Natural H.suis infection in pigs evoked increased expression levels of IL-17 mRNA in the antrum and IL-10 mRNA in the fundus. In contrast to mice, H.suis-stimulated porcine monocyte-derived dendritic cells were unable to express MHCII molecules on their cell surface. These semimature DCs induced proliferation of T-cells, which showed an increased expression of TGF-β and FoxP3 mRNA levels. Helicobacter suis might evade host immune responses by skewing toward a Treg-biased response.

Effects of arsenic on porcine dendritic cells in vitro

Exposure to arsenic (As) is an ongoing, and in some places increasing, health problem. Still, however, the effects of As exposure on the immune system are not well understood. Dendritic cells (DC) are a critical immune cell that bridges the innate and adaptive immune systems. To determine the impact of inorganic (i)As exposure on DC, the effects of (geo)anthropogenically relevant levels of NaAsO2 on the function of porcine monocyte-derived DC (MoDC) were evaluated in an in vitro model. The results showed a low dose of iAs reduced the phagocytic capacity of MoDC. Furthermore, although surface expression of DC activation markers, such as major histocompatibility complex (MHC)-II, CD80/86, CD40 and CD25, were only slightly changed, MoDC T-cell proliferation-inducing capacity was remarkably diminished by iAs treatment. Additionally, iAs induced significant interleukin (IL)-6 secretion by MoDC after 12- or 24-h incubation, whereas IL-1β secretion was only significantly up-regulated after 12 h. The secretion patterns of IL-8, tumor necrosis factor α (TNFα and IL-10 by iAs-treated MoDC were almost similar to that by mock-treated MoDC. Considering the broad roles of DC in immunobiology, this finding deepens the understanding of molecular mechanisms/functional consequences underpinning the immunopathology, inflammation, and increases in infection arising from As exposure.

Generation of potent porcine monocyte-derived dendritic cells (MoDCs) by modified culture protocol

In vitro derivation of dendritic cells (DCs) is an alternative approach to overcome the low frequency of primary DCs and the difficulty of isolation techniques for studying DC immunobiology. To date, the conventional culture protocol of porcine monocyte-derived DCs (MoDCs) has been widely used. However, this protocol is not practical due to the requirement of a substantial number of blood monocytes, and the process often interferes with DC maturation. To improve in vitro porcine MoDC generation, we modified the previous conventional DC generation protocol, based on the human and mouse primary DC culture system, and compared phenotypic and functional features of MoDCs derived from the modified protocol to the conventional protocol. The modified protocol consumed fewer monocytes but generated higher CD1+ cells with DC-like morphology and the ability of maturation. In addition, MoDCs from the modified protocol exhibited increased antigen uptake and IFN-γ production in response to LPS stimulation. Our findings indicate that the modified protocol is expedient and reliable for generating potent MoDCs that substitute for primary DCs. This will be a valuable platform for future research in antigen delivery, vaccines and immunotherapy in pigs, as well as relevant veterinary species.