Parturition and Dexamethasone Effects on CpG Methylation Patterns of
IFNγ and IL4 Promoters in Bovine CD4+ T-cells
M. A. Paibomesai1, B. Hussey, M. Nino-Soto, B.A. Mallard1.
1
Department of Pathobiology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
Introduction
The transition period is time of stress, transition, high energy demand and sub-optimal immune
response. It is accompanied by many changes including hormonal, behaviour, management and
transition into high lactation (Lorraine M Sordillo et al. 2009). Some of the hormonal changes
which occur around calving include rapid decrease in progesterone and increase in
gluccocorticoids and estrogen (Kimura et al. 1999). It is around this period that there is a high
occurrence of both infectious and metabolic disease, which may cause financial loss to the
producer through loss milk production and cost of treatment.
The immune response plays a role in suspectibility of an individual to disease and is effected by
many external and internal factors. Changes in immune cell population and functionality of both
the innate and adaptive immune response occur during the transition period and these could leave
individuals more susceptible to disease during this period (Kim et al. 2005). More specifically,
the adaptive type 1 and type 2 immune responses experience shifts during this period, which is in
part dependent upon CD4+ cell phenotypes that is controlled by epigenetic modifications(Shaferweaver et al. 1999; Karrow et al. 2011). A balance between type 1 and type 2 responses is
essential for a board based disease resistance (Bonnie A Mallard & Wilkie 2007). It is shown in
many mammalian species, including human and mice, that pregnancy is a type 2 dominated
period with parturition inducing a type 1 dominance. Additionally, although there are numerous
studies that focus on changes in peripartum IR, the casual mechanisms of the observed
immunodepression remains largely unknown, particularly any epigenetic contributions.
Epigenetics is the study of modifications to DNA which influence gene expression but do not
change the overall DNA sequence, these modifications include DNA CpG methylation and
histone modifications (Wilson et al. 2009). Typically, methylation occurs on CpG motifs and has
a repressive effect on gene expression, where as DNA demethylation, or the loss of methylation
at CpG motifs, enhances gene expression. Epigenetic modifications are thought to be the link
between enivornmental influence on genetics (Petronis 2010).
The aim of this study was to assess the effects of parturition and dexamethasone (Dex), a
synthetic glucocorticoid, on bovine CD4+ T-cell cytokine production and DNA methylation
patterns of IFNG and IL4 promoters in vitro.
Materials & Methods
Animals
Holstein dairy cows were housed at the University of Guelph dairy research farm. Blood
collection was performed on 5 dairy cows (n=5) four weeks prior to calving and four days postcalving from the same cows for peripartum analysis. The dates for prepartum collection were
chosen based on the predicted calving date. The Dex treatment blood samples were collected
from 3 dairy cows (n=3) in mid-lactation (~100 days in milk), which were different cows from
the peripartum analysis. All animal handling was approved by the Animal Care Committee of the
University of Guelph (AUP #04R063).
Blood Collection and Blood Mononuclear Cell (BMC) Cell Isolation
Blood (80-100ml) was collected by caudal vein venipuncture in 10mL EDTA vacutainers (BD ,
Franklin Lakes, NJ). Cells were isolated using Histopaque 1107 (Sigma, Oakville ON) as per the
manufacturer’s instructions. Viable BMCs were counted using a hemocytometer and tryptan blue
exclusion dye (Sigma, Oakville ON).
CD4+ T-Lymphocyte Selection
CD4+ T-cells were isolated using the MiniMACS system (Miltnyi Biotech, Auburn CA) as per
the manufacturer’s instructions. CD4+ cells were labelled using with 100ul of mouse anti-bovine
CD4+ antibody (ILA-11, VMRD, diluted 500 fold, 4°C, 30 min) and subsequently labelled with
goat anti-mouse IgG coated magnetic microbeads (20µl per 1×107 cells, 4°C, 15 min). The cell
suspension (500µl) was added to the magnet bound column, washed three times and eluted with
1mL of MACS Buffer. The column separation was then repeated a second time to improve purity.
Purity was as confirmed by flow cytometry as >99% (data not shown).
