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Grouping Pig-Specific Responses to Mitogen with Similar Responder Animals
may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal
Model
J. Alex Pasternak
1,2
, Siew Hon Ng
1,2
, Tobias Käser
1
, François Meurens and Heather L. Wilson
1*
1
Vaccine and Infectious Disease Organization, home of the International Vaccine Centre (VIDO-InterVac),
2
University of Saskatchewan, 120 Veterinary Road,
Saskatoon, SK, S7N 5E3, Canada
*
Corresponding author: Heather L. Wilson, VIDO-InterVac, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, S7N 5E3, Canada, Tel: +1-(306)
966-1537; Fax: +1-(306) 966-7478; E-mail: heather.wilson@usask.ca
Rec date: 28 Apr 2014; Acc date: 19 Jun 2014; Pub date: 24 Jun 2014
Copyright: © 2014 J. Alex Pasternak, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Pig peripheral blood-derived mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs)
stimulated with mitogens ex vivo can show significant animal-to-animal variation lead to difficulty in interpreting
responses in an out-bred animal species. Mixed-cell populations were stimulated ex vivo with 2.5 μg/ml Con A or 2.5
ng/ml PMA plus 250 ng/ml ionomycin (PMAi; (LPCMs only)) or media alone for 72 hours. Supernatants were then
tested for cytokine production using a Bioplex assay for porcine IFNα, IFNγ, IL-10, and IL-12. Unstimulated PBMCs
had significant levels of IL-10 and the median value for this group decreased in the presence of Con A. Con A did,
however, induce production of IFNα and IFNγ, but not IL-12 in this cell population. In contrast, unstimulated and Con
A-stimulated LPMCs produced negligible IL-10, IFNα, IFNγ, and the majority of animals’ LPMCs showed negligible
IL-12 production in response to Con A. In contrast, LPMCs stimulated with PMAi produced IFNγ suggesting cytokine
production is mitogen–specific response. When we tracked animal-specific responses, we observed that discrete
subsets of animal’s PBMCs responded to Con A with significantly increased or decreased IL-10 production relative
to unstimulated cells. Further, in the LPMCs, some cells produced no IL-12 in response to Con A but showed
augmented production in response to PMAi, while others showed production of IL-12 in response to Con A but no
response to PMAi. Flow cytometric analysis showed that the PBMCs were a mixture of CD3+ T cells>CD21+ B
cells>CD172+ myeloid cells whereas the LPMCs consisted of mainly Cytotoxic T cells and Natural Killer cells. The
percentage of CD8α+CD4+ antigen-experienced T cells was greater in the LPMCs relative to the PBMCs. As
expected in an out-bred species, animal-specific differences in cytokine production in response to stimulants exist
and may confound interpretation of results unless tracked individually.
Introduction
Because of the increased demand for swine and their byproducts,
pig farming has become a major agricultural industry with barns
processing large numbers of pigs. Thus, the pig industry is vulnerable
to outbreaks of disease which can have enormous economic impact
worldwide [1]. The industry has been proactive in its search for
effective prophylactic strategies, therapeutic treatments and vaccines
to control and/or prevent diseases [2-5]. Studies designed to
understand the porcine immune system and its underlying functional
responses to drugs, mitogens, and immunomodulators are critical to
the design of effective treatments and/or vaccines to protect against
disease.
The Gut-Associated Lymphoid Tissue (GALT) is comprised of
organized inductive sites (Peyer’s Patches (PP), isolated lymphoid
follicles, and draining lymph nodes) and effector sites (non-organized
lymphoid tissue diffusely distributed throughout the Lamina Propria
(LP) [6]. In the theory of the common mucosal immune system,
activated T and B cells get sensitized at specific mucosal sites such as
inductive sites in the GALT. They leave the site of initial antigen
encounter via the lymphatics, transit through the circulatory system,
and migrate to mucosal effector sites such as the LP, where B cells
continue to expand and differentiate [7-8]. As well as homing back to
their site of origin, lymphocytes also seed other mucosa sites which
may be tremendously valuable in protecting the host against further
infections [7,9-11]. In the gut, the LP lies beneath the basement
membrane and is comprised of most of the components of the
immune system such as B cells, plasma cells, and macrophages,
dendritic cells (DCs) and T cells [12-13]. Because leukocytes from the
LP originally circulated through the blood and because PBMCs are
also composed of large numbers of B cells, plasma cells, T cells and
myeloid cells, we will compare the mitogenic responses of the LP cells
to the mitogenic response from Peripheral Blood Mononuclear Cells
(PBMC) population to discern if the responses are conserved across
tissues or differ widely in these distinct mixed-cell populations [12].
