- Research article
- Open Access
PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-naïve regulatory T cells to proliferate
© Rodríguez-Gómez et al.; licensee BioMed Central. 2015
- Received: 25 September 2014
- Accepted: 28 April 2015
- Published: 20 May 2015
In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)+ and N-protein− moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein+ moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein− moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3+ regulatory T cells present within CD4+ T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4+ T cells showed differences in regard to proliferation and frequency of Foxp3+ T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.
- Feline Immunodeficiency Virus
- Foxp3 Expression
- Median Fluorescence Intensity
- Swine Leukocyte Antigen
- Autologous PBLs
Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating diseases for the swine industry world-wide [1,2]. The causative agent, PRRS-virus (PRRSV), belongs to the genus Arterivirus and comprises two different genotypes designated as type 1 (PRRSV-1) and type 2 (PRRSV-2) (formerly European and American genotypes, respectively) [3,4]. PRRSV is able to persist in the host for a long period of time [5-7] supported by a delayed onset of specific humoral and cellular immune responses [8,9].
The mechanisms of this delay are still under investigation. One possible reason could be a decreased stimulation of CD4+ T cells by antigen presenting cells (APCs) which play a central role in T-cell activation via the expression of MHC-II and CD80/86 . In order to study the role of these molecules in PRRSV infection a frequently used model are in vitro generated cultures of monocyte-derived dendritic cells (moDCs). However, published findings on expression levels of MHC-II and CD80/86 expression in PRRSV-infected moDC cultures have led to conflicting results. Some reports claim no changes  or a decreased [12-14] expression of swine leukocyte antigen (SLA)-DR and a decrease  or increase  of CD80/86 expression on infected moDCs with either PRRSV-1 or PRRSV-2 strains.
Another possible explanation for the ineffective adaptive immune response may be the activation or induction of regulatory T cells (Tregs) by PRRSV. Indeed, it has been shown that several viruses such as the human immunodeficiency virus, hepatitis C virus or feline immunodeficiency virus use the induction of Tregs in order to suppress or evade the immune response by the host (reviewed in ). Since the first description of porcine Tregs  and the analysis of their suppressive capabilities  much work was devoted to the study of this T-cell subset during PRRSV infection. Some reports showed an in vitro induction of Tregs within peripheral blood lymphocytes (PBLs) after co-cultivation with PRRSV-2 infected moDCs [18,19]. However, this was not the case when several PRRSV-1 strains  and a high-virulent PRRSV-2 strain (VR2385) were tested . Ex vivo analyzes of Tregs following PRRSV-2 infection of nine week old pigs also indicated an increase of CD4+CD8α+Foxp3+ Tregs .
Due to the controversial results of the effect of PRRSV-infection onto APCs and onto their MHC-II and CD80/86 expression, one of the aims of this study was to assess changes on these two molecules using moDCs infected with PRRSV-2 and PRRSV-1 strains. In comparison to previous reports, we focus on differences between PRRSV-infected and non-infected moDCs present in the same microculture. Furthermore, by making use of the same culture system we revisited the effect of PRRSV-infected moDCs on Foxp3 expression and proliferation of CD4+ T cells in co-culture experiments.
Animals and peripheral blood mononuclear cells (PBMCs) isolation
Six-month old crossbred (Large White X Landrace X Pietrain) pigs from an abattoir served as blood donors for the isolation of PBMCs. The general health status of all animals was controlled before transportation and after arrival at the slaughter plant and all animals appeared clinically healthy. The pigs were subjected to electric high voltage anesthesia followed by exsanguination. This procedure is in accordance to the Austrian Animal Welfare Slaughter Regulation. Heparinized blood was collected at the moment of slaughter.
PBMCs were isolated by density centrifugation with Pancoll (density 1.077 g/mL; PAN Biotech, Aidenbach, Germany) as described elsewhere . Collected PBMCs were washed and resuspended in complete medium (CM; RPMI 1640 with stable L-Glutamine supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 IU/mL penicillin and 100 μg/mL streptomycin; all from PAN Biotech) in a 75 cm2 tissue culture flask (Greiner Bio-One, Kremsmünster, Austria) for subsequent generation of moDCs.