Cell Culture
For Peripartum Analysis: The isolated CD4+ T-cells were cultured at 2.5x106cells/mL in 200μl
RPMI media (RPMI with 300mg/l glutamine, 10% fetal calf serum and 1/250 dilution of
penicillin/streptomycin) in a 96 round bottom plate for 24 hours (5% CO2, 37°C). Half of the
plated cells were stimulated with the mitogen Concanavalin A (ConA, 2.5µg/mL) while the other
half of the cells served as unstimulated controls.
For Dex Analysis: CD4+ T lymphocytes were cultured in a 96 well round-bottom cell culture
plate (37°C, 5% CO2, 72 hours) at a concentration of 2.5×106cells/ml in Phenol red free +
Glutamine RPMI (Invitrogen, Burlington ON) and 10% Charcoal Stripped FCS (Invitrogen,
Burlington ON; T cell media), in 200µl aliquots. Before aliquoting all cells were stimulated with
2.5ug/ml ConA with half also receiving stimulation with 10µM Dex, a dose shown in
preliminary experiments to cause the maximum decrease of CD4+ T-cell proliferation in vitro
(data not shown).
Enzyme Linked Immunosorbance Assay (ELISA)
Supernatant from the CD4+ T-cell cultures (above) were collected to evaluate cytokine (IFNγ
and IL4) production (unstimlulated, ConA, or ConA plus Dex treatments). Supernatant was
collected (150µl) from each well after either 24 or 72 hours of incubation, pooled and then stored
at -20°C for ELISA.
To determine IFNγ concentration a bovine IFNγ ELISA kit (Mabtech, Cincinnati, OH) was used
as per manufacturer’s instructions. For the IL4 ELISA, Immulon 2HB flat-bottom 96 well plates
(Fisher Canada, Nepean, ON) were coated for 48 hrs at 4°C with 100 µl/well of a 1 µg/µl
dilution of mouse anti-bovine IL4 antibody (AbD Serotec - MorphoSys US Inc, Raleigh, NC
USA) in carbonate-bicarbonate buffer pH 9.6. After coating, the coating solution was aspirated
and 200 µl of blocking buffer (PBS pH 7.4 + 3% Tween 20) were added to each plate for 90 min
incubation at room temperature (RT). Samples and standards were added after removal of the
blocking buffer. A recombinant bovine IL4 (AbD Serotec - MorphoSys US Inc, Raleigh, NC
USA) was used as positive control starting with a 40,000pg/ml dilution to prepare a 2,000 pg/ml
working dilution that was serially diluted from 1/2 to 1/256. Blocking buffer was used as
negative control. All controls and sample dilutions were added to plates in duplicate and
incubated for 150 min at RT on a shaker. Plates were washed four times with 300 µl/well of
washing buffer (PBS pH 7.4 + 0.05% Tween 20) in an ELx405 Autoplate Washer (BioTek
Instruments, Inc., Winooski VT, USA). For antibody detection, 100 µl/well of a 1/8,000 dilution
of biotinylated mouse antibovine IL-4 monoclonal antibody (AbD Serotec - MorphoSys US Inc,
Raleigh, NC USA) in washing buffer were added and plates were incubated for 60 min at RT on
a shaker. Next, plates were washed four times as previously described and 100 µl/well of a
1/10,000 dilution of streptavidin-HRP conjugate (Invitrogen, Burlington Ontario) in washing
buffer were added and plates were incubated for 45 min at RT on a shaker. After incubation with
the conjugate, plates were washed five times as previously described and 100 µl/well of 3,3’,5,5’
tetramethyl benzidine (TMB) substrate (IDEXX laboratories Inc, Westbrook, ME, USA) were
added to each well and incubated in the dark for 45 min on a shaker. After incubation, 100
µl/well of 1 M H2SO4 were added to stop the reaction. Individual well OD’s were obtained at
450 nm using an EL808 plate reader (Biotek Instruments Inc) and the KCjunior software
package (Bio-Tek Instruments, Inc., Winooski, VT, USA).
