- Email: [email protected]
Comparison of Th1 and Th2 cytokines production in ovine lymph nodes during early pregnancy
Accepted Manuscript Comparison of Th1 and Th2 cytokines production in ovine lymph nodes during early pregnancy Ling Yang, Pengda Wang, Hao Mi, Wan Lv, Baoliang Liu, Jingsong Du, Leying Zhang PII:
S0093-691X(18)30268-1
DOI:
10.1016/j.theriogenology.2018.10.004
Reference:
THE 14729
To appear in:
Theriogenology
Received Date: 28 May 2018 Revised Date:
1 October 2018
Accepted Date: 2 October 2018
Please cite this article as: Yang L, Wang P, Mi H, Lv W, Liu B, Du J, Zhang L, Comparison of Th1 and Th2 cytokines production in ovine lymph nodes during early pregnancy, Theriogenology (2018), doi: https://doi.org/10.1016/j.theriogenology.2018.10.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Comparison of Th1 and Th2 cytokines production in ovine lymph nodes
2
during early pregnancy
3
Running head: Th1 and Th2 cytokines production
RI PT
4 5 6
Ling Yanga*, Pengda Wanga, Hao Mia, Wan Lva, Baoliang Liua, Jingsong Dub, Leying Zhanga
8
a
9
Engineering, Handan 056021, China
SC
7
M AN U
Department of Animal Science, College of Life Sciences and Food Engineering, Hebei University of
10
b
11
*Corresponding author: Tel/fax: +86 310 8576856. E-mail address: [email protected] (Ling
12
Yang).
TE D EP
14
AC C
13
Handan Agricultural Service Association, Handan 056002, China
1
ACCEPTED MANUSCRIPT 15
ABSTRACT As a fetal allograft to the mother, early conceptus regulates the intrauterine immune and
17
systemic immune responses during early pregnancy in sheep. However, expression of T
18
helper 1 (Th1) and Th2 cytokines in maternal lymph nodes is unclear during early pregnancy
19
in sheep. In this study, inguinal lymph nodes were obtained on day 16 of the estrous cycle and
20
on days 13, 16 and 25 of pregnancy (n = 4 for each group) in ewes, and qRT-PCR, western
21
blot and immunohistochemistry were used to compare the expression of Th1 and Th2
22
cytokines in the lymph nodes. Our results showed that there were the highest levels of Th1
23
cytokines (IFN-γ, TNF-β and IL-2) and Th2 cytokines (IL-4, IL-5, IL-6 and IL-10) in the
24
lymph nodes on day 13 or 16 of pregnancy. Furthermore, there were a downregulation of
25
TNF-β and IL-2 and an upregulation of IL-5 and IL-10 on day 25 of pregnancy compared
26
with that in nonpregnancy, with no significant difference in the expression of IFN-γ, IL-4, and
27
IL-6
28
immunohistochemistry results showed that the IL-2 and IL-10 proteins were limited to the
29
subcapsular sinus and trabeculae in the cortex, lymph sinus. In conclusion, early pregnancy
30
exerted its effects on the lymph node and induced a Th2-biased response, which was essential
31
for a normal pregnancy in sheep.
32
Keywords: Lymph node; Pregnancy; Sheep; T helper cytokine
SC
M AN U
TE D
the
ewes
on
day
25
of
pregnancy
EP
between
AC C
33
RI PT
16
2
and
nonpregnancy.
The
ACCEPTED MANUSCRIPT 34
1. Introduction T helper (Th) cells are subdivided into Th1 and Th2 cells according to the cytokines that
36
they secrete. Th1 cells induce cell-mediated cytotoxicity and inflammatory responses via
37
interleukin (IL-2), interferon-gamma (IFN-γ) and tumor necrosis factor beta (TNF-β),
38
whereas the Th2 cytokines are anti-inflammatory cytokines, such as IL-4, IL-5, IL-6 and
39
IL-10 [1]. Th2 cytokines enhance humoral response, and there is a distinct Th2-bias in a
40
normal pregnancy. However, Th1 cytokines are generally detrimental to normal pregnancy,
41
and significantly higher concentrations of Th1 cytokines are produced in the abortion group
42
[2,3]. A significantly increase of the Th1 cytokine is the underlying immune etiology for the
43
reproductive failures [4]. We previously observed that IFN-γ was attenuated, and IL-4, IL-5,
44
IL-6, IL-10 and IL-13 were augmented in the peripheral blood mononuclear cells (PBMCs)
45
during early pregnancy in cattle [5].
TE D
M AN U
SC
RI PT
35
Lymph nodes are located all over the body in mammals, and their function is to filter and
47
scan the lymph for antigens. The immune reaction to pathogenic antigens, harmless antigens
48
and tolerance is initiated in the lymph nodes [6]. The weights of the lumbar and renal lymph
49
nodes, inguinal lymph nodes increase during pregnancy in mice, and the hormonal effects and
50
the presence of fetal antigens contribute to the changes in the lymph node weight [7]. It has
51
been reported that the count of large pyroninophilic and plasma cells rises significantly in the
52
iliac nodes during early pregnancy in rat [8]. Our previous study revealed that the expression
53
of the progesterone receptor and the progesterone-induced blocking factor is upregulated in
54
the lymph nodes during early pregnancy in ewes [9]. We hypothesized that the expression of
55
Th1 and Th2 cytokines within maternal lymph nodes would be impacted in response to
AC C
EP
46
3
ACCEPTED MANUSCRIPT pregnancy, as a component of pregnancy-induced immunomodulation. Therefore, the
57
objective of this study was to determine the expression of Th1 cytokines (IL-2, IFN-γ and
58
TNF-β) and Th2 cytokines (IL-4, IL-5, IL-6 and IL-10) in the lymph nodes from nonpregnant
59
and early pregnant ewes.
60
2. Materials and methods
61
2.1 Animals and experimental design
SC
RI PT
56
Small-tail Han ewes with approximately 18 months of age were housed at the farm of
63
Handan Boyuan Animal Husbandry Co., Ltd. in China, and all procedures were approved by
64
the Hebei University of Engineering Animal Care and Use Committee. All experiments were
65
conducted following the guidelines of the National Standards for Laboratory Animals of
66
China (GB 14925-2010). The ewes with normal oestrous cycles were observed daily for
67
estrus using vasectomized rams, mated twice with intact rams in a 12-h interval after the
68
detection of sexual receptivity. Thirty-two ewes were randomly assigned into four groups
69
prior to breeding, resulting in 4 pregnant ewes in each group harvested during pregnancy and
70
4 non-pregnant ewes as non-mated controls. The first day of coitus was counted as day 0 of
71
pregnancy or non-pregnancy. The ewes assigned to the nonpregnant group were not mated
72
with an intact ram. The inguinal lymph nodes were obtained from the ewes on days 13, 16
73
and 25 of pregnancy, as well as day 16 of the estrous cycle at the time of slaughter. Pregnancy
74
was confirmed through observing the presence of a conceptus in the uterus. The transverse
75
pieces of the lymph nodes (0.3 cm3) were fixed in fresh 4% (w/v) paraformaldehyde in
76
phosphate buffer saline (PBS) buffer (pH 7.4), and the remaining portions of the lymph nodes
AC C
EP
TE D
M AN U
62
4
ACCEPTED MANUSCRIPT were frozen in liquid nitrogen for subsequent quantitative real time PCR (qRT-PCR) and
78
western blot analysis.
79
2.2. RNA extraction and qRT-PCR assay
RI PT
77
The samples were crushed into fine powders in liquid nitrogen, and the powders were
81
dissolved in TRIzol (Invitrogen, California, USA), and total RNA was extracted according to
82
the manufacturer’s instructions. A FastQuant RT kit (Tiangen Biotech Co., Ltd., Beijing) was
83
used to synthesize the cDNA, and a SuperReal PreMix Plus kit (Tiangen Biotech) was
84
employed for qRT-PCR. The primer sequences for Th1 cytokines (IFN-γ, TNF-β, and IL-2),
85
Th2 cytokines (IL-4, IL-5, IL-6, and IL-10), and GAPDH were designed and synthesized by
86
Shanghai
87
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) at NCBI. PCR amplification efficiency of each pair
88
of primers was assessed before quantification, and was found to be in an acceptable range
89
(between 0.9 and 1.1). The primer product was sequenced to check for specificity, and the
90
expression of the targeted genes was determined by a Bio-rad CFX96 real-time PCR system
91
with 20 µl. The PCR amplifications were carried out at 95 °C for 10 sec, 55-58 °C (55 °C for
92
IL-5 and IL-6, 58 °C for IFN-γ, TNF-β, IL-4, IL-2 and IL-10) for 20 sec, and 72 °C for 25 sec,
93
and the number of PCR cycles was 40. The GAPDH PCR reaction was the same as IFN-γ,
94
TNF-β, IL-2, IL-4, IL-5, IL-6, and IL-10, respectively. The relative expression values for the
95
qRT-PCR assay were calculated by the 2-∆∆Ct analysis method, with GAPDH as the
96
endogenous control [10]. The relative expression value was set to 1 for the group on day 16 of
97
the estrous cycle.
Biotech
Co.,
Ltd.
