Alteration of immune responses in bovine

endometrial cells under hyperthermia conditions

March, 2021

Shunsuke SAKAI

Graduate School of

Environmental and Life Science

(Doctor’s Course)

OKAYAMA UNIVERSITY

CONTENTS

PREFACE-----------------------------------------------------------------------------------------1

ACKNOWLEDGEMENT-----------------------------------------------------------------------2

CHAPTER 1 GENERAL INTRODUCTION--------------------------3

CHAPTER 2

INTRODUCTION-----------------------------------------6

MATHERIALS AND METHODS ----------------------7

RESULTS-------------------------------------------------16

DISCUSSION--------------------------------------------33

SUMMARY----------------------------------------------35

CHAPTER 3

INTRODUCTION---------------------------------------36

MATHERIALS AND METHODS --------------------38

RESULTS-------------------------------------------------43

DISCUSSION--------------------------------------------56

SUMMARY----------------------------------------------58

CHAPTER 4

INTRODUCTION---------------------------------------59

MATHERIALS AND METHODS --------------------60

RESULTS-------------------------------------------------62

DISCUSSION--------------------------------------------76

SUMMARY----------------------------------------------78

CHAPTER 5 CONCLUSIONS-----------------------------------------79

REFERENCES----------------------------------------------------------------------------------81

ABSTRACT IN JAPANEASE----------------------------------------------------------------91

1

PREFACE

The experiments described in this dissertation were carried out at the Graduate school

of Environmental and Life Science (Doctor’s course), Okayama University, Japan, from

April 2018 to March 2021, under the supervision of Professor Koji KIMURA.

This dissertation has not been submitted previously in whole or in part to a council,

university or any other professional institution for degree, diploma or other professional

qualifications.

Shunsuke SAKAI

March, 2021

2

ACKNOWLEDGMENTS

I wish to my express my deep gratitude to Kiyoshi OKUDA, DVM, PhD., Professor

emeritus of Okayama University, Okayama, and President of Obihiro University of

Agriculture and Veterinary Medicine, Obihiro, Japan, and Koji KIMURA, Ph.D.,

Professor of Graduate School of Environmental and Life Science, Okayama, University,

Japan, and Toshimitsu HATABU, Ph.D., Associate Professor of Graduate School of

Environmental and Life Science, Okayama, University, for their guidance,

encouragement, constructive criticism, and excellent supervision and for providing me

the opportunity to conduct this study. I also wish to thank Mrs. Keiko SETOGAWA,

Mariko KUSE, Ph.D., Mr. Takashi NOKUBO, Mr. Tetsuya AOYAMA, and Yuki

YAMAMOTO, DMV, Ph.D., Associate Professor of Graduate School of Environmental

and Life Science, Okayama, University, for their supports, advices, and encouragement

to conduct the present study. It is a pleasure to express my thanks to all the members of

Laboratory of Animal Reproductive Physiology and my parents for helping me during

carrying out this study.

3

CHAPTOR 1

GENERAL INTRODUCTION

Effect of Heat Stress (HS) on the livestock industry

Because of the global warming, the effect of HS on the livestock industry in all over

the world is a special concern. Indeed, the conception rate and milk yield of cows in

Europe, America, and Japan decrease in hot seasons, resulting in the large economic

loss to dairy industry [1, 2]. In addition, the global population is expected to reach 9.6

billion by 2050 and many people are facing food shortage [3]. The demand for livestock

product, such as meat and milk, will increase in developing country which is mainly

located in tropical and subtropical area. Therefore, breeding of cattle in tropical and

subtropical regions is needed [3, 4]. However, since the appreciate temperature range

for breeding of cattle is 5°C to 25°C, it is hard to breed cattle in tropical and subtropical

regions [5]. Taken together, the effect of HS on the livestock industry is a special

concern not only in the developed country but also in the developing country.

Effect of HS on the physiological function in cows

Heat Stress (HS) is defined as the sum of external factors, such as temperature,

humidity, and wind, in the review by Hansen [6]. The degree of HS is quantified as

temperature humidity index (THI) which is calculated based on the following formula

(1) [7]. According to the several reports, THI is categorized as follows: THI < 68 as no

stress, 68 ≤ THI < 72 as mild stress, 72 ≤ THI < 80 as moderate stress, and THI ≥ 80 as

severe stress. To adapt HS conditions, cows suppress the systemic physiological

function such as voluntary reduction of feed intake [6], panting [8], dysregulation of the

hypothalamic-pituitary-ovarian axis [9], and so on. These changes of physiological

4

functions under HS conditions decrease the conception rate and milk yield of cows [1,

10, 11].

THI = (0.8 × T) + [(RH / 100) × (T – 14.4)] + 46.4 (1)

T: dry bulb temperature (°C), RH: relative humidity (%)

Local effect of hyperthermia under HS conditions on cellular function

A lot of researches about the effect of HS on the physiological function of cows focus

on the values of THI [7, 12-15]. On the other hand, Nabenishi et al. reports that there

are positive correlations between the THI values and body temperature of cows during

the hot period, but not during the cool period [16]. Then, they suggested that rise of

body temperature is one of causes for the low conception rate of cows in hot seasons.

The increase of body temperature is systemically sensed and regulated by the nervous

system, whereas local hyperthermia is also sensed at the cellular level via transient

receptor potential [17]. Indeed, local hyperthermia affects cellular functions such as the

mobility of membrane phospholipid [18], the production of reactive oxygen species [19],

arrest of cell growth [20], and induction of apoptosis [19]. Under HS conditions, the

vaginal temperature of cow increases to approximately 40.5°C [16] and the temperature

of uterus is same as that of vagina [21]. Therefore, HS might affect not only the

systemic function but also the local function of uterus in cows. In fact, we previously

found that uteri in hot season adapts to HS at cellular level and the hyperthermia locally

upregulates the expression of enzymes involved in prostaglandin (PG) synthesis,

resulting in the disorder of PG production [22]. Since bovine endometrium has multiple

functions other than PG production for adequate reproductive performance, we

hypothesize that hyperthermia influences the other functions of bovine endometrial cells

5

under HS conditions.

Effect of HS on immune responses in cows

After the parturition, almost all cows undergo bacterial infection in uterus, followed

by the endometritis [23, 24]. Because endometritis negatively affects not only uterine

but also ovarian function [24, 25], rapid recovery from endometritis in cows are

necessary before there can be subsequent pregnancies. However, cows are at a high risk

of endometritis in the summer and that its symptoms last longer than in the other

seasons [26, 27]. For the recovery from endometritis, the systemic and local immune

responses at whole body and uterus, respectively, play important roles for elimination of

bacteria and tissue repair [28, 29]. The effect of HS on the systemic immune responses

is already discussed [28], whereas the effect of hyperthermia on the local immune

responses in uterus has been unclear.

Aim of present study

In the present studies, firstly, we investigated whether hyperthermia affects the

immune responses in bovine endometrial cells at cellular levels. Secondly, we clarified

the mechanisms of the alteration of immune responses in bovine endometrial epithelial

and stromal cells, individually.

6

CHAPTOR 2

Effect of hyperthermia on the immune responses

in bovine endometrial cells

INTRODUCTION

The bovine endometrium is a major contributor to the regulation of reproduction and

is involved in processes such as the estrous cycle, implantation, and placenta formation

[30-32]. For the endometrium to correctly perform its functions, the uterine lumen is

kept aseptic by the cervix and endometrial epithelial cells, which represent anatomical

barriers of innate immunity [33, 34]. However, because the bovine cervix becomes

softer and the endometrial epithelial layer is exfoliated in the postpartum period, the

uterine anatomical barrier functions are disrupted, resulting in bacterial infections in the

bovine endometrium [23, 24]. Indeed, in the postpartum period, approximately 20% of

cows develop clinical endometritis, which is defined as the presence of a purulent

uterine discharge detectable in the vagina [24]. Moreover, 70% of postpartum cows

develop subclinical endometritis, which is characterized by inflammation of the

endometrium in the absence of the purulent uterine discharge even after the repair of the

endometrium [35]. Because endometritis negatively affects not only uterine function but

also ovarian function [24, 25], recovery from endometritis in cows are necessary before

there can be subsequent pregnancies.

The majority of pathogens that cause endometritis are gram-negative bacteria, such as

Escherichia coli [36, 37]. In the bovine endometrium, the response to E. coli and its

pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), is dependent

on the pattern-recognition receptors, including the complex of Toll-like receptor 4

(TLR4), MD-2, and CD14 [38]. After the detection of LPS by this complex, bovine

7

endometrial epithelial and stromal cells secrete chemokine, such as MCP1, IL-6, and

IL-8 [38, 39] to recruit immune cells to the infection site [40, 41]. MCP1 and IL-6

attract macrophages (MΦs) [42, 43], whereas IL-8 recruits neutrophils [44]. In the case

of bovine endometritis, because almost all of E. coli are present in the uterine lumen and

endometrial epithelial layer, immune cells need to be recruited there to eliminate the

bacteria and promote recovery from endometritis [40]. Particularly, the rapid attraction

of neutrophils contributes to the elimination of bacteria [36, 38]. In addition, the MΦs

are attracted following neutrophils and are not only involved in bacterial elimination but

also the secretion of antimicrobial peptides, the orchestration of inflammation, the

activation of other immune cells (T- and B-cells) and tissue repair [28, 29, 38, 45, 46].

Moreover, it has been reported that cows are at a high risk of endometritis in summer

and that its symptoms last longer than in the other seasons [26, 27]. Therefore, we

hypothesized that local effect of hyperthermia might be involved in the innate immune

response in the bovine endometrium at cellular level. To clarify this hypothesis, we

examined the chemokine production in bovine endometrial epithelial and stromal cells

in vitro under hyperthermia conditions.

MATERIALS AND METHODS

Collection of uteri

Apparently healthy uteri from cows without a visible conceptus were obtained from a

local abattoir (Okayama Meat Center and Tsuyama Meat Center) within 10-20 min of

exsanguination and immediately transported to the laboratory, where they were

submerged in ice-cold physiological saline. The stages of the estrous cycle were

confirmed by macroscopic observation of the ovaries as described previously [47].

8

Isolation and culture of endometrial cells

Uteri at Stage I and Stage IV were used for the isolation and culture of endometrial

cells. The epithelial and stromal cells from the bovine endometrium were enzymatically

separated and cultured according to procedures described previously [48, 49]. The

collected epithelial and stromal cells were separately resuspended in culture medium

(DMEM/Ham’s F-12, 1 : 1 (v/v); Invitrogen, Carlsbad, CA, USA) supplemented with

10% (v/v) bovine serum (Invitrogen), 20 µg/mL gentamicin (Sigma-Aldrich, St. Louis,

MO, USA) and 2 µg/mL amphotericin B (Sigma-Aldrich). To culture these epithelial

and stromal cells, 1 mL containing 1 × 105 viable cells was seeded in each well of

24-well dishes (Greuner Bio-One, Frickenhausen, Germany) or 75 cm2 culture flasks

(Greuner Bio-One), and the cells were cultured at 38.5°C in a humidified atmosphere of

5% CO2 in air. When the cells were confluent, the culture medium was replaced with

fresh DMEM/Ham’s F-12 supplemented with 0.1% (w/v) BSA, 5 ng/mL sodium

selenite (Sigma-Aldrich), 0.5 mM ascorbic acid (Wako Pure Chemical Industries, Osaka,

Japan), 5mg/mL transferrin (Sigma-Aldrich), 2 mg/mL insulin (Sigma-Aldrich) and 20

mg/mL gentamicin. Then, these cells were used in the following experiments.

