Sánchez‑Carvajal et al. Vet Res (2021) 52:12 https://doi.org/10.1186/s13567-020-00882-x RESEARCH ARTICLE Open Access 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 Jose María Sánchez‑Carvajal1*† , Inés Ruedas‑Torres1*†, Librado Carrasco1, Francisco José Pallarés1, Enric Mateu2,3, Irene Magdalena Rodríguez‑Gómez1† and Jaime Gómez‑Laguna1† 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 apopto‑ sis 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. Keywords: PRRSV-1, virulent strain, regulated cell death, lung, cleaved caspase-8 Introduction During the last two decades, several outbreaks caused by virulent Porcine reproductive and respiratory syndrome virus-1 (PRRSV-1) strains have been reported along *Correspondence: v42sancj@uco.es; iruedas@uco.es † Jose María Sánchez-Carvajal and Inés Ruedas-Torres contributed equally Europe, leading to high mortality, fever, acute respiratory as first authors clinical signs as well as severe damage in both lung and † Irene Magdalena Rodríguez-Gómez and Jaime Gómez-Laguna shared primary lymphoid organs [1–6]. Lung lesion can range senior authorship 1 Department of Anatomy and Comparative Pathology and Toxicology, from the typical interstitial pneumonia observed with Faculty of Veterinary Medicine, University of Córdoba, 14014 Córdoba, low virulent strains to suppurative bronchopneumonia Spain after infection with virulent strains, usually associated Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 2 of 14 with secondary bacterial infection [3, 5, 7]. In this line, between different RCD pathways and, as a result, innate although apoptosis and necrosis have been well charac- immune signalling events are well activated even upon terised in lymphoid organs of virulent PRRSV-1-infected virus inhibitors [26, 34]. animals [8–11], limited information is available regarding It is well-known that PRRSV elicits apoptosis, necrosis the onset of cell death phenomena in the lung. and necrosis like-apoptosis in lungs and lymphoid organs PRRSV encompasses two different viral species, Betar- causing a detrimental effect of the host immune system terivirus suid 1 (formerly PRRSV-1) and Betarterivirus [11, 17, 35–40]. Nevertheless, the study of RCD in the suid 2 (formerly PRRSV-2) [12], and belongs to the genus lung associated with virulent strains is scarce and just Betaarterivirus (family Arteriviridae, order Nidovirales). focused on detection of cell death by TUNEL [11, 41]. PRRSV has a restricted tropism for cells of the monocyte/ The objective of this study was to evaluate RCD events in macrophage lineage [13], being the pulmonary alveolar the lung of pigs infected with PRRSV-1 strains of different macrophage (PAM) the target cell for viral replication virulence and their impact on pulmonary macrophage and CD163 scavenger receptor the essential mediator subpopulations as well as its contribution to lung lesion for viral internalization and disassembly [14, 15]. PRRSV development. We hypothesised that strain dependent is also able to replicate in other pulmonary macrophage induction of RCD might directly impact on the impair- subpopulations such as interstitial and intravascular ment of the innate and adaptive immune responses at macrophages (PIMs) [13, 16, 17]. Whereas PAMs repre- lung level, decreasing pulmonary macrophages, hence sent the first line of defence against pathogen invasion increasing the susceptibility of infected pigs to secondary [18, 19], PIMs and interstitial macrophages play a pivotal infections. role in the production of pro-inflammatory cytokines compromising their basic functions, including cytokine Material and methods and chemokines production, polarisation, phagocytosis Animals and experimental design and antigen presentation upon PRRSV infection [20–24]. Animals and samples used in this study belong to a large Regulated cell death (RCD) is considered a sophis- project carried out in order to investigate the pathogen- ticated molecular mechanism which can take place in esis of PRRSV-1 strains of different virulence [42]. Briefly, physiological conditions, and then referred to as pro- a total of sixty-five 4-week-old piglets (Landrace x Large grammed cell death, or after extracellular or intracellular White) were assigned to three different experimen- perturbations, comprising different modalities such as tal groups: (i) control group (n = 15), (ii) 3249-infected intrinsic and extrinsic apoptosis, necroptosis, pyroptosis group (n = 25) and (iii) Lena-infected group (n = 25). and NETotic cell death, among others [25]. Thus, dur- All pigs included in the study were tested for relevant ing the course of viral infections, RCD can be activated pathogens, such as PRRSV, porcine circovirus type 2 and to interrupt viral replication and eliminate virus-infected Mycoplasma hyopneumoniae by ELISA and PCR assays cells [26]. Apoptosis is an evolutionary conserved mech- (IDEXX PRRS X3 Ab Test, IDEXX Laboratories, S.L., anism of RCD that can be started by either external or Barcelona, Spain; in-house PCR against porcine circovi- internal stimuli and requires the activation of cysteine- rus type 2 [43] and Mycoplasma hyopneumoniae [44]), aspartic proteases known as caspases, which results in being negative for all of them at the beginning of the the