Activation of the extrinsic apoptotic pathway in the thymus of piglets infected with PRRSV-1 strains of different virulence

Highlights

• Thymi of pigs infected with PRRSV-1 strains of different virulence were evaluated.

• Thymi of pigs infected with Lena virulent strain showed more severe thymic atrophy.

• Expression of TUNEL and cCasp3 was higher in thymi of Lena infected pigs.

• Extrinsic pathway was upregulated in thymi of both strains infected pigs.

Abstract

In the last decade, the outbreaks caused by virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains from both PRRSV-1 and PRRSV-2 have considerably increased. PRRSV is able to modulate the host’s immune response through the induction of apoptosis of cells in lymphoid organs like thymus, increasing the susceptibility to secondary infectious agents. The present study aimed to compare the impact of two PRRSV-1 strains, a field low virulent strain (3249 strain) and a virulent strain (Lena strain), in the thymus of infected pigs, focusing on clinical signs, histological analysis, viraemia, thymus viral load and the study of the different routes of apoptosis phenomena by immunohistochemistry. Sera and thymus samples were collected from infected animals with 3249 strain, Lena strain and mock-infected animals at 1, 3, 6, 8 and 13 days post-infection (dpi). Lena-infected animals showed severe clinical disease, high sera and thymus viral loads with evident thymic atrophy since 6 dpi, matching with PRRSV-N protein, TUNEL and cCasp3 expression in the thymic cortex. In both infected groups, there was an increase in the number of cells expressing molecules related to the extrinsic pathway of apoptosis (cCasp8 and Fas) in cortex and medulla, showing an important role in the apoptosis induction produced in thymus of PRRSV-infected piglets. The extensive apoptosis in the thymus through this pathway would lead to a decrease in the number of mature T lymphocytes and the sustained release of viral particles, which may explain the greater severity of the clinical signs observed in Lena-infected pigs.

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causal agent of the, probably, most economically important disease for the swine industry worldwide. Its virulence and tremendous antigenic diversity have given rise to consider the two classical genotypes of the virus, type 1 or European genotype (PRRSV-1) and type 2 or North American genotype (PRRSV-2), as two independent viral species. Thus, PRRSV has been recently reclassified within the new genus Betaarterivirus (family Arteriviridae, order Nidovirales) as Betaarterivirus suid 1 (former PRRSV-1) and Betaarterivirus suid 2 (former PRRSV-2) (Gorbalenya et al., 2018). PRRSV-1 is divided into four phylogenetical subtypes: pan-European subtype 1, Eastern European subtypes 2 and 3, and a tentative subtype 4 with strains from Latvia and Belarus (Stadejek et al., 2013). In case of PRRSV-2, nine different lineages are recognised (Brar et al., 2014).

From 2007 onwards, several outbreaks, caused by the so-called highly pathogenic PRRSV strains (HP-PRRSV), were reported in Asia and related to PRRSV-2 strains (Tong et al., 2007; Zhou et al., 2008; Wu et al., 2009; Zhou and Yang, 2010). Almost simultaneously, virulent PRRSV-1 strains were also reported in Europe (Karniychuk et al., 2010; Sinn et al., 2016; Canelli et al., 2017; Renson et al., 2017). Since then, the comparative study of the immunopathogenesis of strains of different virulence has gained special interest.

The thymus is the primary lymphoid organ where the development and maturation of T cells take place (Pearse, 2006; Savino, 2006). During this process, thymocytes receive suitable signals for migration, proliferation and differentiation from cortical and medullary epithelial cells, macrophages and dentritic cells, which act as thymic antigen presenting cells (APCs) (Savino, 2006). A negative selection is performed, resulting in the apoptosis of self-reactive thymocytes and allowing positively selected thymocytes to progress in their differentiation status (Savino, 2006; Miller, 2011). PRRSV, and with special emphasis virulent strains, has the capability to replicate in the thymus of infected animals inducing intense death of thymocytes, mainly due to apoptosis phenomena, and leading to thymus atrophy in most cases (Wang et al., 2011; He et al., 2012; Guo et al., 2013; Wang et al., 2014, 2015; Amarilla et al., 2016; Ogno et al., 2019). The presence of elevated numbers of apoptotic cells in the thymus of PRRSV-infected pigs suggests the possibility that virus-infected APCs cannot interact properly with developing thymocytes, which leads to a failure in the positive selection process and a reduction in the total number of mature T cells (Li et al., 2014; Wang et al., 2015). This fact deteriorates the host’s immune system by increasing its susceptibility to secondary infectious agents.

Apoptosis or “programmed cell death” is a highly regulated mechanism of cellular death that involves a complex network of biochemical pathways, which disturb cell metabolism and physiology (Hetts, 1998; Barranco et al., 2011). Three pathways of apoptosis have been already described, namely, the extrinsic pathway, the intrinsic pathway and the perforin/granzyme pathway (Danial and Korsmeyer, 2004; Elmore, 2007). The extrinsic or death receptor pathway is triggered by the binding of extracellular death receptors with their specific soluble ligands (highlighting FasR/FasL and TNF-α/TNFR1, as best characterised models), which then induce the activation of the initiator caspase-8 (Elmore, 2007; Gump and Thorburn, 2011; Miller and Fox, 2004). The intrinsic or mitochondrial pathway is directly initiated as a response to cellular stressors (such as DNA damage, toxics, radiation, hypoxia, hyperthermia or viral infections) and is regulated by Bcl-2 family members. Other molecules, besides Bcl-2 family members, such as inducible nitric oxide synthase (iNOS), have been showed to induce apoptosis by increasing the release of cytochrome c from the mitochondria and activating the initiator caspase-9 (Wu et al., 2002; Elmore, 2007; Miller and Fox, 2004; Nagata, 2018). Perforin/granzyme pathway or T-cell mediated cytotoxicity can produce apoptosis through the release of granzyme A and granzyme B granules (Danial and Korsmeyer, 2004; Elmore, 2007). These three pathways of apoptosis merge in the activation of executioner caspases (caspase-3 and caspase-7), which irreversibly leads to DNA fragmentation, degradation of vital cell proteins and formation of apoptotic bodies (Resendes et al., 2004; Elmore, 2007). Apoptotic bodies are quickly phagocytosed by macrophages and surrounding cells, avoiding the inflammatory reaction, main feature that allows distinguishing apoptosis and necrosis (Hetts, 1998; Elmore, 2007). Knowing the activated pathways of apoptosis by viral infections is crucial for understanding the pathogenic mechanisms whereby viruses exert their action.

Our previous studies have demonstrated a higher induction of apoptosis phenomena by a virulent strain compared to low virulent strains (Amarilla et al., 2016). Nevertheless, the mechanisms involved in the development of such apoptosis in the thymus of PRRSV-infected pigs have not been totally elucidated. Thus, the present study aimed to compare the impact of two PRRSV-1 strains, a field low virulent strain (3249 strain) and a virulent strain (Lena strain), in the thymus of infected pigs, focusing on clinical signs, histological analysis, viraemia, thymus viral load and the study of the different routes of apoptosis phenomena.