Porcine Monocytes Research Articles

ASFV infection induces macrophage necroptosis and releases proinflammatory cytokine by ZBP1-RIPK3-MLKL necrosome activation.

African swine fever (ASF) is an infectious disease characterized by hemorrhagic fever, which is highly pathogenic and causes severe mortality in domestic pigs. It is caused by the African swine fever virus (ASFV). ASFV is a large DNA virus and primarily infects porcine monocyte macrophages. The interaction between ASFV and host macrophages is the major reason for gross pathological lesions caused by ASFV. Necroptosis is an inflammatory programmed cell death and plays an important immune role during virus infection. However, whether and how ASFV induces macrophage necroptosis and the effect of necroptosis signaling on host immunity and ASFV infection remains unknown. This study uncovered that ASFV infection activates the necroptosis signaling in vivo and macrophage necroptosis in vitro. Further evidence showed that ASFV infection upregulates the expression of ZBP1 and RIPK3 to consist of the ZBP1-RIPK3-MLKL necrosome and further activates macrophage necroptosis. Subsequently, multiple Z-DNA sequences were predicted to be present in the ASFV genome. The Z-DNA signals were further confirmed to be present and colocalized with ZBP1 in the cytoplasm and nucleus of ASFV-infected cells. Moreover, ZBP1-mediated macrophage necroptosis provoked the extracellular release of proinflammatory cytokines, including TNF-α and IL-1β induced by ASFV infection. Finally, we demonstrated that ZBP1-mediated necroptosis signaling inhibits ASFV replication in host macrophages. Our findings uncovered a novel mechanism by which ASFV induces macrophage necroptosis by facilitating Z-DNA accumulation and ZBP1 necrosome assembly, providing significant insights into the pathogenesis of ASFV infection.

Porcine Monocyte DNA Traps Formed during Infection with Pathogenic Clostridioides difficile Strains.

Clostridioides (Clostridium) difficile is an enteric pathogen of several mammalian species including man, frequently involving nosocomial resurgence, following oral administration of broad-spectrum antibiotics, but also with human-to-human infection occurring, and neonatal pigs with zoonotic transmission. To date, the immune response to C. difficile has mostly focused on neutrophils and cytokine/chemokines, particularly in human infection. The neonatal pig is now recognized as a valuable model for human infection. We show that porcine monocytes respond to C. difficile differently compared with many other bacterial infections. Infection of porcine monocytes with human C. difficile strains CD630 (Ribotype 078) or R20291 (Ribotype 027) for 3 or 24 h post-infection (pi) resulted in a lack of oxidative burst or nitrite ion production when compared to uninfected controls (p > 0.05). The survival dynamics of both CD630 and R20291 in monocytes were similar with intracellular bacterial numbers being similar at 3 h pi and 24 h pi (p > 0.05). However, we show that porcine monocytes entrap C. difficile via extracellular DNA traps. This process began as early as 3 h pi, and at 24 h pi the nuclei appeared to be depleted of DNA, although extracellular DNA was associated with the cell membrane. Our preliminary study also suggests that entrapment of C. difficile by extracellular DNA may occur via a process of monocyte etosis.

Porcine milk exosomes modulate the immune functions of CD14+ monocytes in vitro

Exosomes mediate near and long-distance intercellular communication by transferring their molecular cargo to recipient cells, altering their biological response. Milk exosomes (MEx) are internalized by immune cells and exert immunomodulatory functions in vitro. Porcine MEx can accumulate in the small intestine, rich in macrophages. No information is available on the immunomodulatory ability of porcine MEx on porcine monocytes, which are known precursors of gut macrophages. Therefore, this study aims at (1) assessing the in vitro uptake of porcine MEx by porcine monocytes (CD14+), and (2) evaluating the in vitro impact of porcine MEx on porcine monocytes immune functions. MEx were purified by ultracentrifugation and size exclusion chromatography. The monocytes’ internalization of PKH26-labeled MEx was examined using fluorescence microscopy. Monocytes were incubated with increasing exosome concentrations and their apoptosis and viability were measured. Lastly, the ability of MEx to modulate the cells’ immune activities was evaluated by measuring monocytes’ phagocytosis, the capacity of killing bacteria, chemotaxis, and reactive oxygen species (ROS) production. MEx were internalized by porcine monocytes in vitro. They also decreased their chemotaxis and phagocytosis, and increased ROS production. Altogether, this study provides insights into the role that MEx might play in pigs’ immunity by demonstrating that MEx are internalized by porcine monocytes in vitro and exert immunomodulatory effects on inflammatory functions.