During PBMC isolation process, plasma samples were collected and tested for antibodies against PRRSV (IDEXX PRRS X3 Ab Test, IDEXX Europe B.V., Hoofddorp, The Netherlands) by an external laboratory for veterinary diagnostics (LaboVet, Vienna, Austria). Only PBMCs from PRRSV antibody-negative donors were used in the subsequent experiments.
Generation of moDCs
Monocytes were separated from PBMCs by plastic adherence for 90 min at 37 °C in 5% CO2. Afterwards, PBLs were removed and immediately frozen for long-term storage at −150 °C as described by Leitner et al. . The remaining plastic-adherent cells were washed twice with CM. MoDCs were generated as previously described by Carrasco et al.  with minor modifications. Briefly, plastic-adherent monocytes were cultured in CM supplemented with 40 ng/mL of recombinant porcine (rp) GM-CSF (R&D Systems, Minneapolis, MN, USA) and 40 ng/mL of rpIL-4 (R&D Systems) at 37 °C in 5% CO2. After three days the medium was replaced by fresh cytokine-supplemented CM. Seven days after the start of in vitro cultivation moDCs were harvested with a cell scraper (Greiner Bio-One).
Two different PRRSV strains belonging to type 1 and 2 of PRRSV were used in this study. A European PRRSV-1 field isolate was propagated on MA-104 cells. This isolate showed 87% homology with the European genotype subtype 1 Lelystad virus based on genome position 12280–13460 (NCBI GenBank accession number KM657092). Five days post-infection, the supernatant was collected, cleared by centrifugation and passed through a 0.45 μm filter membrane. The PRRSV-2 strain NVSL 97–7895 was rescued after electroporation of 5 × 106 HEK293T cells (0.1 kV, 0.95 mF, 0.2 mm gap cuvette) with 1 μg capped SP6 transcripts from an AclI linearized modified full length clone pFL12  generously provided by Fernando Osorio, University of Nebraska. Three days post transfection the supernatant was collected and cleared by centrifugation. Since NVSL 97–7895 displayed reduced growth kinetics on MA-104 cells, the subclone MARC-145  was used for virus production. Therefore, 5 × 106 MARC-145 cells seeded in 10 cm cell culture dishes were infected with the progeny virus. Five days after infection the supernatant was harvested and used to infect fresh MARC-145 cell cultures. When severe cytopathic effect was observed, the supernatant was collected by centrifugation to serve as virus stock. Virus stocks were stored at −80 °C as aliquots. One aliquot of each strain was thawed and viral titers were determined by titration on MA-104 cells for the PRRSV-1 strain and MARC-145 cells for the PRRSV-2 strain. Despite the initial propagation of the PRRSV-1 strain on MA-104 cells this virus showed comparable titers on both MA-104 and MARC-145 cells (1 × 104 focus forming units on MA-104 and 2 × 104 focus forming units on MARC-145 cells). Multiplicity of infection (MOI) values were calculated from titers on MA-104 cells for the PRRSV-1 strain and MARC-145 cells for the PRRSV-2 strain. All experiments were performed with the same batch of viruses.
Flow cytometry (FCM)
FCM analysis was carried out on a FACSCanto™ II (BD Biosciences, San Jose, CA, USA). Data were processed by using FACSDiva software (Version 6.1.3., BD Biosciences) and FlowJo software (Version 7.6, Treestar, Ashland, OR, USA). Details on the staining procedures are given below. Incubation steps for labeling of extracellular antigens, including secondary reagents, lasted for 15 min at 4 °C in the fridge and 30 min under the same conditions for intracellular antigens. For washing steps, performed after each incubation, a buffer containing PBS and 2% FCS was used for extracellular antigens. Prior to the use of Live/Dead® Fixable Near-IR (Life Technologies, Carlsbad, CA, USA, see below for details) cells were washed in pure PBS. Wherever appropriate, isotype-matched control samples or fluorescence minus one samples were included in all staining procedures outlined below.