Isolation of bovine monocytes

Blood samples were collected from healthy Japanese black cows (n=12) aseptically

by venipuncture of the vena jugularis externa into EDTA vacutainer tubes (TERUMO,

Tokyo, Japan). All procedures were approved by the Animal Care and Use Committee,

Okayama University, and were conducted in accordance with the Policy on the Care and

Use of the Laboratory Animals, Okayama University (OKU-2018340). Blood was

9

centrifuged (900 × g, 30 min at 25°C) and separated into plasma, buffy coat and red

blood cell sediment. The buffy coat, containing monocytes was resuspended in culture

medium (DMEM/Ham’s F-12, 1:1 (v/v)) supplemented with 10% (v/v) bovine serum,

20 µg/mL gentamicin, and 2 µg/mL amphotericin B and subsequently seeded in 25 cm2

culture flasks (Greuner Bio-One). After an overnight incubation, the culture medium

containing unattached cells was removed.

Differentiation of monocytes into macrophage-like cell (MΦs)

Monocytes were stimulated with 100 ng/ml bovine INFγ (Gift from Dr. Shigeki

Inumaru) for 5 days to induce differentiation into MΦs.

Migration assay

Migration assays were performed in 24-well plates with ThinCert (Greiner Bio One).

The upper and lower wells were separated by a polycarbonate membrane (8 μm pore

size). The lower wells contained 900 μl medium, including 300 µl isotonic Percoll. The

upper wells contained MΦs (10 × 105 cell/well), which were stained with Dil

(Invitrogen) for 3 h at 38.5°C. These wells were incubated in a humidified atmosphere

of 5% CO2 in air. At the end of the incubation, un-migrated MΦs on the upper -side of

the polycarbonate membrane were removed from the inserts with a cotton-tipped swab.

The MΦs that migrated to the lower -side of the polycarbonate membrane were counted

using a fluorescence microscope (OLYMPUS, Tokyo, Japan).

Total RNA extraction and quantitative RT-PCR

Total RNA was extracted from the cultured cells using RNAiso Plus (TaKaRa Bio,

10

Otsu, Japan) according to the manufacturer’s directions. The optical density A260/280

of RNA samples was measured by NanoDrop LITE (Thermo Fisher Scientific, MA,

USA) and its value was approximately 2. One microgram of each total RNA was

reverse transcribed using ReverTra Ace qPCR RT Master Mix with gDNA remover

(TOYOBO, Osaka, Japan). Quantification of MCP1, IL6, CXCL8, CCL3, CCL5,

CX3CL1, CXCL10, CXCL11, TLR4, MD2, CD14, IL6 Receptor, CCR2, and GAPDH

mRNA expression was carried out following a previously described method with some

modifications [50]. Briefly, we quantified the expression of each mRNA using Brilliant

III Ultra-Fast SYBR Green QPCR Master Mix With Low ROX (Agilent Technologies,

Santa Clara, CA, USA) starting with 2 ng of reverse-transcribed total RNA. The

expression of GAPDH was used as an internal control determined using NormFinder.

For quantification of the mRNA expression levels, PCR was performed under the

following conditions with the AriaMx Real Time PCR System (Agilent Technologies):

95°C for 30 sec, followed by 45 cycles of 95°C for 10 sec, 56°C or 60°C for 10 sec, and

72°C for 15 sec with continuous fluorescence measurement. The melting curve was

analyzed under the following conditions to check the specificity of the PCR products:

95°C for 1 min, 56°C or 60°C to 95°C with 0.5°C interval, 5 sec soak time. Serial

dilutions (20-20000000 copies) of each PCR product extracted from the agarose gel

were used as a standard to analyze each mRNA expression level. The sequence of each

primer and the annealing temperatures are listed in Table 1.

Enzyme immunoassay (EIA)

The concentrations of MCP1 and IL-6 in supernatants were measured by EIA using a

Bovine MCP1 ELISA Reagent Set (GWB-KAAO76; GenWay Biotech, Inc, San Diego,

11

CA, USA) and Bovine IL-6 ELISA Reagent Kit (ESS0029; Thermo Fisher Scientific),

respectively. The concentration of IL-8 in the culture medium was determined by EIA as

described previously [51]. The standard curve ranges of MCP1, IL-6, and IL-8 were

0.156-10 ng/ml, 0.039-10 ng/ml, and 0.078-20 ng/ml, respectively. The median effective

dose (ED50) values of the assays for MCP1, IL-6, and IL-8 were 1.25 ng/ml, 0.625

ng/ml, and 1.25 ng/ml, respectively. The intra- and inter-assay coefficients of variation,

on average, for MCP1, IL-6, and IL-8 were 7.5% and 5.8%, 3.52% and 1.12%, 8.39%

and 13.52%, respectively.

DNA assay

The DNA content of cultured endometrial cells was measured as described previously

[52]. Briefly, after cell disruption with an ultrasonic homogenizer, the cell lysates were

incubated with Hoechst H 33258 (Sigma-Aldrich). After incubation for 10 min, the

fluorescence of each sample and standard was measured with a microplate fluorometer

(Fluoroskan Ascent, Labsystems, Thermo Fisher Scientific). The standard curve ranged

from 0.625 to 30 µg/ml, and the ED50 of the assay was 10 µg/ml.

Fluorescence immunohistochemistry

The paraffin-embedded endometrial tissues were sectioned (4 μm), and placed on

silane-coated glass slides (Dako A/S, Glostrup, Denmark). After deparaffinization with

xylene, the sections were subjected to antigen retrieval via heating in 0.01M citrate

buffer (pH 6.0) using a microwave at 600W for 15 min. The sections were then

incubated in 2.5% normal horse serum (Vector Laboratories, Burlingame, CA) at room

temperature for 20 min, which was followed by incubation at 4°C overnight with

12

CD172A monoclonal antibody DH59B (BOV2049, Washington State University

Monoclonal Antibody Center, Pullman WA) diluted in PBS at 1:100. The sections were

then treated at room temperature for 1 h with an appropriate fluorescent second antibody,

the Alexa Fluor 488 goat anti-rabbit IgG antibody (1:500 dilution of A11005) (Life

Technologies, Grand Island, NY). The sections were washed with PBS, and covered

with Prolong Gold with DAPI (Life Technologies). The sections were photographed

using a FSX100 Bio Image Navigator (Olympus, Tokyo, Japan).

May-Grunwald-Giemsa stain

The localization of neutrophil in bovine endometrium was assessed by

May-Grunwald-Giemsa stain according to the manufacture’s instruction. Briefly, bovine

endometrial tissues were fixed by 10% formaldehyde and embedded in paraffin. The

paraffin-embedded endometrial tissues were ssectioned (4 μm) and placed on

silane-coated glass slides (Dako). After deparaffinization with xylene, the sections were

subjected to May-Grunwald solution (131-12811, Wako Pure Chemical Industries) at

room temperature for 5 min. Subsequently, these sections were stained by Giemsa

solution (1.09204.0103, Merck KGaA, Darmstadt, Germany) at room temperature for

10 min. At the end of stain, the sections were rinsed in deionized water and decolorized

by 0.1% acetic acid, followed by taking photographs using a FSX100 Bio Imaging

Navigator (OLYMPUS).

Experimental design

Experiment 1: Effect of hyperthermia and LPS on the cell viability in bovine

endometrial epithelial and stromal cells

13

Bovine endometrial epithelial and stromal cells were cultured under control or

hyperthermia conditions [22]. The control samples were cultured at 38.5°C for 34 h,

followed by the LPS challenge (0 or 1 µg/ml) (LPS from Escherichia coli O55:B5;

Sigma-Aldrich, L6529) for 12 h at 38.5°C. The hyperthermia samples were cultured at

40.5°C for 10 h, allowed to recover at 38.5°C for 14 h, and then cultured at 40.5°C for

10 h again, this was followed by the LPS challenge (0 or 1 µg/ml) for 12 h at 40.5°C.

Since 1 µg/ml LPS significantly upregulates inflammatory mediator mRNA expression

and production in bovine endometrial cells [53, 54] and reflects the concentrations in

the uterine lumen of infected cattle [55, 56], we performed one dose of the LPS

challenge (1 µg/ml) in the present study. After the LPS challenge, the cells were

harvested to measure the DNA content.

Experiment 2: Effect of hyperthermia and LPS on the mRNA expression of

chemokines in cultured bovine endometrial epithelial and stromal cells

Endometrial epithelial and stromal cells were cultured as described Experiment 1. In

this experiment, to measure the gene expression, these cells were exposed to LPS (0 or

1 µg/ml) for 6 h. We confirmed that the mRNA expression of chemokines was higher at

6 h compared to 3 h and 12 h (data not shown). After LPS treatment, the cells were

harvested to measure the mRNA expression of IL6, MCP1, and CXCL8, CCL3, CCL5,

CX3CL1, CXCL10, CXCL11. In addition, to confirm whether hyperthermia treatment

was effective, we evaluated the heat shock protein 70 and confirmed its increase in both

types of endometrial cells (data not shown).

Experiment 3: Effect of hyperthermia and LPS on the production of IL-6, MCP1 and

14

IL-8 in cultured bovine endometrial epithelial and stromal cells

Endometrial epithelial and stromal cells were cultured as described Experiment 1.

After LPS treatment for 12 h [57, 58], the medium was collected in a 1.5 mL tube and

immediately frozen and stored at -30°C until the measurement of the concentrations of

MCP1, IL-6, and IL-8 by EIA. To normalize the concentration of each chemokines, the

cells were harvested and measured the DNA content.

Experiment 4: Effect of hyperthermia and LPS on the mRNA expression of TLR4,

CD14 and MD2 in cultured bovine endometrial epithelial and stromal cells

Endometrial epithelial and stromal cells were cultured as described Experiment 2.

Since CD14 and MD-2 are related to the recognition of the LPS in cooperation with

TLR4 [38, 59, 60], the mRNA expression of TLR4, MD2, and CD14 was measured.

Experiment 5: Effect of hyperthermia on the mRNA expression of IL6 Receptor and

CCR2 in MΦs and their migration ability

First, to confirm the isolation of the monocytes and their differentiation into MΦs, the

monocytes were stained using specific and non-specific esterase (Muto Pure Chemicals

Co., LTD, Tokyo, Japan), and the morphological change and CD80 mRNA expression

of the differentiated cells were investigated. The differentiated MΦs were incubated at

38.5°C or 40.5°C following a previously described method [22]. After incubation, MΦs

were harvested to measure the mRNA expression of IL6 Receptor and CCR2.