degradation of cellular components, DNA fragmenta- experiment. After a week of acclimation, 3249- and Lena- tion and formation of apoptotic bodies, which are then infected groups were intranasally inoculated with 2 ml of mainly phagocytosed by macrophages [27–30]. In this the low virulent 3249 strain or the virulent Lena strain cascade of events, caspase-8 and caspase-9 play a key role (1 × 105 TCID50) diluted in sterile RPMI 1640 medium as initiator caspases, triggering off extrinsic or intrinsic (ThermoFisher Scientific, Barcelona Spain), respec- apoptosis, respectively. In both cases, initiator caspases tively. The field isolate 3249 strain (subtype 1 PRRSV-1) will ultimately activate the executioner caspases, such was first isolated from the serum of a piglet with pneu- as caspase-3, for dismantling of dying cells [25, 31]. In monia belonging to a PRRSV-positive Spanish farm in recent years, other mechanisms of RCD, such as necrop- 2005 [45]. Lena strain (subtype 3 PRRSV-1) was isolated tosis or pyroptosis [25, 32], which also play a pivotal role from a PRRSV-positive herd with a high mortality rate, in antiviral responses, have been well described [26, 33, reproductive failure and respiratory disorders in 2007 in 34]. Thus, when viruses try to manipulate RCD for its Belarus [3]. Piglets from control group were inoculated own advantage by inhibiting caspases-3, -8 and -9 signal- with porcine alveolar macrophages supernatant diluted ling pathways, pyroptosis and necroptosis may be elicited in sterile RPMI 1640 medium (ThermoFisher Scientific) by the host to destroy infective and non-infective cells, similarly to the inoculum. At 1, 3, 6, 8 and 13 days post- restricting viral replication and limiting reservoir cells inoculation (dpi), 3 animals from the control group and for viruses [32, 33]. Thereby, there are many crosstalk 5 animals from both infected groups were humanely Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 3 of 14 euthanised. At necropsy, gross lung lesions were Signaling Technology, Inc., MA, USA) were performed recorded by the same pathologist as described elsewhere by using commercial kits according to the manufacturer’s [46]. Samples from cranial, middle and caudal lobes of protocols. In the case of TUNEL assay, sixty-one out of the right lung were collected and conserved at – 80 ºC sixty-five animals were selected according to lung histo- for the analysis of PRRSV viral load. In addition, samples pathology score as detailed in Table 1. from the middle lobe of the right lung were fixed in 10% The avidin–biotin-peroxidase complex technique was neutral buffered formalin for the histopathological and performed to detect PRRSV-N-protein, CD163, Argin- immunohistochemical analyses. ase-1 (Arg-1), cleaved Caspase-8 (cCasp8) and cleaved Caspase-9 (cCasp9). Briefly, sections were dewaxed in PRRSV lung viral load xylene and rehydrated followed by endogenous peroxi- dase inhibition with 3% ­H2O2 in methanol for 30 min lung tissue by using TRIzol™ LS Reagent and Nucle- RNA was isolated and purified from a homogenate of in the darkness. Thereafter, slides were exposed to dif- oSpin® RNA virus columns kit according to manufac- ferent antigen retrieval treatments as summarised in turer’s protocols (Macherey–Nagel, Düren, Germany). Table 2. After PBS washes (pH 7.4) and incubation with 100 µL of 2% bovine serum albumin (BSA), monoclonal using VetMAX™ PRRSV EU/NA 2.0 kit (Thermo Fisher Then, viral load in the lung was quantified by RT-qPCR primary antibodies (mAb) against PRRSV-N-protein, CD163, Arg-1, cCasp8 and cCasp9 were incubated over- MyiQ™2 Two-Color Real-Time PCR Detection Sys- Scientific, Barcelona, Spain). RNA was amplified in the night at 4 ºC in a humid chamber. Afterwards, sections tem (Bio-Rad, Hercules, CA, USA) under the following were washed with PBS and incubated with a biotinylated cycling conditions: 50 °C for 5 min, 95 °C for 10 min fol- goat anti-mouse secondary antibody (diluted 1 in 100 in lowed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. In 2% BSA) (Dako, Glostrup, Denmark) for 30 min at room order to not overestimate the number of PRRSV viral temperature. Then, the avidin–biotin-peroxidase com- particles in the lung, results for viral load were expressed plex (ABC Vector Elite, Vector Laboratories, Burlingame, by changes in cycle threshold (Ct) as previously described CA, USA) was applied and incubated for 1 h at room tion of the NovaRED™ substrate kit (Vector Laboratories, [47, 54]. An inter-run calibrator sample with a known temperature. Immunolabelling was visualised by applica- number of PRRSV copies was introduced in each experi- ment to self-control inter-run variation. The area under Burlingame, CA, USA). Sections were counterstained ing alcohols and mounted with Eukitt® (Orsatec GmbH, the curve (AUC) for viral load in the lung was calculated with Harris’s haematoxylin, dehydrated in graded ascend- using the trapezoidal approach [48]. Bobingen, Germany). Antibody specificity was verified Histopathology of lung tissue by substituting the primary antibody by isotype matched Four-micron tissue sections were stained with haematox- reagents of irrelevant specificity. For negative controls, ylin and eosin (H&E) and blindly scored by two patholo- the