Phenotyping of moDCs
Antibody panels used for FCM analyzes
Source of primary Ab
Phenotyping of moDCs
Assessment of moDC infection rate
CD163, CD169, SLA-DR and CD80/86 expression on infected moDCs
two step biotin-streptavidinh
two step biotin-streptavidini
Analysis of moDC infection rate
After seven days of cultivation, moDCs were seeded into round-bottomed 96-well plates (Greiner Bio-One) at 2 × 105 cells per well and infected with PRRSV-1 or PRRSV-2 strains at an MOI of 0.1 at 37 °C in 5% CO2. After two hours, the inoculum was removed and cells were washed twice with CM. Separate microcultures with mock-infected moDCs were included in each experiment. Cultivation of moDCs was continued until 12, 24 and 48 hours post-inoculation (hpi). At these time points moDCs were harvested and analyzed for PRRSV-N-protein expression (see below for details). In addition, moDCs were infected with different MOI (0.1, 0.02 and 0.004) and harvested after 24 hpi. Harvested moDCs were labelled with anti-CD172a mAbs (see Table 1 for details on staining strategy) and isotype-specific secondary antibodies in combination with Live/Dead® Fixable Near-IR during a second incubation step. Subsequently, moDCs were fixed and permeabilized by the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) according to manufacturer’s instructions. Thereafter, cells were incubated with anti-PRRSV-N-protein mAbs (clone P10/b1) . This antibody had been purified and conjugated to Alexa488 as described elsewhere .
Expression of CD163, CD169, SLA-DR and CD80/86 on infected moDCs
MoDCs were infected with PRRSV-1 or PRRSV-2 strains at an MOI of 0.02 at 37 °C in 5% CO2 under the conditions described above. After 24 hpi, moDCs were harvested and labelled with anti-CD172a mAbs and anti-CD80/86 immunoglobulin fusion proteins (see Table 1 for details) followed by incubation with secondary reagents. Free binding sites of secondary antibodies were blocked with whole mouse IgG (1 μg per sample, ChromPure, Jackson ImmunoResearch, West Grove, PA, USA). Thereafter, mouse anti-porcine SLA-DR-PE mAbs and Live/Dead® Fixable Near-IR were added. In parallel samples CD80/86-specific fusion proteins and SLA-DR-specific mAbs were replaced by mAbs against CD163 and CD169. Details on the labeling strategy of CD163 and CD169 are provided in Table 1. Lastly, cells were fixed and permeabilized and labelled for PRRSV-N-protein expression as described above.
Co-culture of PRRSV-infected moDC and sorted CD4+ T cells
Round-bottomed 96-well plates (Greiner Bio-One) were coated with 1.5 μg/mL of anti-CD3 mAbs (clone PPT7, IgG1) as stated elsewhere . Free mAbs were removed by washing the plates three times with PBS. MoDCs cultivated for 7 days were harvested and transferred either into CD3-coated plates or non-coated plates. MoDC microcultures were either mock-infected or infected with PRRSV-2 or PRRSV-1 at an MOI of 0.02 for 2 h (as described above). In parallel, autologous PBLs were defrosted and labelled with mouse anti-porcine CD4 antibody (74-12-4, IgG2b; ) for 15 min at 4 °C in the fridge. Cells were washed with PBS (supplemented with 0.5% of bovine serum albumin). Cell pellets were resuspended in buffer and anti-mouse IgG2a + b MicroBeads were added (Miltenyi Biotec, Bergisch Gladbach, Germany). After incubation for 15 min at 4 °C in the fridge, cells were washed and up to 108 cells resuspended in 500 μL of cold buffer. The cell suspension was applied onto LS columns (Miltenyi Biotec) for positive selection of CD4+ T cells by use of a Quadro-MACS system (Miltenyi Biotec). Purity of sorted CD4+ T cells was between 96.7 and 98.2% (mean of 97.7%). Subsequently, sorted CD4+ T cells were labelled with a proliferation dye (CellTrace™ Violet Cell Proliferation Kit, Life Technologies) as described elsewhere . MoDCs and violet labelled sorted CD4+ T cells were seeded at a ratio of 1:10 in CD3-coated and non-coated plates and co-cultured for four days at 37 °C and 5% CO2. For each experiment, randomly selected microcultures were harvested already after 24 hpi to analyze the frequency of PRRSV-infected moDCs present in co-cultures.