To investigate whether hyperthermia affects the migration ability of MΦs, migration

assays were performed. As control samples, the 24-well plates with ThinCerts

15

containing the Dil-stained MΦs in the upper well were cultured at 38.5°C for 34 h,

which was followed by the addition of 50 ng/ml MCP1 (Bovine MCP1, PBP024,

BIO-RAD Laboratories, Berkeley, CA, USA) in the lower well and further incubation

for 12 h at 38.5°C. As hyperthermia samples, the 24-well plates with ThinCerts that

contained the Dil-stained MΦs in the upper well were cultured at 40.5°C for 10 h and

allowed to recover at 38.5°C for 14 h; they were then cultured at 40.5°C for 10 h again,

which was followed by the addition of 50 ng/ml MCP1 in the lower well and further

incubation for 12 h at 40.5°C. After incubation, the migrated MΦs were counted using a

fluorescence microscope.

To confirm whether the endometrial stromal cells under the hyperthermia condition

attracted a large number of MΦs, a migration assay was performed with the culture

supernatant of the endometrial stromal cells. The endometrial stromal cells were

cultured in 75 cm2 culture flasks with 10 ml of BSA free culture medium at under the

same conditions as described in Experiment 3. After this culture medium was

concentrated to 600 μl with Centricut (U-10, cutoff value of molecular weight: 10000,

KURABO Industries LTD., Okayama, Japan), the migration assay was performed at

38.5°C for 12 h. Since LPS was also concentrated in this experiment and it might affect

the migration ability of the MΦs, the migration assay was performed with the medium

which contain the concentrated LPS.

Experiment 6: Localization of MΦs and neutrophils in bovine endometrium in

summer and winter

For the measurement of the length of the MΦs from epithelial layer, endometrial

tissues (Stage III) were collected from 6 cows in the winter (January - February and

16

November - December, 2017) and 6 cows in the summer (July - August, 2017). The

average temperatures in Okayama during the sampling periods were 6.4°C in the winter

and 28.4°C in the summer, respectively. The temperature data was obtained from the

Japan Meteorological Agency. The MΦs and neutrophils were stained, then 20 positive

immunostaining of MΦs and 10 positive immunostaining of neutrophils were selected

randomly. The distance of these MΦs and neutrophils, which are perpendicular to the

epithelial layer, was measured via cellSens imaging software (OLYMPUS).

Statistical analysis

All experimental data is shown as the mean ± S.E.M. All the data was tested for

normal distribution, and the homogeneity of variance determined whether it was

parametric or non-parametric data. The statistical analysis was performed using

GraphPad Prism (GraphPad Software, La Jolla, CA, USA). First, the data from

Experiment 1-4 and migration assay using MCP1 were assessed using two-way

ANOVA. When an interaction between the additive reagents and hyperthermia

treatment was not detected, each data set was assessed using Tukey-Kramer test. The

data of migration assay using the culture supernatant was assessed using one-way

ANOVA. Moreover, Student’s t-test was conducted on the data for CD80, IL6 Receptor,

CCR2 mRNA expression, and Experiment 6. Probabilities less than 5% (P<0.05) were

considered statistically significant.

RESULTS

Experiment 1: Effect of hyperthermia and LPS on the cell viability in bovine

endometrial epithelial and stromal cells

17

As shown in Figure 1, neither hyperthermia nor LPS affected the cell viability of the

bovine endometria epithelial and stromal cells (Figure 1A, B)

Experiment 2: Effect of hyperthermia and LPS on the mRNA expression of

chemokines in cultured bovine endometrial epithelial and stromal cells

In Experiment 2, we investigated whether hyperthermia affects chemokine mRNA

expression in bovine endometrial epithelial and stromal cells.

In endometrial epithelial cells, LPS significantly (P<0.05) increased the mRNA

expression of IL6, MCP1, CXCL8, CCL5, CX3CL1, and CXCL10 (Figure 2A-C and

2E-G). Moreover, hyperthermia significantly (P<0.05) suppressed the mRNA

expression of IL6 and MCP1 regardless of the presence of LPS stimulation (Figure 2A,

C). The mRNA expression of CXCL8 was significantly (P<0.05) suppressed under the

hyperthermia conditions in the absence of LPS but hyperthermia did not affect the

mRNA expression of CXCL8 in the presence of LPS (Figure 2B). The mRNA

expression of CCL3, CCL5, CX3CL1, and CXCL10 was not influenced under

hyperthermia conditions (Figure 2D-G). In addition, the mRNA expression of CCL3,

CCL5, CX3CL1, and CXCL10 was lower than the mRNA expression of MCP1 (Figure

2C-G).

In contrast, in endometrial stromal cells, LPS significantly (P<0.05) increased the

mRNA expression of IL6, MCP1, CXCL8, CCL3, CCL5 and CX3CL1 (Figure 2H-M).

In the absence of LPS, hyperthermia significantly (P<0.05) enhanced the mRNA

expression of IL6, MCP1 and CXCL8 (Figure 2H-J), while, the mRNA expression of

IL6, MCP1, CXCL8, and CCL5 was enhanced by hyperthermia only in the presence of

LPS (Figure 2H-J and 2L). The mRNA expression of CCL3, CX3CL1, and CXCL10 was

18

not affected by hyperthermia regardless of the presence or absence of LPS (Figure 2K

and 2M, N).

In both types of endometrial cells, CXCL11 mRNA expression was undetectable in

both the presence and absence of LPS (data not shown).

Experiment 3: Effect of hyperthermia and LPS on the production of IL-6, MCP1

and IL-8 in cultured bovine endometrial epithelial and stromal cells

As shown in Figure 3, we examined the effect of hyperthermia on chemokine

secretion in bovine endometrial epithelial and stromal cells.

In the endometrial epithelial cells cultured at 38.5°C, the production of IL-6 was

significantly (P<0.05) stimulated by LPS; however, it decreased to an undetectable level

(<0.056 ng/ml) under hyperthermia conditions (Figure 3A). IL-8 production by

endometrial epithelial cells was significantly (P<0.05) induced by LPS in both the

control and hyperthermia conditions, then hyperthermia did not alter the effect of LPS

on IL-8 production (Figure 3B). MCP1 in endometrial epithelial cells was at an

undetectable level in all treatment groups.

In bovine endometrial stromal cells, the production of IL-6, MCP1, and IL-8 was

significantly (P<0.05) increased by LPS. Moreover, hyperthermia significantly (P<0.05)

enhanced these increases in endometrial stromal cells (Figure 3C-E).

Experiment 4: Effect of hyperthermia and LPS on the mRNA expression of TLR4,

CD14 and MD2 in cultured bovine endometrial epithelial and stromal cells

In Experiment 4, to determine whether hyperthermia affects the recognition of LPS in

bovine endometrial epithelial and stromal cells, we examined the mRNA expression of

19

the pattern-recognition receptor and its related factors.

As shown in Figure 4, while LPS did not affect the mRNA expression of TLR4,

hyperthermia significantly (P<0.05) increased it in endometrial epithelial and stromal

cells (Figure 4A, D). On the other hand, neither hyperthermia nor LPS affected the

mRNA expression of CD14 and MD2 in both types of bovine endometrial cells (Figure

4B, C, E, and F).

Experiment 5: Effect of hyperthermia on the mRNA expression of IL6 Receptor

and CCR2 in MΦs and their migration ability

In this experiment, the effect of hyperthermia on the migration ability of MΦs was

investigated.

After the specific and non-specific esterase staining, we confirmed that more than

80% of the attached cells in the culture flasks were monocytes (Figure 5A). In addition,

as shown in Figure 5, IFNγ-stimulated monocytes appeared pseudopodia and increased

the CD80 mRNA expression (Figure 5B and C).

As shown in Figure 5, hyperthermia did not affect the mRNA expression of IL6

Receptor and CCR2 in the MΦs (Figure 5D and E). MCP1 significantly (P<0.05)

attracted MΦs under both the control and hyperthermia conditions; however, the

migration ability of the MΦs was not affected by hyperthermia (Figure 5F). In addition,

the culture supernatant of the endometrial stromal cells under the hyperthermia

condition significantly (P<0.05) attracted MΦs compared with their supernatant under

the control condition. Moreover, LPS did not affect the migration ability of the MΦs

(Figure 5G).

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Experiment 6: Localization of MΦs and neutrophils in bovine endometrium in

summer and winter

The localization of neutrophils in bovine endometrium did not differ between summer

and winter (Figure 6A-C). MΦs were sparsely localized through the bovine

endometrium in the summer whereas they were localized in the sub-epithelial stroma in

the winter (Figure 6D and E). The distance of the MΦs from the epithelial layer was

significantly (P<0.05) longer in the summer than in the winter (Figure 6F).

21

Genes Forward and reverse primers Accession No. Annealing temperature

5'-CGCCTGCTGCTATACATTCA-3'

5'-GCTCAAGGCTTTGGAGTTTG-3'

5'-AACCACTGCTGGTCTTCTGG-3'

5'-GGTCAGTGTTTGTGGCTGGA-3'

5'-GGCTGTTGCTCTCTTGGCAG-3'

5'-CTGAATTTTCACAGTGTGGCCC-3'

5'-GCTGACTATTTTGAGACCAG-3'

5'-GGTCGGTGATGTATTCCT-3'

5'-AGGAGTATTTCTACACCAGCAG-3'

5'-GCGTTGATGTACTCTCGCA-3'

5'-CAACACGGTGTGAACAAG-3'

5'-CTTGTGGTCTAGGTGCTT-3'

5'-CTGCAAGTCAATCCTGC-3'

5'-TGCAGGAGTAGTAGCAGCT-3'

5'-CAAGCATGAGTGTGAAGG-3'

5'-CTTTCTCAATATCTGCCAC-3'

5'-TGCCTGAGAACCGAGAGTTG-3'

5'-ATATGGGGATGTTGTCGGGG-3'

5'-GGGAAGCCGTGGAATACTCTAT-3'

5'-CCCCTGAAGGAGAATTGTATTG-3'

5'-CAGCCGACAACCAGAGAGAG-3'

5'-TAGACCAGTCAGGCTTCGGA-3'

5'-CATTAGGCGAGGAGGAAGCA-3'

5'-CTGTGGCATTGTCCTCTGGT-3'

5'-TACTTGCCCTTGTATCTCCG-3'

5'-GTATCATTGCCATCCATCTTC-3'

5'-CTAATCAGCACCTGACCTGG-3'

5'-GTCTGCGTATTGCAGCATTT-3'

5'-CTCTCAAGGGCATTCTAGGC-3'

5'-TGACAAAGTGGTCGTTGAGG-3'GAPDH NM_001034034.2

CX3CL1 XM_002694885.5

CXCL10 NM_001046551.2

CXCL11 NM_001113173.1

NM_001046517.1

CD80 NM_001206439.1

NM_174008.1

NM_001110785.1

XM_015459671.1

MD2

TLR4

CXCL8

IL6

MCP1

Table 1. Sequences of primers used for quantitative RT-PCR analysis

NM_175827.2

NM_174006.2

NM_173923.2

NM_173925.2

CCL5

CCL3 NM_174511.2

60℃

60℃

60℃

56℃

60℃

NM_174198

CCR2

IL6 Receptor

CD14

56℃

56℃

60℃

60℃

60℃

60℃

56℃

60℃

60℃

60℃

22

FIGURE 1

Effect of hyperthermia and LPS on the cell viability of bovine endometrial epithelial

(A) and stromal (B) cells (mean±S.E.M., n=8). Cell viability was determined as the

relative portion of the endometrial cells in the absence of LPS at 38.5°C. The open and

closed bars indicate the control and hyperthermia conditions, respectively.

