Activation of regulated cell death in the lung of piglets infected with virulent PRRSV-1 Lena strain occurs earlier and mediated by cleaved Caspase-8

Abstract

PRRSV-1 virulent strains cause high fever, marked respiratory disease and severe lesions in lung and lymphoid organs. Regulated cell death (RCD), such as apoptosis, necroptosis and pyroptosis, is triggered by the host to interrupt viral replication eliminating infected cells, however, although it seems to play a central role in the immunopathogenesis of PRRSV, there are significant gaps regarding their sequence and activation upon PRRSV-infection. The present study evaluated RCD events by means of caspases expression in the lung of PRRSV-1-infected pigs and their impact on pulmonary macrophage subpopulations and lung lesion. Conventional piglets were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6, 8 and 13 dpi. Lena-infected piglets showed severe and early lung damage with a high frequency of PRRSV-N-protein+ cells, depletion of CD163+ cells and high viral load in the lung. The number of TUNEL+ cells was significantly higher than cCasp3+ cells in Lena-infected piglets during the first week post-infection. cCasp8 and to a lesser extent cCasp9 were activated by both PRRSV-1 strains after one week post-infection together with a replenishment of both CD163+ and Arg-1+ pulmonary macrophages. These results highlight the induction of other forms of RCD beyond apoptosis, such as, necroptosis and pyroptosis during the first week post-infection followed by the activation of, mainly, extrinsic apoptosis during the second week post-infection. The recovery of CD163+ macrophages at the end of the study represents an attempt to restore pulmonary macrophage subpopulations lost during the early stages of the infection but also a macrophage polarisation into M2 macrophages.

Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1

“…GO terms such as “cytokine-mediated signaling pathway,” “response to bacterium,” “regulation of cytokine production,” “IFN-I signaling pathway,” and “cellular response to tumor necrosis factor” were enriched in this cluster. According to our previous results and those from others, these inflammatory-related pathways contribute to the severe clinical outcomes and lung lesions observed in Lena-infected piglets, but also to an early upregulation of pro-inflammatory cytokines associated with a high viral load and the induction of regulated cell death ( 14 , 39 – 42 ). These results confirm that the dynamics and extent of virulent Lena strain replication are critical for the disease outcome, inducing a strong and early inflammatory response in the lung.…”

Activation of T-bet, FOXP3, and EOMES in Target Organs From Piglets Infected With the Virulent PRRSV-1 Lena Strain

“…Curiously, in our study, a peak of the FASL gene was demonstrated in the lung and tracheobronchial lymph node from virulent Lena-infected piglets parallel to the expression of EOMES , which was also shown through immunohistochemical expression of Fas. The interaction of Fas/FasL mediates the cleavage of caspase-8, a mediator of extrinsic apoptotic pathway ( 65 ), whose activation has been previously described in the lung and thymus from animals infected with the virulent Lena strain ( 20 , 21 ). Moreover, EOMES, together with T-bet, could also be co-expressed in naïve and effector CD8 + T cells, which promotes IFN-γ, perforin, and granzyme B expressions ( 3 , 66 ).…”

“…The activation of the Th1 and effector CD8 phenotypes by the upregulation of the T-bet gene is also supported in our study by the overexpression of the cytokine TNFA in the tracheobronchial lymph node and lung of virulent Lena-infected animals at the end of the study. This pro-inflammatory cytokine has been associated with apoptosis and other cell death phenomena in the context of PRRSV infection, specifically during infection with the virulent Lena strain, not only in the lung but also in the thymus ( 20 , 21 ), helping to explain the severity of the lesions associated with this strain. Whereas IFN-γ + and TNF-α + cells were mainly located in lymphocytes from the medullar area in the tracheobronchial lymph node and thymus, pointing out lymphoid lineage (expressing the T-bet gene) as the major source of IFN-γ and TNF-α in these organs, the expressions of these cytokines in the lung were mostly evidenced in interstitial and pulmonary alveolar macrophages, mainly associated with local inflammatory response.…”

“…The Lena strain, classified as a virulent PRRSV-1 strain, has been shown to exhibit a strong inflammatory immune response in the tracheobronchial lymph node and the lung, together with high severity of lesions ( 17 , 18 ). Moreover, several studies from our researcher group have shown high rates of cell death in the lung and thymus of pigs infected with virulent PRRSV-1 strains ( 19 – 21 ), which could be partially linked with cytotoxic activity ( 2 , 11 – 13 ).…”

In Situ Hybridization of PRRSV-1 Combined with Digital Image Analysis in Lung Tissues of Pigs Challenged with PRRSV-1

“…These methods—widely used in human and veterinary pathology—allow the examination of the histopathological changes and to observe the amount, the distribution, as well as the cell tropism of the given pathogen within the tissues. These features can be useful to assess the pathological role of PRRSV in natural cases of porcine respiratory disease complex [ 32 ], but the methods are more commonly used in experimental settings, where PRRSV is the only pathogen [ 33 ].…”