The magnitude of the proliferative response of PBMCs to the T cell
mitogen concanavalin A (Con A) has long been used as an indirect
measure of the responsiveness of the immune system to antigenic
stimulation [13]. Phorbol 12-myristate 13-acetate (PMA) activates
Protein Kinase C (PKC) while Ionomycin is a calcium ionophore and
together (PMAi) they bypass the T-cell receptor (TCR) complex,
trigger T-cell activation and signal through several intracellular
signaling pathways [14]. While PMAi and Con A activate T cells it is
important to remember that these mitogens can also act on
monocytes, DCs and other members of a mixed-cell population so
cytokine production cannot be automatically attributed to the T cell
population [13,15-18]. Cytokine production can be influenced by the
site of cell isolation, the cellular composition and the mitogen used.
Vaccines & Vaccination
Pasternak, et al., J Vaccines Vaccin 2014, 5:4
http://dx.doi.org/10.4172/2157-7560.1000242
Research Article Open Access
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242
We investigated production of a several cytokines reflecting the
immune response on viral infections (Interferon (IFN)α) as well as a
T-helper type (Th) 1 (IL-12, IFNγ) or Th2-type (IL-10) immune
response in pig PBMCs and LP mononuclear cells (LPMCs). Myeloid
cells may also be a source of IL-10 production in pigs [19-20]. Type I
IFNα, is critical to promote immunity against viruses and can be
triggered in many cell types, especially plasmacytoid dendritic cells
[21-22]. IFNα can induce T-cell activation or long-term survival,
production of IFNγ and Th1 differentiation [23]. IFNα can promote
DC differentiation, maturation and immunostimulatory functions
and, through production of both type I interferons and IL-6, IFNα can
induce human B cells to differentiate into plasma cells and produce
immunoglobulin [24-25].
IL-12 is a multi-functional cytokine that bridges the early
nonspecific innate resistance and the subsequent antigen-specific
adaptive immunity via a Th1 response [26]. IL-12 is naturally
produced by dendritic cells [27-29] and macrophages [30] and can
stimulate T-cell growth and differentiation of naïve T cells into Th1
cells [31].
CD4+ Th1 cells are required for response against intracellular
infections; they produce IFNγ and promote both macrophage and B-
cell activation [32-34]. Th2 cells synthesize IL-4, IL-10 and IL-13
cytokines, they are responsible for protection against extracellular
pathogens and they provide optimal help for antibody production and
promote both mast cell growth and eosinophil differentiation [35-37].
Th1 and Th2 development is mutually exclusive as expression of either
IFNγ or IL-4 antagonizes expression of the other [38-39]. Th17 cells
also help protect against extracellular pathogens, they are potent
inducers of tissue inflammation and have been associated with the
pathogenesis autoimmune diseases [40]. These cells have recently been
observed in pigs [41].
Studies in two-month old pigs showed that the porcine immune
system is sufficiently mature to be able to selectively control the
response of cytokine producing cells to mitogenic stimulation [42]. In
this study, cytokine production from PBMCs and LPMCs was
determined in 24 and 15 pigs, respectively. We measured baseline and
stimulated cytokine production in the presence of the Con A or PMAi.
We investigated the response to polyclonal T-cell activators rather
than an antigen-specific immune response because we aim to
investigate the capacity of pig leukocytes to produce cytokines without
interference from other components of the immune system. The aim
of the present study was to demonstrate how mixed-cell populations
taken from discrete regions of the body produce distinct cytokine
profiles in response to Con A or PMAi and that animal-specific
responses can differ widely. We then tracked the animal-specific
responses to Con A and PMAi to discern whether grouping similar
responder animals facilitates the interpretation of results obtained in
out-bred animal model.
Materials and methods
Animal use and ethics
This work was approved by the University of Saskatchewan’s
Animal Research Ethics Board, and adhered to the Canadian Council
on Animal Care guidelines for humane animal use. Seven-week old
Landrace cross piglets were obtained from several litters from the
Prairie Swine Centre, Inc. (PSCI), Saskatoon, SK, Canada.
PBMC isolation and stimulation: PBMCs were isolated from 49 day
old pigs by Ficoll gradient centrifugation following the protocol
described by Buchanan et al. [43]. Stimulation of PBMCs were
performed in 96-well, round-bottom plates (Nunc, Naperville, Ill.,
USA) using AIM V medium supplemented with 10% fetal bovine
serum (FBS) (Invitrogen, Burlington, ON, CA), 2 mM L -glutamine,
50 μM 2-mercaptoethanol and 10 μg/ml polymyxin B sulfate (Sigma-
Aldrich, Oakville, ON, Canada; for all) as described before [12]. Cells
were ex vivo stimulated with 2.5 μg/ml Con A (Sigma-Aldrich), 2.5
ng/ml, PMA (Sigma-Aldrich) plus 250 ng/ml ionomycin (Sigma-
Aldrich) or media alone for 72 hours. For each treatment, 5x105 cells
were cultured in triplicate wells in 200 μl total volume. Culture
supernatants were harvested and stored at –20 °C until assayed for
IL-10, IL-12, IFNα and IFNγ.
