key: cord-1007994-2u8bm2fz
authors: Sánchez-Carvajal, J.M.; Rodríguez-Gómez, I.M.; Ruedas-Torres, I.; Larenas-Muñoz, F.; Díaz, I.; Revilla, C.; Mateu, E.; Domínguez, J.; Martín-Valls, G.; Barranco, I.; Pallarés, F.J.; Carrasco, L.; Gómez-Laguna, J.
title: Activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of PRRSV-1 infection with the virulent strain Lena
date: 2020-06-02
journal: Vet Microbiol
DOI: 10.1016/j.vetmic.2020.108744
sha: d185284c60ffb44c2df734cf442cac428501969f
doc_id: 1007994
cord_uid: 2u8bm2fz

Porcine reproductive and respiratory syndrome virus (PRRSV) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. Pulmonary alveolar macrophages (PAMs) are the main cells supporting PRRSV replication, with CD163 as the essential receptor for viral infection. Although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for PRRSV virulent strains. This research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by PRRSV-1 strains of different virulence. Conventional pigs were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6 and 8 dpi. Lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of IL-6 and IFN-γ in sera compared to 3249-infected pigs. Extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in Lena-infected pigs. Lung viral load and PRRSV-N-protein(+) cells were always higher in Lena-infected animals. PRRSV-N-protein(+) cells were linked to a marked drop of CD163(+) macrophages. The number of CD14(+) and iNOS(+) cells gradually increased along PRRSV-1 infection, being more evident in Lena-infected pigs. The frequency of CD200R1(+) and FoxP3(+) cells peaked late in both PRRSV-1 strains, with a strong correlation between CD200R1(+) cells and lung injury in Lena-infected pigs. These results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response.

Porcine reproductive and respiratory syndrome virus (PRRSV) encompasses two species, Betaarterivirus suid 1 and Betaarterivirus suid 2 (formerly, PRRSV-1 and PRRSV-2, respectively) (Gorbalenya et al., 2018) , which present a wide inter-and intraspecies viral diversity (Balka et al., 2018; Shi et al., 2010; Stadejek et al., 2013) . Since 2006, different outbreaks characterised by high morbidity and mortality rates, fever, haemorrhages, severe lesions in lung and, eventually, in other organs such as thymus or lymph nodes, have been reported associated with virulent PRRSV-1 strains (Canelli et al., 2017; Karniychuk et al., 2010; Morgan et al., 2013 Morgan et al., , 2016 Ogno et al., 2019; Sinn et al., 2016; Weesendorp et al., 2013) . Several contradictory results about viraemia, tissue viral load, early virus clearance, low frequencies of PRRSV-specific IFN-γ secreting cells or PRRSV neutralizing antibodies have been reported after infection with PRRSV-1 virulent strains. However, there is consensus on the fact that some strains are more virulent than others (Canelli et al., 2017; Ferrari et al., 2018; Frydas et al., 2013; Geldhof et al., 2012; Morgan et al., 2013; Renson et al., 2017; Stadejek et al., 2017; Weesendorp et al., 2013 Weesendorp et al., , 2014 .

PRRSV replicates predominantly in the lung, causing a mild to severe interstitial pneumonia which may be complicated to suppurative bronchopneumonia due to the increased lung sensitisation to bacterial infections associated with the damage and impairment of the different pulmonary macrophage subpopulations (pulmonary alveolar macrophages, PAMs; pulmonary intravascular macrophage, PIMs; and interstitial macrophages) (Brockmeier et al., 2017; Thanawongnuwech et al., 2000) . PAMs are the main cellular target of PRRSV, although interstitial and intravascular macrophages can be infected too (Bordet et al., 2018; Duan et al., 1997; Gómez-Laguna et al., 2010) , with J o u r n a l P r e -p r o o f the nucleocapsid protein N (PRRSV-N-protein) as the most abundant viral protein during PRRSV infection (Rowland et al., 1999) . PAMs express high levels of CD163 scavenger receptor (Sánchez et al., 1999; Van Gorp et al., 2008) which is essential to support PRRSV internalisation and disassembly interacting with GP2 and GP4 viral proteins (Burkard et al., 2017; Das et al., 2010; Whitworth et al., 2016) . A soluble form of CD163 (sCD163), that may be released from tissue macrophages and monocytes, has been identified in plasma as potential biomarker for macrophage activity and inflammation (Costa-Hurtado et al., 2013; Møller, 2012; Pasternak et al., 2019) .