36 (P3-1). Marine-sulfated polysaccharides extracts exhibit contrasted time-dependent immunomodulatory and antiviral properties on porcine monocytes and alveolar macrophages

36 (P3-1). Marine-sulfated polysaccharides extracts exhibit contrasted time-dependent immunomodulatory and antiviral properties on porcine monocytes and alveolar macrophages

Butyrate modulation of inflammatory responses to lipopolysaccharide in porcine monocytes

Abstract Butyrate is a short-chain fatty acid produced through microbial fermentation of dietary fibers in the lower intestinal tract. Butyrate production is associated with enhanced mucosal immunity, increased barrier integrity, and modulation of immune cells. Butyrate driven immunomodulation occurs through a variety of mechanisms, including histone deacetylase (HDAC) inhibitor activity and inhibition of NF-kB signaling. Butyrate impacts lipopolysaccharide (LPS)-induced cytokine production and gene expression by porcine monocytes in a dose-dependent manner. To investigate the mechanisms driving butyrate modulation of porcine monocyte responses to LPS, peripheral monocytes were cultured with LPS +/− butyrate. After incubation, cells were processed for collection of cytoplasmic and nuclear fractions. HDAC activity in nuclear fractions and NF-kB signaling state (IkBa, p-IkBa, p65, p-p65) in cytoplasmic fractions was assessed. Butyrate did not inhibit HDAC activity after 1 hour (h) but at 24 h, butyrate exposure resulted in inhibition of HDAC activity regardless of LPS exposure, in a dose-dependent manner. Neither the low nor high concentration of butyrate altered IkBa, p-IkBa, p65, or p-p65 status, though p-IkBa and p-p65 status was dependent on LPS exposure. Overall, the impact of butyrate on HDAC activity was time and dose-dependent, and butyrate had little to no effect on NF-kB signaling induced by LPS exposure. Continued investigation is warranted to understand the potential mechanisms driving butyrate modulation of monocyte responses to LPS to enhance abilities to modulate immune status. USDA-ARS CRIS 005

PCV2 Uptake by Porcine Monocytes Is Strain-Dependent and Is Associated with Amino Acid Characteristics on the Capsid Surface.

Porcine circovirus type 2 (PCV2) is associated with several economically important diseases that are described as PCV2-associated diseases (PCVADs). PCV2 is replicating in lymphoblasts, and PCV2 particles are taken up by monocytes without effective replication or complete degradation. Glycosaminoglycans (GAGs) have been demonstrated to be important receptors for PCV2 binding and entry in T-lymphocytes and continuous cell lines. The objective of this study was to determine whether differences exist in viral uptake and outcome among six PCV2 strains from different disease outbreaks in primary porcine monocytes: Stoon-1010 (PCV2a; PMWS), 1121 (PCV2a; abortion), 1147 (PCV2b; PDNS), 09V448 (PCV2d-1; PCVAD with high viral load in lymphoid tissues [PCVADhigh]), DE222-13 (PCV2d-2; PCVADhigh), and 19V245 (PCV2d-2; PCVADhigh). The uptake of PCV2 in peripheral blood monocytes was different among the PCV2 strains. A large number of PCV2 particles were found in the monocytes for Stoon-1010, DE222-13, and 19V245, while a low number was found for 1121, 1147, and 09V448. Competition with, and removal of GAGs on the cell surface, demonstrated an important role of chondroitin sulfate (CS) and dermatan sulfate (DS) in PCV2 entry into monocytes. The mapping of positively/negatively charged amino acids exposed on the surface of PCV2 capsids revealed that their number and distribution could have an impact on the binding of the capsids to GAGs, and the internalization into monocytes. Based on the distribution of positively charged amino acids on PCV2 capsids, phosphacan was hypothesized, and further demonstrated, as an effective candidate to mediate virus attachment to, and internalization in, monocytes. IMPORTANCE PCV2 is present on almost every pig farm in the world and is associated with a high number of diseases (PCV2-associated diseases [PCVADs]). It causes severe economic losses. Although vaccination is successfully applied in the field, there are still a lot of unanswered questions on the pathogenesis of PCV2 infections. This article reports on the uptake difference of various PCV2 strains by peripheral blood monocytes, and reveals the mechanism of the strong viral uptake ability of monocytes of Piétrain pigs. We further demonstrated that: (i) GAGs mediate the uptake of PCV2 particles by monocytes, (ii) positively charged three-wings-windmill-like amino acid patterns on the capsid outer surface are activating PCV2 uptake, and (iii) phosphacan is one of the potential candidates for PCV2 internalization. These results provide new insights into the mechanisms involved in PCVAD and contribute to a better understanding of PCV2 evolution. This may lead to the development of resistant pigs.

