Jaime Gómez-Laguna

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.

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.

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.

Use of real-time and classic polymerase chain reaction assays for the diagnosis of porcine tuberculosis in formalin-fixed, paraffin-embedded tissues.

The current study was carried out to set up a fast and specific technique for porcine tuberculosis diagnosis in formalin-fixed, paraffin-embedded tissues. A retrospective study was carried out using 54 samples fixed in 10% neutral buffered formalin from 29 slaughtered Iberian pigs. Most of the pigs showed tissue samples positive to immunohistochemical staining (70.4%), and mycobacteria were detected within or near the necrotic cores of the lesions. However, diagnosis by this technique was time-consuming and tedious because of the paucibacillar nature of porcine tuberculous lesions. Classic polymerase chain reaction (PCR) was unsuccessful in mycobacteria genome amplification in all of the examined samples; however, real-time PCR amplified the mycobacteria genome in 23 of 29 examined pigs, identifying the Mycobacterium tuberculosis complex in all but one, which amplified Mycobacterium avium complex. Moreover, when reamplification of the DNA was performed, classic PCR amplified the mycobacteria genome in all the examined pigs (29/29), identifying the M. tuberculosis complex in 28 of 29 studied pigs and M. avium complex in only 1 pig. Results of the current study point out that both real-time and classic PCR assays, with genome reamplification, represent sensitive, fast, and specific diagnostic tools for porcine tuberculosis in formalin-fixed, paraffin-embedded tissues.

Cytokines transcript levels in lung and lymphoid organs during genotype 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection.

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases of swine. PRRS virus (PRRSV) infection in the pig is characterized by a weak or absent host innate immune response. The underlying mechanisms of PRRSV pathogenesis are still unclear. The analysis of transcript levels represents an alternative to immunoassays for the detection of cytokines that sometimes are difficult to detect due to their low amounts. This study sets out to determine the differences in pathogenesis and the immune response between lung, tonsil, tracheobronchial lymph node (Tb-LN) and retropharyngeal LN (Rf-LN) of PRRSV 2982 strain infected pigs. PRRSV strain 2982 avoided the onset of an effective innate immune response, especially in PRRSV main target (lung) and reservoir (tonsil) organs. PRRSV lead to an impaired expression of IFN-α and TNF-α gene expression, which finally induced a weak and delayed adaptive immune response through an inefficient IL-12 and IFN-γ expression. Finally, PRRSV replication favored the expression of the anti-inflammatory IL-10 cytokine in infected pigs.

Impact of PRRSV on activation and viability of antigen presenting cells

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine industry. The causal agent, PRRS-virus (PRRSV), is able to evade the host immune response and survive in the organism causing transient infections. Despite all scientific efforts, there are still some gaps in the knowledge of the pathogenesis of this disease. Antigen presenting cells (APCs), as initiators of the immune response, are located in the first line of defense against microorganisms, and are responsible for antigen recognition, processing and presentation. Dendritic cells (DCs) are the main type of APC involved in antigen presentation and they are susceptible to PRRSV infection. Thus, PRRSV replication in DCs may trigger off different mechanisms to impair the onset of a host effective immune response against the virus. On the one side, PRRSV may impair the basic functions of DCs by regulating the expression of major histocompatibility complex class II and CD80/86. Other strategy followed by the virus is the induction of cell death of APCs by apoptosis, necrosis or both of them. The impairment and/or cell death of APCs could lead to a failure in the onset of an efficient immune response, as long as cells could not properly activate T cells. Future aspects to take into account are also discussed in this review.

A comparative study of the local cytokine response in the lungs of pigs experimentally infected with different PRRSV-1 strains: upregulation of IL-1α in highly pathogenic strain induced lesions.

Porcine reproductive and respiratory syndrome viruses (PRRSV) show high genetic differences both among and within genotypes. Recently, several highly pathogenic PRRSV (HP-PRRSV) strains have been described. This study compares and characterizes the production of cytokines by pulmonary macrophages in pigs experimentally infected with four different PRRSV-1 strains: two low-virulent strains, Lelystad (LV) and a British field strain (215-06); a HP strain (SU1-bel) from Belarus and the attenuated vaccine strain DV (Porcilis(®) PRRS). Animals were clinically monitored and post-mortem examinations were performed at 3, 7 and 35 days post-infection (dpi). Lung samples were processed for histopathological and immunohistochemical studies by using specific antibodies against PRRSV, IL1-α, IL-6, TNF-α, IL-10 and IFN-γ. SU1-bel infected animals presented the highest mean scores for clinical observations, gross and microscopic lesions as well as for PRRSV expression compared with the other infected groups (p≤0.027). These animals displayed the highest expression of IL1-α at 7dpi, together with the highest score for lung pathology, whereas LV, 215-06 and DV inoculated animals only showed a transient enhancement in some of these cytokines. SU1-bel-infected pigs showed a positive correlation between the amount of PRRSV antigen and IL-1α expression (r=0.645, p<0.001). The highest expression of IL-10 was detected in 215-06-infected animals (p≤0.004), with a positive correlation with the numbers of virus-infected cells (r=0.375, p≤0.013). In conclusion, the HP-PRRSV SU1-bel strain replicated more efficiently in the lung of infected animals and induced a higher expression of IL-1α than the other PRRSV-1-infected groups, which may have played a key role in the onset of the clinical signs and interstitial pneumonia.

Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues

Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases.