Elsevier

Cytokine

Volume 126, February 2020, 154883
Cytokine

Fetal cytokine response to porcine reproductive and respiratory syndrome virus-2 infection

https://doi.org/10.1016/j.cyto.2019.154883Get rights and content

Highlights

  • Fetal immune response during maternal PRRSV infection is largely restricted to infected fetuses.

  • Over expression of CCL5 is associated with disease progression and reduced fetal viability.

  • Differences were found between gene expression and abundance of type 1, but not type 2, interferons.

  • Regulation of cell cycle progression is significantly altered in highly PRRSV-infected tissue.

Abstract

To understand the fetal immune response to porcine reproductive and respiratory virus-2 (PRRSV) and to evaluate the association with fetal viability, pregnant gilts were challenged on gestation day 85 and euthanized 21 days post infection. Based on preservation status and viral load in serum and thymus, fetuses were classified as either uninfected-viable (UNIF), high viral load viable (HV-VIA), or high viral load meconium stained (HV-MEC) and were compared with age matched control (CON) fetuses derived from mock infected gilts. Gene expression of IFNB, IFNG, CCL2, CCL5, CXCL10 and IL10, were all found to be significantly upregulated in the thymus and spleen of both high viral load groups. UNIF fetuses remained largely unaffected, with only small upregulations in IFNA and IL10 in the thymus, and IFNA, CCL5 and CXCL10 in the spleen. Regarding fetal viability, expression of CCL5 was significantly elevated in the thymus and spleen of HV-MEC compared to HV-VIA fetuses. The concentrations of IFNα, IFNγ, TNFα and CCL2 were elevated in the sera of all infected fetuses, whereas IFNβ was below the detection limit in all fetal sera. Additional gene expression analysis in the thymus showed significant downregulation of CDK1, CDK2 and CDK4, and upregulation of the inhibitor CDKN1A, suggesting altered regulation of cell cycle progression. Collectively, these results show near complete compartmentalization of the fetal immune response to infected fetuses and suggest this immune response is not a major contributor to fetal death.

Introduction

Under normal conditions the developing fetus is protected from external threats by the placental barrier, which prevents the transfer of most microorganisms present in the dam. Although fetal sterility throughout pregnancy has been challenged by evidence of a low biomass commensal microflora, this concept remains controversial [1]. There is however, little disagreement with regards to the consequence of fetal infection with pathogenic organisms. This is particularly true in swine, where despite the significant barrier posed by epitheliochorial placentation, numerous viral pathogens are known to infect the fetus and cause significant mortality and economic loss [2], [3], [4]. While some of these pathogens, such as porcine parvovirus, can be easily controlled through effective vaccination programs [5] others remain a significant burden within the swine industry.

With minimal progress toward a broadly heterologous vaccine or any other effective long-term control mechanism, the impact of porcine reproductive and respiratory virus (PRRSV) on the pig fetus remains a significant challenge for the swine industry. Much of the difficulty encountered with this pathogen stems from its capacity for manipulating the host immune system, a topic that has been extensively studied both in vitro and in vivo. However, the overwhelming majority of this work has focused on post natal swine and, as a result, comparatively little is known about how the virus interacts with the fetal immune system. The late-gestation pig fetus has been shown to be capable of mounting an immune response to PRRSV infection [6], [7]. However, in spite of this immune response, viral strains such as NVSL 97-7985 and KS06-72109 which typically cause only minor morbidity in postnatal animals [8] lead to significant mortality among infected fetuses [9]. In addition, fetuses that do survive to parturition may be congenitally infected and are at greater neonatal susceptibility to other pathogens [10] suggesting that development of the immune system is substantially compromised and unable to clear infection following in utero infection. The mechanism by which PRRSV causes such exaggerated effects following fetal infection remains elusive. With little evidence of pathology in the form of lesions found during examination of fetal tissues [11], much of the work in the area has focused on the placenta. The severity of reproductive PRRS has in the past been associated with viral replication and its effects on the placenta [12], [13], however, more recent examination into the temporal progression of placental pathology following infection has cast doubt on this relationship [14].

In humans, fetal meconium staining, and in particular meconium aspiration, has been associated with the presence of placental lesions [15] as well as hypoxia and abnormal cardiac function [16] and more generally fetal distress. In the PRRSV infected fetus, meconium staining has been associated with higher viral load in both the thymus and serum [17], apoptotic cell numbers in the maternal-fetal interface [18], as well as the presence of fetal and umbilical lesions [14], and is generally thought to be an early indicator of fetal death. In the present study, we probed the relationship between meconium staining, as an indicator of disease progression and reduced viability, and select elements of the fetal immune response. We focused on the expression of cytokines in fetal thymus which is thought to be the primary site of PRRSV viral replication [11], as well in fetal spleen. We then compared this transcriptional response to the systemic accumulation of select cytokines. Finally, we examined expression of cyclin dependant kinases due to their ability to regulate the cell cycle and post-transcriptionally interfere in the production of Interferon beta.

Section snippets

Pregnant gilt challenge model

Fetal spleen, thymus and serum were obtained from a large-scale challenge experiment [19], for which highly detailed methodology has previously been published [20], [21]. In short, a total of 130 pregnant Landrace gilts were obtained from a PRRSV free nucleus herd at day 80 of gestation and housed in a level II containment. At day 85, 111 of these animals were inoculated with a total of 1 × 105 TCID50 of PRRSV-2 strain NVSL 97-7895 delivered 50/50 intramuscularly and intranasally, with 19

Thymic cytokine gene expression

To evaluate the fetal capacity to respond to PRRSV infection and determine if this response was associated with reduced fetal viability, the relative expression of eight cytokines in the fetal thymus was measured (Fig. 1), with the expectation that expression profiles of HV-VIA and HV-MEC would be distinct from each other as well as from UNIF and CON groups. The acute pro-inflammatory cytokine tumor necrosis factor alpha (TNF) was elevated in the HV-VIA group by 1.9 fold relative to CON but

Discussion

Although the host immune response to PRRSV infection has been extensively studied in the post-natal pig, the fetal response to this virus remains inadequately characterized. The present study is, to our knowledge, the first to evaluate the immune response across a variety of phenotypes representing resistant (UNIF), resilient (HV-VIA) and susceptible (HV-MEC) fetuses following maternal infection with PRRSV. Our results show a robust fetal immune response occurs among infected fetuses, as well

Conclusions

Collectively our results show that the late gestions pig fetus is capable of mounting a robust immune response following vertical transmission of PRRSV and find this response is highly compartmentalized, noting only minor increases in select cytokines within uninfected animals. Contrary to expectation, we find limited variation in the immune response associated with disease progression and reduced fetal viability as defined by meconium staining. The primary exception was the over expression of

Author’s contributions

JAP performed fetal selection, conducted the assays, analysis and drafted the manuscript. JCSH planned and led the large scale PRRSV infection trial. DJM and JCSH supervised experimental design. All authors read and revised the final manuscript.

CRediT authorship contribution statement

J. Alex Pasternak: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data curation, Writing - original draft, Visualization. Daniel J. MacPhee: Conceptualization, Resources, Writing - review & editing, Supervision, Funding acquisition. John C.S. Harding: Conceptualization, Methodology, Validation, Resources, Data curation, Writing - review & editing, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The samples used in this experiment were derived from a large scale multi-institutional challenge experiment involving numerous researchers and staff from the University of Saskatchewan and University of Alberta. The authors would like to collectively acknowledge this group, particularly former HQP responsible for phenotyping fetuses. In addition, we with to acknowledge Dr. Joan Lunney for her review of the manuscript and provision of material support including access to the anti-CCL2

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