African Swine Fever Virus Exhibits Distinct Replication Defects in Different Cell Types.

African swine fever virus (ASFV) causes one of the most devastating diseases affecting pigs and wild suids, a worldwide epizootic situation exacerbated in recent years due to the lack of vaccine or effective treatment. ASFV has a restricted cell tropism, and is prone to replicate in porcine monocytes and alveolar macrophages with high efficiency. Here, the replication capabilities of ASFV were examined in swine pulmonary alveolar macrophages (PAMs) and compared with 3D4/21, PK-15, MA-104 and Marc-145 cell lines using PCR, qPCR and Western blot with monoclonal antibodies against the viral p30 and p72 proteins. The results showed that ASFV has a variety of infection characteristics in PAMs and showed four cell lines with distinct defects during virus early transcription-translation, genome replication and late protein synthesis. Furthermore, an antiviral role of the stress granule pathway was revealed against ASFV, and ASFV infection inhibited stress granule formation in PAMs but not 3D4/21. These results will help to deepen our knowledge on ASFV infection and to develop ASFV susceptible cell lines.

Immunomodulatory effects of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) on porcine monocytes (CD14 +) immune response in vitro

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 long-chain polyunsaturated fatty acids (n-3 PUFA) found mostly in fish oil. They have been commonly used as dietary integrators in human and animal nutrition, modulating the immune system, mostly by exerting anti-inflammatory activities as demonstrated by in vivo and in vitro studies. The precise mechanisms of action at the background of EPA and DHA immunomodulatory activity are still not fully elucidated. Moreover, no information on their effects on porcine monocytes immune response is available yet. To cover this gap, the study aimed to evaluate DHA and EPA's in vitro impact on porcine monocytes (CD14 +) defensive functions. Briefly, monocytes were isolated from the blood of twenty-six healthy pigs, using a magnetic-activated cell sorting technique (MACS). Monocytes were first treated with increasing concentrations of DHA and EPA (25, 50, 100 and 200 µM) and apoptosis and viability were measured to assess potential cytotoxic effects. Once determined EPA and DHA subtoxic working concentrations (25, 50 and 100 µM), their effects on chemotaxis, phagocytosis and total, intracellular and extracellular reactive oxygen species (ROS) production were evaluated. DHA and EPA only decreased porcine monocytes viability at the highest concentration (200 µM), but their apoptosis was unaffected. DHA (100 µM) decreased the cells’ chemotaxis, while EPA (25 µM) increased their intracellular ROS production after 60 min under non-inflammatory or resting conditions and at 90 min under pro-inflammatory conditions (PMA challenge). EPA (50 µM) decreased monocytes’ intracellular ROS levels only under resting conditions at 30 min. No effects were observed on porcine monocytes phagocytic capacity. In conclusion, this study demonstrates that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes chemotaxis and potentiating their oxidative burst, respectively. Thus, our results suggest these n-3 PUFA might exert both anti-inflammatory and/or immune-enhancing effects in pigs.

Marine-Sulfated Polysaccharides Extracts Exhibit Contrasted Time-Dependent Immunomodulatory and Antiviral Properties on Porcine Monocytes and Alveolar Macrophages

