Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

doi:10.1016/S0165-2427(03)00059-X

Veterinary Immunology and Immunopathology

Volume 94, Issues 1–2, 15 July 2003, Pages 35-45

https://doi.org/10.1016/S0165-2427(03)00059-X Get rights and content

Abstract

To investigate cytokine alterations in pigs infected in-utero with porcine reproductive and respiratory syndrome virus (PRRSV), constitutive mRNA expression by peripheral blood mononuclear cells (PBMCs) was measured. PBMC from in-utero PRRSV-infected pigs displayed significantly increased IL-6, IL-10, and IFN-γ mRNA expression at 0 and 14 days of age compared with age-matched control pigs. There were no significant differences in IL-2, IL-4, and IL-12 mRNA expression between in-utero PRRSV-infected and control pigs. However, the IL-10/IL-12 ratio was significantly increased in in-utero PRRSV-infected pigs at 0 and 14 days of age, suggesting the imbalance of IL-10 and IL-12 mRNA production. The abnormal mRNA expression of cytokines in in-utero PRRSV-infected pigs occurred concurrently with a significant decrease in the CD4⁺/CD8⁺ T-cell ratio in peripheral blood. PRRSV was not isolated from the sera of pigs at 9 weeks of age that had been viremic at 0 and 14 days old. Delayed type hypersensitivity (DTH) responses to Tuberculin and analysis of cytokine mRNA expression by PBMC showed that cell-mediated immune response and cytokine message profiles in pigs infected in-utero with PRRSV had returned to levels similar to those of control pigs by 9 weeks of age. We conclude that in-utero infection with PRRSV results in significant alteration of cytokine mRNA expression that may cause transient immunomodulation. However, at 10 weeks of age the pigs’ immune responses seemed to recover. This may help to understand the immunopathogenesis of in-utero PRRSV infection and the increased susceptibility to secondary bacterial pathogens in neonatal piglets.

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single-stranded, positive RNA virus of the Arteriviridae group (Benfield et al., 1992). When porcine reproductive and respiratory syndrome (PRRS) was first observed, it was characterized by excessive rates of late-term abortion, stillbirths and mummified fetuses (Dial et al., 1990). Live-born piglets were often born weak and displayed respiratory distress or clinical signs of secondary bacterial pathogens (Dial et al., 1990, Done and Paton, 1995, Keffaber et al., 1992, Stephenson et al., 1993). Once the reproductive disease outbreak subsides, a PRRSV and secondary bacterial disease complex of weaned pigs may continue for years (Stephenson et al., 1993), suggesting PRRSV infection might have immunosuppressive effects on pigs.

We have previously reported on a novel disease model that reproduces the immunosuppressive effects of PRRSV (Feng et al., 2001). Suckling piglets from sows infected at day 98 gestation were found to be highly susceptible to Streptococcus suis type 2 infection. Pathologically the most striking lesions were in the thymus, which displayed a severe depletion of cortical thymocytes. We also found that in-utero infection with PRRSV caused changes in thymocyte subpopulations and peripheral blood T-lymphocyte subsets highlighted by a significantly decreased CD4⁺/CD8⁺ ratio (Feng et al., 2002). These results suggest that in-utero infection with PRRSV can modulate immune functions in piglets.

The nature and role of the humoral and cell-mediated immune responses against PRRSV have not been fully characterized. Cytokines are integral components of the immune response and important for cell-mediated and humoral immunity. Information about the cytokine response during PRRSV infection may be crucial for us to understand the host immune response to the virus and potentially the pathogenesis of the disease. In the present study, we investigated constitutive cytokine patterns in peripheral blood mononuclear cells (PBMCs) of piglets infected in-utero with PRRSV. We demonstrated that IL-6, IL-10, and IFN-γ mRNA expressions were significantly increased at 0 and 14 days of age. We also showed that the IL-10/IL-12 ratio was significantly increased, suggesting an imbalance of IL-10 and IL-12 mRNA expression in PRRSV-infected pigs.

Section snippets

Animals and experimental design

Eight PRRSV-free pregnant sows were purchased from a geographically isolated unvaccinated herd which had been free of clinical signs of PRRSV infection and had remained seronegative to PRRSV for the past 3 years. Five sows were inoculated with strain SD 23983 of PRRSV (gift from Dr. Benfield, South Dakota State University) by intranasal instillation of 2 ml inoculum containing 10^3.2 50% tissue culture infective doses (TCID50) virus as previously described (Kim et al., 1993) at 98 days gestation.

