Francisco J. Pallarés

Cytokine Expression by Macrophages in the Lung of Pigs Infected with the Porcine Reproductive and Respiratory Syndrome Virus

Summary Porcine reproductive and respiratory syndrome (PRRS) is caused by a virus that predominantly replicates in alveolar macrophages. The aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by the PRRS virus (PRRSV). Expression of interleukin (IL) 1α, IL-6 and tumour necrosis factor (TNF)-α correlated with the severity of pulmonary pathology and the numbers of pulmonary macrophages. Significant correlations were observed between PRRSV infection and the expression of IL-10, between the expression of IL-12p40 and interferon (IFN)-γ, and between the expression of TNF-α and IFN-γ. These findings suggest that PRRSV modulates the immune response by the up-regulation of IL-10, which may in turn reduce expression of cytokines involved in viral clearance (e.g. IFN-α, IFN-γ, IL-12p40 and TNF-α). The results also suggest that expression of IFN-γ is stimulated by IL-12p40 and TNF-α, but not by IFN-α. All of these cytokines were expressed mainly by septal macrophages with weaker expression by alveolar macrophages, lymphocytes and neutrophils. There appears to be differential activation of septal and alveolar macrophages in PRRSV infection, with septal macrophages being the major source of cytokines.

Metabolites and tissue distribution of resveratrol in the pig

SCOPE trans-Resveratrol (RES) and/(or) its metabolites exert many effects in vivo. Our aim was to study the metabolism and tissue distribution of RES using the pig, a mammal physiologically close to humans. METHODS AND RESULTS Forty-seven tissues, organs and fluids were analyzed 6 h after intragastric RES administration (5.9 mg/kg body weight) using HPLC-MS/MS. Twelve RES and seven dihydroresveratrol (DH-RES) metabolites were detected. DH-RES was the main metabolite in cecum, colon and rectum, whereas RES-3-O-glucuronide was the most abundant one in fluids and organs. Approximately 74.5% of the total RES administered was recovered in the form of RES, DH-RES and derived metabolites (65.1% along the gastrointestinal tract, 7.7% in urine, 1.2% in bile and 0.5% in organs). We report here, for the first time, the occurrence of RES ribosyl-sulfate derivative, DH-RES diglucuronide, DH-RES sulfoglucuronide and DH-RES disulfate as well as the metabolic profile of RES and DH-RES in the aorta, lymph, lymph node, ovaries, uterus, cerebellum, pancreas, urinary bladder tissue, fat and muscle. CONCLUSION This study contributes to the clarification of the metabolism and tissue distribution of RES and could help to further understand the mechanisms underlying its effects.

Immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs

Abstract Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) impairs local pulmonary immune responses by damaging the mucociliary transport system, impairing the function of porcine alveolar macrophages andinducing apoptosis of immune cells. An imbalance between pro- and anti-inflammatory cytokines, including tumour necrosis factor-α and interleukin-10, in PRRS may impair the immune response of the lung. Pulmonary macrophage subpopulations have a range of susceptibilities to different PRRSV strains and different capacities to express cytokines. Infection with PRRSV decreases the bactericidal activity of macrophages, which increases susceptibility to secondary bacterial infections. PRRSV infection is associated with an increase in concentrations of haptoglobin, which may interact with the virus receptor (CD163) and induce the synthesis of anti-inflammatory mediators. The balance between pro- and anti-inflammatory cytokines modulates the expression of CD163, which may affect the pathogenicity and replication of the virus in different tissues. With the emergence of highly pathogenic PRRSV, there is a need for more information on the immunopathogenesis of different strains of PRRS, particularly to develop more effective vaccines.

Acute phase response in porcine reproductive and respiratory syndrome virus infection.

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.

Changes in lymphocyte subsets and cytokines during European porcine reproductive and respiratory syndrome: increased expression of IL-12 and IL-10 and proliferation of CD4-CD8high.

The changes in peripheral blood mononuclear cells (PBMCs) have been studied in several reports in an attempt to determine the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, how these changes are evoked after PRRSV infection has not yet been clarified. The aim of this study was to analyze the changes seen in lymphocyte subsets and immunomodulatory cytokine expression in pigs after an acute experimental infection with a European PRRSV field isolate. Pigs were inoculated intramuscularly with PRRSV field isolate 2982. Samples from blood, medial retropharyngeal and tracheobronchial lymph nodes, and spleen were collected at different time points for flow cytometry studies and for cytokine expression by ELISA. CD21(+) cell counts increased in PBMCs and tracheobronchial lymph node cells from 17 to 24 dpi, coinciding with an increase in PRRSV-specific antibody titer in blood. CD3(+) T-cell counts increased mainly due to an enhancement of CD4(-)CD8(high) and CD4(+)CD8(+) T cells. CD4(-)CD8(low) T cells were decreased in all organs studied, whereas CD4(+)CD8(-) T cells decreased only in the spleen. The drop in viremia correlated with an enhancement of CD4(-)CD8(high) T cells, and with a higher expression of interleukin-10 (IL-10) and interleukin-12 p40 (IL-12 p40). No efficient interferon-gamma (IFN-gamma) response was detected during the acute phase of the infection, and the expression of interferon-alpha (IFN-alpha) was late and reached its maximum expression once the viremia decreased. These results point to IL-10 and IL-12 as cytokines that might play a significant role in the PRRSV immune response, as may CD4(-)CD8(high) T cells.

Pharmacokinetic study of trans-resveratrol in adult pigs.