Simple SummaryAlgal extracts have a real potential in terms of animal health, strengthening the interest in this natural resource. In pigs, respiratory complex syndrome significantly alters the wellbeing of the animals and threatens the economical sustainability of the sector. In the current study, we assessed various marine-sulfated polysaccharides (MSP®) extracts on two relevant cell populations in pigs, i.e., porcine monocytes and alveolar macrophages. Then, we analyzed the impact of the extracts on the infections of the cells by two important viruses. A modulation of the inflammatory response as well as some inhibitions of viral replication were observed. The type of effect observed was dependent on the extract, the experiment set-up, and the virus. The results obtained prompt us to further decipher the effects of algal extracts on the porcine health and open the door to future experiments, particularly in vivo experiments.Porcine respiratory complex syndrome has a strong economic impact on the swine breeding sector, as well as a clear repercussion on the wellbeing of the animals, leading to overuse of antimicrobial molecules. Algal extracts used in short-term treatments are empirically recognized by farmers as having a positive effect on pigs’ health, however, their mechanisms of action are not well known and more research is needed. Herein we studied the short and median term impact of three algal extracts, in vitro, on the pro-inflammatory and antiviral responses of porcine primary blood monocytes and alveolar macrophages, as well as the susceptibility of the treated cells to infection by Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) and the Aujeszky’s Disease Virus (ADV). All extracts presented a pro-inflammatory short-term effect, associated for two of them, with an inhibition of the PRRSV replication. Conversely, the three extracts presented an anti-inflammatory median term effect, with no impact on PRRSV replication. The observed immune modulation prompts us to test, in vivo, the anti-PRRSV action of algal extracts and strengthen the interest for this natural resource.

Synovial fluid profile dictates nanoparticle uptake into cartilage - implications of the protein corona for novel arthritis treatments

Drug delivery strategies for joint diseases need to overcome the negatively charged cartilage matrix. Previous studies have extensively investigated particle approaches to increase uptake efficiency by harnessing the anionic charge of the cartilage but have neglected to address potential interactions with the protein-rich biological environment of the joint space. We aimed to evaluate the effects of hard protein coronas derived from osteoarthritis (OA) and rheumatoid arthritis (RA) patient synovial fluids as well as the commonly used fetal calf serum (FCS) on nanoparticle (NP) uptake into tissues and cells. We developed a NP panel with varying PEGylation and incubated them with synovial fluid from either OA, RA patients or FCS. We evaluated the effects of the formed NP-biocorona complex uptake into the porcine articular cartilage explants, chondrocytes and monocyte cell lines and primary patient FLS cells. Proteins composing hard biocoronas were identified using a quantitative proteomics approach. Formed biocoronas majorly impacted NP uptake into cartilage tissue and dictated their uptake in chondrocytes and monocytes. The most suitable NP for potential OA applications was identified. A variety of proteins that were found on all NPs, irrespective of surface modifications. NP-, and protein-specific differences were also observed between the groups, and candidate proteins were identified that could account for the observed differences. This study demonstrates the impact of protein coronas from OA and RA patient synovial fluids on NP uptake into cartilage, emphasizing the importance of biological microenvironment considerations for successful translation of drug delivery vehicles into clinics.

D-Alanylation of Lipoteichoic Acids in Streptococcus suis Reduces Association With Leukocytes in Porcine Blood.

Streptococcus suis (S. suis) is a common swine pathogen but also poses a threat to human health in causing meningitis and severe cases of streptococcal toxic shock-like syndrome (STSLS). Therefore, it is crucial to understand how S. suis interacts with the host immune system during bacteremia. As S. suis has the ability to introduce d-alanine into its lipoteichoic acids (LTAs), we investigated the working hypothesis that cell wall modification by LTA d-alanylation influences the interaction of S. suis with porcine blood immune cells. We created an isogenic mutant of S. suis strain 10 by in-frame deletion of the d-alanine d-alanyl carrier ligase (DltA). d-alanylation of LTAs was associated with reduced phagocytosis of S. suis by porcine granulocytes, reduced deposition of complement factor C3 on the bacterial surface, increased hydrophobicity of streptococci, and increased resistance to cationic antimicrobial peptides (CAMPs). At the same time, survival of S. suis was not significantly increased by LTA d-alanylation in whole blood of conventional piglets with specific IgG. However, we found a distinct cytokine pattern as IL-1β but not tumor necrosis factor (TNF)-α levels were significantly reduced in blood infected with the ΔdltA mutant. In contrast to TNF-α, activation and secretion of IL-1β are inflammasome-dependent, suggesting a possible influence of LTA d-alanylation on inflammasome regulation. Especially in the absence of specific antibodies, the association of S. suis with porcine monocytes was reduced by d-alanylation of its LTAs. This dltA-dependent phenotype was also observed with a non-encapsulated dltA double mutant indicating that it is independent of capsular polysaccharides. High antibody levels caused high levels of S. suis—monocyte—association followed by inflammatory cell death and strong production of both IL-1β and TNF-α, while the influence of LTA d-alanylation of the streptococci became less visible. In summary, the results of this study expand previous findings on d-alanylation of LTAs in S. suis and suggest that this pathogen specifically modulates association with blood leukocytes through this modification of its surface.