LPMC isolation and stimulation
Three inch segments of Peyer’s Patch-free jejunum were inverted
and washed in Calcium-free Krebs-Henseleit-bicarbonate (KHB; 119
mM NaCl, 4.8 mM KCl, 1.2 mM MgSO
4
, 1.2 mM KH
2
PO
4
, 25 mM
NaHCO
3
, pH 7.4) + 2.5 mM CaCl
2
(Sigma-Aldrich for all) and dabbed
onto wet paper towel to remove intestinal contents and mucous. The
intestine was sealed via ligature then inflated by injection of KHB +
CaCl
2
into the lumen and the other end was also sealed via ligature.
The inverted segments were incubated in KHB buffer+ 5 mM EDTA
(Sigma-Aldrich) for 20 minutes at 37
°
C and 150 RPM to slough off the
majority of epithelial cells and intra-epithelial lymphocytes. Segments
were rinsed in KHB+EDTA buffer then transferred to a clean flask
containing 100 ml RPMI Complete (RPMI 1640 (Invitrogen, Gibco
#21870-076) plus 10% FBS (Invitrogen), 50 μg/ml -Mercaptoethanol
(Sigma-Aldrich), 1:100 Antibiotic/antimycotic (Invitrogen, Gibco
#15240-062); 1:100 HEPES (Invitrogen, Gibco #15630-080), 1:100
MEM Non-Essential Amino Acids (Invitrogen, Gibco #11140-050)
and 2 mM L-Glutamine (Invitrogen, Gibco #25030-081)) with 100
U/ml Collagenase (C9263, Sigma-Aldrich) for 45 minutes at 37°C and
150 RPM to remove LPMCs. The intestinal segment was washed with
RPMI Complete to remove cells of interest, then tissue was discarded.
The cells were pelleted out of solution at 350 g for 5 min then
resuspended in 10 ml RPMI Complete. Cells were filtered through a 40
μm Nylon Cell Strainer (BD Falcon, Durham, NC, USA). Cells were
counted via Trypan Blue Exclusion assay using a hemocytometer and
resuspended at a final concentration of 0.5x107 live cells/ml. Cell
stimulations was performed as indicated above.
Flow cytometric analysis
PBMCs or LPMCs (1x106) were stained for 10 min in a 30 μl
volume of PBS with 2% FBS (Invitrogen) using the primary antibodies
at concentrations previously titrated in our lab as follows: anti-CD3
(Southern Biotech (4510-01; 1.6 μg/ml; Mouse IgG1; Birmingham, AL
35209, USA)), anti-CD4 (VMRD (74-12-4; 33.3 μg/ml; Mouse IgG2b;
Pullman, WA 99163, USA)), anti-CD8α (AbD Serotec (MCA1223; 3.3
μg/ml; Mouse IgG2a; Raleigh, NC, USA)), anti-CD21 (BD Biosciences,
San Jose, CA, USA) (555421; 0.83 μg/ml; Mouse IgG1; Mississauga,
ON, L5N 0B3, CA)) and anti-CD172 (VMRD (74-22-15A; 0.53 μg/ml;
Mouse IgG2b)). Cells were washed twice with 200 μl of PBS + 2% FBS
(Invitrogen) then pelleted by gently centrifugation for 3 min. Cells
were then stained with fluorescent secondary antibodies against goat
anti mouse IgG1 (APC, Southern Biotech 1070-11S), IgG2a (PE,
Southern Biotech 1082-09) and IgG2b (FITC, Southern Biotech
1092-02) at 0.8 μg/ml. Cells were washed twice as indicated above then
Citation:
Pasternak JA, Ng S, Kaser T, Meurens F, Wilson HL (2014) Grouping Pig-Specific Responses to Mitogen with Similar Responder
Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model . J Vaccines Vaccin 5: 242. doi:
10.4172/2157-7560.1000242
Page 2 of 9
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242
suspended in 200 μl PBS + 2% FBS (Invitrogen) and analysed on a
FACSCalibur Flow Cytometer using CellQuest Pro (BD Biosciences).
Final gating and analysis was conducted using FlowJo software version
7.6 (Tree Star, Ashland, OR, USA) with results presented as a
percentage of gated leukocytes except the CD8α+CD4+ T cells which
were presented as a percentage of all CD4+ T cells.
Bioplex cytokine assays: Bioplex bead coupling was performed as
per the manufacturer’s instructions. The reagents were as follows:
Coating antibody: monoclonal anti-porcine IFNα (Gene Tex
GTX11408), Detection antibody: monoclonal anti-porcine IFNα biotin
(PBL 27105-1), Standard: recombinant porcine IFNα (Genetech),
Bead: region 45 (BioRad MC10045-01). Coating antibody: monoclonal
anti-porcine IFNγ (Fisher ENMP700), Detection antibody:
monoclonal anti-porcine IFNγ (Fisher ENPP700; biotinylated in-
house), Standard: recombinant porcine IFNγ (clone 2-2-1;
biotinylated in-house), Bead: region 43 (BioRad MC10043-01).