PRRSV is known to modulate the host immune response by inducing changes in the frequencies of immune cell subsets in blood (Dwivedi et al., 2012; Ferrari et al., 2018; Morgan et al., 2013; Weesendorp et al., 2013) and in tissues (Gómez-Laguna et al., 2010; Rodríguez-Gómez et al., 2013) , leading to an enhanced susceptibility to secondary bacterial infections (Karniychuk et al., 2010; Renson et al., 2017; Sinn et al., 2016 ). An early decrease in the frequency of monocytes, NK cells or cytotoxic T cells linked to a strong inflammatory response in target organs has been described upon experimental infection with PRRSV-1 virulent strains (Ferrari et al., 2018; Morgan et al., 2013; Weesendorp et al., 2013; . In addition, some studies indicate an early overproduction of pro-inflammatory cytokines, such as IFN-γ, IL-1β or IL-8, as the main source of pulmonary injury after infection with virulent PRRSV-1 strains (Amarilla et al., 2015; Morgan et al., 2013; Renson et al., 2017; Weesendorp et al., 2014) . Nevertheless, other potential mechanisms, such as an imbalance among pro-and anti-inflammatory responses, might predispose to secondary infections contributing to the onset of the porcine respiratory disease complex (PRDC) Van Gucht et al., 2004) .

In this context, overproduction of nitric oxide (NO), mainly triggered by inducible NO synthase (iNOS) (Akaike and Maeda, 2000) , or upregulation of CD14, as the primary lipopolysaccharide (LPS) receptor (Zanoni and Granucci, 2013) , could contribute to lung inflammation upon infection with PRRSV (Chen et al., 2014; Lee and Kleiboeker, 2005; Van Gucht et al., 2004 Yan et al., 2017) . By contrast, the transcription factor forkhead box protein 3 (FoxP3), is an essential transcription factor for the development of regulatory T cells (Tregs) and hence, a useful marker to detect them. This subset could be involved in supressing the activation of other T-cell populations (Käser et al., 2008) .

CD200 receptor 1 (CD200R1), expressed on myeloid cells and B cell subsets (Poderoso et al., 2019) , is an inhibitory surface receptor that might deliver inhibitory signals dampening the activation of cells which express it (Vaine and Soberman, 2014) . Thus, both immune markers might play an important role inhibiting the production of proinflammatory cytokines (Elmore et al., 2014; Nedumpun et al., 2018; Singh et al., 2019; Vaine and Soberman, 2014; Wang et al., 2018) , lessening the exuberant lung injury observed with virulent PRRSV-1 strains. Whereas all these markers may play a key role in PRRSV virulence, there are scarce studies analysing their role in the context of the lung lesion. Therefore, the systemic immune response and immunopathology of lung are evaluated in this study with the goal of exploring the role of selected immune markers in the pro-and anti-inflammatory priming of the lung to secondary bacteria after infection with a virulent PRRSV-1 strain (subtype 3, Lena strain) in comparison with a low virulent PRRSV-1 strain (subtype 1, 3249 strain).

J o u r n a l P r e -p r o o f

The low virulent 3249 strain (subtype 1 PRRSV-1) was isolated from the serum of a piglet with pneumonia from a PRRSV-positive herd located in Spain in 2005 . The virulent Lena strain (subtype 3 PRRSV-1) is considered as the prototype of PRRSV-1 virulent strains. Lena strain isolation was performed from lung homogenates obtained from weak born piglets from a PRRSV-positive herd from Belarus in 2007 with a high mortality rate, reproductive failure and respiratory disorders (Karniychuk et al., 2010) . Viral stocks were produced from the 4 th passage of each strain on PAMs, titrated by means of immunoperoxidase monolayer assay and expressed as tissue culture infectious doses 50 (TCID50)/mL (3249 strain: 10 5.79 TCID50/mL; Lena strain: 10 5.66 TCID50/mL).

The animals and samples used in this study were part of a project to investigate the pathogenesis of the infection with PRRSV-1 strains of different virulence . Briefly, fifty-two 4-week-old male and female piglets (Landrace x Large White crossbred) were obtained from a historically PRRSV-negative farm. All pigs were negative for porcine circovirus type 2 (PCV2), PRRSV and Mycoplasma hyopneumoniae by ELISA and PCR assays (Mattsson et al., 1995; Sibila et al., 2004) .

Piglets were blocked by weight and sex and randomly assigned to three different groups and housed in separate pens: Lena group (n=20), 3249 group (n=20) and control group (n=12). After an acclimation period of seven days, piglets were intranasally inoculated with either the low virulent 3249 strain or the virulent Lena strain (both used at 1x10 5 TCID50/mL, 1 mL/nostril, using the MAD Nasal™ Intranasal Mucosal Atomization Device, Teleflex, Alcalá de Henares, Madrid, Spain). The control group was mock- 

Commencing 1 day prior to inoculation, piglets were daily monitored to evaluate clinical signs (liveliness, respiratory symptoms and anorexia) and rectal temperature.

Quantification of clinical signs was performed by applying the following clinical score: liveliness (score 0, no abnormalities; score 1, reduced liveliness but with response to external stimuli; score 2, pig prostration; score 3, agonic pig); respiratory symptoms (score 0, no abnormalities; score 1, mild dyspnoea; score 2, evident dyspnoea; score 3, evident dyspnoea with tachypnoea; score 4, evident dyspnoea, tachypnoea and cyanosis); and anorexia (score 0, eating without abnormality; score 1, sporadic frequency of eating; score 2, no eating). The sum of these scores represented the total clinical score per animal and per day. Rectal temperatures > 40.5 °C were considered hyperthermia. At necropsy, gross lung lesions were recorded and scored by the same pathologist (Halbur et al., 1996) . Afterwards, samples from apical, medial and caudal lobes from the right lung were collected and fixed in 10 % neutral-buffered formalin (Fisher Scientific Ltd., Loughborough, UK) for histopathological and immunohistochemical studies.

Four-micron tissue sections were stained with haematoxylin and eosin and blindly graded by two pathologists for the histopathological evaluation. The severity of histopathological J o u r n a l P r e -p r o o f lesions in the lung was scored as previously described by Halbur et al. (1996) : 0, no microscopic lesions; 1, mild interstitial pneumonia; 2, moderate multifocal interstitial pneumonia; 3, moderate diffuse interstitial pneumonia; and 4, severe interstitial pneumonia. In addition, a similar score was developed considering the diagnosis of suppurative bronchopneumonia : 0, no microscopic lesions; 1, mild bronchopneumonia; 2, moderate multifocal bronchopneumonia; 3, moderate diffuse bronchopneumonia; and 4, severe bronchopneumonia. Altogether, the final score included the total of both, the interstitial pneumonia score and the bronchopneumonia score, being 8 points the maximum possible score.

RNA was isolated from sera using NucleoSpin ® RNA virus (Macherey-Nagel, Düren, Germany) according to manufacturer's instructions. For lung, RNA was purified from tissue homogenate using a combined procedure with TRIzol™ (Thermo Fisher Scientific, Serial 10-fold dilutions of 3249 or Lena ORF7 RT-PCR products with known quantities, ranging from 10 8 to 10 2 genomic copies/mL were used as standards to generate a standard curve and, therefore, to determine the PRRSV genomic copies in sera and lung. The RT-qPCR efficiency (E) was estimated for each strain by a linear regression model. The E value was calculated from the slope of the standard curve according to equation:

Also, a set of eight serial 10-fold dilutions of know TCID50/mL (starting from 10 6 TCID50/mL) was included in order to determine a relation between Ct-values, genomic copies/mL and TCID50/mL. An inter-run calibrator sample with a known number of PRRSV copies was introduced in each experiment to self-control inter-run variation. The area under the curve (AUC) for viremia and lung viral load was calculated using the trapezoidal approach (Greenbaum et al., 2001) . Results of viral load in sera and lung are showed in equivalent TCID 50 (eq TCID50) per mL. Results were expressed in pg/mL for IFN-γ, IL-6 and IL-10, and ng/mL for LBP and J o u r n a l P r e -p r o o f sCD163. The minimum detectable concentrations were 2 pg/mL for IFN-γ, 45 pg/mL for IL-6, 3 pg/mL for IL-10, 1.6 ng/mL for LBP and 23.4 ng/mL for sCD163.

