Up-Regulation of Immune Checkpoints in the Thymus of PRRSV-1-Infected Piglets in a Virulence-Dependent Fashion

Abstract

Virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains, such as the Lena strain, have demonstrated a higher thymus tropism than low virulent strains. Virulent PRRSV strains lead to severe thymus atrophy, which could be related to marked immune dysregulation. Impairment of T-cell functions through immune checkpoints has been postulated as a strategy executed by PRRSV to subvert the immune response, however, its role in the thymus, a primary lymphoid organ, has not been studied yet. Therefore, the goal of this study was to evaluate the expression of selected immune checkpoints (PD1/PDL1, CTLA4, TIM3, LAG3, CD200R1 and IDO1) in the thymus of piglets infected with two different PRRSV-1 strains. Thymus samples from piglets infected with the low virulent 3249 strain, the virulent Lena strain and mock-infected were collected at 1, 3, 6, 8 and 13 days post-infection (dpi) to analyze PRRSV viral load, relative quantification and immunohistochemical staining of immune checkpoints. PD1/PDL1, CTLA4, TIM3, LAG3 and IDO1 immune checkpoints were significantly up-regulated in the thymus of PRRSV infected piglets, especially in those infected with the virulent Lena strain from 6 dpi onwards. This up-regulation was associated with disease progression, high viral load and cell death. Co-expression of these molecules can affect T-cell development, maturation and selection, negatively regulating the host immune response against PRRSV.

PRRSV-1 induced lung lesion is associated with an imbalance between costimulatory and coinhibitory immune checkpoints

“…Previous studies have already pointed to an up-regulation of PD1/PDL1 axis in PRRSV-1 and PRRSV-2 infections ( Richmond et al, 2015a , b ; Chaudhari et al, 2020 , 2021 ; Ruedas-Torres et al, 2021b ), with decreased levels of apoptosis and anergy in porcine lymphocytes deficient in PD1 ( Richmond et al, 2015b ). In our study, virulent Lena-infected piglets showed a remarkable up-regulation of PDL1 from 6 dpi onwards at lung level in association with a more severe inflammatory response.…”

“…Thus, the lack of costimulatory signals or exuberant coinhibitory signals lead to anergy or tolerance ( Sckisel et al, 2015 ). Many coinhibitory molecules such as CTLA4, PD1/PDL1, LAG3, CD200:CD200R have been evaluated during acute viral infection in pigs, suggesting an effect of these molecules to prevent overstimulation of T cell and/or immunopathology in peripheral organs ( Yue et al, 2014 ; Richmond et al, 2015a , b ; Yue et al, 2015 ; Cai et al, 2020 ; Ruedas-Torres et al, 2021b ). However, to the authors’ knowledge, few studies have considered the expression of costimulatory and coinhibitory molecules in target organs upon PRRSV infection ( Yue et al, 2014 ; Richmond et al, 2015a , b ; Yue et al, 2015 ; Cai et al, 2020 ; Ruedas-Torres et al, 2021b ).…”

“…Many coinhibitory molecules such as CTLA4, PD1/PDL1, LAG3, CD200:CD200R have been evaluated during acute viral infection in pigs, suggesting an effect of these molecules to prevent overstimulation of T cell and/or immunopathology in peripheral organs ( Yue et al, 2014 ; Richmond et al, 2015a , b ; Yue et al, 2015 ; Cai et al, 2020 ; Ruedas-Torres et al, 2021b ). However, to the authors’ knowledge, few studies have considered the expression of costimulatory and coinhibitory molecules in target organs upon PRRSV infection ( Yue et al, 2014 ; Richmond et al, 2015a , b ; Yue et al, 2015 ; Cai et al, 2020 ; Ruedas-Torres et al, 2021b ). Thus, the present work evaluates the relative expression of costimulatory ( CD28 , CD226 , TNFRSF9 , SELL , ICOS and CD40 ) and coinhibitory ( CTLA4 , TIGIT , PD1/PDL1 axis, TIM3 , LAG3 , and IDO1 ) molecules in lung and tracheobronchial lymph node upon an experimental infection with two PRRSV-1 strains of different virulence, virulent Lena and low virulent 3249 strains, during the first 2 weeks post-infection.…”

“…Up-regulation of these molecules was higher in the virulent CSFV strain and would contribute to the lymphopenia observed after CSFV infection ( Auray et al, 2020 ). Besides playing a potential role in apoptosis phenomena, PDL1 has been hypothesized to interact with CD80 molecule competing or inhibiting its interaction with CD28 and, thus, leading to an inhibitory milieu which affects T-cells activation ( Yue et al, 2015 ; Ruedas-Torres et al, 2021b ). In the tracheobronchial lymph node from both infected groups an increase in the expression of PD/PDL1 axis was observed at the end of the study, which could lead to a disorder on T-cell activation.…”

“…The role of PD1/PDL1 axis in lymphocyte anergy and apoptosis phenomena in pigs was demonstrated by the same authors in a parallel study ( Richmond et al, 2015b ). Moreover, a marked up-regulation of PDL1 and CTLA4 genes has been also observed in the thymus of piglets infected with the virulent PRRSV-1 Lena strain ( Ruedas-Torres et al, 2021b ). In this context, transcriptional genome analysis performed by our research group and others have also described an overexpression of some of these costimulatory and coinhibitory molecules in target organs from PRRSV-1 and PRRSV-2 infected pigs ( Chaudhari et al, 2020 , 2021 ; Fleming et al, 2020 ; Sánchez-Carvajal et al, 2021a ).…”