Clinical signs and virus isolation

Piglets born to PRRSV-infected sows were weak and had rough hair coats. Some exhibited fever, eyelid edema, forehead edema, conjunctivitis, inappetence, and dyspnea during the first 2 weeks of life. After 6 weeks, the pigs from PRRSV-infected sows were clinically normal. Control pigs showed no adverse clinical signs throughout the experimental period. PRRSV was isolated from the sera of all pigs born to PRRSV-infected sows at 0 and 14 days of age (Table 1). However, at 67 days of age PRRSV was

Discussion

The purpose of this study was to investigate the cytokine mRNA expression profiles produced by PBMC from piglets infected in-utero with PRRSV. This report extends our previous studies in which we demonstrated that in-utero infection with PRRSV increased piglet susceptibility to S. suis infection (Feng et al., 2001) and caused abnormal changes in thymocyte and T-lymphocyte subsets (Feng et al., 2002). We report herein that IL-6, IL-10, and INF-γ mRNA levels are significantly increased in

Acknowledgements

We thank Dr. Jack Britt for his critiques and Dr. Zuckermann for his generous gift of biotinylated porcine CD8 MAb. We also thank Mrs. Kimberly Ainge, Dr. Kristie Karli, Dr. Kelly Gambino, Dr. Stacy Jones, and Dr. Jan Shaw for taking care of the newborn piglets. This report was funded by North Carolina Pork Producers Association, College of Veterinary Medicine, and College of Agriculture and Life Sciences, North Carolina State University.

