Evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus.

The host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) from infected pigs is currently poorly understood. To better understand the dynamics of host-pathogen interactions, seventy-five of 100 pigs infected with PRRSV-JA142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). Blood, lung, bronchoalveolar lavage (BAL) and bronchial lymph node (BLN) samples were collected to evaluate the cellular immune responses. The humoral responses were evaluated by measuring the levels of anti-PRRSV IgG and serum virus-neutralizing (SVN) antibodies. Consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. At peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived DC/macrophage and conventional DC frequencies were increased, and these effects coincided with the early induction of local T-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, BAL, and BLN as early as 10 dpc. Conversely, the systemic T-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. Taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining PRRSV through the early induction of T-cell responses at the sites of virus replication.

Effect of polymorphisms in porcine guanylate-binding proteins on host resistance to PRRSV infection in experimentally challenged pigs.

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.

In vitro immune responses of porcine alveolar macrophages reflect host immune responses against porcine reproductive and respiratory syndrome viruses.

BACKGROUND: Currently, an in vitro immunogenicity screening system for the immunological assessment of potential porcine reproductive and respiratory syndrome virus (PRRSV) vaccine candidates is highly desired. Thus, in the present study, two genetically divergent PRRSVs were characterized in vitro and in vivo to identify an in vitro system and immunological markers that predict the host immune response. Porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) collected from PRRSV-negative pigs were used for in vitro immunological evaluation, and the response of these cells to VR2332c or JA142c were compared with those elicited in pigs challenged with the same viruses. RESULTS: Compared with VR2332c or mock infection, JA142c induced increased levels of type I interferons and pro-inflammatory cytokines (TNF-α, IL-1α/ß, IL-6, IL-8, and IL-12) in PAMs, and these elevated levels were comparable to the cytokine induction observed in PRRSV-challenged pigs. Furthermore, significantly greater numbers of activated CD4+ T cells, type I helper T cells, cytotoxic T cells and total IFN-γ+ cells were observed in JA142c-challenged pigs than in VR2332c- or mock-challenged pigs. CONCLUSIONS: Based on these results, the innate immune response patterns (particularly IFN-α, TNF-α and IL-12) to specific PRRSV strains in PAMs might reflect those elicited by the same viruses in pigs.

Immunological Compatibility of Bone Tissues from Alpha-1,3-galactosyltransferase Knockout Pig for Xenotransplantation.

We investigated whether the lack of galactosyltransferase (α-Gal) expression in bone tissue is associated with reduced immune response of human peripheral blood mononuclear cells (PBMCs) against pig bone tissue. When human PBMC obtained from heparinized blood of healthy volunteers was stimulated with bone extracts of pigs with α-1,3-galactosyltransferase knock out (α-Gal KO), the proliferation of human PBMCs and production of proinflammatory cytokines such as TNF-α and IL-1ß were significantly reduced compared to those stimulated with bone extracts of wild type (WT) pigs. In addition, activation of CD4+ helper T cells and production of IL-2, IFN-γ, and IL-17 were reduced upon stimulation with bone tissue extracts from α-Gal KO pigs. This is possibly due to the lowered activities of the NF-κB, p38, ERK, and JNK signaling pathways. Our findings can be used to evaluate the compatibility of bone tissues from α-Gal KO pigs with human bone grafting as novel natural biomaterials, thereby increasing the feasibility of future clinical applications.

A hot-water extract of Sanguisorba officinalis ameliorates endotoxin-induced septic shock by inhibiting inflammasome activation.

The inflammasome is a multiprotein signaling complex that mediates inflammatory innate immune responses through caspase 1 activation and subsequent IL-1ß secretion. However, because its aberrant activation often leads to inflammatory diseases, targeting the inflammasome holds promise for the treatment of inflammation-related diseases. In this study, it was found that a hot-water extract of Sanguisorba officinalis (HSO) suppresses inflammasome activation triggered by adenosine 5'-triphosphate, nigericin, microbial pathogens, and double stranded DNA in bone marrow-derived macrophages. HSO was found to significantly suppress IL-1ß production in a dose-dependent manner; this effect correlated well with small amounts of caspase 1 and little ASC pyroptosome formation in HSO-treated cells. The anti-inflammatory activity of HSO was further confirmed in a mouse model of endotoxin-induced septic shock. Oral administration of HSO reduced IL-1ß titers in the serum and peritoneal cavity, increasing the survival rate. Taken together, our results suggest that HSO is an inhibits inflammasome activation through nucleotide-binding domain and leucine-rich repeat pyrin domain 3, nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and absent in melanoma 2 pathways, and may be useful for treatment of inflammasome-mediated diseases.