(Table
1),
and
assessed
by
BLAST
AC C
EP
TE D
Sangon
M AN U
SC
80
5
ACCEPTED MANUSCRIPT 98
2.3. Western blot
Total proteins in the lymph samples were extracted by RIPA Lysis Buffer (Biosharp,
100
BL504A). The protein concentration was measured using a BCA Protein Assay kit with
101
bovine serum albumin as the standard (Tiangen Biotech). Equal amounts of total protein (10
102
µg/lane) were separated using 12% SDS-PAGE, and the proteins were transferred to 0.22 µm
103
polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). IFN-γ, TNF-β, IL-2,
104
IL-4, IL-5, IL-6 and IL-10 were detected by western blot analysis using a mouse anti-IFN-γ
105
monoclonal antibody (Abcam, ab27919, 1:1000), a mouse anti-TNF-β monoclonal antibody
106
(Santa Cruz Biotechnology, Inc., SC-28345, 1:1000), a rabbit anti-IL-2 polyclonal antibody
107
(Abcam, ab193807, 1:1000), a mouse anti-IL-4 monoclonal antibody (Bio-Techne, MAB2468,
108
1:1000), a mouse anti-IL-5 monoclonal antibody (Santa Cruz Biotechnology, SC-8433,
109
1:1000), a rabbit anti-IL-6 polyclonal antibody (Abcam, ab193853, 1:1000) and a mouse
110
anti-IL-10 monoclonal antibody (Santa Cruz Biotechnology, SC-32815, 1:1000), respectively.
111
Secondary goat anti-mouse IgG-HRP (Biosharp, BL001A) and goat anti-rabbit IgG-HRP
112
(Biosharp, BL003A) were diluted to 1:2000. Pro-light HRP chemiluminescence detection
113
reagent (Tiangen Biotech) was used to detect the immunoreactive bands. Sample loading was
114
monitored with an anti-GAPDH antibody (Santa Cruz Biotechnology, sc-20357) at a dilution
115
of 1:1000, and secondary goat anti-mouse IgG-HRP was diluted to 1:2000. The intensity of
116
blots were quantified using Quantity One V452 (Bio-Rad Laboratories), and the relative
117
levels were calculated using the internal control protein (GAPDH). The expression of GAPDH
118
protein was measured by western blot, and there was no difference among the four groups.
AC C
EP
TE D
M AN U
SC
RI PT
99
6
ACCEPTED MANUSCRIPT 119
2.4. Immunohistochemistry analysis
The fixed lymph samples were embedded in paraffin, and the paraffin-embedded sections
121
were deparaffinized in xylene, and rehydrated in ethanol. The rehydrated sections were treated
122
with 3% H2O2 to quench the endogenous peroxidase activity, and reduced non-specific
123
binding with 5% normal goat serum in PBS. Immunohistochemical localization of IL-2 and
124
IL-10 in the lymph tissue was performed using the rabbit anti-IL-2 polyclonal antibody
125
(Abcam, ab193807, 1:100) and the mouse anti-IL-10 monoclonal antibody (Santa Cruz
126
Biotechnology, SC-32815, 1:100), respectively. Negative controls were treated with
127
antiserum-specific isotype instead of the IL-2 or IL-10 antibody at the same protein
128
concentration. A DAB kit (Tiangen Biotech) was used to visualize the antibody binding sites
129
in the tissue sections. Finally, the images were captured using a light microscope (Nikon
130
Eclipse E800, Japan) and a digital camera (AxioCam ERc 5s), and the intensity of staining
131
and density of stained cells was analyzed through the images. The immunostaining intensity
132
of the different lymph samples was scored by two different investigators in a blinded fashion,
133
according to the following scale: 0, no staining; 1, weak staining; 2, moderate staining; 3,
134
strong staining [11].
135
2.5. Statistical analyses
AC C
EP
TE D
M AN U
SC
RI PT
120
136
The data for the relative expression levels of IFN-γ, TNF-β, IL-2, IL-4, IL-5, IL-6 and
137
IL-10 mRNA and proteins were analyzed using a completely randomized design with four
138
animals per group via the Proc Mixed model of SAS (Version 9.1; SAS Institute, Cary, NC).
139
For the lymph nodes from different stages of gestation or pregnancy status, the model 7
ACCEPTED MANUSCRIPT contained the random effect of the ewe and the fixed effects of the stage of gestation,
141
pregnancy status and the interaction between the stage of gestation and pregnancy status. The
142
comparisons among the relative expression levels of the different groups were performed
143
using the Duncan method and controlling the experimentwise type ± error equal to 0.05. The
144
data are presented as least squares means. Groups were considered significantly different at P
145
< 0.05.
146
3. Results
147
3.1. Relative expression levels of Th1 cytokines mRNA and proteins in the lymph nodes
M AN U
SC
RI PT
140
The qRT-PCR assay and western blot revealed (Fig. 1, Fig. 2) that the relative
149
expression levels of Th1 cytokines (IFN-γ, TNF-β and IL-2) mRNA and proteins were the
150
highest in the lymph nodes on day 16 of pregnancy among nonpregnant and pregnant ewes (P
151
< 0.05), but there was no significant difference in the expression levels of IFN-γ mRNA and
152
protein in the lymph nodes among nonpregnant, days 13 and 25 pregnant ewes (P > 0.05).
153
Furthermore, the relative expression levels of TNF-β, IL-2 mRNA and proteins were the
154
lowest in the lymph nodes on day 25 of pregnancy among nonpregnant and pregnant ewes (P
155
< 0.05), but there was no significant difference in the expression levels of TNF-β, IL-2
156
mRNA and proteins between nonpregnant and day 13 pregnant ewes (P > 0.05; Fig. 1, Fig.
157
2).
158
3.2. Relative expression levels of Th2 cytokines mRNA and proteins in the lymph nodes
AC C
EP
TE D
148
159
The relative expression levels of IL-4 mRNA and protein were the highest on day 13 of
160
pregnancy among nonpregnant and pregnant ewes (P < 0.05), and there was a decline from
8
ACCEPTED MANUSCRIPT day 13 to 25 of pregnancy, but there was no significant difference between nonpregnant ewes
162
and day 25 of pregnant ewes (P > 0.05; Fig. 1, Fig. 2). The peaks of relative expression levels
163
of IL-5, IL-6, IL-10 mRNA and proteins in the lymph nodes were on day 16 of pregnancy (P
164
< 0.05), and the relative expression levels of IL-5 and IL-10 mRNA and proteins were lower
165
in the lymph nodes on day 16 of the estrous cycle than that on day 25 of pregnancy (P < 0.05;
166
Fig. 1, Fig. 2).
167
3.3. The immunohistochemistry for IL-2 and IL-10 proteins in the lymph nodes
SC
RI PT
161
The immunohistochemistry for IL-2 and IL-10 proteins was limited to the subcapsular
169
sinus and trabeculae in the cortex, lymph sinus, but there was almost no immunostained in the
170
lymphoid nodules and medullary cords (Fig. 3). The staining intensity for IL-2 in the
171
subcapsular sinus, trabeculae and lymph sinus was 0, 2+, 2+, 2+, and 0 for the negative
172
control, the lymph nodes from day 16 of the estrous cycle, and the lymph nodes from days 14,
173
16, and 25 of pregnancy, respectively, and the staining intensity for IL-10 was 0, 0, 2+, 2+,
174
and 2+ for the negative control, the lymph nodes from day 16 of the estrous cycle, and the
175
lymph nodes from days 14, 16, and 25 of pregnancy, respectively (Fig. 3).
176
4. Discussion
AC C
EP
TE D
M AN U
168
177
Th1 cytokines, including IL-2, IFN-γ and TNF-β, are implicated in cell-mediated
178
cytotoxicity and inflammatory responses. IFN-γ is the only member of the type II class of
179
interferon, has antiviral, immunoregulatory, and anti-tumor properties [12]. TNF-β is also
180
known as lymphotoxin-alpha (LT-α), has a significant impact on the maintenance of immune
181
system, including the development of secondary lymphoid organs [13]. TNF-β plays an
182
essential role in the innate immune regulation, and can prevent the tumor growth and destroy
9
ACCEPTED MANUSCRIPT the cancerous cell lines [14]. IL-2 is a type of cytokine signaling molecule which has key
184
roles in the functions of immune system, is primarily via its direct effects on T cells
185
differentiation [15]. IL-2 promotes the differentiation of certain immature T cells into
186
regulatory T cells, which modulates the immune system, maintains tolerance to self-antigens,
187
and prevents autoimmune disease [16]. In general, Th1 cytokines are related with the
188
reproductive failures [4].
RI PT
183
A raise in foetal mortality is along with an increased level of IFN-γ mRNA in the placenta
190
of pregnant cows after inoculation with Neospora caninum (a coccidian parasite) [17], and
191
our previous study revealed that relative level of IFN-γ mRNA is dropped sharply in the
192
PBMCs on days 18 and 30 pregnancy in cattle [5]. TNF-β is associated with preeclampsia and
193
poor fetal growth in the European Americans [18]. There is a high level of IL-2 in the
194
endometriosis group in nonpregnant mice, which indicates that high level of IL-2 lead to
195
infertility in mice [19]. Our results revealed that there was a decrease in the expression of
196
IFN-γ, TNF-β and IL-2 in lymph nodes on day 25 of pregnancy (Fig. 1, Fig. 2), which
197
suggested that downregulation of IFN-γ, TNF-β and IL-2 in the lymph nodes on day 25 of
198
pregnancy may be beneficial for ovine fetal implantation and pregnancy maintenance.
AC C
EP
TE D
M AN U
SC
189
199
TNF-β is involved in the development of secondary lymphoid organs [13] and
200
lymphocyte activation [20]. There is a high level of IL-2 in blood serum at the second week of
201
gestation compared with that in anestrus and diestrus in bitches, and the upregulation of
202
expression of IL-2 has been considered necessary for the development and maintenance of
203
pregnancy [21]. It has been reported that IL-2 plays key roles in the immune response through
204
promoting naive CD4+ T cell differentiation into Th1 and Th2 cells [22]. As the primary
10
ACCEPTED MANUSCRIPT pregnancy recognition signal, interferon-tau (IFNT) is implicated in maternal systemic
206
immune regulation through blood circulation and lymph circulation via the PBMCs in the
207
bovine [23,24]. It has been reported that there is an upregulation in the expression of IFN-γ in
208
CD4+ T cell lines stimulated by IFNT in vitro in cattle [25]. Our results indicated that there
209
was an upregulation of IFN-γ, TNF-β and IL-2 in the lymph nodes on day 16 of pregnancy
210
(Fig. 1, Fig. 2). Therefore, it was suggested that upregulation of IFN-γ, TNF-β and IL-2 may
211
be related with the high level of IFNT on day 16 of pregnancy in sheep, and involved the
212
development and immune regulation of maternal lymph nodes during early pregnancy in
213
sheep.
M AN U
SC
RI PT
205
IL-4, IL-5, IL-6 and IL-10 are Th2 cytokines that enhance humoral response. IL-4 is a key
215
regulator in humoral and adaptive immunity, induces the differentiation of naive helper T
216
cells (Th0 cells) into Th2 cells, and Th2 cells subsequently produce additional IL-4 in a
217
positive feedback loop, and IL-4 also decreases the production of Th1 cells and IFN-γ [26].
218
IL-5 is an interleukin produced by Th2 cell and mast cell, can stimulate B cell growth and
219
increase
220
anti-inflammatory myokine, and plays a major role in suppressing the immune system. IL-10
221
exerts its effects on the immunoregulation through down-regulated expression of Th1
222
cytokines, major histocompatibility complex class II antigens, and blocks the NF-κB activity.
223
In a normal pregnancy, there is a distinct Th2-bias.
TE D
214
secretion.
IL-6
acts
as
both
pro-inflammatory
and
AC C
EP
immunoglobulin
224
It has been reported that the IFNT from the fetus increases the expression of IL-4 mRNA
225
by the effector T cells in cattle [25], and progesterone (P4) can significantly upregulate the
226
expression of IL-4 in the PBMCs in pregnant cows [27]. IL-6 plays a contributing role in
11
ACCEPTED MANUSCRIPT stimulation of the trophoblast invasion, regulation of the endometrial function and
228
implantation [28]. The endometrial IL-6 mRNA increases during early pregnancy in pigs,
229
which stimulates the attachment and proliferation of the trophoblast cells in vitro, and is
230
beneficial for the successful implantation [29]. IL-6 is produced by extravillous and
231
cytotrophoblast, and involved in regulating the migration, invasion, trophoblast differentiation
232
and proliferation of the placental cells, which is important for the normal placenta
233
development and successful pregnancy [30]. We found that the expression levels was higher
234
for IL-4 on days 13 and 16 of pregnancy, and for IL-6 on day 16 of pregnancy (Fig. 1, Fig. 2),
235
which indicated that upregulation of IL-4 and IL-6 during early pregnancy is implicated in
236
immune regulation of maternal lymph nodes, and the higher levels of IL-4 and IL-6 on day 16
237
of pregnancy may be due to the high serum concentration of IFNT and P4. However, our
238
results also showed that IL-4 and IL-6 were downregulated in the lymph nodes from day 16 to
239
day 25 of pregnancy (Fig. 1, Fig. 2). It has been reported that the serum concentration of IL-4
240
is elevated in early gestation, and decreases between 30 and 40 days of gestation owing to the
241
high concentration of prolactin in bitches [31], which is almost consistent with our results in
242
the ovine lymph nodes. It has been reported that there is an increased IL-6 level in the
243
maternal plasma in preterm delivery patients during pregnancy [28], which indicated that high
244
level of IL-6 in the lymph nodes may be harmful after early pregnancy in ewes.
AC C
EP
TE D
M AN U
SC
RI PT
227
245
It has been reported that the dendritic cells treated with placental growth factor lead to
246
the suppression of naive CD4+ T cell proliferation, but increase the IL-5 secretion by the
247
CD4+ T cell, which is helpful for a normal pregnancy in humans [32]. Pregnancy-specific
248
glycoprotein enhances the proliferation of IL-5-secreting cells in vivo, which is involved in
12
ACCEPTED MANUSCRIPT the successful pregnancy [33]. There is a low spontaneous in vitro secretion of the IL-5 in
250
preeclampsia compared with normal pregnancy, which indicates that there is a decrease in
251
systemic Th2 immunity in preeclampsia in humans [34]. It has been reported that treatment
252
with recombinant IL-10 can prevent the development of preeclampsia during pregnancy in
253
mice [35], and IL-10 plays a pivotal role in regulating the maternal immune tolerance for the
254
survival of an allogeneic fetus [36]. It has been reported that IL-10 is expressed in peripheral
255
blood immune cells, which is responsive to IFNT before the maternal recognition period in
256
cattle [23]. Our results showed that the expression levels of IL-5 and IL-10 were the highest
257
in ovine lymph nodes on day 16 of pregnancy (Fig. 1, Fig. 2), suggesting that the peak of
258
IL-10 may be induced by fetal IFNT on day 16 of pregnancy. Furthermore, IL-5 and IL-10
259
were upregulated in the lymph nodes, which were indicated that upregulation of IL-5 and
260
IL-10 may be necessary for immune regulation of maternal lymph node during early
261
pregnancy.
TE D
M AN U
SC
RI PT
249
Lymph nodes are present widely throughout the body, and are vital for the adaptive
263
immune system [37]. Lymph node is enclosed by a fibrous capsule, and divided into an outer
264
cortex and an inner medulla. Lymph nodes are major sites of B, T lymphocytes, and other
265
white blood cells. As a part of the circulatory system, lymph nodes are linked by the
266
lymphatic vessels. Lymph enters the convex through the subcapsular sinus and trabeculae
267
around lymphoid nodules, and flows into the medulla through the lymph sinus around the
268
medullary cord. All lymphocytes leave the lymph nodes via the efferent lymphatics or the
269
blood system, which are involved in the systemic immunoregulation [6]. Our
270
immunohistochemistry results showed that the immunostaining for IL-2 and IL-10 proteins
AC C
EP
262
13
ACCEPTED MANUSCRIPT were limited to the subcapsular sinus and trabeculae in the cortex, lymph sinus (Fig. 3). The
272
lymph fluid contains lymphocytes, and lymph circulates to the lymph node via subcapsular
273
sinus, trabecular sinuses and lymph sinus. It was obvious that the early conceptus exerted its
274
effects through the lymph circulation, which induced to changes in the expression of IL-2 and
275
IL-10 in the subcapsular sinus, trabeculae and lymph sinus.
RI PT
271
In conclusion, there was the highest expression of Th1 and Th2 cytokines in the lymph
277
nodes on day 13 or 16 of pregnancy. Furthermore, TNF-β and IL-2 were downregulated, and
278
IL-5 and IL-10 were upregulated on day 25 of pregnancy compared with that in nonpregnancy.
279
There was no significant difference in the expression of IFN-γ, IL-4, and IL-6 between day 25
280
of pregnancy and nonpregnancy. The immunohistochemistry results showed that the IL-2 and
281
IL-10 proteins were limited to the subcapsular sinus and trabeculae in the cortex, lymph sinus.
282
Therefore, we suggested that early pregnancy exerted its effect on the lymph nodes to regulate
283
the production of Th1 and Th2 cytokines, and induced a Th2-based response in the maternal
284
lymph nodes, which was essential for a normal pregnancy in sheep.
285
Conflict of interest statement
287
M AN U
TE D
EP
The authors declare no personal conflict of interest.
AC C
286
SC
276
Acknowledgments
288
This work was supported by the Science and Technology R&D Project of Hebei
289
Province, China (16236605D-2), and the Technology Research Project of Higher Education
290
Institutions of Hebei Province, China (ZD2016069).
291
References
292
[1] Ott TL, Gifford CA. Effects of early conceptus signals on circulating immune cells:
14
ACCEPTED MANUSCRIPT 293
lessons from domestic ruminants. Am J Reprod Immunol 2010; 64:245-54. [2] Raghupathy R, Makhseed M, Azizieh F, Omu A, Gupta M, Farhat R. Cytokine
295
production by maternal lymphocytes during normal human pregnancy and in unexplained
296
recurrent spontaneous abortion. Hum Reprod 2000;15:713-8.
RI PT
294
[3] Zhang L, Xia Y, Tang F, Li SJ, Yang L, Wang B. The regulation of intrauterine immune
298
cytokines and chemokines during early pregnancy in the bovine. Large Anim Rev
299
2015;21:23-31.
SC
297
[4] Ng SC, Gilman-Sachs A, Thaker P, Beaman KD, Beer AE, Kwak-Kim J. Expression of
301
intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion,
302
implantation failures after IVF/ET or normal pregnancy. Am J Reprod Immunol
303
2002;48:77-86.
M AN U
300
[5] Yang L, Wang Y, Ma X, Wang S, Zhang L. Changes in expression of Th1 and Th2
305
cytokines in bovine peripheral blood mononuclear cells during early pregnancy. Indian J
306
Anim Res 2016;50:466-70.
309 310 311 312
EP
308
[6] Buettner M, Bode U. Lymph node dissection--understanding the immunological function of lymph nodes. Clin Exp Immunol 2012; 169:205-12.
AC C
307
TE D
304
[7] Hetherington CM, Humber DP. The effect of pregnancy on lymph node weight in the mouse. J Immunogenet 1977;4:271-6. [8] Mosley JG, McLean JM, Gibbs AC. The response of iliac lymph nodes to the fetal allograft. J Anat 1975;119:619-23.
313
[9] Yang L, Zang S, Bai Y, Yao X, Zhang L. Effect of early pregnancy on the expression of
314
progesterone receptor and progesterone-induced blocking factor in ovine lymph node.
15
ACCEPTED MANUSCRIPT 315 316 317
Theriogenology 2017;93:78-83. [10] Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques 2005;39:75-85. [11] Yu H, Jin GZ, Liu K, Dong H, Yu H, Duan JC, Li Z, Dong W, Cong WM, Yang JH.