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Interleukin 4 (IL-4) plays a major role in T-lymphocyte development and is thought to be a central regulator as a cofactor in resting B-lymphocyte proliferation. Primary infection with porcine reproductive and respiratory syndrome virus (PRRSV) induces minimal IL-4 production, whereas an IL-4 response occurs in the peripheral blood of piglets reinfected by PRRSV. The locations and interaction partners for the massive volume of IL-4 triggered by PRRSV reinfection remain unclear. This study aimed to investigate the characteristics of IL-4 secretion and location changes in peripheral immune organs induced by PRRSV infection and reinfection. Our results show that PRRSV reinfection induced higher levels of IL-4 mRNA and protein expression in the peripheral immune organs (e.g., lymph node and spleen) and peripheral blood compared with PRRSV primary infection. Importantly, we found that, following PRRSV reinfection, an obvious large-scale migration of IL-4 occurred in the lymph nodes. During PRRSV primary infection, IL-4 was mainly concentrated around the lymphoid follicles and paracortical regions of the lymph node and also located in the marginal area and periarterial lymphatic sheath region of the spleen. During PRRSV reinfection, the now abundant IL-4 gathered into the lymphoid follicles of the lymph node and spleen. Notably, IL-4 changed its location state from scattered and sparse during primary infection to clinging to B lymphocytes in the lymphoid follicles during reinfection. During reinfection, IL-4 was often co-localized with T and B lymphocytes; furthermore, the percentages of several T lymphocyte subsets, N protein-specific antibody levels, and viral load in the peripheral blood or lymph tissues underwent remarkable variation. Another important finding of this study was that the numbers of B lymphocytes and T lymphocytes in the lymphoid nodes were significantly reduced after PRRSV infection or reinfection, presumably due to PRRSV-induced acute bone marrow failure and autophagy in thymic epithelial cells. This study revealed the characteristics of IL-4 migration and distribution in the peripheral lymph organs induced by PRRSV reinfection and provides valuable clues for further exploration of the interactions between IL-4, B lymphocytes, and T lymphocytes during PRRSV infection and reinfection.
Immunoregulatory signal FoxP3, cytokine gene expression and IFN-γ cell responsiveness upon porcine reproductive and respiratory syndrome virus (PRRSV) natural infection
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The study aims at evaluating gene expression of pro-inflammatory (IL-1β, IL-8, TNF-α), pro-immune (IFN-γ), anti-inflammatory (IL-10) cytokines and of the immunoregulatory signal FoxP3 in association with PRRSV-specific IFN-γ secreting cell (SC) responsiveness upon PRRSV natural infection. Forty PRRSV-negative pigs were assigned to two groups: 20 pigs were vaccinated at 3 weeks of age (weaning) against PRRSV (V-PRRSV) with a modified live virus vaccine (MLV) and 20 pigs were kept non-vaccinated (NV) as controls. Blood samples were collected at 3 (vaccination), 6, 8, 10, 12, 14, and 16 weeks of age.
Natural infection occurred from 8 weeks of age onward in both groups and viremia lasted 8 weeks.
In the early phase of infection, pro-inflammatory cytokines (IL-1β, IL-8, TNF-α) showed a delayed increase concomitant with the peak of viremia in both groups. In both groups, IL-10 peaked at 12 weeks in association with the increase of pro-inflammatory cytokines. Conversely, in vaccinated pigs (V-PRRSV), IFN-γ showed higher gene expression during the early phase of infection and a more intense secreting cell (SC) response in the late phase. Differently, gene expression of the transcription factor FoxP3, expressed by T regulatory lymphocytes (Tregs), increased significantly in controls only and was associated with the rise of the viral load. Moreover, FoxP3 levels remained significantly higher during the late phase of infection and paralleled with lower levels of IFN-γ SC detected by ELISPOT.
The expression/production of immunoregulatory signals involved in Treg activation could be a promising marker to study the immunobiology of PRRSV infection.
Innate and adaptive immunity against Porcine Reproductive and Respiratory Syndrome Virus
2015, Veterinary Immunology and Immunopathology
Many highly effective vaccines have been produced against viruses whose virulent infection elicits strong and durable protective immunity. In these cases, characterization of immune effector mechanisms and identification of protective epitopes/immunogens has been informative for the development of successful vaccine programs. Diseases in which the immune system does not rapidly clear the acute infection and/or convalescent immunity does not provide highly effective protection against secondary challenge pose a major hurdle for clinicians and scientists. Porcine reproductive and respiratory syndrome virus (PRRSV) falls primarily into this category, though not entirely. PRRSV causes a prolonged infection, though the host eventually clears the virus. Neutralizing antibodies can provide passive protection when present prior to challenge, though infection can be controlled in the absence of detectable neutralizing antibodies. In addition, primed pigs (through natural exposure or vaccination with a modified-live vaccine) show some protection against secondary challenge. While peripheral PRRSV-specific T cell responses have been examined, their direct contribution to antibody-mediated immunity and viral clearance have not been fully elucidated. The innate immune response following PRRSV infection, particularly the antiviral type I interferon response, is meager, but when provided exogenously, IFN-α enhances PRRSV immunity and viral control. Overall, the quality of immunity induced by natural PRRSV infection is not ideal for informing vaccine development programs.
The epitopes necessary for protection may be identified through natural exposure or modified-live vaccines and subsequently applied to vaccine delivery platforms to accelerate induction of protective immunity following vaccination. Collectively, further work to identify protective B and T cell epitopes and mechanisms by which PRRSV eludes innate immunity will enhance our ability to develop more effective methods to control and eliminate PRRS disease.
Modulation of innate immune signaling by nonstructural protein 1 (nsp1) in the family Arteriviridae