Potential use of lactic acid bacteria Leuconostoc mesenteroides as a probiotic for the removal of Pb(II) toxicity.

It has been demonstrated that certain lactic acid bacteria (LAB) can sequester metal ions by binding them to their surfaces. In the present study, lead (Pb)-resistant LAB were isolated from kimchi, a Korean fermented food. A total of 96 different LAB strains were isolated, and 52 strains showed lead resistance. Among them, an LAB strain-96 (L-96) identified as Leuconostoc mesenteroides showed remarkable Pb resistance and removal capacity. The maximum adsorption capacity of this strain calculated using the Langmuir isotherm was 60.6 mg Pb/g. In an in vivo experiment, young male mice were provided with water (A), Pb-water (B), or Pb-water+ L-96 (C) during puberty. Lower glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in Pb-exposed male mice that received strain L-96 as a probiotic were suggestive of reduced hepatotoxicity. Moreover, feces from mice treated with L-96 contained more Pb than feces from untreated mice. Increased Pb elimination likely reduced internal accumulation, and this hypothesis was supported by significantly lower Pb concentrations in kidneys and testes of the mice treated with strain L-96. The motility and ATP content of epididymal spermatozoa were partially restored if strain L-96 was administered. In conclusion, isolated L-96 LAB had lead-biosorption activity and efficiently detoxified lead-poisoned male mice, resulting in recovering male reproductive function. These results suggest the potential use of LAB as a probiotic to protect humans from the adverse effects of Pb exposure.

A hot water extract of Aralia cordata activates bone marrow-derived macrophages via a myeloid differentiation protein 88-dependent pathway and protects mice from bacterial infection.

In traditional Asian medicine, Aralia cordata (AC) is a known as a pain reliever and anti-inflammatory drug. Although several of its biological activities have been reported, the immunomodulatory effects of a hot water extract of AC (HAC) have not yet been described. The aim of this study was to investigate whether HAC modulates the activation of macrophages, which play important roles in innate immune responses against microbial pathogens, and if so, to determine the molecular mechanisms by which HAC mediates this process. It was found that HAC activates bone marrow-derived macrophages (BMDM) and increases amounts of nitric oxide and proinflammatory cytokines in a dose-dependent manner. In addition, HAC was found to induce phosphorylation of NF-κB and mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinases, extracellular signal-regulated kinases and p38. Interestingly, these effects were absent in BMDM prepared from myeloid differentiation protein 88-knockout mice. Polysaccharides from HAC exerted stronger immunostimulatory effects than HAC itself. Furthermore, orally administered HAC clearly enhanced clearance of the intracellular pathogen Listeria monocytogenes by boosting innate immune responses. These results demonstrate that HAC exerts immunostimulatory effects through the TLR/MyD88 and NF-κB/MAPK signal transduction pathways.

Effect of polymorphisms in the GBP1, Mx1 and CD163 genes on host responses to PRRSV infection in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is the most economically important disease to the swine industry, and effective prevention strategy for this disease is still required. Guanylate-binding protein 1 (GBP1) and myxovirus resistance protein 1 (Mx1) are two important proteins belonging to the GTPase superfamily that have been previously described to show antiviral effects. CD163 is considered the most important receptor for PRRSV attachment and internalization. Therefore, the aim of the present study was to evaluate the effects of these genes on host resistance against PRRSV infection in conjunction with the host immune response following PRRSV challenge. The results showed that pigs with AG genotype for the GBP1 exon2 exhibited a significantly higher average daily weight gain (ADWG) and lower average viremia than AA or GG genotype. Furthermore, pigs harbouring the AG genotype for the GBP1 gene presented greater CD4(+)CD25(+) and CD8(+)CD25(+) T cell populations at 4 and 18 days post challenge (dpc), respectively, as compared with other genotypes whereas pigs with CC genotype for the CD163 gene displayed significantly higher nucleocapsid-specific antibody titers at 11dpc. However, pigs with a single 11-bp deletion or insertion in the Mx1 gene did not show significant differences in either weight gain or viremia. Based on these results, we concluded that GBP1 is most significantly associated with resistance against PRRSV infection and efficient T cell activation in pigs.