319
Twist2 is a valuable prognostic biomarker for colorectal cancer. World J Gastroenterol
320
2013;19:2404-11.
323 324
SC
322
[12] Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol 2004;75:163-89.
M AN U
321
RI PT
318
[13] Ruddle NH. Lymphotoxin and TNF: how it all began-a tribute to the travelers. Cytokine Growth Factor Rev 2014;25:83-9.
[14] Fernandes MT, Dejardin E, dos Santos NR. Context-dependent roles for lymphotoxin-β
326
receptor signaling in cancer development. Biochim Biophys Acta 2016;1865:204-19.
327
[15] Liao W, Lin JX, Leonard WJ. IL-2 family cytokines: new insights into the complex roles
328
of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol
329
2011;23:598-604.
EP
TE D
325
[16] Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK.
331
Reciprocal developmental pathways for the generation of pathogenic effector TH17 and
332
regulatory T cells. Nature 2006;441:235-8.
AC C
330
333
[17] Cantón GJ, Katzer F, Maley SW, Bartley PM, Benavides-Silván J, Palarea-Albaladejo J,
334
Pang Y, Smith SH, Rocchi M, Buxton D, Innes EA, Chianini F. Cytokine expression in
335
the placenta of pregnant cattle after inoculation with Neospora caninum. Vet Immunol
336
Immunopathol 2014;161:77-89.
16
ACCEPTED MANUSCRIPT 337
[18] Harmon QE, Engel SM, Wu MC, Moran TM, Luo J. Stuebe AM, Avery CL, Olshan AF.
338
Polymorphisms in inflammatory genes are associated with term small for gestational age
339
and preeclampsia. Am J Reprod Immunol 2014;71:472-84. [19] Bilotas MA, Olivares CN, Ricci AG, Baston JI, Bengochea TS, Meresman GF, Barañao
341
RI. Interplay between Endometriosis and Pregnancy in a Mouse Model. PLoS One
342
2015;10:e0124900.
SC
344
[20] Bauer J, Namineni S, Reisinger F, Zöller J, Yuan D, Heikenwälder M. Lymphotoxin, NF-ĸB, and cancer: the dark side of cytokines. Dig Dis 2012;30:453-68.
M AN U
343
RI PT
340
[21] Maciel GS, Uscategui RR, de Almeida VT, Oliveira ME, Feliciano MA, Vicente WR.
346
Quantity of IL-2, IL-4, IL-10, INF-γ, TNF-α and KC-like cytokines in serum of bitches
347
with pyometra in different stages of oestrous cycle and pregnancy. Reprod Domest Anim
348
2014;49:701-4.
349 350
TE D
345
[22] Liao W, Lin JX, Leonard WJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 2013;38:13-25. [23] Shirasuna K, Matsumoto H, Kobayashi E, Nitta A, Haneda S, Matsui M, Kawashima C,
352
Kida K, Shimizu T, Miyamoto A. Upregulation of interferon-stimulated genes and
353
interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows. J
354
Reprod Dev 2012;58:84-90.
356
AC C
355
EP
351
[24] Yang L, Zhang LY, Qiao HY, Liu N, Wang YX, Li SJ. Maternal immune regulation by conceptus during early pregnancy in the bovine. Asian J Anim Vet Adv 2014;9:610-20.
357
[25] Tuo W, MacMillan H, Günter N, Bazer FW, Brown WC. Upregulation of interleukin-4
358
and IFN-gamma expression by IFN-tau, a member of the type I IFN family. J Interferon
17
ACCEPTED MANUSCRIPT 359 360 361
Cytokine Res 1999;19:179-87. [26] Sokol CL, Barton GM, Farr AG, Medzhitov R. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat Immunol 2008;9:310-8. [27] Maeda Y, Ohtsuka H, Tomioka M, Oikawa M. Effect of progesterone on Th1/Th2/Th17
363
and regulatory T cell-related genes in peripheral blood mononuclear cells during
364
pregnancy in cows. Vet Res Commun 2013;37: 43-9.
RI PT
362
[28] Dibble S, Andersen A, Lassen MR, Cunanan J, Hoppensteadt D, Fareed J. Inflammatory
366
and procoagulant cytokine levels during pregnancy as predictors of adverse obstetrical
367
complications. Clin Appl Thromb Hemost 2014;20:152-8.
M AN U
SC
365
[29] Blitek A, Morawska E, Ziecik AJ. Regulation of expression and role of leukemia
369
inhibitory factor and interleukin-6 in the uterus of early pregnant pigs. Theriogenology
370
2012;78:951-64.
TE D
368
[30] Goyal P, Brünnert D, Ehrhardt J, Bredow M, Piccenini S, Zygmunt M. Cytokine IL-6
372
secretion by trophoblasts regulated via sphingosine-1-phosphate receptor 2 involving
373
Rho/Rho-kinase and Rac1 signaling pathways. Mol Hum Reprod 2013;19:528-38.
EP
371
[31] Pantaleo M, Piccinno M, Roncetti M, Mutinati M, Rizzo A, Sciorsci RL. Evaluation of
375
serum concentrations of interleukin (IL)-4, IL-10, and IL-12 during pregnancy in bitches.
376
Theriogenology 2013;79:970-3.
AC C
374
377
[32] Lin YL, Liang YC, Chiang BL. Placental growth factor down-regulates type 1 T helper
378
immune response by modulating the function of dendritic cells. J Leukoc Biol
379
2007;82:1473-80.
380
[33] Martínez FF, Knubel CP, Sánchez MC, Cervi L, Motrán CC. Pregnancy-specific
18
ACCEPTED MANUSCRIPT 381
glycoprotein 1a activates dendritic cells to provide signals for Th17-, Th2-, and Treg-cell
382
polarization. Eur J Immunol 2012;42:1573-84. [34] Jonsson Y, Matthiesen L, Berg G, Ernerudh J, Nieminen K, Ekerfelt C. Indications of an
384
altered immune balance in preeclampsia: a decrease in in vitro secretion of IL-5 and
385
IL-10 from blood mononuclear cells and in blood basophil counts compared with normal
386
pregnancy. J Reprod Immunol 2005;66:69-84.
RI PT
383
[35] Chatterjee P, Chiasson VL, Seerangan G, Tobin RP, Kopriva SE, Newell-Rogers MK,
388
Mitchell BM. Cotreatment with interleukin 4 and interleukin 10 modulates immune cells
389
and prevents hypertension in pregnant mice. Am J Hypertens 2015;28:135-42.
393 394
M AN U
[37] Willard-Mack CL. Normal structure, function, and histology of lymph nodes. Toxicol
TE D
392
Immunol 2015;73:487-500.
Pathol 2006;34:409-24.
EP
391
[36] Cheng SB, Sharma S. Interleukin-10: a pleiotropic regulator in pregnancy. Am J Reprod
AC C
390
SC
387
19
ACCEPTED MANUSCRIPT
Figure Legends
396
Fig. 1. Relative expression values of Th1 cells cytokines (IL-2, IFN-γ, and TNF-β) and Th2
397
cytokines (IL-4, IL-5, IL-6, and IL-10) mRNA in the lymph nodes measured by qRT-PCR.
398
Note: DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of
399
pregnancy; DP25 = Day 25 of pregnancy. Significant differences (P < 0.05) are indicated by
400
different letters within the same column.
RI PT
395
SC
401
Fig. 2. Expression of Th1 cells cytokines (IL-2, IFN-γ, and TNF-β) and Th2 cytokines (IL-4,
403
IL-5, IL-6, and IL-10) proteins in the lymph nodes analyzed by western blot analysis. Note:
404
DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of
405
pregnancy; DP25 = Day 25 of pregnancy. Significant differences (P < 0.05) are indicated by
406
different superscript letters within the same color column.
407
Fig. 3. Immunohistochemical localization of IL-2 and IL-10 proteins in lymph nodes. Lymph
408
node is divided into the cortex and the medulla. Lymph enters the convex through the
409
subcapsular sinus (SS) and trabeculae (TR) around the lymphoid nodules (LN), and flows into
410
the medulla through the lymph sinus (LS) around the medullary cord (MC). Note: DN16 =
411
Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of pregnancy;
412
DP25 = Day 25 of pregnancy. Bar = 20 µm.
AC C
EP
TE D
M AN U
402
20
ACCEPTED MANUSCRIPT Table 1 Primers used for qRT-PCR Gene
Primer
Sequence
Size (bp)
Forward AAACCTGAACACCAGAGAGAT IL-2
117 GCCTTTACTGTCGCATCA
Forward TTGAACGGCAGCTCTGAGAA IFN-γ
RI PT
Reverse
124
Reverse
TTGGCGACAGGTCATTCATC
SC
Forward CCACTGACGGGCTTTACCT TNF-β
141
TGATGGCAGAGAGGATGTTG
M AN U
Reverse
Forward CCAAAGAACGCAACTGAGAA IL-4 Reverse
120
GCTGCTGAGATTCCTGTCAA
Forward CATCTGCGTTTGACCTTGG IL-5
139
AGTTCCCATCACCTATCAGCA
TE D
Reverse
Forward CGAGTTTGAGGGAAATCAGG IL-6
Reverse
118
GTCAGTGTGTGTGGCTGGAG
EP
Forward CTCTGTTGCCTGGTCTTCCT
IL-10
AC C
Reverse
169
TGTTCAGTTGGTCCTTCATTTG
Forward GGGTCATCATCTCTGCACCT
GAPDH
Reverse
176 GGTCATAAGTCCCTCCACGA
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights Downregulation of tumor necrosis factor beta and IL-2 in the lymph node. Upregulation of IL-5 and IL-10 in the lymph node. IL-2 and IL-10 were localized in the subcapsular sinus and trabeculae in the cortex,
AC C
EP
TE D
M AN U
SC
RI PT
medullary sinuses.