Epithelial cells

A

LPS

020406080

100120140

Cel

l via

bili

ty

(%)

Stromal cells

B

020406080

100120140

Cel

l via

bili

ty

(%)

- + LPS - +

23

Epithelial cells

*

A B

C

E

D

G

IL6

mR

NA

0

2

4

6

8

CXC

L8m

RN

A

0

1

2

3

4

CC

L3m

RN

A

0

0.0005

0.001

0.0015

0.002

0.0025

MC

P1

mR

NA

0

0.02

0.04

0.06

0.08

CC

L5m

RN

A

0

0.01

0.02

0.03

0.04

CX

3C

L1m

RN

A

0

0.0004

0.0008

0.0012

0.0016

CXC

L10

mR

NA

0

0.00005

0.0001

0.00015

0.0002

0.00025

F

a

b

xy

a

b

x

y

a

b

x

y

a

b

x

y

a

b

x

y

a

b

x

y

*

*

*

*

LPS - + LPS - +

LPS

LPS

LPS

LPS

LPS

- + - +

- + - +

- +

24

FIGURE 2

Effect of hyperthermia and LPS on the mRNA expression of IL6 (A, H), CXCL8 (B,

I), MCP1 (C, J), CCL3 (D, K), CCL5 (E, L), CX3CL1 (F, M) and CXCL10 (G, N) in

cultured bovine endometrial epithelial and stromal cells (mean±S.E.M., n=6-10).

Each mRNA expression level was indicated as their copy number normalized by the

expression of GAPDH. The open and closed bars indicate the control and

IL6

mR

NA

0

1

2

3

CXC

L8m

RN

A

0

4

8

12

CC

L3m

RN

A

0

0.05

0.1

0.15

0.2

0.25

MC

P1

mR

NA

0

1

2

3

4

5

6

CX

3C

L1m

RN

A

0

0.004

0.008

0.012

0.016

CC

L5m

RN

A

0

1

2

3

4

5

CXC

L10

mR

NA

0

0.001

0.002

0.003

Stromal cells

H I

J

L

K

N

M

ab

x

y

a

bx

y

a

bx

y

a

bx

y

a b

x

y

a

b

x

y

*

*

*

*

*

*

*

LPS - + LPS - +

LPS

LPS

LPS

LPS

LPS

- + - +

- + - +

- +

25

hyperthermia conditions, respectively.

a, b: Significant differences among groups cultured at 38.5°C (P < 0.05).

x, y: Significant differences among groups cultured at 40.5°C (P < 0.05).

*: Significant differences between 38.5°C and 40.5°C in the presence or absence of

LPS (P < 0.05).

26

FIGURE 3

Effect of hyperthermia and LPS on the production of IL-6 (A, C), IL-8 (B, D), and

MCP1 (E) in cultured bovine endometrial epithelial and stromal cells (mean±S.E.M.,

n=8). The open and closed bars indicate the control and hyperthermia conditions,

respectively.

a, b: Significant differences among groups cultured at 38.5°C (P < 0.05).

x, y: Significant differences among groups cultured at 40.5°C (P < 0.05).

*: Significant differences between 38.5°C and 40.5°C in the absence or presence of

LPS (P < 0.05).

0

0.1

0.2

0.3

IL-6

pro

du

ctio

n(n

g/µ

g D

NA

)

Not Detect

Not Detect

ab*

*

0

0.2

0.4

0.6

IL-8

pro

du

ctio

n(n

g/µ

g D

NA

)

a

b

x

y

0

0.4

0.8

1.2

1.6

2

IL-6

pro

du

ctio

n(n

g/µ

g D

NA

)

a bx

y

*

*

0

1

2

3

4

5IL

-8 p

rod

uct

ion

(ng

/µg

DN

A)

abx

y

*

*

0

2

4

6

8

MC

P1

pro

du

ctio

n(n

g/µ

g D

NA

)

abx

y

*

*

A B

C D

E

Epithelial cells

Stromal cells

LPS - + LPS - +

LPS - + LPS - +

LPS - +

27

FIGURE 4

Effect of hyperthermia and LPS on the mRNA expression of TLR4 (A, D), MD2 (B,

E), and CD14 (C, F) in cultured bovine endometrial epithelial and stromal cells (mean

±S.E.M., n=10). Each mRNA expression level was indicated as their copy number

normalized by the expression of GAPDH. The open and closed bars indicate the control

and hyperthermia conditions, respectively.

*: Significant differences between 38.5°C and 40.5°C in the absence or presence of

LPS (P < 0.05).

0

0.4

0.8

1.2

1.6

2

0

1

2

3

4

0

1

2

3

0

0.4

0.8

1.2

1.6

2

0

0.4

0.8

1.2

1.6

2

0

2

4

6

8

10

TLR

4m

RN

ATL

R4

mR

NA

MD

2m

RN

AM

D2

mR

NA

CD

14

mR

NA

CD

14

mR

NA

**

* *

A B

C

D E

F

Epithelial cells

Stromal cells

LPS - +

LPS - +LPS - +

LPS - +

LPS - + LPS - +

28

FIGURE 5

Evaluation of the isolation of monocytes (A) and differentiation of them into MΦs (B,

C). Effect of hyperthermia on the mRNA expression of IL6 Receptor (D) and CCR2 (E)

in MΦs and migration ability of MΦs (F) (mean±S.E.M., n=5). The effect of culture

supernatant of the endometrial stromal cells on attraction of MΦs (G). The concentrated

Nu

mb

er o

f m

igra

ted

MΦ

s

CC

R2

mR

NA

IL6

Rec

epto

rm

RN

A

D E

F

0

0.04

0.08

0.12

0.16

0

0.0004

0.0008

0.0012

0

200

400

600

a x

b y

G

Control HS Control HS

Control MCP1 Nu

mb

er o

f m

igra

ted

MΦ

s

Control LPS

a a

b

c

0

100

200

300

400

500

600

700

800

900

38.5℃ 40.5℃

Supernatant

0

0.2

0.4

0.6

0.8

1

CD

80

mR

NA

Control IFNγ

a

b

A B

C20 µm 50 µm

29

supernatant of endometrial stromal cells under control and hyperthermia conditions was

indicated as 38.5°C and 40.5°C, respectively. Each mRNA expression level was

indicated as their copy number normalized by the expression of GAPDH. The open and

closed bars indicate the control and hyperthermia conditions, respectively.

a, b: Significant differences among groups cultured at 38.5°C (P < 0.05).

x, y: Significant differences among groups cultured at 40.5°C (P < 0.05).

30

31

FIGURE 6

The localization of neutrophils and MΦs in bovine endometrium in summer and

winter. Arrow heads indicate the neutrophils and MΦs (A, B, D and E). The distance

from epithelial layer to neutrophils and MΦs (C and F) (mean±S.E.M., n=6).

Different superscripts indicate significant difference (P<0.05)

D E

F

0

40

80

120

160

a

b

Winter Summer

Dis

tan

ce o

f M

Φs

fro

mep

ith

elia

l lay

er (

μm

)

100 µm 100 µm

32

FIGURE 7

Illustration depicting our conclusion. Hyperthermia increased IL-6, MCP1, and IL-8

production in endometrial stromal cells but suppressed IL-6 production in endometrial

epithelial cells. This disruption of chemokine production in both types of endometrial

cells might cause the alteration of MΦs localization in the endometrium under

hyperthermia conditions.

IL-8

IL-6

IL-8 MCP1

IL-6

IL-8

IL-6

IL-8 MCP1

IL-6

Normal Condition Heat Stress Condition

Epithelial cells (Epithelial layer)

Stromal cells (Stromal layer)

TLR4

Macrophage (MΦ)

Neutrophil

MD-2

E. coli

Lipopolysaccharide (LPS)

CD14

33

DISCUSSION

One type of bacteria that cause endometritis is E. coli, and it localizes in the uterine

lumen and beneath the epithelial layer in the infected uterus [40] To eliminate these

bacteria, immune cells need to be recruited to the epithelial layer [40] and neutrophils

play an important role in the elimination of bacteria, especially [36, 38]. Neutrophils in

peripheral blood recognize the gradient of IL-8 concentrations and are chemoattracted

to the infection site [61, 62]. In the present study, hyperthermia enhanced IL-8

production in bovine endometrial stromal cells (Figure 3D) but did not affect it in

endometrial epithelial cells (Figure 3B). Because IL-8 production in endometrial

epithelial cells, a major site for the recognition of the LPS produced by E. coli, was not

decreased by hyperthermia, the recruitment of neutrophils might not be influenced by

hyperthermia. Indeed, the localization of neutrophils in bovine endometrium was not

changed in summer and winter (Figure 6A, B and C).

Following the recruitment of neutrophils, MΦs are attracted by some chemokines

secreted by endometrial cells. These attracted MΦs contribute to the elimination of

bacteria [36], the orchestration of inflammation [28], the activation of T-cells and

B-cells [29, 46], and tissue repair [38]. In the current study, hyperthermia enhanced the

MCP1 production induced by LPS in endometrial stromal cells (Figure 3E). However,

in endometrial epithelial cells, MCP1 production was not detected under either the

control or hyperthermia conditions regardless of the presence of LPS. Therefore, in the

endometrial epithelial cells, MCP1 might not contribute to the recruitment of MΦs to

endometrial sub-epithelial stroma. Moreover, hyperthermia enhanced the IL-6

production in the endometrial stromal cells (Figure 3C) but suppressed it in the

endometrial epithelial cells (Figure 3A). In addition, hyperthermia enhanced CCL5

34

mRNA expression induced by LPS but did not affect the mRNA expression of CCL3,

CX3CL1, and CXCL10 in the endometrial stromal cells (Figure 3K-N). Moreover, the

supernatant of endometrial stromal cells cultured under hyperthermia conditions

attracted a large number of MΦs (Figure 5G). Taken together hyperthermia might affect

the localization of MΦs in endometrium. Indeed, MΦs were mainly localized

sub-epithelial stroma in winter, whereas they were sparsely localized throughout the

endometrium in summer (Figure 6D-F). However, it is still unclear whether this change

of MΦ localization under hyperthermia condition causes the prolongation of

endometritis symptoms in summer. Therefore, further in vivo study is necessary.

Interestingly, we found that hyperthermia had opposite effects on chemokine

production between endometrial epithelial and stromal cells. We presumed that

difference of bacterial sensing by its specific receptor was involved in this opposite

reaction in both types of endometria cells. However, hyperthermia did not affect the

mechanism for recognition of LPS because hyperthermia upregulated only TLR4 but did

not MD-2 and CD14 in bovine endometrial epithelial and stromal cells (Figure 4A-F).