Genomic DNA (gDNA) Extraction
After collection of the culture supernatant , PBS was added (200ul) to the CD4+ T-cells
remaining in the culture plate. The cell suspension was mixed and washed (300g, 5min, rt) and
either stored at -80°C for future DNA extraction or cells went directly to DNA extraction
preformed using DNeasy® Tissue Kit (Qiagen, Mississauga ON) as per manufacturer
instructions.
Bisulphite Treatment and PCR amplification
To evaluate DNA methylation, gDNA was bisulphite treated using EZ DNA methylation kit
(Zymo Research, Orange CA) following the manufacturer instructions. Specific primers (Table
1) for both converted and unconverted promoter regions of bovine IFNG (GI:23821137) and IL4
(GI:555892) genes were designed using the BiSearch Software (Tusnady et al, 2005). The
promoter region selected for IFNG contained six CpG sites and IL4 contained five CpG sites.
PCR amplification of the selected regions was performed using Platinum® Taq polymerase
(Invitrogen Canada Inc., Burlington, ON, Canada) in 20 µl reactions using 2 µl of template
converted or unconverted DNA, 2 µl of 10X PCR buffer, 0.6 µl of 50 mM MgCl2, 0.5 µl of 10
mM dNTPs (Invitrogen Canada Inc., Burlington, ON, Canada) and 1 µl of the respective forward
and reverse primer at a concentration of 15 mM. For both IL4 and IFNG promoter analysis, a
touch-down PCR program was used with annealing temperature going from 60 to 54°C in the
first part of the program after a denaturation step at 95°C for 2 min, and 6 cycles of 95°C/30 secs
– 60°C/30 secs – 72°C/45secs, followed by 23 cycles of 95°C/30 secs – 54°C/30 secs – 72°C/45
secs and a final extension step of 72°C for 20min. PCR products were run on a 2% agarose gel
for band size verification. For IFNG and IL4, gel extraction was performed using Invitrogen’s
PureLink Quick Gel Extraction Kit (Invitrogen Canada Inc., Burlington, ON, Canada) on the
band corresponding to the IL4 promoter region (684bp) or IFNG (609bp).
Cloning and Sequence Analysis
Cloning was performed using TOPO TA Cloning Kit (Invitrogen, Burlington ON) as per the
manufacturers’ instructions. Two LB plates per sample were prepared at different concentrations
(20ul & 40ul cell suspension) and incubated at 37°C overnight. Ten individual colonies were
selected between both plates for each treatment and cultured in 5ml LB liquid broth overnight.
Plasmids were extracted (GenElute Plasmid Miniprep Kit, Sigma) and insertion of IFNG and IL4
was verified by PCR and gel electrophoresis (1.5% agar). PCR conditions were as follows:
denaturation at 95°C for 10 min, 34 cycles of 95°C/45 secs – 59°C/45 secs – 72°C/45 secs and
extension at 72ºC for 20 min. Verified insert containing plasmid preparations were sequenced
(Robarts Research Institute, London ON). Sequences were annotated and edited in BioEdit
(http://www.mbio.ncsu.edu/BioEdit/BioEdit.html) and analysed using BiQ Analyser software
(Bock et al. 2005). Seven clones per condition in the peripartum period were collected and 10
clones per condition were collected for the Dex treatment analysis.
Statistical Analysis
Statistical significance was reported at p≤ 0.05, highly significant at p≤ 0.01 and a trend at p≤0.1.
Treatment effect of Dex on ConA stimulated CD4+ T-cells on cytokine production, as measured
by ELISA, was calculated with a two-tailed, paired t-test using the program R 2.11.1 (Team
2010)
Significance of ELISA data was determine with an ANOVA between the four treatment groups
(prepartum unstimulated, prepartum ConA stimulated, postpartum unstimulated, postpartum
ConA stimulated) using R 2.11.1 (Team 2010) for both IFNG and IL4.
Analysis of percent methylation in the IFNG by comparison of the six CpG sites within the
promoter region for each of the treatments. The overall change in methylation from prepartum to
postpartum samples were calculated by taking the difference of overall CpG methylated sites
between stimulated and unstimulated divided by the difference of CpG unmethylated sites. This
same procedure was completed on five CpG sites of the IL4 promoter region. Bioinformatic
analysis for DNA element identification and conservation estimates were conducted in MultiTF
(Ovcharenko et al., 2008).