S0093-691X(18)30268-1
DOI:
10.1016/j.theriogenology.2018.10.004
Reference:
THE 14729
To appear in:
Theriogenology
Received Date: 28 May 2018 Revised Date:
1 October 2018
Accepted Date: 2 October 2018
Please cite this article as: Yang L, Wang P, Mi H, Lv W, Liu B, Du J, Zhang L, Comparison of Th1 and Th2 cytokines production in ovine lymph nodes during early pregnancy, Theriogenology (2018), doi: https://doi.org/10.1016/j.theriogenology.2018.10.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Comparison of Th1 and Th2 cytokines production in ovine lymph nodes
2
during early pregnancy
3
Running head: Th1 and Th2 cytokines production
RI PT
4 5 6
Ling Yanga*, Pengda Wanga, Hao Mia, Wan Lva, Baoliang Liua, Jingsong Dub, Leying Zhanga
8
a
9
Engineering, Handan 056021, China
SC
7
M AN U
Department of Animal Science, College of Life Sciences and Food Engineering, Hebei University of
10
b
11
*Corresponding author: Tel/fax: +86 310 8576856. E-mail address: [email protected] (Ling
12
Yang).
TE D EP
14
AC C
13
Handan Agricultural Service Association, Handan 056002, China
1
ACCEPTED MANUSCRIPT 15
ABSTRACT As a fetal allograft to the mother, early conceptus regulates the intrauterine immune and
17
systemic immune responses during early pregnancy in sheep. However, expression of T
18
helper 1 (Th1) and Th2 cytokines in maternal lymph nodes is unclear during early pregnancy
19
in sheep. In this study, inguinal lymph nodes were obtained on day 16 of the estrous cycle and
20
on days 13, 16 and 25 of pregnancy (n = 4 for each group) in ewes, and qRT-PCR, western
21
blot and immunohistochemistry were used to compare the expression of Th1 and Th2
22
cytokines in the lymph nodes. Our results showed that there were the highest levels of Th1
23
cytokines (IFN-γ, TNF-β and IL-2) and Th2 cytokines (IL-4, IL-5, IL-6 and IL-10) in the
24
lymph nodes on day 13 or 16 of pregnancy. Furthermore, there were a downregulation of
25
TNF-β and IL-2 and an upregulation of IL-5 and IL-10 on day 25 of pregnancy compared
26
with that in nonpregnancy, with no significant difference in the expression of IFN-γ, IL-4, and
27
IL-6
28
immunohistochemistry results showed that the IL-2 and IL-10 proteins were limited to the
29
subcapsular sinus and trabeculae in the cortex, lymph sinus. In conclusion, early pregnancy
30
exerted its effects on the lymph node and induced a Th2-biased response, which was essential
31
for a normal pregnancy in sheep.
32
Keywords: Lymph node; Pregnancy; Sheep; T helper cytokine
SC
M AN U
TE D
the
ewes
on
day
25
of
pregnancy
EP
between
AC C
33
RI PT
16
2
and
nonpregnancy.
The
ACCEPTED MANUSCRIPT 34
1. Introduction T helper (Th) cells are subdivided into Th1 and Th2 cells according to the cytokines that
36
they secrete. Th1 cells induce cell-mediated cytotoxicity and inflammatory responses via
37
interleukin (IL-2), interferon-gamma (IFN-γ) and tumor necrosis factor beta (TNF-β),
38
whereas the Th2 cytokines are anti-inflammatory cytokines, such as IL-4, IL-5, IL-6 and
39
IL-10 [1]. Th2 cytokines enhance humoral response, and there is a distinct Th2-bias in a
40
normal pregnancy. However, Th1 cytokines are generally detrimental to normal pregnancy,
41
and significantly higher concentrations of Th1 cytokines are produced in the abortion group
42
[2,3]. A significantly increase of the Th1 cytokine is the underlying immune etiology for the
43
reproductive failures [4]. We previously observed that IFN-γ was attenuated, and IL-4, IL-5,
44
IL-6, IL-10 and IL-13 were augmented in the peripheral blood mononuclear cells (PBMCs)
45
during early pregnancy in cattle [5].
TE D
M AN U
SC
RI PT
35
Lymph nodes are located all over the body in mammals, and their function is to filter and
47
scan the lymph for antigens. The immune reaction to pathogenic antigens, harmless antigens
48
and tolerance is initiated in the lymph nodes [6]. The weights of the lumbar and renal lymph
49
nodes, inguinal lymph nodes increase during pregnancy in mice, and the hormonal effects and
50
the presence of fetal antigens contribute to the changes in the lymph node weight [7]. It has
51
been reported that the count of large pyroninophilic and plasma cells rises significantly in the
52
iliac nodes during early pregnancy in rat [8]. Our previous study revealed that the expression
53
of the progesterone receptor and the progesterone-induced blocking factor is upregulated in
54
the lymph nodes during early pregnancy in ewes [9]. We hypothesized that the expression of
55
Th1 and Th2 cytokines within maternal lymph nodes would be impacted in response to
AC C
EP
46
3
ACCEPTED MANUSCRIPT pregnancy, as a component of pregnancy-induced immunomodulation. Therefore, the
57
objective of this study was to determine the expression of Th1 cytokines (IL-2, IFN-γ and
58
TNF-β) and Th2 cytokines (IL-4, IL-5, IL-6 and IL-10) in the lymph nodes from nonpregnant
59
and early pregnant ewes.
60
2. Materials and methods
61
2.1 Animals and experimental design
SC
RI PT
56
Small-tail Han ewes with approximately 18 months of age were housed at the farm of
63
Handan Boyuan Animal Husbandry Co., Ltd. in China, and all procedures were approved by
64
the Hebei University of Engineering Animal Care and Use Committee. All experiments were
65
conducted following the guidelines of the National Standards for Laboratory Animals of
66
China (GB 14925-2010). The ewes with normal oestrous cycles were observed daily for
67
estrus using vasectomized rams, mated twice with intact rams in a 12-h interval after the
68
detection of sexual receptivity. Thirty-two ewes were randomly assigned into four groups
69
prior to breeding, resulting in 4 pregnant ewes in each group harvested during pregnancy and
70
4 non-pregnant ewes as non-mated controls. The first day of coitus was counted as day 0 of
71
pregnancy or non-pregnancy. The ewes assigned to the nonpregnant group were not mated
72
with an intact ram. The inguinal lymph nodes were obtained from the ewes on days 13, 16
73
and 25 of pregnancy, as well as day 16 of the estrous cycle at the time of slaughter. Pregnancy
74
was confirmed through observing the presence of a conceptus in the uterus. The transverse
75
pieces of the lymph nodes (0.3 cm3) were fixed in fresh 4% (w/v) paraformaldehyde in
76
phosphate buffer saline (PBS) buffer (pH 7.4), and the remaining portions of the lymph nodes
AC C
EP
TE D
M AN U
62
4
ACCEPTED MANUSCRIPT were frozen in liquid nitrogen for subsequent quantitative real time PCR (qRT-PCR) and
78
western blot analysis.
79
2.2. RNA extraction and qRT-PCR assay
RI PT
77
The samples were crushed into fine powders in liquid nitrogen, and the powders were
81
dissolved in TRIzol (Invitrogen, California, USA), and total RNA was extracted according to
82
the manufacturer’s instructions. A FastQuant RT kit (Tiangen Biotech Co., Ltd., Beijing) was
83
used to synthesize the cDNA, and a SuperReal PreMix Plus kit (Tiangen Biotech) was
84
employed for qRT-PCR. The primer sequences for Th1 cytokines (IFN-γ, TNF-β, and IL-2),
85
Th2 cytokines (IL-4, IL-5, IL-6, and IL-10), and GAPDH were designed and synthesized by
86
Shanghai
87
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) at NCBI. PCR amplification efficiency of each pair
88
of primers was assessed before quantification, and was found to be in an acceptable range
89
(between 0.9 and 1.1). The primer product was sequenced to check for specificity, and the
90
expression of the targeted genes was determined by a Bio-rad CFX96 real-time PCR system
91
with 20 µl. The PCR amplifications were carried out at 95 °C for 10 sec, 55-58 °C (55 °C for
92
IL-5 and IL-6, 58 °C for IFN-γ, TNF-β, IL-4, IL-2 and IL-10) for 20 sec, and 72 °C for 25 sec,
93
and the number of PCR cycles was 40. The GAPDH PCR reaction was the same as IFN-γ,
94
TNF-β, IL-2, IL-4, IL-5, IL-6, and IL-10, respectively. The relative expression values for the
95
qRT-PCR assay were calculated by the 2-∆∆Ct analysis method, with GAPDH as the
96
endogenous control [10]. The relative expression value was set to 1 for the group on day 16 of
97
the estrous cycle.
Biotech
Co.,
Ltd.
(Table
1),
and
assessed
by
BLAST
AC C
EP
TE D
Sangon
M AN U
SC
80
5
ACCEPTED MANUSCRIPT 98
2.3. Western blot
Total proteins in the lymph samples were extracted by RIPA Lysis Buffer (Biosharp,
100
BL504A). The protein concentration was measured using a BCA Protein Assay kit with
101
bovine serum albumin as the standard (Tiangen Biotech). Equal amounts of total protein (10
102
µg/lane) were separated using 12% SDS-PAGE, and the proteins were transferred to 0.22 µm
103
polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). IFN-γ, TNF-β, IL-2,
104
IL-4, IL-5, IL-6 and IL-10 were detected by western blot analysis using a mouse anti-IFN-γ
105
monoclonal antibody (Abcam, ab27919, 1:1000), a mouse anti-TNF-β monoclonal antibody
106
(Santa Cruz Biotechnology, Inc., SC-28345, 1:1000), a rabbit anti-IL-2 polyclonal antibody
107
(Abcam, ab193807, 1:1000), a mouse anti-IL-4 monoclonal antibody (Bio-Techne, MAB2468,
108
1:1000), a mouse anti-IL-5 monoclonal antibody (Santa Cruz Biotechnology, SC-8433,
109
1:1000), a rabbit anti-IL-6 polyclonal antibody (Abcam, ab193853, 1:1000) and a mouse
110
anti-IL-10 monoclonal antibody (Santa Cruz Biotechnology, SC-32815, 1:1000), respectively.