Coating antibody: monoclonal anti-porcine IL-10 (Invitrogen
ASC0104), Detection antibody: monoclonal anti-porcine IL-10 biotin
(Invitrogen ASC9109), Standard: recombinant porcine IL-10
(Invitrogen PSC0104), Bead: region 28 (BioRad MC10028-01).
Coating antibody: monoclonal anti-porcine IL-12 (Kingfisher
MA0413S-C), Detection antibody: monoclonal anti-porcine IL-12
biotin (R&D BAM9122), Standard: recombinant porcine IL-12 (R&D
912-PL-026), Bead: region 36 (BioRad MC10036-01). The multiplex
assay was carried out in a 96 well Grener Bio-One Fluotrac 200 96F
black (VWR, #82050-754) which allows washing and retention of the
Luminex beads. The porcine IFNα, porcine IFNγ, porcine IL-10 and
porcine IL-12 protein standards were added to the wells at 50 μl per
well at a final concentration of 200 pg/ml, 2000 pg/ml, 5000 pg/ml and
5000 pg/ml, respectively. The PBMC supernatants were prediluted 1:3
and added to the wells at 50 μl per well. The 4 beadsets conjugated
with the IFNα, IFNγ, IL-10 and IL-12 antigens were vortexed for 30
seconds followed by sonication for another 30 seconds to ensure total
bead dispersal. The bead density was adjusted to 1200 beads per μl in
PBS-BN (1x PBS + 1% BSA (Sigma-Aldrich) + 0.05 % sodium azide
(Sigma-Aldrich), pH 7.4) and 1 μl of each beadset was added to 49 μl
of the PBSA + 1% New Zealand Pig Serum (Sigma-Aldrich P3484) +
0.05 % sodium azide (Sigma-Aldrich) which was then added to each
well. The plate was sealed with plate sealer (Thermo Fisher Scientific,
#12565491) and covered with foil lid. The plate was agitated at 800
rpm for 1 hour at room temperature. After 1 hour incubation with
serum, the plate was washed using the Bio-Plex ProII Wash Station
(Bio-Rad; soak 60 s, wash with 300 μl PBST). A 50 μl of biotin cocktail
consisting of biotinylated porcine IFNα (PBL 27105-1; 1/5000;
biotinylated in-house), biotinylated porcine IFNγ (Fisher ENPP700;
1/300; biotinylated in-house), biotinylated porcine IL-10 (Invitrogen
ASC9109; 0.5 μg/ml) and biotinylated porcine IL-12 (R&D BAM9122;
0.5 μg/ml) was added to each well. The plate was again sealed, covered
and agitated at 800 rpm for 30 minutes at room temperature then
washed again as indicated above. A 50 μl of Streptavidin RPE
(Cedarlane PJRS20; diluted to 5 μg/ml) was added to each well. The
plate was again sealed, covered and agitated at 800 rpm for 30 minutes
at room temperature and washed as indicated above. A 100 μl of 1x
Tris-EDTA was added to each well and then the plate was vortexed for
5 minutes before reading on the Luminex100 xMAP™ instrument
following the manufacturer's instructions and as described in
(Anderson et al., 2011). The instrument was set up to read beadsets in
regions 45, 43, 28 and 36 for IFNα, IFNγ, IL-10 and IL-12,
respectively. A minimum of 60 events per beadset were read and the
median value obtained for each reaction event per beadset. For all
samples the multiplex assay MFI data was corrected for subtracting the
background levels.
Statistical analysis
The outcome data from this study were not normally distributed
and therefore, differences among experimental groups were tested
using Kruskal-Wallis analysis and medians were compared using
Dunn’s test. Differences were considered significant if p<0.05. All
statistical analyses and graphing were formed using GraphPad Prism 5
software (GraphPad Software, San Diego, CA).
Results and Discussion
Influence of Con A on cytokine production from PBMCs
Con A is a T-cell mitogenic lectin that has been used extensively to
evaluate lymphocyte activation responses [42,44]. As well as their role
in T cell activation, Con A also can bind to and activate monocytes. In
fact some studies show that monocytes are required for T cells to
proliferate in response to Con A [45-46] while previously work in our
lab showed that sorted porcine T cells proliferate well after Con A
stimulation even in the absence of myeloid cells (unpublished
observations from one of the authors). When we investigated IL-10
and IL-12 production from PBMCs isolated from pigs, we observed
that a number of the unstimulated cells had high baseline IL-10
production and that Con A stimulation did not significantly increase
the median IL-10 value (Figure 1a; left side). In contrast, unstimulated
PBMCs did not show a baseline IL-12 production nor did they show
augmented IL-12 production in response to Con A (Figure 1b; left
side). Käser et al. (2008) showed that purified blood-derived pig CD4+
T cells produced IL-10 in response to Con A and they further specified
that the IL-10 producing population was predominately
CD4+CD25dim T cells, not T regulatory cells (CD4+CD25high) [47].