primary antibody was replaced by BSA blocking gists for histopathological evaluation. The severity of solution. lung lesions for the interstitial pneumonia was graded The labelled cells were counted in 25 non-overlapping according to Halbur et al. [46], as follows: no microscopic selected high magnification fields of 0.2 mm2 (Olympus lesions, 0; mild interstitial pneumonia, 1; moderate mul- BX51, Olympus Iberia SAU, L’Hospitalet de Llobregat, tifocal interstitial pneumonia, 2; moderate diffuse inter- Barcelona, Spain). The number of positive cells per ­mm2 stitial pneumonia, 3; and severe interstitial pneumonia, (cells/mm2) was expressed as the mean of the score for 4. Moreover, a similar score was conducted as previously each animal within each group. Immunolabelled cells described by Rodríguez-Gómez et al. [42] to evaluate suppurative bronchopneumonia: no microscopic lesions, 0; mild bronchopneumonia, 1; moderate multifocal bron- Table 1 Number of pigs from each group subjected chopneumonia, 2; moderate diffuse bronchopneumonia, to TUNEL assay at 1, 3, 6, 8 and 13 days post-infection (dpi) 3; and severe bronchopneumonia, 4. The sum of both Dpi Control 3249 Lena interstitial pneumonia and bronchopneumonia scores were considered the final score. 1 3 4 4 3 3 5 5 Immunohistochemistry assays 6 2 5 5 Terminal dUTP Nick End-Labelling (TUNEL) assay (In 8 2 5 5 Situ Cell Death Detection Kit, POD, Roche, Mannheim, 13 3 5 5 Germany) and cleaved Caspase-3 (cCasp3) (SignalStain 3249: field strain of low virulence. Apoptosis Cleaved Caspase-3 IHC Detection Kit, Cell Lena: virulent PRRSV-1 strain. Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 4 of 14 Table 2 Summary of immunohistochemical methodology Specificity Type Commercial origin Blocking solution Dilution Antigen retrieval of antibody TUNEL N.A In Situ Cell Death Detection Kit, POD, Roche, N.A N.A Proteinase K in heat incubator Germany cCasp3 (Asp175) mAb SignalStain Apoptosis Cleaved Caspase-3 IHC 5% NGS 1:500 pH 6 citrate buffer in microwave Detection Kit, Cell Signaling, USA PRRSV (SDOW 17) mAb Rural Technologies, Brookings, SD, USA 2% BSA 1:500 Protease XIV in water bath CD163* (2A10/11) mAb INIA, Madrid, Spain 2% BSA Neat pH 3.2 citrate buffer microwave Arginase-1 pAb ThermoFisher Scientific, Barcelona, Spain 2% BSA 1:100 pH 8.5 citrate buffer microwave cCasp8 (Asp391) mAb Cell Signaling; Danvers, MA, USA 2% BSA 1:100 Proteinase K in heat incubator cCasp9 (Asp330) mAb Cell Signaling; Danvers, MA, USA 2% BSA 1:50 Proteinase K in heat incubator mAb: monoclonal antibody, pAb: polyclonal antibody, N.A.: not applicable, NGS: Normal goat serum, BSA: Bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany); Proteinase K (Roche, Basel, Switzerland), 15 min at 37 °C in heat incubator; Protease Type XIV (Sigma-Aldrich), 8 min at 37 °C in water bath. *CD163 mAb was kindly provided by Dr. J. Domínguez, INIA, Madrid, Spain. were morphologically identified as PAMs, PIMs, intersti- syncytial cells (Figure 1B). This lesion had a stronger and tial macrophages, lymphocytes, or neutrophils. The AUC earlier onset in pigs inoculated with virulent Lena strain for cCasp3, cCasp8 and cCasp9 was calculated using the (Figures 1B, C). Moreover, extensive foci of suppurative trapezoidal approach [48]. bronchopneumonia composed of neutrophils, cells debris and mucus filling the bronchial, bronchiolar, and alveolar Statistical analyses lumen were observed from 6 dpi onwards, particularly in Data analyses and figures were performed by using those animals infected with Lena strain. Multifocal clumps GraphPad Prism software version 7.0 (GraphPad soft- of purple amorphous material which were proved to be ware, San Diego, CA, USA) and InkScape software ver- free chromatin by Feulgen technique [42], were observed sion 0.92.3 (InkScape, Brooklyn, NY, USA). Differences in the alveoli of some of the Lena-infected pigs with higher between groups were evaluated for approximate normal- bronchopneumonia scores. ity of distribution by the D’Agostino & Pearson omnibus normality test, followed by the Mann Whitney’s U test. Lung viral load was higher in Lena‑infected pigs Correlation coefficients were assessed by the Spearman At day 0, all animals were negative by RT-qPCR, remaining and Pearson tests and were considered relevant if r > 0.6 pigs from control group negative throughout the study. As or r < -0.6 and P < 0.05. For all data, a P value lower than early as 1 dpi, two out of five piglets in both infected groups 0.05 was considered statistically significant and repre- were PRRSV-1 positive; from this time-point onwards, viral sented as “*”P ≤ 0.05, “**”P ≤ 0.01 and “***”P ≤ 0.001. copies were detected in all infected piglets. Viral load was higher in Lena- compared to 3249-infected pigs (P < 0.01 at 3 dpi; P < 0.05 at 6 and 8 dpi), reaching the peak at 6 dpi Results in Lena (Ct 18.9 ± 0.9), whereas 3249 group peaked at 8 dpi Lena‑infected pigs displayed severe lung lesions compared (Ct 22.9 ± 2.5). At the end of the study, viral load in the lung to 3249‑infected pigs was comparable in both infected groups, Lena and 3249 Gross lesions and histopathology were thoroughly (Figure 2A). The AUC for viral load in lung (median) was described by Rodríguez-Gómez et al. [42]. At necropsy, 