These results suggest that there is respective different mechanism(s) for the opposite

reaction of chemokine production in endometrial epithelial and stromal cells under

hyperthermia condition. Indeed, similar phenomena are observed in the several types of

neighboring cells. In the bovine endometrial epithelial and stromal cells, Trueperella

pyogenes induces cytolysis of endometrial stromal cells but not of endometrial

epithelial cells, because endometrial stromal cells contain more cholesterol than

endometrial epithelial cells [63]. In addition, under the ischemic and hypoxic stress

conditions, the neuronal cells undergo the apoptosis but astrocytes have tolerate,

nevertheless they are neighboring cells, because astrocytes have unique endoplasmic

35

reticulum stress transducers [64]. Therefore, to get the clue for the improvement of the

local effect of hyperthermia on chemokine production, further studies on the cellular

mechanism(s) in endometrial epithelial and stromal cells, individually, are necessary.

SUMMARY

Our present results suggested that hyperthermia caused alteration of the chemokine

production in bovine endometrial epithelial and stromal cells with the respective

different mechanism. This change of chemokine production might be affect the

recruitment of MΦs and alter their localization in the endometrium in summer (Figure

7).

36

CHAPTER 3

Hyperthermia enhanced IL-6 production via endoplasmic stress (ER

stress) in bovine endometrial stromal cells

INTRODUCTION

In response to the invasion of bacteria into uterus in the postpartum period, bovine

endometrial epithelial and stromal cells secrete the chemokines, such as MCP1, IL-6,

and IL-8, to recruitment the immune cells into infection site. In Chapter 2, we revealed

that hyperthermia suppressed the IL-6 production induced by LPS in endometrial

epithelial cells but enhanced it in endometrial stromal cells. To reveal the mechanism

for this opposite reaction in endometrial epithelial and stromal cells against

hyperthermia, we focused on the LPS recognition by TLR4; however, it was not

involved in the effect of hyperthermia on the alteration of IL-6 production in both types

of endometrial cells. Therefore, in Chapter 3, we investigated the other cellular

mechanisms for the change of IL-6 secretion in bovine endometrial epithelial and

stromal cells under hyperthermia conditions.

The endoplasmic reticulum (ER) is an organelle in which large amount of proteins is

synthesized, folded, and modified. Several external stresses induce the accumulation of

misfolded proteins in ER and disrupt the homeostasis of ER, which is called as ER

stress [65, 66]. In response to ER stress, three distinct sensors, Inositol requiring 1

(IRE1), PKR-like endoplasmic reticulum kinase (PERK), and Activating transcription

factor 6 (ATF6), are activated [67-69]. Under the non-stress conditions, each sensor

molecule forms a complex with BiP, an ER chaperon, to keep them inactive. However,

under stress conditions, BiP dissociates from these sensors and shows its chaperon

function to modify the misfolded proteins, followed by the activation of IRE1, PERK,

and ATF6 [67, 70, 71]. Especially, in the downstream of IRE1, X-box binding protein 1

37

(XBP1) is spliced and activated, resulting in the upregulation of BiP expression [67].

The activation of PERK suppresses the de novo protein translation [72] and induces the

apoptosis via activating transcription factor 4 (ATF4) [73]. In addition, it is reported that

ER stress contributes to the immune response in several types of cells [74, 75]. Both

IRE1 and PERK regulate NF-κB activation and alter the inflammatory cytokine

expression, such as IL-6 [76-80].

Hyperthermia causes the several alterations at cellular level, such as the mobility of

membrane phospholipid [18], the production of reactive oxygen species [19] , arrest of

cell growth [20] , and induction of cell death [19] , resulting in the disruption of cellular

homeostasis. These alterations are caused by the protein denaturation and aggregation

under hyperthermia conditions [81, 82]. In response to this denaturation of proteins

induced by hyperthermia, heat shock protein (HSP), one of the chaperon proteins, is

increased in cells and it controls the cellular homeostasis. This cellular reaction under

hyperthermia conditions is similar to the ER chaperons which are increased during ER

stress. Moreover, hyperthermia stimulates the three distinct ER stress sensors and

induces the ER stress in several types of cells [81, 82].

Therefore, in Chapter 3, we hypothesized that hyperthermia induces ER stress,

resulting in the alteration of IL-6 production in two types of bovine endometrial cells.

To test this hypothesis, we investigated 1) whether hyperthermia stimulated the ER

stress in bovine endometrial epithelial and stromal cells, 2) the effect of ER stress

inhibitor and activator on the IL6 mRNA expression and IL-6 production in bovine

endometrial stromal cells, and 3) the effect of ER stress inhibitor on the IL6 mRNA

expression in bovine endometrial epithelial cells.

38

MATERIALS AND METHODS

Collection of bovine uteri, endometrial cell isolation, and cell culture

Collection of bovine uteri, endometrial cell isolation, and cell culture were conducted

as described in MATERIALS AND METHODS of chapter 2 (Page 9-10). For the

analysis of each mRNA expression and IL-6 concentration, both types of endometrial

cells were cultured in 24-well culture plates. For the analysis of each protein expression,

two types of endometrial cells were cultured in 75 cm2 flasks.

Total RNA extraction and quantitative RT-PCR

Total RNA extraction and quantitative RT-PCR were conducted as described in

MATERIALS AND METHODS of chapter 2 (Page 11-12). Specific primers for BiP,

un-spliced XBP1 (uXBP1), spliced XBP1 (sXBP1), ATF4, IL6, and GAPDH mRNA

were showed in Table 2.

Protein fractionation and extraction

For protein analysis of BiP in both types of endometrial cells, total cell lysates were

extracted. After culture, endometrial epithelial and stromal cells were collected using

homogenization buffer (300 mM sucrose, 32.5 mM Tris-HCl, 2mM EDTA, 20 mM NaF,

10 mM Sodium Orthovanadate(V), protease inhibitor cocktail (1697498; Roch,

Mannheim, Germany), pH 7.4) and centrifuged at 2330×g for 10 min. After the

centrifugation, two types of endometrial cells were lysed in lysis buffer (20 mM

Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 20 mM NaF,

10 mM Sodium Orthovanadate(V), and protease inhibitor cocktail). Their protein

concentrations were determined by BCA method [83].

39

Western blotting

Total cell lysates, nuclei fraction, and cytoplasmic fraction were mixed with SDS

gel-loading buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 1% β mercaptoethanol,

pH 6.8) and heated at 95°C for 10 min. Each sample (50 µg protein/lane) was separated

on 9% SDS-PAGE, and then transferred to a PVDF membrane (RPN303F; GE

Healthcare., Little Chalfont, Buckinghamshire, UK). The membrane was washed in

TBS-T (0.1% Tween 20 in TBS (25 mM Tris-HCl, 137 mM NaCl, pH 7.5)), followed by

the incubation in 5% nonfat dry milk in TBS-T for 1 h at room temperature. After

washing, the membranes were incubated with BiP antibody diluted at 1:1000 (#3183,

Cell Signaling Technology, MA, USA) and β-actin antibody diluted at 1:10000 (A2228,

Sigma-Aldrich) for overnight at 4°C. After washing with TBS-T, the membranes were

incubated with the appropriate secondary antibodies for 1 h at room temperature. For

the analysis of BiP, the membrane was incubated with HRP-linked donkey anti-rabbit Ig

(NA934; Amersham Biosciences Corp., San Francisco, CA, USA) diluted at 1:10000.

For analysis of β-actin, the membranes were incubated with HRP-linked sheep

anti-mouse IgG (NA931; Amersham Biosciences Corp.) diluted at 1:20000 for β-actin.

After washing again with TBS-T, the signal was detected by chemiluminescence using

Immobilon Western Chemiluminescent HRP Substrate (P36599; Millipore, Billerica,

MA, USA). The protein expression of β-actin was used as an internal control for the

experiments using total cell lysates. The intensity of the immunological reaction in the

endometrial cells was estimated by measuring the optical density in the defined area by

computerized densitometry using Inage LabTM Software version 4.0 (Bio-Rad

Laboratories, Inc, Berkeley, CA, USA).

40

Enzyme immunoassay (EIA)

The concentration of IL-6 in supernatant of endometrial stromal cells was measured

by EIA as described in MATERIALS AND METHODS of chapter 2 (Page 12-13). The

intra- and inter-assay coefficients of variation, on average, for IL-6 were 7.5% and 5.8%,

respectively.

DNA assay

DNA assay was conducted as described in MATERIALS AND METHODS of chapter

2 (Page 13).

Experimental design

Experiment 1: Effects of hyperthermia on the ER stress in the endometrial epithelial

and stromal cells

Bovine endometrial epithelial and stromal cells were cultured under control or

hyperthermia conditions, as described in MATERIALS AND METHODS of chapter 2.

Briefly, as the non-hyperthermia treatment, endometrial epithelial and stromal cells

were cultured at 38.5°C for 34 h, followed by the LPS challenge (1 µg/ml) (LPS from

Escherichia coli O55:B5; Sigma-Aldrich, L6529) for 6 h at 38.5°C. As the hyperthermia

treatment, endometrial epithelial and stromal cells were cultured at 40.5°C for 10 h,

allowed to recover at 38.5°C for 14 h, and then cultured at 40.5°C for 10 h again, this

was followed by the LPS challenge (1 µg/ml) for 6 h at 40.5°C. After the LPS challenge,

the cells were harvested to measure the mRNA expression of BiP, uXBP1, sXBP1, and

ATF4 and to measurement the protein expression of BiP.

41

Experiment 2: Effect of ER stress inhibitors on the mRNA expression of IL6 in the

endometrial epithelial and stromal cells cultured under hyperthermia conditions

Bovine endometrial epithelial and stromal cells were cultured under control or HS

conditions with each ER stress inhibitor, Salubrinal (SML0951, Sigma-Aldrich),

Toyocamycin (17371, Cayman Chemical Company, MI, USA), and KIRA6 (19151,

Cayman Chemical Company). In the present study, Salubrinal, Toyocamycin, and

KIRA6 were used as inhibitors for PERK, RNase domain of IRE1, and Kinase domain

of IRE1, respectively. As the non-hyperthermia treatment, endometrial epithelial and

stromal cells were cultured at 38.5°C for 34 h with Salubrinal (0, 0.4, and 4 mM),

Toyocamycin (0, 0.01, and 0.1 µM), or KIRA6 (0, 0.01, and 0.1 µM), followed by the

LPS challenge (1 µg/ml) in the presence or absence of each ER stress inhibitor for 6 h at

38.5°C. As the hyperthermia treatment, endometrial epithelial and stromal cells were

cultured at 40.5°C for 10 h, allowed to recover at 38.5°C for 14 h, and then cultured at

40.5°C for 10 h again, with each ER stress inhibitor, this was followed by the LPS

challenge (1 µg/ml) in the presence or absence of each ER stress inhibitor for 6 h at

40.5°C. After LPS and each ER stress inhibitor treatment, the cells were harvested to

measure the mRNA expression of IL6.