Our data also shows partial agreement with Andersson et al. (2007)
who showed that pig PBMCs showed induced IL-10 gene expression
but no change in IL-12 gene expression in response to Con A [48].
Further, our data showed that in pig PBMCs, Con A significantly
induced production of IFNα (p<0.0001; Figure 1c; left side) and IFNγ
(p<0.0001; Figure 1d; left side) relative to unstimulated cells. These
data show agreement with Chuang et al. 2009 who showed, using
ELISPOT analysis, that human PBMCs produced low levels of IFNγ-
secreting cells in response to Con A [49]. Donaldson et al, 2005 used a
custom-designed innate immunity microarray to investigate Con A
stimulation of PBMC in cattle over a 24 hour period. They saw
induction of the gene coding for IFNγ in response to Con A which
shows agreement with our study (although we evaluated changes in
protein expression) [50]. However, our data also contrasted with
results from numerous other studies. For instance, Wilkinson et al
looked at transcriptomic profile of porcine PBMCs to Con A
stimulation for up to 68 hours [51]. They comment that one of the
genes most up-regulated in response to Con A was TNFRSF9 (also
known as 4-1BB) which encodes a receptor that signals to maintain T
cell proliferation and promote the release of Th1 cytokines [51].
Despite this, their study did not show that pig PBMCs responded to
Con A with induced expression of IFNγ, the classical Th1 cytokine.
Also in contrast to our data, their transcriptomic analysis failed to
show induced IL-10, IL-12 or IFNα expression in response to Con A.
Raskova et al, 2005 determined that Con A alone had no stimulatory
effect on IFNγ- secreting cells in pig PBMCs relative to unstimulated
cells [42]. Together these responses show that PBMC production of
Citation:
Pasternak JA, Ng S, Kaser T, Meurens F, Wilson HL (2014) Grouping Pig-Specific Responses to Mitogen with Similar Responder
Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model . J Vaccines Vaccin 5: 242. doi:
10.4172/2157-7560.1000242
Page 3 of 9
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242
cytokines in response to Con A may not be consistent across all species
and/or may be highly sensitive to timing of analysis, concentration of
Con A, or method of analysis. Differences in response across the
literature may be due to inconsistent mitogen concentrations and/or
ages of animals under investigation [52].
Figure 1: Porcine PBMCs and LPMCs show distinct cytokine production profiles in response to Con A or PMAi. IL-10 (a), IL-12 (b), IFNα (c)
and IFNγ (d) cytokine concentrations were obtained at once using a BioPlex assay in seven week old pigs. Data shown are presented as the
mean of duplicate concentrations for individual biological replicates and the horizontal line represents the median value for the group. ***
p<0.001; **** p<0.0001
Influence of Con A on cytokine production from LPMCs
The level of cytokine production in response to Con A within the
LPMCs was quite different from what was observed in the PBMC
population. For instance, unstimulated and Con A-stimulated LPM
cells failed to produce IL-10 and LPMCs failed to induce IFNα and
IFNγ production in response to Con A (Figure 1a, 1c, 1d; right side).
These data suggest that CD8α+ LP T cells are poor producers of these
cytokines in response to Con A. While no PBMCs showed induced
expression of IL-12 in response to Con A, four of the fifteen pigs
showed Con A-induced IL-12 expression (Figure 1b; right side).
Because IL-12 is produced by dendritic cells and monocyte/
macrophages and it promotes IFNγ production in blood-derived NK
cells and T cells [53-54] , one may expect that if IL-12 is produced
within the LPMCs (Figure 1b), that there would likely be a rise in the
production of IFNγ, which was not observed (Figure 1d). However,
Cella et al. 2009 also showed that while IL-12 induced IFNγ in blood
NK cells, it failed to induce IFNγ in intestinal NK cells suggesting that
cells obtained from different locations in the body do not respond
Citation: Pasternak JA, Ng S, Kaser T, Meurens F, Wilson HL (2014) Grouping Pig-Specific Responses to Mitogen with Similar Responder
Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model . J Vaccines Vaccin 5: 242. doi:
10.4172/2157-7560.1000242
Page 4 of 9
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242
uniformly to mitogens [55]. Further, our data shows agreement with
Chuang et al. who showed that ELISPOT analysis of human gut
LPMCs showed a negligible increase in the number of IFNγ-secreting
cells in response to Con A [49]. In contrast, others show that intestinal
LPMCs from normal nonhuman primates responded to Con A with
increased mRNA coding for IFNγ [56]. Whether the corresponding
proteins levels also changed was not evaluated. Collectively, our data
shows that in pigs, Con A-induced cytokine productions from distinct
mixed-cell populations are not conserved.