300.0 and 261.2 for Lena and 3249 groups, respectively. In tan-mottled areas, atelectasis, rubbery consistency, and both infected groups, statistical analysis revealed a posi- consolidated areas were observed in the lungs from both tive significant correlation between lung viral load and the PRRSV-1-infected groups with a marked increase from microscopic score (r = 0.7 and P < 0.001, for 3249 group; 6 dpi onwards (Figure 1A–C). From this time-point and r = 0.6 and P < 0.01; for Lena group). until the end of the study, lung lesions were more intense in Lena- than in 3249-infected pigs and consisted of severe Lena‑infected pigs presented the highest number of interstitial pneumonia and extensive consolidated areas in PRRSV‑N‑protein+ cells and the lowest number of CD163+ cranial and middle lung lobes (Figure 1A–C). The main cells histopathological lesion in the lung of both infected groups Figure 2 shows the expression of PRRSV-N-protein in consisted of a mild to moderate interstitial pneumonia the lung of each experimental group along the infec- characterised by thickening of the alveolar septa due to tion. The immunolabelling of PRRSV-N-protein was infiltrating lymphocytes and macrophages with occasional Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 5 of 14 Figure 1 Macroscopic and microscopic lung findings. Pictures show the macroscopic lung appearance A at necropsy of representative control (left), 3249 (middle) and Lena (right) animals euthanised at 13 dpi. Lungs from 3249 (middle) and Lena (right) infected pigs shown tan-mottled areas, atelectasis, rubbery consistency, but also consolidated areas are observed in cranial apical and middle lung lobes in Lena-infected pigs (right) (white arrows). Photomicrographs of the middle lung lobe B from control (left), 3249 (middle) and Lena (right) pigs euthanised at 13 dpi (H&E; bars 50 μm). Lung from 3249 (middle) and Lena (right) representative infected piglets show a moderate interstitial pneumonia characterised by thickening of the alveolar septa due to infiltrating lymphocytes and macrophages. Moreover, Lena-infected lung (right) exhibits extensive foci of suppurative bronchopneumonia with neutrophils, cellular debris, and multifocal chromatin clumps (arrow-head). Graphs display macroscopic C and microscopic D lung lesion score of each animal along the study. Bars indicate median values for each group. mainly observed in PAMs and to a lesser extent in PIMs Both Lena- and 3249-infected groups followed a similar and interstitial macrophages (Figures 2C, D). Multifocal kinetics for PRRSV-N-protein+ cells, reaching a peak at 6 clusters of PRRSV-N-protein+ macrophages surrounded dpi. At 13 dpi, the frequency of PRRSV-N-protein+ cells by apoptotic bodies were observed within areas of bron- markedly dropped in both infected groups. The high- chopneumonia in Lena-infected piglets (Figure 2D). est number of PRRSV-N-protein+ cells was observed in Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 6 of 14 Figure 2 PRRSV kinetics of viral load and immunohistochemical expression of PRRSV-N-protein, CD163 and Arg-1 in the lung. Graphs display the kinetics of viral load in the lung for Lena and 3249 strains. Viral load is represented by changes in the cycle threshold (Ct) A and the number of PRRSV-N-protein positive cells/mm2 B in the lung. Scatter dot plot shows individual values from each animal, bars and dashed lines indicate the median for each group and time points. Columns show the median with range of PAMs and septal macrophages (PIMs and interstitial macrophages). Photomicrographs of the middle lung lobe show the expression of PRRSV-N-protein in a representative 3249- C and Lena-infected pig D euthanised at 6 dpi (bar, 50 μm). Insets show in detail PRRSV-N-protein+ expression and viral particles (bar, 20 μm). Photomicrographs of the middle lung lobe show the expression of CD163 in a representative control E and Lena-infected F pig euthanised at 6 dpi (bar, 50 μm). Insets show in detail the immunolabelling of CD163 in the cell membrane and cytoplasm of PAMs (bar, 20 μm). Graph displays the number of C ­ D163+ (primary + 2 axis) and Arg-1 (secondary axis) cells/mm in the lung for each group and time point (G). Scatter dot plots show individual values from each animal, bars and dashed lines indicate the median for each group and time point. Columns represent the median with range of PAMs and septal macrophages. Photomicrograph of the middle lung lobe show the expression of Arg-1+ PAMs in a representative Lena-infected pig at 13 dpi H (bar, 50 μm). Inset shows in detail Arg-1 expression in PAMs (bar, 20 μm). Black arrows represent PAMs, and black arrow-heads interstitial macrophages. Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 7 of 14 lung tissue from Lena-infected piglets at 6 dpi, showing animals from 3 dpi onwards (P < 0.05), as well as com- a significant increase when compared to 3249-infected pared with 3249-infected pigs at 3 (P < 0.05), 6 (P < 0.01), pigs (P < 0.05) (Figure 2B). No positive cells were detected and 8 dpi (P < 0.05) (Figure 4A). For 3249-infected pigs, in control pigs throughout the experiment (Figure 2B). ­ UNEL+ cells took place later at 8 and 13 dpi, the rise in T A significant statistical correlation between PRRSV-N- as compared to control pigs (P < 0.05) (Figure 4A). The protein expression and lung viral load was found in both highest expression was detected at 8 dpi in both PRRSV-1 infected groups (r = 0.6 and P < 0.01, for 3249 group; and infected groups. At 13 dpi a marked dropped in the total r = 0.8 and P < 0.001, for Lena group). number of ­TUNEL+ cells, but still higher than control The expression of the scavenger receptor CD163 was animals, was observed in both infected groups. A nega- detected in the cell membrane and cytoplasm of PAMs, tive correlation was revealed among ­TUNEL+ cells and interstitial macrophages and, occasionally, PIMs (Fig- ­CD163+ cells (r = -0.6 and P < 0.001) in Lena group. ure 2E, F). Control animals exhibited a steady frequency of ­CD163+ cells during the whole study, whereas there Casp3 followed a similar kinetics in Lena‑ was a continuous drop in the number of C ­ D163+ cells and 3249‑infected groups, being more intense for Lena from the beginning until 8 dpi, followed by a sharp rise in group the frequency of ­CD163+ cells at 13 dpi in both PRRSV- cCasp3 was detected in the nuclei and cytoplasm of 1-infected groups (Figure 2G). The decrease was more macrophages, lymphocytes, neutrophils and apoptotic intense in Lena-infected animals when compared to 3249 bodies of both control and infected piglets (Figs. 3C- (Figure 2G). Of note, PAMs were the subpopulation of 3D). ­cCasp3+ interstitial macrophages, PIMs and in pulmonary macrophages which underwent the great- lesser extent lymphocytes were mainly located in areas est reduction in the number of ­CD163+ cells. Moreo- of interstitial pneumonia, whereas c­Casp3+ PAMs and ver, there was a significant negative correlation between neutrophils were commonly observed infiltrating foci of PRRSV-N-protein+ and ­ CD163+ cells (r = -0.6 and bronchopneumonia (Figure 3D). The kinetics of ­cCasp3+ P < 0.0005) in Lena-infected piglets. expression was similar in both PRRSV-1-infected groups, To confirm macrophage polarisation into M2-alterna- rising gradually from 3 dpi (Lena) or 6 dpi (3249) until tive activated macrophages at 13 dpi, Arg-1, a M2 mac- the end of the study and showing a significant increase rophage marker, was performed. Arg-1 immunolabelling when compared to control group (P < 0.05 at 3, 6, 8 and was detected mainly in the cytoplasm of PAMs placed 13 dpi) (Figure 4B). This increase in cCasp3 cells was inside or surrounding bronchopneumonia foci, but also earlier, as above mentioned, and more intense in Lena- in PIMs and interstitial macrophages in a lesser extent infected pigs showing a peak at 8 dpi, whereas the maxi- (Figure 2H). There was a significantly higher frequency mum expression in 3249-infected pigs was observed at of Arg-1+ macrophages in both Lena and 3249-infected 13 dpi (Figure 4B). By contrast, the number of ­cCasp3+ groups when compared with control group at 13 dpi cells remained at low levels in the control group along the (Figure 2G). study. In 3249-infected group, a positive correlation was found between cCasp3 expression and viral load in the TUNEL labelling was highly expressed in the lung lung (r = 0.6 and P < 0.01) as well as, between ­cCasp3+ of Lena‑infected animals cells and PRRSV-N-protein+ cells (r = 0.7 and P < 0.001). TUNEL assay was conducted to detect in situ localization The AUC for cCasp3 (median) in 3249 and Lena groups of DNA fragmentation. TUNEL labelling was detected was 12.1 and 26.9, respectively (Figure 4E, F). in the nuclei and cytoplasm of PAMs, PIMs, interstitial macrophages, free apoptotic bodies and, occasionally, A positive correlation between the frequency of cCasp8+ cells neutrophils, in both control and infected groups (Fig- and cCasp9+ cells was observed in both PRRSV‑1 infected ­ UNEL+ interstitial macrophages and PIMs ures 3A, B). T groups were mainly observed in areas of interstitial pneumonia The immunolabelling of cCasp8 was primarily detected (Figure 3A) whereas ­TUNEL+ PAMs were identified in in the cytoplasm of interstitial macrophages as well as foci of suppurative bronchopneumonia with cell debris PIMs, and in a lesser extent in PAMs and lymphocytes and mucus filling the lumen of bronchioles and alveoli (Figures 5A, B). The frequency of c­ Casp8+ cells in Lena- (Figure 3B). However, due to the aggregation of ­TUNEL+ infected pigs, showed a curve with a progressive increase, cells, mostly in areas of bronchopneumonia, it was not reaching a peak at 8 dpi and decreasing onwards (Fig- always possible to differentiate subsets of labelled cells, ure 4C). This rise was significantly higher when com- thus, in this case the results are expressed as the total pared with the control group at 8 and 13 dpi (P < 0.05). number of T ­ UNEL+ cells. The frequency of T ­ UNEL+ By contrast, the number of c­ Casp8+ cells in 3249 group cells was higher in Lena-infected pigs than in control remained at baseline or below control group with a Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 8 of 14 Figure 3 Immunohistochemistry for TUNEL and cCasp3 in the lung. TUNEL expression in a representative 3249- A and Lena-infected pig B euthanised at 8 dpi (bar, 50 μm). Insets show in detail the immunolabelling of TUNEL in the nuclei and cytoplasm (bar, 20 μm). PAMs (black arrow), interstitial