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Arteriviruses infect immune cells and may cause persistence in infected hosts. Inefficient induction of pro-inflammatory cytokines and type I IFNs are observed during infection of this group of viruses, suggesting that they may have evolved to escape the host immune surveillance for efficient survival. Recent studies have identified viral proteins regulating the innate immune signaling, and among these, nsp1 (nonstructural protein 1) is the most potent IFN antagonist. For porcine reproductive and respiratory syndrome virus (PRRSV), individual subunits (nsp1α and nsp1β) of nsp1 suppress type I IFN production. In particular, PRRSV-nsp1α degrades CREB (cyclic AMP responsive element binding)-binding protein (CBP), a key component of the IFN enhanceosome, whereas PRRSV-nsp1β degrades karyopherin-α1 which is known to mediate the nuclear import of ISGF3 (interferon-stimulated gene factor 3). All individual subunits of nsp1 of PRRSV, equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) appear to contain IFN suppressive activities. As with PRRSV-nsp1α, CBP degradation is evident by LDV-nsp1α and partly by SHFV-nsp1γ. This review summarizes the biogenesis and the role of individual subunits of nsp1 of arteriviruses for innate immune modulation.
Age-associated differential production of IFN-γ, IL-10 and GM-CSF by porcine alveolar macrophages in response to lipopolysaccharide
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The aim of the present study was to investigate the age-related production variation of T helper (Th)-type cytokines (IL-2, IL-4, IFN-γ and IL-10), granulocyte macrophage-colony stimulating factor (GM-CSF) and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated porcine alveolar macrophages (AMs) in a time-dependent manner. For this purpose, AMs were isolated from 5-days (newborn), 40-days (post-weaned) and 120-days (young) old pigs. Cells were incubated for 24 h in the absence or presence of increasing concentrations of LPS (0.0, 0.01, 1.0, 5.0 and 10.0 μg/mL).
IL-10, IFN-γ and GM-CSF mRNA expression was upregulated in a dose-dependent manner for all age groups (P < 0.05). Age-related differences included a significantly increased IL-10 mRNA and protein production in newborn piglets compared to post-weaned and young pigs. IL-10 production pattern was similar with a higher peak between 12 and 36 h post-induction in all age groups. In contrast, IFN-γ mRNA and protein level was significantly elevated in young pigs 12 h and 24 h post-induction, respectively, while the time course production of IFN-γ was mostly consistent in newborn and post-weaned piglets. GM-CSF mRNA expression was significantly lower in newborn piglets than in post-weaned and young pigs. The kinetic of GM-CSF expression peaked at 12 h in young and post-weaned pigs and at 24 h in newborn piglets. IL-4 mRNA levels were very low and no apparent change of IL-2 expression was observed following LPS stimulation in all age groups. Only very low levels of NO were detected in the cell supernatants of young pigs. Collectively, these studies suggest age-related differences in time-dependent production of IL-10, IFN-γ and GM-CSF by porcine AMs with potential immunoregulatory consequences to be explored further.
Plasmids expressing porcine interferon gamma up-regulate pro-inflammatory cytokine and co-stimulatory molecule expression which are suppressed by porcine reproductive and respiratory syndrome virus
2013, Veterinary Immunology and Immunopathology
Citation Excerpt :
In contrast to the reduced pro-inflammatory cytokine and co-stimulatory molecule expression, monocytes inoculated with PRRSV demonstrated significantly increased IL-10 and increased TGFβ expression in response to LPS stimulation (Table 2 and Fig. 4). PRRSV induction of IL-10 and TGFβ expression has been demonstrated in vitro in PBMC and myeloid APC inoculated with the virus (Chang et al., 2008; Charerntantanakul and Kasinrerk, 2010, 2012; Flores-Mendoza et al., 2008; Genini et al., 2008; Park et al., 2008; Royaee et al., 2004; Suradhat and Thanawongnuwech, 2003; Suradhat et al., 2003), and in vivo in serum, bronchoalveolar lavage fluid, and lymphoid tissues of PRRSV-infected pigs (Chung and Chae, 2003; Diaz et al., 2005, 2006; Feng et al., 2003; Johnsen et al., 2002; Labarque et al., 2003). PRRSV up-regulation of IL-10 expression has been demonstrated to contribute to the reduced expression of some pro-inflammatory immune parameters, e.g. IFNγ, TNFα, CD80, and CD86 as examined by IL-10 knockdown using porcine IL-10-specific antisense oligodeoxynucleotides and plasmids expressing porcine IL-10 short hairpin RNA (Charerntantanakul and Kasinrerk, 2010, 2012).
Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the pro-inflammatory immune response following infection of myeloid antigen-presenting cells. A reduced pro-inflammatory immune response modulates PRRSV replication, clinical disease, and persistent infection of the virus. Numerous efforts have been made to enhance the pro-inflammatory immune response to PRRSV, but only a few attempts have so far elicited satisfactory results. The present study aims to evaluate in vitro the potential of plasmids expressing porcine interferon gamma (pcDNA-IFNγ) to enhance the expression of pro-inflammatory immune parameters in PRRSV-inoculated monocytes. Naïve blood monocytes from eight PRRSV-seronegative pigs were inoculated with PRRSV and subsequently transfected with pcDNA-IFNγ or pcDNA (empty plasmid vector) and stimulated with lipopolysaccharide (LPS). The mRNA expression levels of IFNγ, interleukin-1 beta (IL-1β), IL-10, IL-12p40, tumor necrosis factor alpha (TNFα), transforming growth factor beta (TGFβ), CD80, and CD86 were evaluated by real-time PCR. The IFNγ, IL-10, and TNFα protein production was determined by ELISA. Compared with PRRSV-inoculated monocyte control, transfection with pcDNA-IFNγ, but not pcDNA, significantly enhanced IFNγ, TNFα, CD80, and CD86 mRNA expression, and IFNγ and TNFα protein production. A slight increase in IL-1β and IL-12p40 mRNA expression was also observed. Neither pcDNA-IFNγ nor pcDNA transfection affected IL-10 and TGFβ expression. Our results thus suggest that pcDNA-IFNγ may be an effective immunostimulator for potentiating the pro-inflammatory immune response to PRRSV.

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¹: Present address: Linberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, CB #7295, Chapel Hill, NC 27599, USA.

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Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

Abstract

Introduction

Section snippets

Animals and experimental design

Clinical signs and virus isolation

Discussion

Acknowledgements

Vet. Immunol. Immunopathol.

Vet. Immunol. Immunopathol.

Vet. Immumol. Immunopathol.

Virology

J. Nutr.

Poul. Sci.

Vet. Immunol. Immunopathol.

Vet. Microbiol.

Vet. Immunol. Immunopathol.

Vet. Microbiol.

Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332)

J. Vet. Diagn. Invest.

Persistent fetal infection of porcine reproductive and respiratory syndrome (PRRS) virus

Proc. Am. Assoc. Swine Pract.