Experiment 3: Effect of ER stress inhibitors on the IL-6 secretion in the endometrial

stromal cells cultured under hyperthermia conditions

The bovine endometrial stromal cells were cultured under control and hyperthermia

conditions as same method in Experiment 2; however they were treated by Salubrinal,

Toyocamycin, and KIRA6 in the presence of LPS for 12 h for measurement of the

concentration of IL-6 in culture supernatant by EIA. The concentration of IL-6 was

42

normalized by defending as the IL-6 concentration per µg DNA.

Experiment 4: Effect of ER stress activator, NaF, on the IL6 mRNA expression and

IL-6 production in the endometrial stromal cells cultured under control and

hyperthermia conditions

Bovine endometrial stromal cells were cultured under control or hyperthermia

conditions with NaF (31420-82, Nacalai tesque, Kyoto, Japan) as an ER stress activator.

As the non-hyperthermia treatment, endometrial epithelial and stromal cells were

cultured at 38.5°C for 34 h with NaF (0, 0.1, and 1 mM), followed by the LPS challenge

(1 µg/ml) in the presence or absence of NaF (0, 0.1, and 1 mM) for 6 h at 38.5°C. As the

hyperthermia treatment, endometrial epithelial and stromal cells were cultured with NaF

(0, 0.1, and 1 mM) at 40.5°C for 10 h, allowed to recover at 38.5°C for 14 h, and then

cultured at 40.5°C for 10 h again, this was followed by the LPS challenge (1 µg/ml) in

the presence or absence of NaF (0, 0.1, and 1 mM) for 6 h at 40.5°C. After LPS and

NaF treatment, the cells were harvested to measure the mRNA expression of IL6. In

addition, to measure the IL-6 concentration by EIA, endometrial stromal cells were

cultured under control and hyperthermia conditions as described above; however they

were treated by LPS (1 µg/ml) and NaF (1 mM) for 12 h. The concentration of IL-6 was

normalized by defending as the IL-6 concentration per µg DNA.

Statistical analysis

All experimental data is shown as the mean ± S.E.M. All the data was tested for

normal distribution, and the homogeneity of variance to determine whether it was

43

parametric or non-parametric data. The statistical analysis was performed using

GraphPad Prism (GraphPad Software). Student t-test was conducted on the data from

Experiment 1. The data from Experiment 2-4 was firstly assessed using two-way

ANOVA. When an interaction between the additive reagents and hyperthermia

treatment was not detected, each data set was assessed using one-way ANOVA and

Student’s t-test for multi-group and two-group comparison, respectively. Probabilities

less than 5% (P<0.05) were considered statistically significant.

RESULTS

Experiment 1: Effect of hyperthermia on the ER stress in the endometrial epithelial

and stromal cells

In Experiment 1, to investigate whether hyperthermia induces the ER stress in the

bovine endometrial epithelial and stromal cells, we measured the mRNA expression of

major ER stress markers, such as BiP, ATF4, and sXBP1.

As shown in Figure 8, hyperthermia did not affect the mRNA expression of BiP,

ATF4, and sXBP1 in the bovine endometrial epithelial cells (Figure 8A-C). Whereas, the

in endometrial stromal cells, hyperthermia significantly (P<0.05) increased the mRNA

expression of BiP, sXBP1 and ATF4 (Figure 8E and G).

Furthermore, we revealed that hyperthermia significantly (P<0.05) increased the

protein expression of BiP in endometrial stromal cells (Figure 8H) but not affect in

endometrial epithelial cells (Figure 8D).

Experiment 2: Effect of ER stress inhibitors on the mRNA expression of IL6 in the

endometrial epithelial and stromal cells cultured under hyperthermia conditions

44

In experiment 2, we investigated whether ER stress was involved in the alteration of

IL6 mRNA expression in bovine endometrial epithelial and stromal cells under

hyperthermia conditions.

As shown in Figure 9, in endometrial epithelial cells, hyperthermia significantly

(P<0.05) suppressed the IL6 mRNA expression in the presence of LPS (Figure 9A-C).

Toyocamycin (0.1 µM) and KIRA6 (0.01 and 0.1 µM) significantly (P<0.05)

suppressed the IL6 mRNA expression under control conditions but both of ER stress

inhibitors did not affect IL6 mRNA expression under hyperthermia conditions (Figure

9A and B). In addition, under the Toyocamycin (0.1 µM) and KIRA6 (0.01 and 0.1 µM)

treatment, there was no difference of IL6 mRNA expression between control and

hyperthermia conditions in endometrial epithelial cells (Figure 9A and B). Salubrinal

affected the IL6 mRNA expression under neither control nor hyperthermia conditions in

the endometrial epithelial cells (Figure 9C).

In endometrial stromal cells, all of ER stress inhibitors, Toyocamycin (0.1 µM),

KIRA6 (0.1 µM), and Salubrinal (4 mM), significantly (P<0.05) suppressed the IL6

mRNA expression under hyperthermia conditions (Figure 9D-F). There was no

difference of IL6 mRNA expression between control and hyperthermia conditions under

Toyocamycin (0.01 and 0.1 µM) and KIRA6 (0.01 and 0.1 µM) treatment, however, the

IL6 mRNA expression was significantly (P<0.05) higher under hyperthermia conditions

compared with control conditions in the presence of Salubrinal (0.4 and 4 mM) (Figure

9F). Under control conditions, Toyocamycin (0.1 µM) significantly (P<0.05) suppressed

the IL6 mRNA expression but KIRA6 and Salubrinal did not affect it in endometrial

stromal cells (Figure 9D-F).

45

Experiment 3: Effect of ER stress inhibitors on the IL-6 secretion in the endometrial

stromal cells cultured under hyperthermia conditions

Since, in Figure 1, we showed that hyperthermia was not involved in the expression

of ER stress related factors in the endometrial epithelial cells, we examined the effect of

ER stress inhibitors on the IL-6 production only in endometrial stromal cells under

hyperthermia conditions in Experiment 3.

As shown in Figure 10, Toyocamycin, KIRA6 and Salubrinal significantly (P<0.05)

suppressed the IL-6 production under not only control but also hyperthermia conditions

in the endometrial stromal cells (Figure 10A, B and C). Whereas the IL-6 production

was significantly (P<0.05) higher under hyperthermia compared with control conditions

in the absence of KIRA6 and Toyocamycin, it was not different between control and

hyperthermia conditions in their presence (Figure 10A and B). The IL-6 production was

significantly higher (P<0.05) under hyperthermia condition compared with control

conditions regardless of the presence of Salubrinal (Figure 10C).

Experiment 4: Effect of ER stress activator, NaF, on the IL6 mRNA expression and

IL-6 production in the endometrial stromal cells cultured under control and

hyperthermia conditions

Since hyperthermia did not induce the ER stress in the endometrial epithelial cells, we

investigated the effect of ER stress activator, NaF, on the IL6 mRNA expression and

IL-6 production only in the endometrial stromal cells in Experiment 4.

As shown in Figure 11, NaF (1 mM) significantly (P<0.05) increased the IL6 mRNA

expression under control conditions but did not change it under hyperthermia conditions

in the endometrial stromal cells (Figure 11A). In addition, IL6 mRNA expression was

46

higher (P<0.05) under hyperthermia compared with control conditions in the presence

at lower concentration or absence of NaF (0.1 or 0 mM); however, in the presence of

NaF at higher concentration (1 mM), IL6 mRNA expression under control conditions

increased significantly (P<0.05) and reached to same level of that under hyperthermia

conditions in endometrial stromal cells (Figure 11A).

In endometrial stromal cells, NaF significantly (P<0.05) increased the IL-6

production under control and hyperthermia conditions (Figure 11B). The secretion level

of IL-6 was higher (P<0.05) under hyperthermia conditions than control conditions in

the absence of NaF; however, IL-6 production was not different between under control

and hyperthermia conditions in the presence of NaF (Figure 11B).

47

Genes Forward and reverse primers Accession No. Annealing temperature

5'-GTGCCCACCAAGAAGTCTCA-3'

5'-CTTTCGTCAGGGGTCGTTCA-3'

5'-CAGACTACGTGCACCTCTGC-3'

5'-CTGGGTCCAAGTTGAACAGAAT-3'

5'-GCTGAGTCCGCAGCAGGT-3'

5'-CTGGGTCCAAGTTGAACAGAAT-3'

5'-GAGTTAAGCACCAAAACCTC-3'

5'-ATCCATTTTCTCCACCATCC-3'

5'-AACCACTGCTGGTCTTCTGG-3'

5'-GGTCAGTGTTTGTGGCTGGA-3'

5'-CTCTCAAGGGCATTCTAGGC-3'

5'-TGACAAAGTGGTCGTTGAGG-3'

60℃

GAPDH NM_001034034.2 60℃

sXBP1

uXBP1

BiP

Table 2. Sequences of primers used for quantitative RT-PCR analysis

NM_173923.2

NM_001075148.1

NM_001034727.3

NM_001271737.1

IL6

ATF4 NM_001034342.2

60℃

60℃

60℃

60℃

48

FIGURE 8

Effect of hyperthermia on the mRNA expression of sXBP1 (A and E), ATF4 (B and

F), and BiP (C and G) and BiP protein level (D and H) in endometrial epithelial and

stromal cells in the presence of LPS (mean±S.E.M., n=10-15). The mRNA expression

level of sXBP1 was indicated as their copy number normalized by the expression of

uXBP1 and the mRNA expression level of ATF4 and BiP was indicated as their copy

number normalized by the expression of GAPDH. The protein level of BiP was

Epithelial cells

0

0.05

0.1

0.15

0.2

0.25

sXB

P1

/uX

BP

1m

RN

A

38.5℃ 40.5℃

A

00.020.040.060.080.1

0.120.14

ATF4

mR

NA

38.5℃ 40.5℃

B

0

5

10

15

20

25

BiP

mR

NA

38.5℃ 40.5℃

C

0

0.02

0.04

0.06

0.08

0.1

sXB

P1

/uX

BP

1m

RN

A

38.5℃ 40.5℃

E

0

0.01

0.02

0.03

0.04

ATF

4 m

RN

A

38.5℃ 40.5℃

F

010203040506070

BiP

mR

NA

38.5℃ 40.5℃

G

Stromal cells

a

ba

b

a

b

00.20.40.60.81

1.21.4

BiP

pro

tein

38.5℃ 40.5℃

H

a

b BiP

B actin

0

0.2

0.4

0.6

0.8

1

1.2

BiP

pro

tein

38.5℃ 40.5℃

DBiP

B actin

49

normalized by the protein level of B actin. The open and closed bars indicate the control

and hyperthermia conditions, respectively.

a, b: Significant differences between 38.5°C and 40.5°C (P < 0.05).