macrophages (black arrow-head) as well as ­TUNEL− neutrophils (red arrows). cCasp3 expression in a representative 3249- C and Lena-infected pig D euthanised at 13 dpi (bar, 50 μm). Insets show in detail the expression of cCasp3 in the nuclei and cytoplasm of PAMs (bar, 20 μm). significant ascent at 13 dpi (P < 0.05) (Figure 4C). A wide for Lena group). In addition, a positive correlation was individual variability was observed in both, Lena- and observed in 3249-infected animals between the fre- 3249-infected pigs. quency of c­ Casp9+ cells and lung viral load (r = 0.6 and Regarding cCasp9, its expression was mainly observed P < 0.01), PRRSV-N-protein+ cells (r = 0.6 and P < 0.001) in the cytoplasm of interstitial macrophages and, occa- and ­cCasp3+ cells (r = 0.7 and P < 0.001). The AUC for sionally, PIMs and PAMs (Figures 5C, D). Both, PRRSV- cCasp8 (median) in 3249 and Lena groups was 9 and 1-infected groups displayed a similar kinetics for 20.6, respectively, whereas for cCasp9 (median) was 5.5 ­cCasp9+ cells. Thus, there was a mild gradual rise from in 3249 and 8.3 in Lena groups. 8 dpi until the end of the study, reaching maximum fre- quencies at 13 dpi in both infected groups (P < 0.05) (Fig- Discussion ure 4D). Lena virulent strain induced a higher number of RCD is a key player in the innate response to combat ­cCasp9+ cells compared to 3249 strain at 8 and 13 dpi. viral infection, disrupting replication and destroying One pig infected with 3249 strain at 6 dpi was considered infected cells from the host [26]. Therefore, PRRSV, as an outlier due to an extreme number of c­Casp9+ cells well as many other viruses, have developed strategies (Figure 4D). The frequencies of ­cCasp8+ and ­cCasp9+ to obstruct or delay apoptosis in order to obtain a win- cells remained low and constant in control pigs dur- dow of time in which viral replication, self-assembly and ing the whole study. In both PRRSV-1 infected groups, release can take place [26, 34, 35, 49]. It has been showed a positive correlation was found between the number of that PRRSV-induced apoptosis is reliant on virulence- ­cCasp8+ cells and the number of ­cCasp9+ cells (r = 0.86 strain since PRRSV-1 virulent strains cause apoptosis and and P < 0.001, for 3249 group; and, r = 0.6 and P < 0.01, necrosis with a higher severity in lymphoid organs than Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 9 of 14 Figure 4 Immunohistochemical expression of TUNEL, cCasp3, cCasp8 and cCasp9 in the lung along the experiment. Graph displays the number of ­TUNEL+ in the lung (cells/mm2) (A). Scatter dot plots show individual values from each animal and dashed lines indicate the median for each group and time points. Columns represent the median with range of total positive cells. Graph represents the number of c­ Casp3+ (B), ­cCasp8+ (C) ­cCasp9+ (D) in the lung (cells/mm2). Scatter dot plots show individual values from each animal and dashed lines indicate the median for each group and time point. Columns represent the median with range of PAMs, septal macrophages lymphocytes and neutrophils. One pig infected with 3249 strain at 6 dpi was considered an outlier (empty green triangle). Graph displays the AUC (median) for the frequency of cCasp3, cCasp8 and cCasp9 positive cells in 3249 E and Lena-infected F group. low virulent strains [9–11]. However, the mechanisms apoptotic bodies. Whereas in a previous study we were involved in RCD phenomena used by virulent strains in able to describe the replenishment of C­ D163+ cells in the lung of PRRSV-1 infected pigs have not been eluci- the lung of SU1-bel infected pigs about one month post dated yet. infection [50], this phenomenon was already observed In the present study, a marked depletion in the num- at two weeks post-infection in the present study. This ber of C­ D163+ PAMs in the lung of infected pigs was recovery of ­CD163+ PAMs, together with an increase observed, which may lead to a failure in the phagocy- of Arg-1+ macrophages, coincided with a decrease in tosis by these cells [7] and hence, affect the clearance of the number of PRRSV-N-protein+ cells and viral load in Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 10 of 14 Figure 5 Immunohistochemistry for cCasp8 and cCasp9 in lung tissue. cCasp8 expression in a representative 3249- A and Lena-infected pig B euthanised at 8 dpi (bar, 50 μm). Inset shows in detail the cCasp8 immunolabelling in the cytoplasm of interstitial macrophages (bar, 20 μm). cCasp9 expression in a representative 3249- C and Lena-infected pig D euthanised at 13 dpi (bar, 50 μm). Inset shows in detail the expression of cCasp9 in the cytoplasm of interstitial macrophages (bar, 20 μm). the lung, which may depict an attempt to remove apop- (necroptosis) and pyroptosis are considered as distinct totic cells and cellular debris, restoring tissue damage. approaches of RCD, depending on the signalling pathway Besides Arg-1 upregulation in M2-alternative activated engaged. Apoptosis is a non-lytic and immunologically macrophages [51, 52], the induction of CD163 is, by far, silent manner of cell death; by contrast, programmed a feature of this subset, which could be playing a relevant necrosis and pyroptosis are lytic and highly inflammatory role in tissue wound repair, accelerating