111
Secondary goat anti-mouse IgG-HRP (Biosharp, BL001A) and goat anti-rabbit IgG-HRP
112
(Biosharp, BL003A) were diluted to 1:2000. Pro-light HRP chemiluminescence detection
113
reagent (Tiangen Biotech) was used to detect the immunoreactive bands. Sample loading was
114
monitored with an anti-GAPDH antibody (Santa Cruz Biotechnology, sc-20357) at a dilution
115
of 1:1000, and secondary goat anti-mouse IgG-HRP was diluted to 1:2000. The intensity of
116
blots were quantified using Quantity One V452 (Bio-Rad Laboratories), and the relative
117
levels were calculated using the internal control protein (GAPDH). The expression of GAPDH
118
protein was measured by western blot, and there was no difference among the four groups.
AC C
EP
TE D
M AN U
SC
RI PT
99
6
ACCEPTED MANUSCRIPT 119
2.4. Immunohistochemistry analysis
The fixed lymph samples were embedded in paraffin, and the paraffin-embedded sections
121
were deparaffinized in xylene, and rehydrated in ethanol. The rehydrated sections were treated
122
with 3% H2O2 to quench the endogenous peroxidase activity, and reduced non-specific
123
binding with 5% normal goat serum in PBS. Immunohistochemical localization of IL-2 and
124
IL-10 in the lymph tissue was performed using the rabbit anti-IL-2 polyclonal antibody
125
(Abcam, ab193807, 1:100) and the mouse anti-IL-10 monoclonal antibody (Santa Cruz
126
Biotechnology, SC-32815, 1:100), respectively. Negative controls were treated with
127
antiserum-specific isotype instead of the IL-2 or IL-10 antibody at the same protein
128
concentration. A DAB kit (Tiangen Biotech) was used to visualize the antibody binding sites
129
in the tissue sections. Finally, the images were captured using a light microscope (Nikon
130
Eclipse E800, Japan) and a digital camera (AxioCam ERc 5s), and the intensity of staining
131
and density of stained cells was analyzed through the images. The immunostaining intensity
132
of the different lymph samples was scored by two different investigators in a blinded fashion,
133
according to the following scale: 0, no staining; 1, weak staining; 2, moderate staining; 3,
134
strong staining [11].
135
2.5. Statistical analyses
AC C
EP
TE D
M AN U
SC
RI PT
120
136
The data for the relative expression levels of IFN-γ, TNF-β, IL-2, IL-4, IL-5, IL-6 and
137
IL-10 mRNA and proteins were analyzed using a completely randomized design with four
138
animals per group via the Proc Mixed model of SAS (Version 9.1; SAS Institute, Cary, NC).
139
For the lymph nodes from different stages of gestation or pregnancy status, the model 7
ACCEPTED MANUSCRIPT contained the random effect of the ewe and the fixed effects of the stage of gestation,
141
pregnancy status and the interaction between the stage of gestation and pregnancy status. The
142
comparisons among the relative expression levels of the different groups were performed
143
using the Duncan method and controlling the experimentwise type ± error equal to 0.05. The
144
data are presented as least squares means. Groups were considered significantly different at P
145
< 0.05.
146
3. Results
147
3.1. Relative expression levels of Th1 cytokines mRNA and proteins in the lymph nodes
M AN U
SC
RI PT
140
The qRT-PCR assay and western blot revealed (Fig. 1, Fig. 2) that the relative
149
expression levels of Th1 cytokines (IFN-γ, TNF-β and IL-2) mRNA and proteins were the
150
highest in the lymph nodes on day 16 of pregnancy among nonpregnant and pregnant ewes (P
151
< 0.05), but there was no significant difference in the expression levels of IFN-γ mRNA and
152
protein in the lymph nodes among nonpregnant, days 13 and 25 pregnant ewes (P > 0.05).
153
Furthermore, the relative expression levels of TNF-β, IL-2 mRNA and proteins were the
154
lowest in the lymph nodes on day 25 of pregnancy among nonpregnant and pregnant ewes (P
155
< 0.05), but there was no significant difference in the expression levels of TNF-β, IL-2
156
mRNA and proteins between nonpregnant and day 13 pregnant ewes (P > 0.05; Fig. 1, Fig.
157
2).
158
3.2. Relative expression levels of Th2 cytokines mRNA and proteins in the lymph nodes
AC C
EP
TE D
148
159
The relative expression levels of IL-4 mRNA and protein were the highest on day 13 of
160
pregnancy among nonpregnant and pregnant ewes (P < 0.05), and there was a decline from
8
ACCEPTED MANUSCRIPT day 13 to 25 of pregnancy, but there was no significant difference between nonpregnant ewes
162
and day 25 of pregnant ewes (P > 0.05; Fig. 1, Fig. 2). The peaks of relative expression levels
163
of IL-5, IL-6, IL-10 mRNA and proteins in the lymph nodes were on day 16 of pregnancy (P
164
< 0.05), and the relative expression levels of IL-5 and IL-10 mRNA and proteins were lower
165
in the lymph nodes on day 16 of the estrous cycle than that on day 25 of pregnancy (P < 0.05;
166
Fig. 1, Fig. 2).
167
3.3. The immunohistochemistry for IL-2 and IL-10 proteins in the lymph nodes
SC
RI PT
161
The immunohistochemistry for IL-2 and IL-10 proteins was limited to the subcapsular
169
sinus and trabeculae in the cortex, lymph sinus, but there was almost no immunostained in the
170
lymphoid nodules and medullary cords (Fig. 3). The staining intensity for IL-2 in the
171
subcapsular sinus, trabeculae and lymph sinus was 0, 2+, 2+, 2+, and 0 for the negative
172
control, the lymph nodes from day 16 of the estrous cycle, and the lymph nodes from days 14,
173
16, and 25 of pregnancy, respectively, and the staining intensity for IL-10 was 0, 0, 2+, 2+,
174
and 2+ for the negative control, the lymph nodes from day 16 of the estrous cycle, and the
175
lymph nodes from days 14, 16, and 25 of pregnancy, respectively (Fig. 3).
176
4. Discussion
AC C
EP
TE D
M AN U
168
177
Th1 cytokines, including IL-2, IFN-γ and TNF-β, are implicated in cell-mediated
178
cytotoxicity and inflammatory responses. IFN-γ is the only member of the type II class of
179
interferon, has antiviral, immunoregulatory, and anti-tumor properties [12]. TNF-β is also
180
known as lymphotoxin-alpha (LT-α), has a significant impact on the maintenance of immune
181
system, including the development of secondary lymphoid organs [13]. TNF-β plays an
182
essential role in the innate immune regulation, and can prevent the tumor growth and destroy
9
ACCEPTED MANUSCRIPT the cancerous cell lines [14]. IL-2 is a type of cytokine signaling molecule which has key
184
roles in the functions of immune system, is primarily via its direct effects on T cells
185
differentiation [15]. IL-2 promotes the differentiation of certain immature T cells into
186
regulatory T cells, which modulates the immune system, maintains tolerance to self-antigens,
187
and prevents autoimmune disease [16]. In general, Th1 cytokines are related with the
188
reproductive failures [4].
RI PT
183
A raise in foetal mortality is along with an increased level of IFN-γ mRNA in the placenta
190
of pregnant cows after inoculation with Neospora caninum (a coccidian parasite) [17], and
191
our previous study revealed that relative level of IFN-γ mRNA is dropped sharply in the
192
PBMCs on days 18 and 30 pregnancy in cattle [5]. TNF-β is associated with preeclampsia and
193
poor fetal growth in the European Americans [18]. There is a high level of IL-2 in the
194
endometriosis group in nonpregnant mice, which indicates that high level of IL-2 lead to
195
infertility in mice [19]. Our results revealed that there was a decrease in the expression of
196
IFN-γ, TNF-β and IL-2 in lymph nodes on day 25 of pregnancy (Fig. 1, Fig. 2), which
197
suggested that downregulation of IFN-γ, TNF-β and IL-2 in the lymph nodes on day 25 of
198
pregnancy may be beneficial for ovine fetal implantation and pregnancy maintenance.
AC C
EP
TE D
M AN U
SC
189
199
TNF-β is involved in the development of secondary lymphoid organs [13] and
200
lymphocyte activation [20]. There is a high level of IL-2 in blood serum at the second week of
201
gestation compared with that in anestrus and diestrus in bitches, and the upregulation of
202
expression of IL-2 has been considered necessary for the development and maintenance of
203
pregnancy [21]. It has been reported that IL-2 plays key roles in the immune response through
204
promoting naive CD4+ T cell differentiation into Th1 and Th2 cells [22]. As the primary
10
ACCEPTED MANUSCRIPT pregnancy recognition signal, interferon-tau (IFNT) is implicated in maternal systemic
206
immune regulation through blood circulation and lymph circulation via the PBMCs in the
207
bovine [23,24]. It has been reported that there is an upregulation in the expression of IFN-γ in
208
CD4+ T cell lines stimulated by IFNT in vitro in cattle [25]. Our results indicated that there
209
was an upregulation of IFN-γ, TNF-β and IL-2 in the lymph nodes on day 16 of pregnancy
210
(Fig. 1, Fig. 2). Therefore, it was suggested that upregulation of IFN-γ, TNF-β and IL-2 may
211
be related with the high level of IFNT on day 16 of pregnancy in sheep, and involved the
212
development and immune regulation of maternal lymph nodes during early pregnancy in
213
sheep.