A number of pharmacokinetic studies have shown marked differences in the plasma metabolic profile of resveratrol (RES) between humans and animals and between individuals of the same species, which complicates the identification of the putative bioactive metabolites responsible for the beneficial effects of RES. On the basis of the physiological similarity between pigs and humans, the aim of this work was to characterize the metabolic profile and pharmacokinetics of RES in the plasma of pigs and to compare this to values reported in humans. RES (5.9 mg/kg of body weight) was orally administered to pigs. The following metabolites were identified in plasma using HPLC-MS/MS: RES-diglucuronide (1), two isomers of RES-sulfoglucuronide (2, 3), two isomers of RES-glucuronide (4, 5), RES-sulfate (6), and RES. The most abundant metabolites were 2, 5 (identified as resveratrol 3-O-glucuronide), and 6. The t(max) ranged from 0.9 h for compounds 2 and 5 to 2 h for compound 3. The highest C(max) value was 2223 ng/mL (5.5 μM) for metabolite 5, which was 2.6-, 3.3-, and 12-fold higher than that for metabolites 6, 2, and 3, respectively. Peak plasma levels of RES (53 ng/mL; 0.23 μM) were detected 0.5 h after RES ingestion. Apart from the low levels of RES aglycone, the RES metabolic profile in pigs differs from that found in humans. The identification of the actual active RES metabolites is a challenge that requires more complex studies which should take into account many possible influencing factors such as age, gender, and methodological approaches.

Endogenous Interleukin-12 Is Not Required for Resolution of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection in Mice

ABSTRACT A Th1 immune response involving gamma interferon (IFN-γ) production is required to eliminate Chlamydophila abortusinfections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12−/− and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12−/− mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12−/− mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-γ. The low level of IFN-γ was essential for survival of IL-12−/−infected mice. Both WT and IL-12−/− mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-γ and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge withC. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12−/−mice. The lack of IL-12 resulted in few infiltrating CD4+ T cells in the liver relative to the number in WT mice, although the number of CD8+ T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.

Use of saliva for haptoglobin and C-reactive protein quantifications in porcine respiratory and reproductive syndrome affected pigs in field conditions

The purpose of this study was to study the use of saliva samples as alternative to serum for acute phase protein (APP) quantifications in pigs at field conditions. To this end, haptoglobin (Hp) and C-reactive protein (CRP) concentrations were examined in 100 animals at different ages. Sixty pigs were from a farm with chronic PRRS virus infection and 40 from a specific pathogen free (SPF) farm. A serological study was performed to obtain an overview of the immune status of animals and to evaluate possible concomitant infections in animals with PRRS infection. The results reported in this study showed that both saliva and serum samples had higher APP concentrations in PRRS pigs aged 8-9, 17-18 and 24-25 weeks in conventional herds than SPF pigs of the same age (p<0.05). In addition, increases in APP were obtained with age independently of the health status of the animals. According to the ROC analyses performed, saliva could be a better specimen than serum to quantify Hp and CRP levels in field conditions and may contribute to a more efficient detection of diseased animals at farm level.

Haptoglobin and C-reactive protein as biomarkers in the serum, saliva and meat juice of pigs experimentally infected with porcine reproductive and respiratory syndrome virus

Concentrations of the acute phase proteins haptoglobin (Hp) and C-reactive protein (CRP) were measured in the serum, saliva and meat juice of pigs experimentally infected with a porcine reproductive and respiratory syndrome virus (PRRSv) field isolate. Sixteen PRRSv-free pigs were inoculated IM, killed in groups of four at 7, 14, 21 and 24 days post-inoculation (dpi), and samples of blood, saliva and diaphragmatic muscle were collected. Four non-infected controls were killed at 24 dpi. Significant differences in lung lesions were found between PRRSv-inoculated animals and controls. Changes in the concentrations of Hp and CRP in serum, saliva and meat juice samples were similar, peaking at 21 dpi. The correlations found suggest that the measurement of Hp and CRP in saliva and meat juice could serve as complementary, or possibly alternative, biomarkers of pig herd-health.

Effects of long-term consumption of low doses of resveratrol on diet-induced mild hypercholesterolemia in pigs: a transcriptomic approach to disease prevention

Metabolic and cardiovascular diseases (CVDs) have risen to alarming proportions, and there is a need for therapeutic and preventive measures. The polyphenol resveratrol (RES) protects against CVDs, but in vivo molecular mechanisms responsible for protection are not yet understood. Peripheral blood mononuclear cells (PBMNCs) are involved in the development of atherosclerosis and metabolic disorders. The identification of PBMNCs genes responding to dietary compounds might help to understand the mechanisms underlying the effects of polyphenols. We determined gene expression differences between PBMNCs from pigs fed a high-fat diet manifesting a mild increase of cholesterol and pigs fed a high-fat diet containing low doses of RES. Although the consumption of RES did not modify the levels of cholesterol, microarray analyses indicated that some of the differentially expressed genes, collagens (COL1A, COL3A), lipoprotein lipase (LPL) and fatty-acid binding proteins (FABPs) involved in CVDs and lipid metabolism were up-regulated by the high-fat diet and down-regulated by RES. Reverse transcriptase polymerase chain reaction confirmed that RES and RES-containing grape extract prevented the induction of FABP4 in PBMNCs in female pigs fed a high-fat diet. Low micromolar concentrations of RES and its metabolite dihydroresveratrol exerted a minor but significant reducing effect on the induction of FABP4 expression in human macrophages treated with oxidized low-density lipoprotein. Our results show that the consumption of low doses of RES modulates the expression of genes related to lipid metabolism and metabolic disorders that are affected by a high-fat diet and suggest that some of the circulating RES metabolites may contribute to these effects.