Four-micron sections from lung were dewaxed in xylene and rehydrated in descending grades of alcohol until distilled water. Then, endogenous peroxidase inhibition was performed in a 3% H2O2 solution in methanol for 30 min. Epitope demasking, primary antibodies dilutions and blocking of non-specific binding are detailed in Table 1 .

Monoclonal primary antibodies were incubated overnight at 4 ºC in a humid chamber. Tris buffered saline (pH 7.6) were used as wash and diluent buffers, respectively.

Antibody specificity was verified by exchanging the primary antibody by isotype matched reagents of irrelevant specificity. One negative control which consisted of replacement of the primary antibody by BSA blocking solution was included in each immunohistochemical assay to rule out non-specific bindings.

The number of immunolabelled cells was quantified in 25 non-overlapping selected high magnification fields of 0.2 mm 2 (Olympus BX51, Olympus Iberia SAU, L'Hospitalet de J o u r n a l P r e -p r o o f Llobregat, Barcelona, Spain) and expressed as the mean of the score for each animal per mm 2 . Labelled cells were morphologically identified by differentiating among PAMs, PIMs and interstitial macrophages.

Differences between groups were evaluated for approximate normality of distribution by the D'Agostino and Pearson omnibus normality test followed by the Mann Whitney's U non-parametric mean comparisons test. Correlation coefficients were assessed by the Spearman and Pearson tests and were considered relevant with r > 0.6 and P < 0.05. Data analyses and figures were performed by using GraphPad Prism 7.0 software (GraphPad Prism software 7.0, Inc., San Diego, CA, USA) and InkScape 0.92 software. A P value lower than 0.05 was considered statistically significant and represented as * P ≤ 0.05, ** P ≤ 0.01 *** P ≤ 0.001 and **** P ≤ 0.0001. (Table 2) .

Viraemia and lung viral load were determined by RT-qPCR (efficiency of 99 %; slope = 3.34; detection limit: 1 copy/µl; slope-intercept = 39.5; and high linearity, r = 0.99). All animals were negative by RT-qPCR at day 0 and control pigs remained so all throughout the experiment. In sera, four out of five 3249-infected pigs and all Lena-infected pigs were PRRSV positive as early as 1 dpi. Viraemia was always higher in Lena-than in 3249-infected pigs from 1 to 8 dpi (P < 0.01 at 1, 3, 6 dpi; P<0.05 at 8 dpi), reaching the highest viral load at 6 dpi (1.9x10 7 eq TCID50/mL). The AUC for viremia (mean) in Lena and 3249 group were 44.8 and 33.8 respectively ( Fig. 2A) . The viral load in the lung displayed a similar kinetics to that of serum for both infected groups, reaching the J o u r n a l P r e -p r o o f maximum lung viral loads at 6 dpi in Lena group (1.6x10 7 eq TCID50/mL), whereas 3249 group peaked at 8 dpi (1.9x10 6 eq TCID50/mL) (Fig. 2B) . By contrast to sera, PRRSV-1 was just detected in lung in two out of five animals in both infected groups at 1 dpi, being positive all infected piglets from 3 dpi onwards. The AUC for lung viral load (mean) in

Lena group was 45 and 36.2 for 3249 group (Fig. 2B) . In Lena infected-group the statistical analysis revealed a positive correlation among viraemia, lung viral load, temperature, clinical signs score and the number of PRRSV-N-protein + cells in the lungs (Table 2) . A correlation among lung viral load and viraemia and PRRSV-N-protein + cells was also observed in 3249-infected pigs (r = 0.71, P < 0.0001; and, r = 0.60, P < 0.005, respectively).