Following co-cultivation, cells were harvested and stained with Live/Dead® Fixable Near-IR dye (Life Technologies) according to manufacturer’s instructions. Thereafter, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and subsequently labelled with rat anti-mouse/rat Foxp3-PE mAbs (clone FJK-16 s, IgG2a; eBioscience). This mAb has a well-documented cross-reactivity with porcine Foxp3 [16,32].
Data on SLA-DR, CD80/86 and Foxp3 expression as well as CD4+ T-cell proliferation was analyzed for statistical significance by GraphPad Prism Version 5.01 Software (GraphPad Software, San Diego, CA, USA). Values were evaluated for normality distribution by the Kolmogorov-Smirnov test. Subsequently, data sets were analyzed using the Mann–Whitney U non-parametric test. P values below 0.05 are indicated by an asterisk (*).
Phenotyping of moDCs
Analysis of moDC infection rate
CD163, CD169, SLA-DR and CD80/86 expression on PRRSV-infected moDCs
Co-culture of PRRSV infected moDCs and autologous sorted CD4+ T cells
Porcine moDCs have been used in many studies as an in vitro model to study PRRSV host-pathogen interactions [11-14,18] but results in regard to MHC-II expression, CD80/86 expression and induction of Tregs were often conflicting. One possible explanation to this is the use of different viral strains which often differ considerably in regard to genomic sequence and virulence, even within a particular genotype. However, we hypothesized that these differences might also be related to a suboptimal characterisation of the experimental systems used.
To overcome this, in a first step we performed a series of experiments addressing the phenotype of porcine moDCs with a marker panel consisting of CD172a, CD1, CD14, CD163, CD169, SLA-DR and CD80/86. In accordance with previous descriptions of porcine moDCs [24,33,35] the moDCs in our culture system were CD172highCD1dimCD14+SLA-DR+CD80/86+. We additionally analyzed expression of CD163 and CD169 since the current knowledge suggests that these two molecules are of high relevance for PRRSV-entry into the host cell (reviewed in ). CD163 has been described as a marker for the discrimination of different porcine monocyte subsets (reviewed in ). Domínguez and co-workers also investigated CD163− and CD163+ monocytes for potential differences in regard to their differentiation into moDCs . Both subsets were capable to mature into DCs but moDCs derived from CD163+ monocytes expressed still higher levels of SLA-DR and CD80/86 and were more efficient to induce proliferation in allogeneic T cells than moDCs derived from CD163− monocytes. Of note, these authors reported that both moDC populations did not express CD163 after 7 days of in vitro cultivation. This is in contrast to our results where - albeit at different levels between moDCs from individual animals – CD163 was expressed on the vast majority of analyzed moDCs. The reasons for these discrepancies are speculative, but differences in the cultivation protocol, like the addition of TNF-α on day 5 of cultivation in the work performed by Chamorro et al.  may be of relevance to this.
In regard to CD169 expression monocytes analyzed in this study at day 0 did not express this molecule, which is in agreement with previous results where CD169 could be only detected on monocytes after three days of in vitro stimulation with IFN-α . A moderate expression of CD169 has been reported on moDCs after 7 days of cultivation in the presence of IL-4, GM-CSF and IFN-α  which is different to the very low expression levels observed after 7 days in this study. The increased expression described by Revilla et al.  might be a consequence of the additional IFN-α stimulation as indicated by the results obtained with IFN-α stimulated monocytes .
Despite this overall very low CD169 expression, PRRSV replication, identified by N-protein+ cells, was clearly restricted to a CD163+CD169dim phenotype as shown in Figure 3B. Thereby our results confirm the postulated role of these two molecules for PRRSV binding, internalization and RNA release [34,40-43] and consequently virus replication.
