The low-virulent African swine fever virus (ASFV/NH/P68) induces enhanced expression and production of relevant regulatory cytokines (IFNα, TNFα and IL12p40) on porcine macrophages in comparison to the highly virulent ASFV/L60

The impact of infection by the low-virulent ASFV/NH/P68 (NHV) and the highly virulent ASFV/L60 (L60) isolates on porcine macrophages was assessed through the quantification of IFNα, TNFα, IL12p40, TGFβ and ASFV genes by real-time PCR at 2, 4 and 6 h post-infection. Increased IFNα, TNFα and IL12p40 expression was found in infection with NHV, in which expression of TGFβ was lower than in infection with L60. Principal component analysis showed a positive interaction of cytokines involved in cellular immune mechanisms, namely IFNα and IL12p40 in the NHV infection. Quantification by ELISA confirmed higher production of IFNα, TNFα and IL12p40 in the NHV-infected macrophages. Overall, our studies reinforce and clarify the effect of the NHV infection by targeting cellular and cellular-based immune responses relevant for pig survival against ASFV infection

Effects of Astragalus Polysaccharide on Immune Responses of Porcine PBMC Stimulated with PRRSV or CSFV

BACKGROUND: Astragalus polysaccharide (APS) has been used as an immunomodulator that can enhance immune responses, whereas the immunomodulatory effects of APS on porcine peripheral blood mononuclear cells (PBMCs) exposed to porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) have not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: Porcine PBMCs were cultured in complete RPMI media in the presence of the R98-strain of PRRSV (5×10(4) TCID(50)/ml) or C-strain of CSFV (10(3) TCID(50)/ml) with or without APS. The expression of mRNA for CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), transforming growth factor-β (TGF-β), interleukin 2 (IL-2) and IL-10 was assayed by TaqMan real-time RT-PCR. The expression of mRNA for CD28 and CTLA-4 increased at 24 h after stimulation of PBMCs with CSFV and the increased production of CTLA-4 was confirmed by western blot analysis, whereas the increases were inhibited by the addition of APS. In addition, APS alone upregulated IL-2 and TGF-β mRNA expression in PBMCs and the addition of APS had the capacity to prevent a further increase in IL-2 mRNA expression in PBMCs during CSFV or PRRSV infection, but had no effect on TGF-β mRNA expression. The production of tumor necrosis factor-alpha (TNF-α) increased at 12 h after stimulation with PRRSV or CSFV, but not with PRRSV plus APS or CSFV plus APS, whereas the addition of APS to PBMCs infected with PRRSV or CSFV promoted IL-10 mRNA expression. CONCLUSIONS: We suggested that APS had immunomodulatory effects on cells exposed to PRRSV or CSFV. It might be that APS via different mechanisms affects the activities of immune cells during either PRRSV or CSFV infection. This possibility warrants further studies to evaluate whether APS would be an effective adjuvant in vaccines against PRRSV or CSFV

Understanding PRRSV Infection in Porcine Lung Based on Genome-Wide Transcriptome Response Identified by Deep Sequencing

Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection

Nitric Oxide Not Apoptosis Mediates Differential Killing of Mycobacterium bovis in Bovine Macrophages

To identify the resistance phenotype against Mycobacterium bovis in cattle, we used a bactericidal assay that has been considered a marker of this trait. Three of 24 cows (12.5%) were phenotyped as resistant and 21 as susceptible. Resistance of bovine macrophages (MΦ) to BCG challenge was evaluated for its association with SLC11A1 GT microsatellite polymorphisms within 3′UTR region. Twenty-three cows (95.8%) had a GT(13) genotype, reported as resistant, consequently the SLC11A1polymorphism was not in agreement with our bactericidal assay results. MΦ of cows with resistant or susceptible phenotype were challenged in vitro with virulent M. bovis field strain or BCG, and nitric oxide production, bacterial killing and apoptosis induction were measured in resting and LPS-primed states. M. bovis field strain induced more apoptosis than BCG, although the difference was not significant. Resistant MΦ controlled better the replication of M. bovis (P<0.01), produced more nitric oxide (P<0.05) and were slightly more prone to undergo apoptosis than susceptible cells. LPS pretreatment of MΦ enhanced all the functional parameters analyzed. Inhibition of nitric oxide production with n (G)-monomethyl-L-arginine monoacetate enhanced replication of M. bovis but did not modify apoptosis rates in both resistant and susceptible MΦ. We conclude that nitric oxide production not apoptosis is a major determinant of macrophage resistance to M. bovis infection in cattle and that the influence of SLC11A1 gene 3′UTR polymorphism is not associated with this event

Polyunsaturated fatty acid receptors, GPR40 and GPR120, are expressed in the hypothalamus and control energy homeostasis and inflammation

Abstract Background The consumption of large amounts of dietary fats is one of the most important environmental factors contributing to the development of obesity and metabolic disorders. GPR120 and GPR40 are polyunsaturated fatty acid receptors that exert a number of systemic effects that are beneficial for metabolic and inflammatory diseases. Here, we evaluate the expression and potential role of hypothalamic GPR120 and GPR40 as targets for the treatment of obesity. Methods Male Swiss (6-weeks old), were fed with a high fat diet (HFD, 60% of kcal from fat) for 4 weeks. Next, mice underwent stereotaxic surgery to place an indwelling cannula into the right lateral ventricle. intracerebroventricular (icv)-cannulated mice were treated twice a day for 6 days with 2.0 μL saline or GPR40 and GPR120 agonists: GW9508, TUG1197, or TUG905 (2.0 μL, 1.0 mM). Food intake and body mass were measured during the treatment period. At the end of the experiment, the hypothalamus was collected for real-time PCR analysis. Results We show that both receptors are expressed in the hypothalamus; GPR120 is primarily present in microglia, whereas GPR40 is expressed in neurons. Upon intracerebroventricular treatment, GW9508, a non-specific agonist for both receptors, reduced energy efficiency and the expression of inflammatory genes in the hypothalamus. Reducing GPR120 hypothalamic expression using a lentivirus-based approach resulted in the loss of the anti-inflammatory effect of GW9508 and increased energy efficiency. Intracerebroventricular treatment with the GPR120- and GPR40-specific agonists TUG1197 and TUG905, respectively, resulted in milder effects than those produced by GW9508. Conclusions GPR120 and GPR40 act in concert in the hypothalamus to reduce energy efficiency and regulate the inflammation associated with obesity. The combined activation of both receptors in the hypothalamus results in better metabolic outcomes than the isolated activation of either receptor alone