PRRSV-specific antibodies were first detected at 8 dpi in sera from both PRRSV-1infected groups (non-significant differences in S/P ratios) (data not shown). A significant increase in IFN-γ serum levels was detected after Lena infection at 6 and 8 dpi (maximum mean level of 234 ± 100 pg/mL at 6 dpi) compared to control (P<0.05) and 3249 (P<0.01) groups (Fig. 2C ). Maximum IL-6 levels in serum of 3249 group were observed at 6 dpi (mean of 350 ± 220 pg/mL), whereas pigs belonging to Lena group reached the highest IL-6 levels at 8 dpi (mean of 480 ± 50 pg/mL) (Fig. 2D) . IL-10, LBP or sCD163 were not detected in serum samples from both control and infected groups throughout the study.

Both viraemia and lung viral load displayed a positive statistical correlation with IFN-γ levels, which in turn were also correlated with temperature and the clinical signs score in

Lena infected-pigs (Table 2) .

J o u r n a l P r e -p r o o f

The labelling of PRRSV-N-protein was mainly observed in PAMs and in a lesser extent in interstitial and intravascular macrophages (Figs. 3A-3B) . In Lena-infected pigs, clusters of PRRSV-N-protein + macrophages were observed within foci of bronchopneumonia at 6 and 8 dpi (Fig. 3B inset) . A progressive increase in the number of PRRSV-N-protein + cells was detected throughout the study in both PRRSV-1-infected groups, reaching a peak at 6 and 8 dpi in Lena and 3249-infected piglets, respectively.

This increase was significantly higher in Lena than in 3249 group (P<0.05 at 3, 6 and 8 dpi) (Fig. 3E , primary axis). No positive cells were detected in control pigs. (Table 2) .

The labelling against CD14 was mainly observed in the cell membrane and cytoplasm of monocytes, interstitial and intravascular macrophages and, occasionally, in PAMs (Figs.

4A-4B, insets). Whereas no changes were observed in the number of CD14 + cells in the control group along the study, a gradual increase with maximum expression at 8 dpi was detected in both infected groups (Fig. 4E) . Lena-infected pigs showed the highest frequency of CD14 + cells when compared to control animals (P<0.01) in association with the presence of suppurative bronchopneumonia (Fig. 4B ). CD14 + interstitial and intravascular macrophages were observed infiltrating extensive areas of the interstitium, whereas almost no CD14 + cells were present in the bronchial wall and alveolar lumen.

Interestingly, the number of CD14 + cells in Lena-infected piglets displayed a strong positive correlation with the concentration of IL-6 in sera (Table 2) .

The granular intracytoplasmic immunostaining of iNOS was primarily observed in PAMs and interstitial macrophages in foci of interstitial pneumonia and bronchopneumonia (Figs. 4C-4D). The number of iNOS + cells followed a similar kinetics in both PRRSV-1infected groups, with a progressive increase from 6 dpi onwards, reaching a significant increase by the end of the study (8 dpi) in Lena-infected pigs compared to 3249 (P<0.01) and control groups (P<0.05) (Fig. 4F) .

CD200R1 labelling was detected in the cytoplasm of intravascular and interstitial macrophages located inside or surrounding bronchopneumonia foci, with occasional J o u r n a l P r e -p r o o f expression in PAMs and monocytes (Figs. 5A-5B, insets). Whereas the number of CD200R1 + cells significantly increased in Lena-infected pigs at 6-8 dpi (P<0.05 at 6 dpi with respect to 3249 group; and P<0.01 at 6 dpi and P<0.05 at 8 dpi with respect to control group), this increase was just detected at 8 dpi in 3249-infected pigs (P<0.05 with respect to control group) (Fig. 5E, primary axis) . Control animals presented a scarce number of CD200R1 + cells along the study. For Lena infected-pigs a strong positive correlation was observed among the frequency of CD200R1 + cells and the microscopic lung lesions (Fig.   5E ) ( Table 2) .

FoxP3 yielded a nuclear immunolabelling in lymphocytes mainly located in areas of atelectasis and interstitial pneumonia (Figs. 5C-5D, insets). Although two Lena-infected pigs exhibited a higher number of FoxP3 + cells at 1 dpi, the kinetics of positive cells for this immune markers showed a gradual increase along the study in both Lena-and 3249infected animals, reaching the maximum at 6 dpi (Fig. 5F ). There were no significant differences in the number of FoxpP3 + cells among infected groups. However, a significant increase of FoxP3 + cells was detected at 6 and 8 dpi in Lena-infected pigs compared to control animals (P<0.05).