The discrimination between N-protein+ and N-protein− moDCs also revealed that PRRSV-infected moDCs showed increased expression levels of SLA-DR and CD80/86 compared to non-infected moDCs and this applied to both PRRSV strains tested. Interestingly N-protein− moDCs showed a slight downregulation of CD80/86 expression, when compared to moDCs derived from mock-infected cultures. These changes can be only observed by the distinction of infected versus non-infected cells in the same culture but not by the analysis of the expression of surface molecules for the entire cell population derived from a PRRSV-infected culture. Indeed, this finding may explain why previously published results, as outlined in the introduction, differ so widely in regard to the effect of PRRSV infection on SLA-DR and CD80/86 expression in moDCs. Remarkably, we observed a high expression of both molecules for viruses representing the two major genotypes PRRSV-1 and PRRSV-2. Moreover, our results provide no immediate hints that the expression of SLA-DR and/or CD80/86 is negatively affected by a PRRSV infection, thereby we could not corroborate a potential immuno-evasion of PRRSV by this mechanism.
The induction of Tregs in PRRSV-naïve animals was also postulated as a immuno-evasive mechanism of PRRSV. Silva-Campa et al.  reported an expansion of CD25+Foxp3+ lymphocytes following co-culture of PRRSV-2 infected moDCs and autologous PBLs. By using a similar experimental setup and the same PRRSV-2 strain (NVSL 97–7895) we were not able to identify an increase in Foxp3+CD4+ T cells regardless whether these cells had been stimulated with anti-CD3 mAbs (Figure 4D) or were kept in medium (data not shown). Differences in the stimulation protocol like different MOI rates (0.1, Silva-Campa versus 0.02, this study) and the time span between infection of moDCs and addition of autologous T cells (24 h, Silva-Campa versus 7 h, this study) may account for our negative results. However, as shown in Figure 2C, we did not observe major differences in the frequency of N-protein+ moDCs following infection with an MOI of 0.1 versus 0.02. Similarly, adding CD4+ T cells to moDCs 24 hpi did not change our results (data not shown). It is probably also of relevance that Silva-Campa et al.  used a mouse anti-human Foxp3 specific mAb clone 221D/D3 that in our hands showed no reactivity with porcine Foxp3 . An induction of CD4+CD25+Foxp3+ T cells by moDCs infected with a PRRSV-2 isolate from Thailand was also described by Wongyanin et al. . Again, these results were achieved by a protocol very similar to ours, but could not be confirmed in our experiments. It should be noted that Wongyaning and co-workers worked with a mouse anti-human Foxp3 mAb (clone 236A/E7) which has no documented history of cross-reactivity with porcine Foxp3. Moreover, the authors used a commercial fixation and permeabilization reagent kit that in our hands showed only a poor performance for the identification of antigens located in the nucleus .
Nevertheless, published work from Silva-Campa and co-workers as well as Wongyaning and co-workers also provided hints for increased RNA expression levels of Foxp3 in PRRSV-infected moDC co-cultures  and PBMCs incubated with cell lysate derived from PRRSV-infected MARC cells . In a consecutive study it was also shown that moDCs transfected with N-protein coding plasmids produced IL-10 and also caused increased frequencies of CD4+CD25+Foxp3+ T cells in co-cultures . Therefore, our results do not exclude an influence of PRRSV on Tregs. However, we think that our experiments demonstrate the difficulties in reproducing published results based on in vitro culture systems which are poorly characterised.
Moreover, as illustrated by our findings on differences in expression of SLA-DR and CD80/86 between PRRSV-infected and non-infected moDCs present in the same microculture, our work indicates that every possible effort on a detailed characterisation of the experimental systems used for PRRSV host-pathogen interaction is probably rewarded by more consistent results across different studies.
I.M.R.G. was financially supported by the Council of Economy, Science, Innovation and Employment of the Andalusian Government, Spain (Project CEI10/00039). The work was partially supported by the Spanish Ministry of Education and Science, project number AGL2009-12438/GAN.
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