Influence of PMAi on cytokine production from LPMC
Because Con A failed to induce production of IFNα, IFNγ or IL-10
in LPMCs, we next wanted to evaluate whether these cells responded
to another activator. PMA activates Protein Kinase C while Ionomycin
is a calcium ionophore, which will lead to activation of several
intracellular signaling pathways [14,57-58]. To ensure that PMA/
Ionomycin (PMAi) concentrations were not toxic to the pig LPMCs,
we performed a dose titration and determined that 2.5 ng/ml PMA
plus 250 ng/ml ionomycin concentration induced an average of 15%
proliferation after 3 days (data not shown). Our BioPlex data showed
that LPMCs responded to PMAi with negligible IL-10 and IFNα and
only five animals responded with IL-12 production (Figure 1a, 1c, 1b;
right side). However, LPMCs from all but one pig responded to PMAi
with relatively high production of IFNγ (Figure 1d; right side) which
was significantly higher than the control population (p<0.001). The
subset population in the porcine LPMCs responsible for IFNγ
production showed preferential responsiveness to PMAi but not Con
A further indicating that the site from which cells are isolated (with
what is likely a very distinct cellular composition) and the mitogen
chosen for activation can have profound effect on leukocyte-derived
cytokine production.
Distinct animal-specific responders:
By tracking animal specific responses to stimulation (or baseline
cytokine expression in response to media alone), we see patterns of
responses emerging that are not evident when simply the median
responses are evaluated. Livestock pigs are out-bred animals, means
that within a group there are commonly disparate responses to
treatment. If we track the animal-specific changes in IL-10 cytokine
production within the PBMCs, we observe that cells from some
animals respond to Con A with increased IL-10 production whereas
other respond with decreased IL-10 production relative to the
unstimulated cells (Figure 2a; left side). When we break the group into
responders and non-responders (right side of the graph), we observe
that PBMCs from a number of pigs that showed high baseline IL-10
production (Media (Decr)) showed significantly reduced (p<0.0001)
IL-10 production in response to Con A (Con A (Decr)), while at the
same time PBMCs from other pigs that showed high baseline IL-10
production (Media (Incr)) showed significantly increased (p<0.05)
IL-10 production in response to Con A (Con A (Incr)) (Figure 2a).
Further, PBMCs from some animals failed to show IL-10 production
regardless of whether they were simulated with Con A or not (non-
responders). Our data does not specify whether the IL-10 producing
cells are of myeloid or lymphoid origin.
Similarly, when we evaluated the production of IL-12 in LPMCs, we
observe that the majority of animals had negligible IL-12 production
but that some LPMCs responded to Con A with increased IL-12
production (Figure 2b; responders: left side) but others did not (Figure
2b; non-responders: right side). From the animals that produced IL-12
in response to Con A, with the exception of one animal, they
responded with negligible IL-12 production in response to PMAi
(Figure 2b). As well, cells from animals which failed to produce IL-12
in response to Con A stimulation produced high levels of IL-12 in
response to PMAi. And again, some animals’ cells did not produce
IL-12 in response to either Con A or PMAi (non-responders).
Although the differences in IL-12 production in response to Con A or
PMAi do not meet the criteria of being statistically different from each
other, discrete patterns of expression were evident when were
investigated individual animal responses to mitogen.
If T cells are responsible for IL-10 production, we anticipate that
they are Th2 cells and would necessarily produce negligible IFNγ [59].
We investigated whether the PBMCs from the animals that had high
baseline and Con A-induced expression of IL-10 had reduced
expression of IFNγ. As indicated in the right-side of Figure 2a, there is
a group of animals that show high levels of IL-10 in the absence of Con
A (Figure 2a, Media (Incr) which produced significantly increased
expression of IL-10 in the presence of Con A (Figure 2a, ConA (Incr).
The unstimulated cells from this group of animals (same as Figure
2, filled circles) will be referred to as ‘IL-10 (Media (Incr)’ in Figure 3
and the corresponding Con A-stimulated cells will be called ‘IL-10
(ConA (Incr)’ in Figure 3. Similarly, there is a group of animals that
showed high levels of IL-10 in the absence of Con A (Figure 2a, Media
(Decr) and lower IL-10 production in the presence of ConA (Figure
2a, ConA (Decr). In Figure 3, the unstimulated cells from this group of
animals will be referred to as ‘IL-10 (Media (Decr)’ and the
corresponding Con A-stimulated cells will be called ‘IL-10 (ConA
(Decr))’. We charted the IL-10 production on the left y-axis and the
corresponding IFNγ production was charted on the right y-axis
(Figure 3). For both the ‘IL-10 (Media (Incr))’ and ‘IL-10 (Media
(Decr)) groups, we observed high IL-10 with negligible baseline IFNγ
production suggesting a low IFNγ to IL-10 ratio (i.e. a Th2 type
immune response). The Con A-stimulated cells in the ‘IL-10
(ConA(Incr) group all produced relatively high concentrations of both
IL-10 and IFNγ. Because Th2 cells produce IL-10 and Th1 cells
produce IFNγ, these results indicate that likely two populations of cells
are responsible for production of these cytokines. A single trend was
not observed in the group of Con A-stimulated cells referred to as
‘IL-10 (ConA (Decr)). From this group, 9 /15 animals had negligible
IL-10 production but produced low to moderate levels of IFNγ. The
remaining six animals from this group produced relatively high
concentrations of IL-10 and IFNγ in response to Con A. Together
these data again may indicate two cell types are responsible for
producing the cytokines. Differences in mixed-cell population
responses to a stimulant are not unexpected when we consider that
pigs are an out-bred species with potentially diverse inherent
biological variation. Others have shown that the biological variability
amongst an out-bred population such as humans and cattle are
profound and made it very difficult to generate statistically significant
results [60-62]. It has been suggested that variation in Con A
responsiveness of human peripheral lymphocytes may be partly
related to differences in purification which give rise to cell
preparations containing varying amounts of monocytes [63]. From
these results, we suspect that there are likely at least two subsets of cells
that are responsible for the cytokine production in this mixed cell
population but this would have likely gone unobserved if the animal-
specific responses to mitogen were not tracked and grouped together.