M AN U
SC
RI PT
205
IL-4, IL-5, IL-6 and IL-10 are Th2 cytokines that enhance humoral response. IL-4 is a key
215
regulator in humoral and adaptive immunity, induces the differentiation of naive helper T
216
cells (Th0 cells) into Th2 cells, and Th2 cells subsequently produce additional IL-4 in a
217
positive feedback loop, and IL-4 also decreases the production of Th1 cells and IFN-γ [26].
218
IL-5 is an interleukin produced by Th2 cell and mast cell, can stimulate B cell growth and
219
increase
220
anti-inflammatory myokine, and plays a major role in suppressing the immune system. IL-10
221
exerts its effects on the immunoregulation through down-regulated expression of Th1
222
cytokines, major histocompatibility complex class II antigens, and blocks the NF-κB activity.
223
In a normal pregnancy, there is a distinct Th2-bias.
TE D
214
secretion.
IL-6
acts
as
both
pro-inflammatory
and
AC C
EP
immunoglobulin
224
It has been reported that the IFNT from the fetus increases the expression of IL-4 mRNA
225
by the effector T cells in cattle [25], and progesterone (P4) can significantly upregulate the
226
expression of IL-4 in the PBMCs in pregnant cows [27]. IL-6 plays a contributing role in
11
ACCEPTED MANUSCRIPT stimulation of the trophoblast invasion, regulation of the endometrial function and
228
implantation [28]. The endometrial IL-6 mRNA increases during early pregnancy in pigs,
229
which stimulates the attachment and proliferation of the trophoblast cells in vitro, and is
230
beneficial for the successful implantation [29]. IL-6 is produced by extravillous and
231
cytotrophoblast, and involved in regulating the migration, invasion, trophoblast differentiation
232
and proliferation of the placental cells, which is important for the normal placenta
233
development and successful pregnancy [30]. We found that the expression levels was higher
234
for IL-4 on days 13 and 16 of pregnancy, and for IL-6 on day 16 of pregnancy (Fig. 1, Fig. 2),
235
which indicated that upregulation of IL-4 and IL-6 during early pregnancy is implicated in
236
immune regulation of maternal lymph nodes, and the higher levels of IL-4 and IL-6 on day 16
237
of pregnancy may be due to the high serum concentration of IFNT and P4. However, our
238
results also showed that IL-4 and IL-6 were downregulated in the lymph nodes from day 16 to
239
day 25 of pregnancy (Fig. 1, Fig. 2). It has been reported that the serum concentration of IL-4
240
is elevated in early gestation, and decreases between 30 and 40 days of gestation owing to the
241
high concentration of prolactin in bitches [31], which is almost consistent with our results in
242
the ovine lymph nodes. It has been reported that there is an increased IL-6 level in the
243
maternal plasma in preterm delivery patients during pregnancy [28], which indicated that high
244
level of IL-6 in the lymph nodes may be harmful after early pregnancy in ewes.
AC C
EP
TE D
M AN U
SC
RI PT
227
245
It has been reported that the dendritic cells treated with placental growth factor lead to
246
the suppression of naive CD4+ T cell proliferation, but increase the IL-5 secretion by the
247
CD4+ T cell, which is helpful for a normal pregnancy in humans [32]. Pregnancy-specific
248
glycoprotein enhances the proliferation of IL-5-secreting cells in vivo, which is involved in
12
ACCEPTED MANUSCRIPT the successful pregnancy [33]. There is a low spontaneous in vitro secretion of the IL-5 in
250
preeclampsia compared with normal pregnancy, which indicates that there is a decrease in
251
systemic Th2 immunity in preeclampsia in humans [34]. It has been reported that treatment
252
with recombinant IL-10 can prevent the development of preeclampsia during pregnancy in
253
mice [35], and IL-10 plays a pivotal role in regulating the maternal immune tolerance for the
254
survival of an allogeneic fetus [36]. It has been reported that IL-10 is expressed in peripheral
255
blood immune cells, which is responsive to IFNT before the maternal recognition period in
256
cattle [23]. Our results showed that the expression levels of IL-5 and IL-10 were the highest
257
in ovine lymph nodes on day 16 of pregnancy (Fig. 1, Fig. 2), suggesting that the peak of
258
IL-10 may be induced by fetal IFNT on day 16 of pregnancy. Furthermore, IL-5 and IL-10
259
were upregulated in the lymph nodes, which were indicated that upregulation of IL-5 and
260
IL-10 may be necessary for immune regulation of maternal lymph node during early
261
pregnancy.
TE D
M AN U
SC
RI PT
249
Lymph nodes are present widely throughout the body, and are vital for the adaptive
263
immune system [37]. Lymph node is enclosed by a fibrous capsule, and divided into an outer
264
cortex and an inner medulla. Lymph nodes are major sites of B, T lymphocytes, and other
265
white blood cells. As a part of the circulatory system, lymph nodes are linked by the
266
lymphatic vessels. Lymph enters the convex through the subcapsular sinus and trabeculae
267
around lymphoid nodules, and flows into the medulla through the lymph sinus around the
268
medullary cord. All lymphocytes leave the lymph nodes via the efferent lymphatics or the
269
blood system, which are involved in the systemic immunoregulation [6]. Our
270
immunohistochemistry results showed that the immunostaining for IL-2 and IL-10 proteins
AC C
EP
262
13
ACCEPTED MANUSCRIPT were limited to the subcapsular sinus and trabeculae in the cortex, lymph sinus (Fig. 3). The
272
lymph fluid contains lymphocytes, and lymph circulates to the lymph node via subcapsular
273
sinus, trabecular sinuses and lymph sinus. It was obvious that the early conceptus exerted its
274
effects through the lymph circulation, which induced to changes in the expression of IL-2 and
275
IL-10 in the subcapsular sinus, trabeculae and lymph sinus.
RI PT
271
In conclusion, there was the highest expression of Th1 and Th2 cytokines in the lymph
277
nodes on day 13 or 16 of pregnancy. Furthermore, TNF-β and IL-2 were downregulated, and
278
IL-5 and IL-10 were upregulated on day 25 of pregnancy compared with that in nonpregnancy.
279
There was no significant difference in the expression of IFN-γ, IL-4, and IL-6 between day 25
280
of pregnancy and nonpregnancy. The immunohistochemistry results showed that the IL-2 and
281
IL-10 proteins were limited to the subcapsular sinus and trabeculae in the cortex, lymph sinus.
282
Therefore, we suggested that early pregnancy exerted its effect on the lymph nodes to regulate
283
the production of Th1 and Th2 cytokines, and induced a Th2-based response in the maternal
284
lymph nodes, which was essential for a normal pregnancy in sheep.
285
Conflict of interest statement
287
M AN U
TE D
EP
The authors declare no personal conflict of interest.
AC C
286
SC
276
Acknowledgments
288
This work was supported by the Science and Technology R&D Project of Hebei
289
Province, China (16236605D-2), and the Technology Research Project of Higher Education
290
Institutions of Hebei Province, China (ZD2016069).
291
References
292
[1] Ott TL, Gifford CA. Effects of early conceptus signals on circulating immune cells:
14
ACCEPTED MANUSCRIPT 293
lessons from domestic ruminants. Am J Reprod Immunol 2010; 64:245-54. [2] Raghupathy R, Makhseed M, Azizieh F, Omu A, Gupta M, Farhat R. Cytokine
295
production by maternal lymphocytes during normal human pregnancy and in unexplained
296
recurrent spontaneous abortion. Hum Reprod 2000;15:713-8.
RI PT
294
[3] Zhang L, Xia Y, Tang F, Li SJ, Yang L, Wang B. The regulation of intrauterine immune
298
cytokines and chemokines during early pregnancy in the bovine. Large Anim Rev
299
2015;21:23-31.
SC
297
[4] Ng SC, Gilman-Sachs A, Thaker P, Beaman KD, Beer AE, Kwak-Kim J. Expression of
301
intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion,
302
implantation failures after IVF/ET or normal pregnancy. Am J Reprod Immunol
303
2002;48:77-86.
M AN U
300
[5] Yang L, Wang Y, Ma X, Wang S, Zhang L. Changes in expression of Th1 and Th2
305
cytokines in bovine peripheral blood mononuclear cells during early pregnancy. Indian J
306
Anim Res 2016;50:466-70.
309 310 311 312
EP
308
[6] Buettner M, Bode U. Lymph node dissection--understanding the immunological function of lymph nodes. Clin Exp Immunol 2012; 169:205-12.
AC C
307
TE D
304
[7] Hetherington CM, Humber DP. The effect of pregnancy on lymph node weight in the mouse. J Immunogenet 1977;4:271-6. [8] Mosley JG, McLean JM, Gibbs AC. The response of iliac lymph nodes to the fetal allograft. J Anat 1975;119:619-23.
313
[9] Yang L, Zang S, Bai Y, Yao X, Zhang L. Effect of early pregnancy on the expression of
314
progesterone receptor and progesterone-induced blocking factor in ovine lymph node.
15
ACCEPTED MANUSCRIPT 315 316 317
Theriogenology 2017;93:78-83. [10] Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques 2005;39:75-85. [11] Yu H, Jin GZ, Liu K, Dong H, Yu H, Duan JC, Li Z, Dong W, Cong WM, Yang JH.
319
Twist2 is a valuable prognostic biomarker for colorectal cancer. World J Gastroenterol
320
2013;19:2404-11.