the resolution of processes [25, 56]. In vitro studies have evidenced the inflammation [15, 53, 54]. activation of anti-apoptotic pathways in PAMs and sus- Subsequently, TUNEL assay was carried out to identify ceptible cell lines during the early infection with PRRSV, in situ DNA fragmentation, a feature of both apoptotic followed by the stimulation of apoptosis in late infection as well as necrotic cells [55]. Besides, a cCasp3 immu- [35, 49, 57]. Accordingly, our results highlight a delayed nolabelling method was used to demonstrate the acti- activation of apoptosis up to one week post-infection as vation of the caspase-dependent apoptotic pathway. In a mechanism by which PRRSV could evade the local host our study, the number of T ­ UNEL+ cells was significantly immune response, but also suggest that other forms of higher than the number of c­ Casp3+ cells. This fact was RCD, such as necroptosis and pyroptosis, could be play- observed not only in PRRSV-1-infected but also in non- ing a role during the first week post-infection. infected piglets throughout the study. Thus, this result Pyroptosis may be initiated by inflammasomes, which suggests that an independent cCasp3 pathway of RCD in addition trigger the secretion of proinflammatory could be involved in both pulmonary cell homeostasis of cytokines [58]. PRRSV is able to activate NLRP3 inflam- pigs and clearance of virus-infected cells, since infected masome in PAMs, resulting in the secretion of IL-1β pigs exhibited higher frequencies of T ­UNEL+ cells and a robust inflammatory response [59–61], which than control animals. Apoptosis, programmed necrosis are associated with a severe pathologic lung damage Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 11 of 14 in piglets infected with PRRSV-1 virulent strains, such the lung, which is in agreement with a recent publication as Lena and SU1-bel strains [6, 7, 62]. In the present of our group which describes an unidirectional activa- study, a significant rise in ­TUNEL+ cells was observed tion of apoptosis in the thymus of PRRSV-1 infected pigs in both PRRSV-1-infected pigs, although more pro- through the activation of the extrinsic pathway induced nounced in Lena-infected pigs, from the beginning of by Fas/cCasp8 [9]. Interestingly, different proinflam- the study (3 dpi). This fact may be probably associated matory cytokines may play a significant role not only in with the cytopathic effect of PRRSV, but also with the the inflammatory response but also modulating apopto- strong inflammatory response induced in the lung, espe- sis, underlining the role of TNF-α as a mediator of the cially when the extent of lung injury was greater (8 dpi). extrinsic pathway of apoptosis [27, 28, 30]. In this con- Previously, in a parallel study, we demonstrated that text, our findings highlight the interest of examining dif- Lena-infected animals developed a strong inflamma- ferent target organs and biomarkers to better understand tory response associated with a high serum level of IL-6 the modulation of the host immune response. and IFN-γ, contributing to fever and more severe clini- The immunolabelling of PRRSV-N-protein was cal signs [63]. In this sense, further studies will help to observed in PAMs and to a lesser extent in interstitial elucidate the link between the inflammatory response macrophages and PIMs. By contrast, apoptotic mark- and RCD phenomena in the lung along PRRSV infec- ers such as cCasp3, cCasp8 and cCasp9, were mainly tion. Consequently, the activation of RCD upon virulent observed in interstitial macrophages, PIMs, or lympho- PRRSV-1 infection might play a double-edged sword, in cytes located in lung interstitium and secondly in PAMs. one way, trying to kill faster and earlier infected cells but Thus, whereas apoptosis was mainly activated in intersti- on the other way also causing severe lung injury. tial macrophages and PIMs, other mechanisms of RCD Curiously, nets of free chromatin were observed in besides apoptosis, such as necroptosis and/or pyrop- the alveoli of some Lena-infected pigs, accompanied by tosis, might be involved in the cell death of PAMs. The a moderate neutrophil infiltrate in areas of suppurative enhancement of apoptosis phenomena in non-infected bronchopneumonia. Many viruses, bacteria and fungi cells from the lung could be addressed by two different can stimulate the formation of neutrophils extracellular scenarios: (i) during PRRSV infection there is a strong traps (NETs) in a process called NETosis, another type of influx of interstitial macrophages and monocytes medi- RCD [25, 64–69]. These net-like structures are composed ated by cytokines and chemotactic factors in an attempt of chromatin and equipped with granule protein trapping to replace the lost cell population [21, 72, 73], whose and/or killing extracellular and intracellular microorgan- overwhelming production may lead to an excessive infil- isms to prevent their local spreading [67]; however, bac- tration of cells and hence apoptosis may be triggered as teria can also take advantage of NETs as growth source homeostatic mechanism to control cell population; (ii) [64, 66]. Since NETs and NET precursor neutrophils are apoptosis of uninfected bystander cells is a key element described to be ­TUNEL− [70], and most neutrophils as of PRRSV infection to destroy host immune cells [36, 41, well as the clumps of free chromatin observed in our 60], which have been associated with the release of pro- study were ­TUNEL− and ­cCasp3−, we hypothesised that inflammatory cytokines, such as TNF-α, IL-1α, IL-1β or these clumps could be associated with NETs triggered by IL-6, in the lung of pigs infected with both low virulent neutrophils in foci of suppurative bronchopneumonia. and virulent PRRSV-1 strains [6, 21, 36, 62] as well as NETs formation during infection with virulent PRRSV other porcine viruses [74]. strains might be linked with the higher extent of the lung Taken together, the present study evaluated RCD in damage as well as with the onset of secondary bacterial the lung upon infection with two PRRSV-1 strains of infections and the porcine respiratory disease complex. different virulence. Our results suggest the activation of There is mounting evidence of viruses activating non-apoptotic RCD during the first week post-infection extrinsic and/or intrinsic apoptotic pathways [26]. In the followed by the activation of mainly extrinsic apoptosis, present study, cCasp8 and cCasp9 were activated by Lena and in a lesser extent intrinsic apoptosis, during the sec- and 3249 strains after one week post-infection showing a ond week post-infection. These phenomena were mark- similar kinetics and a correlation among both caspases, edly elicited by Lena strain accompanied by a marked which is consistent with the crosstalk between extrinsic inflammatory response, high viral load, and severe deple- and intrinsic apoptosis pathways already described in tion of ­CD163+ PAMs, causing severe and early lung PRRSV infection [49, 71]. Whereas Lena virulent strain damage. Recovery of C ­ D163+ PAMs, together with an induced a higher number of ­cCasp8+ cells than ­cCasp9+ + increase of Arg-1 PAMs, was observed at two weeks cells, for 3249 strain, the expression of both caspases was post-infection which point out that pulmonary mac- quite similar. These results suggest that Lena virulent rophages were polarised to M2, but also, an attempt to strain is able to induce a higher activation of c­ Casp8+ in reconstitute the pulmonary macrophages subpopulations Sánchez‑Carvajal et al. Vet Res (2021) 52:12 Page 12 of 14 lost during the early stages of the infection. Further stud- and pathogenic characterization of a Russian subtype 2 PRRSV-1 isolate. Vet Microbiol 211:22–28. https​://doi.org/10.1016/j.vetmi​c.2017.09.017 ies should be performed to decipher the role of other 5. Sinn LJ, Klingler E, Lamp B, Brunthaler R, Weissenböck H, Rümenapf pathways and mediators of RCD during PRRSV infection. T, Ladinig A (2016) Emergence of a virulent porcine reproductive and respiratory syndrome virus (PRRSV) 1 strain in Lower Austria. Porc Heal Acknowledgements Manag 2:1–10. https​://doi.org/10.1186/s4081​3-016-0044-z We express our appreciation to Gema Muñoz and Alberto Alcántara for their 6. Weesendorp E, Rebel JMJ, Popma-De Graaf DJ, Fijten HPD, Stockhofe- technical assistance and Dr. Hans Nauwynck for providing us with the PRRSV-1 Zurwieden N (2014) Lung pathogenicity of European genotype 3 strain subtype 3 Lena strain. porcine reproductive and respiratory syndrome virus (PRRSV) differs from that of subtype 1 strains. Vet Microbiol 174:127–138. https​://doi. Authors’ contributions org/10.1016/j.vetmi​c.2014.09.010 JMSC, IRT, IMRG, JGL and LC conceived, designed, performed the project; FJP, 7. Renson P, Rose N, Le Dimna M, Mahé S, Keranflec’h A, Paboeuf F, Belloc IMRG and JGL helped in the animal experiments and sample collection, JMSC C, Le Potier MF, Bourry O (2017) Dynamic changes in bronchoalveolar and IMRG made the lab experiments; JMSC and IRT analysed the data; JMSC macrophages and cytokines during infection of pigs with a highly or and IRT wrote the manuscript IMRG and JGL reviewed the manuscript; LC, EM low pathogenic genotype 1 PRRSV strain. Vet Res 48:15. https​://doi. and JGL supervise the study, and contributed the reagents/materials/analysis org/10.1186/s1356​7-017-0420-y tools. All authors read and approved the final manuscript. 8. Ogno G, Rodríguez-Gómez IM, Canelli E, Ruedas-Torres I, Álvarez B, Domínguez J, Borghetti P, Martelli P, Gómez-Laguna J (2019) Impact of Funding PRRSV strains of different in vivo virulence on the macrophage popula‑ This work was supported by the Spanish Ministry of Economy and Com‑ tion of the thymus. Vet Microbiol 232:137–145. https​://doi.org/10.1016/j. petitiveness (AGL2016-76111-R) and by the Spanish Ministry of Science and vetmi​c.2019.04.016 Innovation (PID2019-109718GB-I00). J. Gómez-Laguna is supported by a 9. Ruedas-Torres I, Rodríguez-Gómez IM, Sánchez-Carvajal JM, Pallares FJ, “Ramón y Cajal” contract of the Spanish Ministry of Economy and Competitive‑ Barranco I, Carrasco L, Gómez-Laguna J (2020) Activation of the extrinsic ness (RyC-2014-16735). apoptotic pathway in the thymus of piglets infected with PRRSV-1 strains of different virulence. Vet Microbiol 243:108639. https​://doi.org/10.1016/j. Availability of data and materials vetmi​c.2020.10863​9 The datasets analysed during the current study, supporting their conclusions, 10. 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