Results & Discussion
Stimulation of bovine CD4+ T-cells with ConA increased IFNγ production significantly between
unstimulated and stimulated cells (p<0.05). The IFNγ production of ConA stimulated CD4+ cells
collected prepartum (961pg/mL) was less than those collected postpartum (1498.7 pg/mL),
approaching significance (p=0.08). ConA stimulated CD4+ cells IL4 decreased from cells
collected prepartum (124pg/mL) to those collected postpartum (90.2pg/mL), this difference was
not significant. These differences were in agreement to studies looking at parturitions effect on
type 1 and type 2 immune responses of human and mice, which is predominately skewed
towards a type 1 immune response around this period (Ishikawa et al. 2004).
The CD4+ T-cells there were used for cytokine quantification were then used for DNA
methylation analysis. The promoter region of the IFNG and IL4 locus were investigated in this
study, as previous studies have shown DNA methylation in promoter region can affect gene
transcription and subsequent protein production. The IFNG promoter region contains six CpG
sites (-334, -291, -220, -85, +57, +72 from the transcription start site[TSS]), four of these sites
were extragenic and two sites contained within the gene. Overall there was a decrease in the
change of methylation upon stimulation with ConA, postpartum samples (-9.5%) had a greater
decrease than prepartum samples (-3%), see Figure 1a. These results are consistent with the
increase in IFNγ upon ConA stimulation and from prepartum to postpartum, as demethylation is
associated with increased transcription and protein production (Wilson et al. 2009). For IFNG
promoter region there was also an overall increase in CpG methylation from prepartum to
postpartum for both stimulated (+9%) and unstimulated(+15.5%) CD4+ T-cells, see Figure 1b.
This was also consistent with the decrease in IFNγ from prepartum to postpartum for stimulated
CD4+ T-cells. Although there was an overall decrease in CpG methylation upon stimulation
there were three sites of interest: CpG site 2 (-291bp), CpG site 3 (-220bp) and CpG site 4 (85bp). CpG site 3(-200) and CpG 4 (-85) contained putative transcription factor binding
sites for Tbet and CREB. Tbet is a master regulatory transcription factor for differentiation
of naïve CD4+ T-cells into Th1 T-cells which predominately produce IFNγ and play a large
role in type 1 immune responses (Wilson et al. 2009). Although these sites are promising
regions of CpG methylation that could influence IFNγ production, further investigation into
other regulatory regions are necessary for dairy cows.
The IL4 promoter region contains 5 CpG sites (-329, +12, +128, +175, +193bp from TSS),
one extragenic and four intragenic regions. Overall, there was an increase in CpG
methylation at this region for both prepartum (+15.3%) and postpartum (+12.6%) samples
upon stimulation with ConA, see Figure 2a. This was not consistent with the increase in
IL4 production, a key type 2 cytokine, that was observed when these cells were stimulated
with ConA. There was also an increase in CpG methylation of the IL4 promoter region from
prepartum to postpartum for both stimulated (+12%) and unstimulated (+9%) cells, as seen
in Figure 2b. Unlike IFNG promoter region there was no observed sites of interest.
Individual variation was apparent in CpG methylation patterns and cytokine concentration
for both stimulation and parturition effects.
Dexamethosone (Dex) was used to determine the effects of gluccocorticoids on bovine
CD4+ T-cell production of type 1 (IFNγ) and type 2 (IL4) cytokines and its influence on
CpG methylation profiles of IFNG and IL4. Isolated bovine CD4+ T-cells were treated with
ConA with a subset of cells being treated with 10µM of Dex. ConA increased IFN γ
(3351pg/mL) and IL4 (1726pg/mL) production and was completely abrogated with Dex
treatmeant for both cytokines (IFNγ [p<0.01]; IL4 [p=0.23]). Analysis of CpG methylation
showed that upon Dex treatment overall IFNG methylation increased by 9% (Figue 3a) and
IL4 decreased by 18% (Figure 3b). This observation was consistent with decreased IFNγ
production, but was not consistent for IL4, with the assumption that DNA methylation
inhibits transcription. Therefore, it can be concluded that promoter region for IFNG may
have some influence on transcription and subsequent protein production. Further
investigation of regions which influence IL4 production is necessary. It should be noted that
for these two cytokines were in inverse to one other for both cytokine production and CpG
methylation of the promoter region of IFNG and IL4.