PRRSV plays a pivotal role in PRDC, modulating the host immune response and favouring secondary bacterial infections Van Gucht et al., 2004) . Virulent PRRSV-1 strains cause more severe clinical signs, higher mortality rates as well as marked lung injury with a higher incidence of bronchopneumonia as opposed to low virulent strains (Amarilla et al., 2015; Canelli et al., 2017; Frydas et al., 2013;  J o u r n a l P r e -p r o o f Gómez-Laguna et al., 2010; Morgan et al., 2013; Renson et al., 2017; Rodríguez-Gómez et al., 2019; Stadejek et al., 2017; Weesendorp et al., 2013) . Accordingly, we hypothesise that severe pulmonary lesions observed along infection with virulent PRRSV-1 strains might be associated with a higher decrease in the amount of PAMs as well as an imbalance between anti-and pro-inflammatory responses with different molecules potentially involved in this process.

As previously described (Renson et al., 2017; Weesendorp et al., 2013) , severe systemic and respiratory symptoms as well as hyperthermia were observed in animals infected with virulent Lena strain, whereas low virulent 3249 strain only caused mild clinical signs and a slightly increase of rectal temperature. Furthermore, virulent Lena strain caused an earlier and stronger onset of lung lesions due to extensive consolidated areas in the apical and medial lobes which were microscopically linked to suppurative bronchopneumonia as well as severe characteristic interstitial pneumonia. On the other hand, PRRSV virulence has been associated with higher virus titre and antibody response in vivo (Brockmeier et al., 2012; Lu et al., 2014) Although Lena virulent strain elicited a quite higher viraemia than the low virulent strain, no differences were observed in the antibody response in the early phase of infection. Similar results have been previously reported by others when comparing Lena with low virulent strains (Renson et al., 2017; Weesendorp et al., 2013) , and confirm that PRRSV-1 virulence and specific non-neutralizing antibodies are not associated in the acute phase of infection.

response when compared with low virulent strains (Amarilla et al., 2015; Liu et al., 2010;  levels of IL-6 at 8 dpi and IFN-γ at 6-8 dpi were detected in the sera of Lena-infected pigs. Increased concentration of IL-6 in plasma is associated with both systemic and respiratory symptoms (Van Reeth and Nauwynck, 2000) and could play a dual role during virus infection: (i) protecting the host from infection and (ii) inducing inflammation and tissue damage when it is overexpressed (Liu et al., 2010) . It is known that IFN-γ is, mostly produced by activated NK cells, NKT cells, γ/δ T cells, cytotoxic T cells and memory T cells (Gerner et al., 2015; Mair et al., 2014) , and participates in regulating the immune and inflammatory responses (Van Reeth and Nauwynck, 2000) . In fact, an early increase of NKT cells has been associated with viraemia peak in piglets infected with PR40 CD14 and iNOS are involved in lung inflammation after infection with PRRSV (Chen et al., 2014; Lee and Kleiboeker, 2005; Van Gucht et al., 2004 Yan et al., 2017) Upregulation of CD14, as lipopolysaccharides (LPS) co-receptor, after infection with PRRSV sensitises the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to bacterial LPS (Van Gucht et al., 2005) . For its part, iNOS is mainly expressed in response to different stimuli, such as cytokines and LPS, playing a role in tissue injury upon production of NO (Chen et al., 2014; Cho and Chae, 2002; Vlahos et al., 2011; Yan et al., 2017) . In this study, an increase in the number of CD14 + cells after PRRSV-1 infection was observed in association with suppurative J o u r n a l P r e -p r o o f bronchopneumonia, which was more evident in Lena-infected piglets at 6 -8 dpi. This increase was mainly due to CD14 + monocytes, interstitial and intravascular macrophages infiltrating extensive areas of the interstitium. The influx of CD14 + monocytes and immature macrophages may be explained by an attempt to replenish the loss of CD163 + macrophages contributing to clearance of cellular debris and resolution of inflammation, restoring the normal lung function. On the other hand, the increase of CD14 + cells implies a higher availability of the LPS-LBP complex receptor, which is likely to sensitise the lung to future secondary bacterial infections making the onset of PRDC easier (Van Gucht et al., 2005) . In the case of iNOS, a significant increase in the number of iNOS + cells was observed in areas of interstitial pneumonia as well as bronchopneumonia in Lena-infected pigs. The induction of iNOS has been associated with both a direct effect of the viral replication or viral components and an indirect effect mediated by cytokines, such as IFNγ, or by LPS (Akaike and Maeda, 2000; Chen et al., 2014; Lee and Kleiboeker, 2005) . Of note, the peak of iNOS in Lena-infected animals appeared in our study just after the peak of PRRSV replication in the lung as well as after the peak of serum IFN-γ, being associated with the maximum lung injury and bronchopneumonia lesion. These factors may play a role in the regulation of iNOS expression along PRRSV infection and its role in lung injury development (Chen et al., 2014; Lee and Kleiboeker, 2005; Yan et al., 2017) .