Citation:
Pasternak JA, Ng S, Kaser T, Meurens F, Wilson HL (2014) Grouping Pig-Specific Responses to Mitogen with Similar Responder
Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model . J Vaccines Vaccin 5: 242. doi:
10.4172/2157-7560.1000242
Page 5 of 9
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242
Figure 2: Distinct profiles of responder and non-responders are evident when animal-specific changes in IL-10 and IL-12 production are
tracked. Animal-specific IL-10 production in PBMCs (a) and IL-12 production in LPMCs (b) in unstimulated cells or in response to Con A or
PMAi were tracked across animals. Data shown are the same as those shown in Figure 1a and 1b but presented as an aligned scatter plot and
animal-specific changes to each stimulant are tracked using a joining line. * p<0.05; **** p<0.0001.
Figure 3: Distinct profiles of responders and non-responders are
evident when the ratio of IL-10:IFNγ in PBMCs are tracked across
animals. The left Y-axis represents IL-10 concentrations and the
right Y-axis represents IFNγ concentrations. Animal-specific
ConA-induced IL-10 and IFNγ production were tracked. Data
shown are the same as those shown in Figure 1a and 1d but
presented as an aligned scatter plot and animal-specific changes to
each cytokine production are tracked using a joining line.
Cellular composition of PBMC and LPMCs:
To gain a better understanding of cells responsible for the cytokine
production above, we investigated the cellular composition of the
PBMC and LPMC populations. Because PBMCs are acquired from the
circulatory system and the LPMCs are derived from a tissue, it is not
surprising that our data shows that the PBMCs are largely comprised
of leukocytes (Figure 4a) but that the leukocytes represent a minority
population in the LPMCs (Figure 4h), with the majority of LPMCs
likely being epithelial or stromal cells. Flow cytometric analysis from
PBMCs and LPMCs from four different pigs indicates that CD172+
myeloid cells represent a moderate percentage of the PBMCs (Figure
4b; 7.2% ± 1.5) but are virtually absent in the LPMCs (Figure 4i; 0.7%
± 0.5). Similarly, the B cells (CD21+) cells represent a major
population within the PBMCs (Figure 4e; 27.8% ± 2.3) but are virtually
absent from the LPMC population (Figure 4l; 0.3% ± 0.3). Within both
the PBMC and the LPMC populations, the CD3+ lymphocytes
comprise a very large portion of the total leukocyte population of cells
(Figure 4d; 56.4% ± 6.4, Figure 4k; 74.0 ± 5.3, respectively). The
percentage of CD3+ T-cell subsets in the PBMCs was as follows:
cytotoxic T cells (CTLs; Figure 4f; 9.1% ± 2.7) and CD4+ T cells
(Figure 4f; 27.8% ± 4.4). The percentage of CD3+ T-cell subsets in the
LPMCs (Figure 4k) were as follows: CTLs (Figure 4m; 54.1% ± 6.6)
which is approximately 6-fold higher than the corresponding PBMC
population and CD4+ T cells (Figure 4m; 2.7% ± 1.0) which is 10-fold
lower than the corresponding PBMC population. In figure 4g and 4n,
histograms show that there is a higher relative increase in the number
of CD8α+CD4+ T cells (which represent antigen-experienced T cells)
in the LPMC population versus the PBMC population. Finally, the
percentage of NK cells was 4.3% ± 3.1 in the PBMCs (Figure 4c) and
13.8% ± 5.8 in the LPMC population (Figure 4j). The total percentage
leukocyte for each subtype for each individual animal is shown in
Figure 4o. There was significantly higher total leucokyes, B cells,
Citation: Pasternak JA, Ng S, Kaser T, Meurens F, Wilson HL (2014) Grouping Pig-Specific Responses to Mitogen with Similar Responder
Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model . J Vaccines Vaccin 5: 242. doi:
10.4172/2157-7560.1000242
Page 6 of 9
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242
Myeloid cells and CD4+ T cell in the PBMCs than in the LPBMC
population (p<0.05).