323 324
SC
322
[12] Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and functions. J Leukoc Biol 2004;75:163-89.
M AN U
321
RI PT
318
[13] Ruddle NH. Lymphotoxin and TNF: how it all began-a tribute to the travelers. Cytokine Growth Factor Rev 2014;25:83-9.
[14] Fernandes MT, Dejardin E, dos Santos NR. Context-dependent roles for lymphotoxin-β
326
receptor signaling in cancer development. Biochim Biophys Acta 2016;1865:204-19.
327
[15] Liao W, Lin JX, Leonard WJ. IL-2 family cytokines: new insights into the complex roles
328
of IL-2 as a broad regulator of T helper cell differentiation. Curr Opin Immunol
329
2011;23:598-604.
EP
TE D
325
[16] Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK.
331
Reciprocal developmental pathways for the generation of pathogenic effector TH17 and
332
regulatory T cells. Nature 2006;441:235-8.
AC C
330
333
[17] Cantón GJ, Katzer F, Maley SW, Bartley PM, Benavides-Silván J, Palarea-Albaladejo J,
334
Pang Y, Smith SH, Rocchi M, Buxton D, Innes EA, Chianini F. Cytokine expression in
335
the placenta of pregnant cattle after inoculation with Neospora caninum. Vet Immunol
336
Immunopathol 2014;161:77-89.
16
ACCEPTED MANUSCRIPT 337
[18] Harmon QE, Engel SM, Wu MC, Moran TM, Luo J. Stuebe AM, Avery CL, Olshan AF.
338
Polymorphisms in inflammatory genes are associated with term small for gestational age
339
and preeclampsia. Am J Reprod Immunol 2014;71:472-84. [19] Bilotas MA, Olivares CN, Ricci AG, Baston JI, Bengochea TS, Meresman GF, Barañao
341
RI. Interplay between Endometriosis and Pregnancy in a Mouse Model. PLoS One
342
2015;10:e0124900.
SC
344
[20] Bauer J, Namineni S, Reisinger F, Zöller J, Yuan D, Heikenwälder M. Lymphotoxin, NF-ĸB, and cancer: the dark side of cytokines. Dig Dis 2012;30:453-68.
M AN U
343
RI PT
340
[21] Maciel GS, Uscategui RR, de Almeida VT, Oliveira ME, Feliciano MA, Vicente WR.
346
Quantity of IL-2, IL-4, IL-10, INF-γ, TNF-α and KC-like cytokines in serum of bitches
347
with pyometra in different stages of oestrous cycle and pregnancy. Reprod Domest Anim
348
2014;49:701-4.
349 350
TE D
345
[22] Liao W, Lin JX, Leonard WJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 2013;38:13-25. [23] Shirasuna K, Matsumoto H, Kobayashi E, Nitta A, Haneda S, Matsui M, Kawashima C,
352
Kida K, Shimizu T, Miyamoto A. Upregulation of interferon-stimulated genes and
353
interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows. J
354
Reprod Dev 2012;58:84-90.
356
AC C
355
EP
351
[24] Yang L, Zhang LY, Qiao HY, Liu N, Wang YX, Li SJ. Maternal immune regulation by conceptus during early pregnancy in the bovine. Asian J Anim Vet Adv 2014;9:610-20.
357
[25] Tuo W, MacMillan H, Günter N, Bazer FW, Brown WC. Upregulation of interleukin-4
358
and IFN-gamma expression by IFN-tau, a member of the type I IFN family. J Interferon
17
ACCEPTED MANUSCRIPT 359 360 361
Cytokine Res 1999;19:179-87. [26] Sokol CL, Barton GM, Farr AG, Medzhitov R. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat Immunol 2008;9:310-8. [27] Maeda Y, Ohtsuka H, Tomioka M, Oikawa M. Effect of progesterone on Th1/Th2/Th17
363
and regulatory T cell-related genes in peripheral blood mononuclear cells during
364
pregnancy in cows. Vet Res Commun 2013;37: 43-9.
RI PT
362
[28] Dibble S, Andersen A, Lassen MR, Cunanan J, Hoppensteadt D, Fareed J. Inflammatory
366
and procoagulant cytokine levels during pregnancy as predictors of adverse obstetrical
367
complications. Clin Appl Thromb Hemost 2014;20:152-8.
M AN U
SC
365
[29] Blitek A, Morawska E, Ziecik AJ. Regulation of expression and role of leukemia
369
inhibitory factor and interleukin-6 in the uterus of early pregnant pigs. Theriogenology
370
2012;78:951-64.
TE D
368
[30] Goyal P, Brünnert D, Ehrhardt J, Bredow M, Piccenini S, Zygmunt M. Cytokine IL-6
372
secretion by trophoblasts regulated via sphingosine-1-phosphate receptor 2 involving
373
Rho/Rho-kinase and Rac1 signaling pathways. Mol Hum Reprod 2013;19:528-38.
EP
371
[31] Pantaleo M, Piccinno M, Roncetti M, Mutinati M, Rizzo A, Sciorsci RL. Evaluation of
375
serum concentrations of interleukin (IL)-4, IL-10, and IL-12 during pregnancy in bitches.
376
Theriogenology 2013;79:970-3.
AC C
374
377
[32] Lin YL, Liang YC, Chiang BL. Placental growth factor down-regulates type 1 T helper
378
immune response by modulating the function of dendritic cells. J Leukoc Biol
379
2007;82:1473-80.
380
[33] Martínez FF, Knubel CP, Sánchez MC, Cervi L, Motrán CC. Pregnancy-specific
18
ACCEPTED MANUSCRIPT 381
glycoprotein 1a activates dendritic cells to provide signals for Th17-, Th2-, and Treg-cell
382
polarization. Eur J Immunol 2012;42:1573-84. [34] Jonsson Y, Matthiesen L, Berg G, Ernerudh J, Nieminen K, Ekerfelt C. Indications of an
384
altered immune balance in preeclampsia: a decrease in in vitro secretion of IL-5 and
385
IL-10 from blood mononuclear cells and in blood basophil counts compared with normal
386
pregnancy. J Reprod Immunol 2005;66:69-84.
RI PT
383
[35] Chatterjee P, Chiasson VL, Seerangan G, Tobin RP, Kopriva SE, Newell-Rogers MK,
388
Mitchell BM. Cotreatment with interleukin 4 and interleukin 10 modulates immune cells
389
and prevents hypertension in pregnant mice. Am J Hypertens 2015;28:135-42.
393 394
M AN U
[37] Willard-Mack CL. Normal structure, function, and histology of lymph nodes. Toxicol
TE D
392
Immunol 2015;73:487-500.
Pathol 2006;34:409-24.
EP
391
[36] Cheng SB, Sharma S. Interleukin-10: a pleiotropic regulator in pregnancy. Am J Reprod
AC C
390
SC
387
19
ACCEPTED MANUSCRIPT
Figure Legends
396
Fig. 1. Relative expression values of Th1 cells cytokines (IL-2, IFN-γ, and TNF-β) and Th2
397
cytokines (IL-4, IL-5, IL-6, and IL-10) mRNA in the lymph nodes measured by qRT-PCR.
398
Note: DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of
399
pregnancy; DP25 = Day 25 of pregnancy. Significant differences (P < 0.05) are indicated by
400
different letters within the same column.
RI PT
395
SC
401
Fig. 2. Expression of Th1 cells cytokines (IL-2, IFN-γ, and TNF-β) and Th2 cytokines (IL-4,
403
IL-5, IL-6, and IL-10) proteins in the lymph nodes analyzed by western blot analysis. Note:
404
DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of
405
pregnancy; DP25 = Day 25 of pregnancy. Significant differences (P < 0.05) are indicated by
406
different superscript letters within the same color column.
407
Fig. 3. Immunohistochemical localization of IL-2 and IL-10 proteins in lymph nodes. Lymph
408
node is divided into the cortex and the medulla. Lymph enters the convex through the
409
subcapsular sinus (SS) and trabeculae (TR) around the lymphoid nodules (LN), and flows into
410
the medulla through the lymph sinus (LS) around the medullary cord (MC). Note: DN16 =
411
Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of pregnancy;
412
DP25 = Day 25 of pregnancy. Bar = 20 µm.
AC C
EP
TE D
M AN U
402
20
ACCEPTED MANUSCRIPT Table 1 Primers used for qRT-PCR Gene
Primer
Sequence
Size (bp)
Forward AAACCTGAACACCAGAGAGAT IL-2
117 GCCTTTACTGTCGCATCA
Forward TTGAACGGCAGCTCTGAGAA IFN-γ
RI PT
Reverse
124
Reverse
TTGGCGACAGGTCATTCATC
SC
Forward CCACTGACGGGCTTTACCT TNF-β
141
TGATGGCAGAGAGGATGTTG
M AN U
Reverse
Forward CCAAAGAACGCAACTGAGAA IL-4 Reverse
120
GCTGCTGAGATTCCTGTCAA
Forward CATCTGCGTTTGACCTTGG IL-5
139
AGTTCCCATCACCTATCAGCA
TE D
Reverse
Forward CGAGTTTGAGGGAAATCAGG IL-6
Reverse
118
GTCAGTGTGTGTGGCTGGAG
EP
Forward CTCTGTTGCCTGGTCTTCCT
IL-10
AC C
Reverse
169
TGTTCAGTTGGTCCTTCATTTG
Forward GGGTCATCATCTCTGCACCT
GAPDH
Reverse
176 GGTCATAAGTCCCTCCACGA
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights Downregulation of tumor necrosis factor beta and IL-2 in the lymph node. Upregulation of IL-5 and IL-10 in the lymph node. IL-2 and IL-10 were localized in the subcapsular sinus and trabeculae in the cortex,
AC C
EP
TE D
M AN U
SC
RI PT
medullary sinuses.