After a cascade of proinflammatory events, the host is able to trigger off the release of anti-inflammatory or regulatory mediators to restrain the extent of the injury. Thus, the role of CD200R1 and FoxP3 was evaluated in the present study. A strong positive correlation was detected among the frequency of CD200R1 + cells and the microscopic score which was mainly associated with a higher severity of typical interstitial pneumonia J o u r n a l P r e -p r o o f and suppurative bronchopneumonia in Lena-infected pigs. Likewise, an increase in the frequency of FoxP3 + cells between 6 and 8 dpi was triggered by both PRRSV-1 strains when the lung injury was higher. CD200R1 has been involved in reducing the expression of pro-inflammatory cytokines in a wide range of inflammatory diseases (Vaine and Soberman, 2014) , nevertheless to the best of the authors' knowledge, the role of CD200R1 in viral diseases of swine is largely unknown. Previous studies in a murine model reported that influenza virus infection induced the upregulation of CD200R1 in macrophages, decreasing their responsiveness and increasing the sensitivity to bacterial infection and finally severe lung injury (Snelgrove et al., 2008; Vaine and Soberman, 2014) . In contrast, CD200/CD200R1 signalling pathway limited type I IFN production during coronavirus infection protecting the host from cytokine storm (Vaine and Soberman, 2014) . In addition, FoxP3 has been reported as a potential inhibitor of the cell- 

The present study dissects the immunopathology of lung injury along an acute infection with PRRSV-1 strains of different virulence, revealing a drop in the number of CD163 + cells together with an enhancement in the expression of CD14 and iNOS as mechanisms involved in the earlier and higher extent of lung lesion in Lena-infected piglets. These changes could sensitise the lung to future secondary bacterial infections. In addition, the increase in the number of CD14 + cells is likely to respond to an attempt to replenish the CD163 + macrophages subset lost along the infection with both PRRSV-1 strains. On the other hand, the increase in the expression of CD200R1 and FoxP3 represents potential pathways activated to contain the inflammatory response.

The authors declare that they have no competing interest. marked microglia activation in the hippocampus and deficits in spatial learning.

Journal of Neuroscience, 34 (6) temperature (B) for control group (gray circles), 3249-infected group (green triangle) and Lena-infected group (red diamonds). "a" indicates a significant difference between the Lena and 3249 and control groups, "b" a significant difference between the Lena and 3249 groups and "c" a significant difference between the 3249 and control groups. P value lower than 0.05 was considered statistically significant and represented as * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. Days post-inoculation, dpi. (C) At necropsy, lungs were scored and processed for histological and immunohistochemical studies. Box plots display the macroscopic lung score for each group (control, gray circles; 3249, green triangles; Lena, red For all data, a P value lower than 0.05 was considered statistically significant and represented as * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ****P ≤ 0.0001. iNOS -NS: not statistically significant or with r < 0.6. Correlation coefficients were considered relevant with r > 0.6 and P < 0.05.