50

FIGURE 9

Effect of each ER stress inhibitor on the mRNA expression of IL6 in cultured bovine

endometrial epithelial (A-C) and stromal (D-F) cells under the hyperthermia conditions

in the presence of LPS (mean±S.E.M., n=10). The mRNA expression level of IL6 was

indicated as their copy number normalized by the expression of GAPDH. The open and

Epithelial cells

Toyocamycin0

0.1

0.2

0.3

0.4

0.5

0 µM 0.01 µM 0.1 µM

IL6

mR

NA a

ab

b

**

A

0

0.1

0.2

0.3

0.4

KIRA6 0 µM 0.01 µM 0.1 µM

IL6

mR

NA

ab b

*B

0

0.1

0.2

0.3

0.4

0.5

Salubrinal 0 mM 0.4 mM 4 mM

IL6

mR

NA

*C

00.05

0.10.15

0.20.25

0.30.35

Toyocamycin 0 µM 0.01 µM 0.1 µM

IL6

mR

NA

Stromal cells

a ab

xx

y

*D

00.05

0.10.15

0.20.25

0.30.35

KIRA6 0 µM 0.01 µM 0.1 µM

IL6

mR

NA

x

xyy

*E

0

0.05

0.1

0.15

0.2

0.25

0.3

Salubrinal 0 mM 0.4 mM 4 mM

IL6

mR

NA

xxy

y

**

*

F

51

closed bars indicate the control and hyperthermia conditions, respectively.

a, b: Significant differences among groups cultured at 38.5°C (P < 0.05).

x, y: Significant differences among groups cultured at 40.5°C (P < 0.05).

*: Significant differences between 38.5°C and 40.5°C in the same concentration of

ER stress inhibitor (P < 0.05).

52

FIGURE 10

Effect of each ER stress inhibitor on the IL-6 production in cultured bovine

endometrial stromal cells under the hyperthermia conditions in the presence of LPS

(A-C) (mean±S.E.M., n=7). The open and closed bars indicate the control and

hyperthermia conditions, respectively.

Stromal cells

0

0.2

0.4

0.6

0.8

IL-6

pro

du

ctio

n(n

g/µ

g D

NA

)

Toyocamycin 0 µM 0.1 µM

*

a

b

x

y

A

00.040.080.120.16

0.2

IL-6

pro

du

ctio

n(n

g/µ

g D

NA

)

KIRA6 0 µM 0.1 µM

a

b

x

y

*B

IL-6

pro

du

ctio

n(n

g/µ

g D

NA

)

Salubrinal 0 mM 4 mM

C

a

b

x

y

*

*

00.10.20.30.40.50.6

53

a, b: Significant differences among groups cultured at 38.5°C (P < 0.05).

x, y: Significant differences among groups cultured at 40.5°C (P < 0.05).

*: Significant differences between 38.5°C and 40.5°C in the same concentration of

ER stress inhibitor (P < 0.05).

54

FIGURE 11

Effect of NaF on the mRNA expression of IL6 (A) and IL-6 production (B) in

endometrial stromal cells under the hyperthermia conditions in the presence of LPS

(mean±S.E.M., n=6-10). The mRNA expression level of IL6 was indicated as their

copy number normalized by the expression of GAPDH. The open and closed bars

indicate the control and hyperthermia conditions, respectively.

a, b: Significant differences among groups cultured at 38.5°C (P < 0.05).

x, y: Significant differences among groups cultured at 40.5°C (P < 0.05).

0

0.1

0.2

0.3

NaF 0 mM 0.1 mM 1 mM

aa

b* *

IL6

mR

NA

A

0

0.1

0.2

0.3

0.4

NaF 0 mM 1 mM

a

bx

y*

IL-6

pro

du

ctio

n(n

g/µ

g D

NA

)

B

55

*: Significant differences between 38.5°C and 40.5°C in the same concentration of

NaF (P < 0.05).

56

DISCUSSION

Endoplasmic reticulum stress is induced by several external factors including the

hyperthermia [7, 78, 81, 82, 84]. Under the ER stress conditions, the expression of BiP,

sXBP1, and ATF4 is increased to deal with the impairment in ER homeostasis [67,

70-73]. In the present study, we revealed that hyperthermia increased the mRNA

expression of BiP, sXBP1, and ATF4 in bovine endometrial stromal cells (Figure 8E-G).

In addition, the protein level of BiP was also increased in endometrial stromal cells

under hyperthermia conditions (Figure 8H). Therefore, our results suggested that

hyperthermia induced the ER stress in bovine endometrial stromal cells.

We also confirmed whether ER stress induced the IL-6 production in endometrial

stromal cells using an ER stress activator, NaF [85]. In in vitro study of ER stress,

tunicamycin and thapsigargin are generally used as an ER stress activator [86-88]. In the

present study, we conducted preliminary experiment and clarified that tunicamycin

induced cell death in endometrial stromal cells (data not shown). In addition,

thapsigargin suppressed the release of Ca2+

from ER, resulting in the activation of ER

stress; however, the release of Ca2+

from ER directly influences cytokine production.

Therefore, we considered that, in the present study, tunicamycin and thapsigargin were

not appropriate for ER stress activators. In the present study, NaF significantly

stimulated the IL6 mRNA expression in the endometrial stromal cells under control

conditions but not affected that under hyperthermia conditions (Figure 11A). In addition,

the IL6 mRNA expression induced by NaF under control conditions was same level

with that under hyperthermia conditions (Figure 11A). Moreover, IL-6 production under

control conditions was significantly enhanced by NaF and reached to same level of that

under hyperthermia conditions (Figure 11B). Taken together, our results suggested that

57

ER stress was involved in the upregulation of IL-6 production in endometrial stromal

cells under hyperthermia conditions.

Under the ER stress conditions, three distinct sensors are activated [64, 67, 69].

Especially, activation of both IRE1 and PERK signal regulates the activity of NFκB and

MAPK, respectively, resulting in the increase of cytokine production, such as IL-6,

MCP1, and IL-8 [76-80, 88]. In the present study, two types of IRE1 signal inhibitors

and PERK signal inhibitor, Toyocamycin, KIRA6, and Salubrinal, suppressed the IL6

mRNA expression induced by hyperthermia in endometrial stromal cells (Figure 9D-F).

These results suggested that IRE1 and PERK signals were involved in the upregulation

of IL6 mRNA expression in endometrial stromal cells under hyperthermia conditions.

Moreover, the IL6 mRNA expression under hyperthermia conditions was suppressed by

Toyocamycin and KIRA6 to the same level as that under control condition; however, in

the presence of Salubrinal (4 µM), the IL6 mRNA expression under hyperthermia

conditions was higher than that under control conditions (Figure 9D-F). In addition,

Toyocamycin, KIRA6, and Salubrinal significantly suppressed the IL-6 production

stimulated by hyperthermia (Figure 10A-C). However, these inhibitors also suppressed

the IL-6 production under control conditions (Figure 10A-C). Since LPS alone

stimulates the ER stress in goat endometrial stromal cells [80], in the present study, ER

stress inhibitors might suppress the ER stress stimulated by LPS, followed by the

decrease of IL-6 production in bovine endometrial stromal cells under control

conditions. Moreover, the IL-6 production under hyperthermia conditions was same

level as that under control conditions in the presence of Toyocamycin and KIRA6;

however, the IL-6 production was still higher under hyperthermia conditions compared

with that under control conditions in the presence of Salubrinal in the endometrial

58

stromal cells (Figure 10A-C). Taken together, our present results suggested that the

IRE1 signal was mainly involved in the increase of IL-6 production under hyperthermia

conditions in endometrial stromal cells.

On the other hand, hyperthermia did not affect the mRNA expression and protein

level of ER stress markers in endometrial epithelial cells (Figure 8A-D). In addition,

Toyocamycin, KIRA6, and Salubrinal did not recover the decrease of IL6 mRNA

expression in endometrial epithelial cells under hyperthermia conditions (Figure 9A-C).

These results suggested that ER stress was not involved in the attenuate effect of

hyperthermia on the IL6 mRNA expression in endometrial epithelial cells. Therefore,

the other factors might be involved in the suppression of IL6 mRNA expression in

endometrial epithelial cells. Since we could not reveal these factors in the present study,

further studies are necessary.

SUMMARY

In conclusion, hyperthermia might induce the ER stress in bovine endometrial stromal

cells but not in endometrial epithelial cells. The induction of ER stress under

hyperthermia conditions might be involved in the upregulation of IL-6 production in the

endometrial stromal cells.

59

CHAPTER 4

Comprehensive analysis of gene expression patterns

in bovine endometrial epithelial and stromal cells

under hyperthermia conditions

INTRODUCTION

Under hyperthermia conditions, IL-6 production was suppressed in bovine

endometrial epithelial cells but enhanced in bovine endometrial stromal cells [89]. In

Chapter 3, we revealed that ER stress induced by hyperthermia was involved in the

upregulation of IL-6 production in bovine endometrial stromal cells. On the other hand,

we did not clarify the mechanism for the downregulation of IL-6 production under

hyperthermia conditions in bovine endometrial epithelial cells.

Hyperthermia affects the various cellular functions via several signal cascades. For

example, the cells are able to sense the hyperthermia by transient receptor potential

(TRP), resulting in the increase of Ca2+

influx [90, 91]. The increase of Ca2+

under

hyperthermia condition affects the cell proliferation cycle and cell death via p53 and

MAPK [92]. In addition, under hyperthermia conditions, accumulation of reactive

oxygen species (ROS) causes the increase of misfolded proteins [19, 93, 94]. In

response to this accumulation of misfolded proteins, heat shock protein (HSP), whose

expression increased under hyperthermia conditions, acts as a chaperon protein to

restore these misfolded proteins [95]. Moreover, hyperthermia activates heat shock

factor 1 (HSF1) as a transcription factor and then it binds to heat shock element (HSE)

[96]. Since some genes, such as IL6 and COX2, contain the HSE in their promoter

region, HSF1 directly regulates their gene expression [96-99].

Cytokine production, such as IL-6, MCP1, and IL-8, is also regulated by various

60

factors. AP-1 activated via MAPK and NFκB are well known as transcription factors for

regulation of cytokine production [100-102]. The activation of MAPK and NFκB is

caused by the ROS accumulation, increase of Ca2+

influx, ER stress, and so on [78, 100,

103]. In addition, the suppression of mTOR activity is involved in the inhibition of

4EBP1, resulting in the decrease of IL-6 production [104]. Moreover, HSF1 acts as a

transcription factor and causes both increase and decrease of IL6 mRNA expression in

some types of cells [98].

As described above, hyperthermia intricately relates with the regulation of cytokine

production, such as IL-6, MCP1, and IL-8. Indeed, in the study of microarray,

mammary epithelial cells of buffalo cultured at 42°C alter the approximately 20000

genes (fold change > 2), including the IL6, TNFα, and NFκB [105].Therefore, to reveal

which factors and pathways were involved in the alteration of IL-6 production in two

types of endometrial cells under hyperthermia conditions, we conducted the

comprehensive analysis of gene expression patterns in bovine endometrial epithelial and

stromal cells under hyperthermia conditions.