Figure 4: Frequency of leukocyte subsets in PBMC and LPMC using
FCM analysis A) shows a representative FCM analysis (n=4) from
PBMCs (left) and LPMC (right) isolated from 6 week old pigs were
analysed on the frequencies of different immune cell subsets using
three-color FCM. Forward scatter (FSC) and side scatter (SSC)
were used to gate on leukocytes (PBMCs (a) and LPMC (h) for
further analysis of myeloid cells (CD172+; b and i), NK cells (CD3-
CD8α+; c and j), B cells (CD21+; e and l) and T cells (CD3+; d and
k). Gated T cells were then further analysed on their CD4/CD8α
expression to determine the frequency of CTLs (CD4-CD8αhigh; f
and m) and CD4+ T cells (f and m). CD8α expression of CD4+ T
cells was also investigated to define the frequency of experienced
CD4+ T cells (CD8α+CD4+; g and n). (o) Summarizes the results
of this FCM analysis of all four analysed animals. Percentages from
the PBMC population are represented by a closed circle and the
percentages from the LPMC populations are represented by an
open box. (p) and (q) is a pie chart showing the relative average
percentage of PBMCs and LPMCs from all 4 animals.
In turn, there was significantly higher CD3+ T cells and CTLS in
the LPMCs than the PBMC population (p<0.05). We note that the data
is extremely consistent across individual animals suggesting that any
variation in functional responsiveness within a specific immune
compartment in the assays above are most probably not caused by
animal-specific differences in the cellular composition.
In figure 4p and 4q, we see a pie chart representing the major cell
populations in the PBMCs and LPMCs. The cell types that comprise
the PBMCs are B cells=CD4 T cells>CTLs>Myeloid cells>NK cells. In
contrast, the cell types that comprise the LPMCs are CTLs>>>NK
cells>>CD4+ T cells>Myeloid cells>B cells. Thus, it is reasonable to
assume that there may indeed be two distinct subsets of cells in the
PBMC population responsible for expressing the IL-10 and IFNγ
(Figure 3) and distinct subset of cells may express IL-12 in response to
ConA or PMAi in the LPMCs (Figure 2b). Techniques such as cell-
sorting may be able to clarify which cell types are responsible.
Conclusion
This study demonstrates that tracking animal-specific responses to
mitogen and grouping them with similar responder animals facilitates
the interpretation of results obtained in an out-bred animal model.
LPMCs from the pigs were primarily CTLs which did not show IL-10,
IL-12, IFNα or IFNγ production in response to Con A, but, they did
produce IFNγ in response to PMAi stimulation. PBMCs were a
mixture of myeloid cells and B and T lymphocytes which produced
IL-10, IFNα and IFNγ in response to Con A. Animal-specific
responses to Con A and PMAi were evident and may be due to
differences in each animal’s capacity to produce cytokine. If such
differences in responses to ex vivo stimulation with mitogen are
evident from pig cells, one begins to understand how challenging it
may be to design an effective vaccine or treatment in out-bred animal
species. Unlike in mice and other rodent models, humans and most
livestock populations are out-bred populations; therefore it is clear
that there is a tremendous advantage in studying treatments or vaccine
responses across a group or herd where the response is likely going to
be heterogeneous. It may be that multiple treatments or vaccination
strategies should be undertaken to protect a greater percentage of the
members of out-bred populations.
HLW conceived of and designed the experiments and wrote the
manuscript. SN and JAP performed the laboratory experiments. TK
and FM offered considerable advice on flow-cytometry technique and
the subsequent data analysis. All authors contributed to the editing of
the manuscript.
Acknowledgments
We gratefully acknowledge financial support from the Alberta
Livestock and Meat Agency, Ontario Pork and the Saskatchewan
Agriculture Development Fund (Saskatchewan Ministry of Agriculture
and the Canada-Saskatchewan Growing Forward bi-lateral
agreement). We would like to thank the members of PSCI for their
excellent animal expertise as well as Donna Dent, Dr. Andrea Ladinig
and Dr. John Harding for their excellent technical assistance with the
BioPlex assays. HLW is an adjunct professor in the Department of
Biochemistry and the School of Public Health at the University of
Saskatchewan.
This manuscript is published with the permission of the Director of
VIDO as journal series no. 704.
Citation:
Pasternak JA, Ng S, Kaser T, Meurens F, Wilson HL (2014) Grouping Pig-Specific Responses to Mitogen with Similar Responder
Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model . J Vaccines Vaccin 5: 242. doi:
10.4172/2157-7560.1000242
Page 7 of 9
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242
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Citation: Pasternak JA, Ng S, Kaser T, Meurens F, Wilson HL (2014) Grouping Pig-Specific Responses to Mitogen with Similar Responder
Animals may Facilitate the Interpretation of Results Obtained in an Out-Bred Animal Model . J Vaccines Vaccin 5: 242. doi:
10.4172/2157-7560.1000242
Page 9 of 9
J Vaccines Vaccin
ISSN:2157-7560 JVV, an open access journal
Volume 5 • Issue 4 • 1000242