Conclusions
The aim of this study was to investigate CpG methylation of key type 1 (IFNγ) and type 2
(IL4) cytokine in dairy cows in the context the transition period. There was observed
significant increase in cytokine production for ConA treatment of cells. This observation
correlated will with CpG methylation patterns for IFNG promoter, but was not the case for
IL4. Parturition had an overall immune response depression that was evident in both
cytokine and CpG methylation data. Dexamethosone also showed immune response
depression for both IFNγ and IL4, with correlated with IFNG promoter CpG methylation,
but did not correlate for the IL4 promoter. Overall, this study demonstrated that epigenetic
mechanisms, such as DNA methylation plays a role in the regulation of bovine cytokines
and that this may be influenced by parturition effects on CD4+ T-cells. Further investigation
on the specific hormonal influences is needed to further understand this critical period for a
dairy cow.
15.0%
Prepartum
Postpartum
10.0%
10.7%
% Change CpG Methylation
5.0%
0.0%
5.2%
-334
-220
-291
-85
2.9%
+72
+57
+1
-1.3%
-3%
-9.5%
-5.7%
-5.0%
-7.2%
-8.6%
-9.7%
-10.0%
-11.4%
-14.3%
-15.9%
-15.0%
-20.0%
-20.0%
-25.0%
Figure 1a. Percent change of CpG methylation between stimulated and unstimulated for IFNG
promoter region of bovine CD4+ T-cells.
30.0%
Unstimulated
28.1%
Stimulated
25.0%
22.1%
21.3%
% Change CpG Methyltion
20.0%
15.0%
14.3%
14.3%
15.5%
10.0%
11.4%
10.3%
5.0%
9.8%
5.7%
5.7%
2.9%
0.0%
-5.0%
-334
-291
-220
9%
1.5%
-85
+1
+57
+72
Figure 1b. Percent change of CpG methylation between postpartum and prepartum for IFNG
promoter region of ConA stimulated and unstimulated bovine CD4+ T-cells.
25.0%
22.9%
21.4%
20.0%
% Change CpG Methylation
18.8%
15.0%
Prepartum
Postpartum
18.3%
15.8%
14.3%
14.3%
15.3%
10.0%
12.6%
5.0%
5.7%
5.7%
1.9%
0.0%
+1
-329
+12
+128
+175
+ 193
-5.0%
Figure 2a. Percent change of CpG methylation between stimulated and unstimulated for IL4
promoter region of bovine CD4+ T-cells.
25.0%
Unstimulated
20.0%
Stimulated
20.0%
% Change CpG Methylation
17.1%
15.0%
15.7%
14.3%
12.6%
12%
10.0%
9.9%
6.6%
5.0%
5.7%
2.9%
1.7%
0.0%
-5.0%
-329
+1
+12
+128
+175
+193
9.3%
Figure 2b. Percent change of CpG methylation between postpartum and prepartum for IL4
promoter region of ConA stimulated and unstimulated bovine CD4+ T-cells.
25.0%
23.0%
% Change of CpG Methylation
20.0%
16.1%
15.0%
10.0%
5.0%
6.0%
6.2%
+57
+72
+9.0%
2.5%
0.2%
0.0%
-334
-291
-220
+1
-85
Figure 3a. Percent change of CpG methylation for IFNG promoter region of 2.5µg/mL ConA+
10µM Dex treated bovine CD4+ T-cells.
5.0%
0.0%
-329
+1
+12
+128
+175
+193
% Change of DNA Methylation
-5.0%
-18.0%
-9.9%
-10.0%
-13.5%
-15.0%
-19.0%
-20.0%
-25.0%
-20.0%
-26.0%
-30.0%
Figure 3b. Percent change of CpG methylation for IFNG promoter region of 2.5µg/mL ConA+
10µM Dex treated bovine CD4+ T-cells.
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