CD200R family receptors are expressed on porcine monocytes and modulate the production of IL-8 and TNF-α triggered by TLR4 or TLR7 in these cells

The inhibitory receptor CD200R1 and its paired activating receptor CD200R1L are involved in the regulation of myeloid cell immune responses. The aim of this study was to analyze their distribution, regulation by cytokines, and function in porcine monocyte subsets. We had previously observed that CD200R1 and CD200R1L genes can generate different protein isoforms through alternative mRNA splicing, therefore in this study, we explored the diversity of transcripts in monocyte subsets, and described several new splicing variants of both CD200R1 and CD200R1L, some of which could be expressed on the porcine monocyte surface. A substantial proportion of CD163-SLAII+ and most CD163+SLAII+ monocytes expressed CD200R1 and CD200R1L receptors, while CD163-SLAII- monocytes did not. CD200R1 and CD200R1L expression was down-regulated in monocytes polarized by IFN-ɣ, a cytokine that induces classical activation of macrophages, while IL-10 which gives rise to regulatory macrophages, increased the expression of CD200R1. Finally, treatment of monocyte subsets with a monoclonal antibody specific for the inhibitory CD200R1 receptor and its splicing variants enhanced TNFα and IL-8 production, induced by TLR4 or TLR7 stimulation, suggesting a modulatory role for these receptors on porcine monocyte functions.

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.

Butyrate alters lipopolysaccharide-induced inflammatory response by porcine monocytes

Abstract Continuous, low-level inflammation can have a negative impact on intestinal health. Butyrate is a short-chain fatty acid produced in the lower intestinal tract by microbial fermentation of dietary fibers and modulates immune cell responses through various mechanisms. Blood monocytes migrate into the intestine and respond to signals, including potential modulation by butyrate or in response to microbial components, such as lipopolysaccharide (LPS). To investigate the impact of butyrate on inflammatory response to LPS, porcine monocytes were either cultured with a combination of LPS and varying butyrate concentrations or pre-treated with butyrate before LPS exposure. Independent of butyrate, LPS stimulation of monocytes induced IL-1β and TNF production. Monocytes cultured just three hours (h) prior to LPS stimulation had reduced overall cytokine production, suggesting rapid phenotypic changes in monocytes. Butyrate had varying effects on the amount of cytokine produced in response to LPS. Specifically, low butyrate concentrations (0.25 mM) limited IL-1β production, but did not impact TNF production. Higher butyrate concentrations (> 4 mM) limited TNF production to LPS stimulation. Pre-treatment of monocytes with butyrate for 3 h prior to LPS stimulation altered IL-1β production. Pre-treatment with 0.25 mM butyrate did not limit IL-1β production, but concentrations > 4 mM did. In conclusion, reduction of LPS-induced cytokine production by porcine monocytes was dependent on butyrate concentration and exposure time relative to LPS exposure. Further work is ongoing to understand the mechanism of butyrate modulation of pig monocytes and inflammatory response to LPS for potential application in vivo.

β-Glucan-Induced IL-10 Secretion by Monocytes Triggers Porcine NK Cell Cytotoxicity.

Beta-glucans are naturally occurring polysaccharides present in cell walls of fungi, yeast, bacteria, cereals, seaweed, and algae. These microbe-associated molecular patterns (MAMPs) possess immunomodulatory properties. In human, it has been suggested that NK cells can be activated by β-glucans. Here, we aimed to elucidate whether β-glucans modulate porcine NK cell responses in vitro and if so, how these effects are mediated. We investigated the effect of two β-glucans, Macrogard and Curdlan, which differ in solubility and structure. Direct addition of β-glucans to purified porcine NK cells did not affect cytotoxicity of these cells against K562 target cells. However, when using PBMC instead of purified NK cells, β-glucan addition significantly increased NK cell-mediated cytotoxicity. This effect depended on factors secreted by CD14+ monocytes upon β-glucan priming. Further analysis showed that monocytes secrete TNF-α, IL-6, and IL-10 upon β-glucan addition. Of these, IL-10 turned out to play a critical role in β-glucan-triggered NK cell cytotoxicity, since depletion of IL-10 completely abrogated the β-glucan-induced increase in cytotoxicity. Furthermore, addition of recombinant IL-10 to purified NK cells was sufficient to enhance cytotoxicity. In conclusion, we show that β-glucans trigger IL-10 secretion by porcine monocytes, which in turn leads to increased NK cell cytotoxicity, and thereby identify IL-10 as a potent stimulus of porcine NK cell cytotoxicity.