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f injury: Cytokines, uncontrolled inflammation, and therapeutic implications

Dynamic changes in inflammatory cytokines in pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus

Attenuation and Immunogenicity of a Live High Pathogenic PRRSV Vaccine Candidate with a 32-Amino Acid Deletion in the nsp2

Interleukin-8, interleukin-1beta, and interferon-gamma levels are linked to PRRS virus clearance

The porcine innate immune system: An update

Detection of Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rRNA gene

Soluble CD163

Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity

Increased pathogenicity of European porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance

Induction of porcine reproductive and respiratory syndrome virus (PRRSV)-specific regulatory T lymphocytes (Treg) in the lungs and tracheobronchial lymph nodes of PRRSVinfected pigs

Impact of PRRSV strains of different in vivo virulence on the macrophage population of the J o u r n a l P r e -p r o o f thymus

Development and application of a porcine specific ELISA for the quantification of soluble CD163

Analysis of the expression of porcine CD200R1 and CD200R1L by using newly developed monoclonal antibodies

Dynamic changes in bronchoalveolar macrophages and cytokines during infection of pigs with a highly or low pathogenic genotype 1 PRRSV strain

Downregulation of antigen-presenting cells in tonsil and lymph nodes of porcine reproductive and respiratory syndrome virusinfected pigs

Virulent Lena strain induced an earlier and stronger downregulation of CD163 in bronchoalveolar lavage cells

The evolution of porcine reproductive and respiratory syndrome virus: Quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332

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

Kinetics of the expression of CD163 and CD107a in the lung and tonsil of pigs after infection with PRRSV-1 strains of different virulence

The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation

The PD-1/PD-L1 axis and virus infections: A delicate balance

Molecular epidemiology of PRRSV: A phylogenetic perspective

Use of a polymerase chain reaction assay and an ELISA to monitor porcine circovirus type 2 infection in pigs from farms with and without postweaning multisystemic wasting syndrome

European genotype of porcine reproductive and respiratory syndrome ( PRRSV ) infects monocyte-derived dendritic cells but does not induce Treg cells

Induction of T helper 3 regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus

Regulatory T Cells in Respiratory Health and Diseases. Pulmonary Medicine

Emergence of a virulent porcine reproductive and respiratory syndrome virus (PRRSV) 1 strain in Lower Austria

A critical function for CD200 in lung immune homeostasis and the severity of influenza infection

Pathogenicity of three genetically diverse strains of PRRSV Type 1 in specific pathogen free pigs

The role of pulmonary intravascular macrophages in porcine reproductive and respiratory syndrome virus infection

The CD200-CD200R1 Inhibitory Signaling Pathway. Immune Regulation and Host-Pathogen Interactions

Sialoadhesin and CD163 join forces during entry of the porcine reproductive and respiratory syndrome virus

The combination of PRRS virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs

Porcine Reproductive and Respiratory Syndrome Virus Infection Increases CD14 Expression and Lipopolysaccharide-Binding Protein in the Lungs of Pigs

Proinflammatory cytokines and viral respiratory disease in pigs

Inhibition of Nox2 Oxidase Activity Ameliorates Influenza A Virus-Induced Lung Inflammation

Recovery from acute lung injury can be regulated via modulation of regulatory T cells and Th17 cells

Comparative analysis of immune responses following experimental infection of pigs with European porcine reproductive and respiratory syndrome virus strains of differing virulence

Lung pathogenicity of European genotype 3 strain porcine reproductive and respiratory syndrome virus (PRRSV) differs from that of subtype 1 strains

Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus

Regulation of iNOS-Derived ROS Generation by HSP90 and Cav-1 in Porcine Reproductive and Respiratory Syndrome Virus-Infected Swine Lung Injury

Porcine reproductive and respiratory syndrome virus; CD200R1, CD200 Receptor 1; iNOS, inducible nitric oxide synthase; FoxP3, forkhead box protein 3; mAb, monoclonal antibody; pAb

Citrate pH 6, microwave heat treatment at 420W for 10 minutes

enzymatic digestion with protease type XIV (Sigma-Aldrich) at 38º C for 8 minutes; Citrate pH 6*, autoclave treatment at 121º C for 10 min

We express our appreciation to Gema Muñoz, Alberto Alcántara and Esmeralda Cano for their technical assistance and Dr. Hans Nauwynck for providing us the PRRSV-1 subtype