Differential induction of innate memory in porcine monocytes by β-glucan or bacillus Calmette-Guerin.

Innate immunomodulation via induction of innate memory is one mechanism to alter the host’s innate immune response to reduce or prevent disease. Microbial products modulate innate responses with immediate and lasting effects. Innate memory is characterized by enhanced (training) or depressed (tolerance) innate immune responses, including pro-inflammatory cytokine production, to secondary exposure following a priming event. To investigate the ability of β-glucans and bacillus Calmette-Guerin to induce innate training or tolerance in pig cells, porcine monocytes were cultured with priming agonist (β-glucans or bacillus Calmette-Guerin) then re-stimulated 5 d later with a heterologous microbial agonist to determine induction of innate memory. Priming with β-glucan from Saccharomyces cerevisiae depressed IL-1β and TNF-α cytokine responses to re-stimulation with LPS, indicative of a tolerized state. However, bacillus Calmette-Guerin priming induced a trained state in porcine monocytes, as LPS re-stimulation enhanced IL-1β and TNF-α gene expression and protein production. We present the first evidence of innate memory in pig monocytes, with bacillus Calmette-Guerin (training) or Saccharomyces cerevisiae β-glucan (tolerance). Induction of a trained or tolerized state in vitro is a first step to identify agonists to alter the innate immune system at the animal level with the intent of enhancing disease resistance.

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.

Expression analysis of LILRB3, &lt;I&gt;LAIR1&lt;/I&gt; and &lt;I&gt;LXRA&lt;/I&gt; genes in porcine monocytes stimulated with Salmonella LPS

Expression analysis of LILRB3, &lt;I&gt;LAIR1&lt;/I&gt; and &lt;I&gt;LXRA&lt;/I&gt; genes in porcine monocytes stimulated with Salmonella LPS

Interaction of historical and modern Sardinian African swine fever viruses with porcine and wild-boar monocytes and monocyte-derived macrophages

African swine fever (ASF) is a contagious viral disease of wild and domestic pigs that is present in many parts of Africa, Asia and Europe, including Sardinia (Italy). Deletions in the EP402R and B602L genes have been found in almost all ASF virus (ASFV) strains circulating in Sardinia from 1990 onwards, and modern Sardinian strains (isolated after 1990) might have acquired some selective advantage compared to historical ones (isolated before 1990). Here, we analysed the host cell responses of wild boars and domestic pigs upon infection with virus variants. Higher intracellular levels of the late protein p72 were detected after infection with the modern strain 22653/14 compared to the historical strain Nu81.2, although both isolates grew at the same rate in both monocytes and monocyte-derived macrophages. Higher cytokine levels in the supernatants of ASFV-infected pig monocytes compared to pig macrophages and wild-boar cells were detected, with no differences between isolates.

Breed Differences in PCV2 Uptake and Disintegration in Porcine Monocytes.

Porcine circovirus type 2 (PCV2) is associated with various diseases which are designated as PCV2-associated diseases (PCVADs). Their severity varies among breeds. In the diseased pigs, virus is present in monocytes, without replication or full degradation. PCV2 entry and viral outcome in primary porcine monocytes and the role of monocytes in PCV2 genetic susceptibility have not been studied. Here, virus uptake and trafficking were analyzed and compared among purebreds Piétrain, Landrace and Large White and hybrid Piétrain × Topigs20. Viral capsids were rapidly internalized into monocytes, followed by a slow disintegration to a residual level. PCV2 uptake was decreased by chlorpromazine, cytochalasin D and dynasore. The internalized capsids followed the endosomal trafficking pathway, ending up in lysosomes. PCV2 genome was nicked by lysosomal DNase II in vitro, but persisted in monocytes in vivo. Monocytes from purebred Piétrain and the hybrid showed a higher level of PCV2 uptake and disintegration, compared to those from Landrace and Large White. In conclusion, PCV2 entry occurs via clathrin-mediated endocytosis. After entry, viral capsids are partially disintegrated, while viral genomes largely escape from the pathway to avoid degradation. The degree of PCV2 uptake and disintegration differ among pig breeds.