MATERIALS AND METHODS

Collection of bovine uteri, endometrial cell isolation, and cell culture

Collection of bovine uteri, endometrial cell isolation, and cell culture were conducted

as described in MATERIALS AND METHODS of chapter 2 (Page 9-10). For the

analysis of the alteration of global gene expression, bovine endometrial epithelial and

stromal cells were cultured in 4-well culture plates under control and hyperthermia

conditions, as described in MATERIALS AND METHODS of chapter 2. Briefly, as the

non-hyperthermia treatment, endometrial epithelial and stromal cells were cultured at

61

38.5°C for 34 h, followed by the LPS challenge (1 µg/ml) (LPS from Escherichia coli

O55:B5; Sigma-Aldrich, L6529) for 6 h at 38.5°C. As the hyperthermia treatment,

endometrial epithelial and stromal cells were cultured at 40.5°C for 10 h, allowed to

recover at 38.5°C for 14 h, and then cultured at 40.5°C for 10 h again, this was followed

by the LPS challenge (1 µg/ml) for 6 h at 40.5°C. The cultured endometrial epithelial

and stromal cells were frozen and stored at -80°C until RNA extraction.

Transcriptome analysis

Three each of endometrial epithelial and stromal cells were collected from cows

which estrous cycle was Stage IV. These two types of endometrial cells were cultured as

described above and used for RNA-seq.

Total RNA extraction was conducted using the RNeasy Mini kit (74134, Qiagen,

Redwood City, CA, USA), according to the manufacturer’s instructions. The optical

density A260/280 of RNA was measured by NanoDrop LITE (Thermo Fisher Scientific)

and its value was approximately 2. The RNA integrity number (RIN) was assessed by

Agilent 2100 Bioanalyzer (Agilent Thechnologies) and its value was approximately 9.

To produce the cDNA libraries, 500 ng total RNA was used for mRNA isolation using

the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), then

the library quality and quantity were determined using the Agilent 2100 Bioanalyzer

and the Kapa Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA),

respectively. Each cDNA library was sequenced on the NextSeq 500 (Illumina) using a

High Output paired end 2 × 75 bp run (NextSeq 500/550 High Output Kit v2.5,

Illumina). After base calling by RTA version 2.4.11, Fastq file was generated using

bcl2fastq version 2.18.0.12 (Illumina), according to the manufacturer’s instructions.

62

Sequence data was filtered to discard adapter sequences, ambiguous nucleotides, and

low-quality sequences. To count sequence reads, the remaining sequence data was

aligned to the Bos Taurus genome sequence (ARS-UCD1.2/bosTau9). The expression

values for each gene and statistical analysis of differentially expressed genes were

determined using mapped sequence data. Filtering, mapping, and subsequent analysis

were performed using the CLC Genomics Workbench software (Qiagen). Statistical

significance was determined by empirical analysis using DGE tool. Differentially

expressed genes (P < 0.05 or Fold Change >1.2) were used for further analyses.

RESULTS

Differentially expressed genes in two types of endometrial cells between under

hyperthermia and control conditions

The results of Principal Component Analysis (PCA) indicated that altered genes

formed the cluster between endometrial epithelial and stromal cells, regardless of the

hyperthermia (Figure 12). In addition, when endometrial epithelial and stromal cells

were separately analyzed, altered genes formed the cluster among each cow but not

formed the cluster between control and hyperthermia conditions (Figure 13A and B). As

shown in the Table 3, we found that the expression of 112 genes and 101 genes was

significantly (P<0.05) increased and decreased, respectively, under hyperthermia

conditions in endometrial epithelial cells. On the other hand, in endometrial stromal

cells, hyperthermia significantly (P<0.05) increased and decreased the expression of

169 genes and 227 genes, respectively. However, in endometrial epithelial and stromal

cells, heat map indicated that altered gene expression by hyperthermia depended on

each cows (Figure 14A and B). These genes were analyzed using a functional

63

annotation tool (DAVID software, https://david.ncifcrf.gov/). As shown in the Table 4,

up-regulated genes in endometrial epithelial cells under hyperthermia conditions were

annotated in protein processing in endoplasmic reticulum, estrogen signaling pathway,

thyroid hormone synthesis, antigen processing and presentation, and TGF-beta signaling

pathway, on the other hand, down-regulated genes in endometrial epithelial cells under

hyperthermia conditions were annotated in metabolic pathways, cytokine-cytokine

receptor interaction, TNF signaling pathway, and insulin resistance. In endometrial

stromal cells, up-regulated genes under hyperthermia conditions were annotated in

protein processing in endoplasmic reticulum, estrogen signaling pathway, TNF

signaling pathway, antigen processing and presentation, and NF-kappa B signaling

pathway, whereas; down-regulated genes under hyperthermia conditions were annotated

in cAMP signaling pathway, metabolic pathways, and neuroactive ligand-receptor

interaction.

Next, when we set the fold-differences as 1.3 times, we could not find genes

differently expressed between the endometrial epithelial and stromal cells. Therefore,

we set the fold-difference as 1.2 times for selection of genes differently expressed

between the endometrial epithelial and stromal cells, and then analyzed them. The heat

map of these genes showed that the gene expression changed differently between two

types of endometrial cells under hyperthermia conditions formed the cluster among each

cow (Figure 14C). In addition, we analyzed these oppositely regulated genes using a

functional annotation tool. As shown in Table 5, genes which were up-regulated in

endometrial epithelial cells but down-regulated in endometrial stromal cells under

hyperthermia condition (312 genes) were annotated in ECM-receptor interaction,

vascular smooth muscle contraction, focal adhesion, PI3K-Akt signaling pathway,

64

TGF-beta signaling pathway, platelet activation, Wnt signaling pathway, signaling

pathways regulating pluripotency of stem cells, calcium signaling pathway, and

cytokine-cytokine receptor interaction. On the other hand, genes which were

down-regulated in endometrial epithelial cells but up-regulated in endometrial stromal

cells under hyperthermia condition (141 genes) were annotated in cytokine-cytokine

receptor interaction, Transcriptional misregulation in cancer, Hematopoietic cell lineage,

Jak-STAT signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway,

ErbB signaling pathway, NF-kappa B signaling pathway, and Bladder cancer.

65

FIGURE 12

The PCA was conducted in the both types of endometrial cells under control and

hyperthermia conditions.

66

FIGURE 13

The PCA was separately conducted in the endometrial epithelial (A) and stromal (B)

cells under control and hyperthermia conditions.

A Epithelial cell

Stromal cellB

67

Cell type Up-regulation Down-regulation

Epithelial cells 112 101

Stromal cells 169 227

Up; Fold change > 1.2, Down; Fold change < -1.2

Table 3. The number of differentially expressed genes in endometrial

epithelial and stromal cells under hyperthermia conditions

68

A

69

B

70

FIGURE 14

Heat map indicated the altered gene expression in endometrial epithelial (A) and

stromal (B) cells under hyperthermia conditions. Heat map indicated the gene

expression changed differently between two types of endometrial cells under

hyperthermia conditions (C).

C

71

72

Term P -Value

ECM-receptor interaction 0.0016

Vascular smooth muscle contraction 0.0076

Focal adhesion 0.0108

PI3K-Akt signaling pathway 0.0294

TGF-beta signaling pathway 0.0326

Platelet activation 0.0376

Wnt signaling pathway 0.0493

Signaling pathways regulating pluripotency of stem cells 0.0519

Calcium signaling pathway 0.0574

Cytokine-cytokine receptor interaction 0.0982

Cytokine-cytokine receptor interaction 0.0013

Transcriptional misregulation in cancer 0.0019

Hematopoietic cell lineage 0.0053

Jak-STAT signaling pathway 0.0059

TNF signaling pathway 0.0093

PI3K-Akt signaling pathway 0.0163

ErbB signaling pathway 0.0281

NF-kappa B signaling pathway 0.0325

Bladder cancer 0.0375

Table 5. Functional annotation (KEGG pathway) of opposite changed genes in endometrial eithelial and stromal cells

under hyperthermia conditions (Fold Change > 1.2)

Epithelial cells (Up) - Stromal calls (Down)

312 genes

Epithelial cells (Down) - Stromal calls (Up)

141 genes

Forcal adohesion

それはストレスに対抗しようとしていると考えられる。

一方、間質は様々な生理活性機能の制御因子が増加している。

これはストレスに対して間質細胞はセンシティブに働いていることを示唆している。

73

FIGURE 15

The effect of hyperthermia on the gene expression related in ER stress pathway in

endometrial epithelial (A) and stromal (B) cells. Stars indicated the genes which were

altered in endometrial epithelial and stromal cells under hyperthermia conditions.

ATF6PERK IRE1

eIF2α

ATF4GADD34

CHOP

WFS1S1P

S2P

p50 ATF6

BiP

TRAF2

ASK1

JNKXBP

AARE ERSE

Misfolded protein

BiP BiP BiP

BiP

GRP94

HSP40

NEF

EDEM

Ero1

PDIs

OS9

XTP3B

TRAP

Sec61

Bap31

UbiquitinLigasecomplex

TRAM

Derlin

Ubx p97

Npl4

Ufd1

HSP70

HSP40

HSP90

sHSF

Proteasome

Folding

Accumulation

ER stress

Ub

ER-associated degradation(ERAD)

NEF

Endoplasmic reticulum

Chaperon

A

Nucleus

ATF6PERK IRE1

eIF2α

ATF4GADD34

CHOP

WFS1S1P

S2P

p50 ATF6

BiP

TRAF2

ASK1

JNKXBP

AARE ERSE

Misfolded protein

BiP BiP BiP

BiP

GRP94

HSP40

NEF

EDEM

Ero1

PDIs

OS9

XTP3B

TRAP

Sec61

Bap31

UbiquitinLigasecomplex

TRAM

Derlin

Ubx p97

Npl4

Ufd1

HSP70

HSP40

NEF

HSP90

sHSF

Proteasome

Folding

Accumulation

ER stress

ER-associated degradation(ERAD)

Ub

Chaperon

BEndoplasmic reticulum

Nucleus

74

MHC/Antigen

Antigen

IL-1β

TNFα

EDA-A1

EDA-A2

LBP

MD-2

TCR

BCR

IL-1R

TNF-R1

EDAR

XEDAR

CD14

TLR4

LcK

Zap

Syk

Lyn

MyD88

IRAK1/4

TRAF6

LAT PLCy1 PKC8

BLNK

BtkPLCy2 PKCβ

RIP1

TRADD

TRAF2/5

TRAF6

TRAF3

TRAF6

TAK1

TAB

CARMA

Bcl10

MALT1

NEMO

IKKα

IKKβ

TIRAP

MyD88

TRAM

TRIF

IRAK1/4

TRAF6

RIP1

TRAF6

p50

p65

IL8

IL1β

TNFα

A20

COX2

MIP1β

MIP2

VCAM1

IL6

A

Cytokine

GFReceptor JAK

STAM

STAT

TC-PTP SHP1

SHP2

GRB

PI3K

SOS

AKT mTOR

Raf

Cell cycle

Cell survival

proliferation

Differentiation

CIS SOCS

Bcl2 MCL1

BclXL PIM1

C-Myc

B

75

FIGURE 16

The effect of hyperthermia on the gene expression related in NFκB (A), Jak-STAT (B),

and TNF (C) signal pathways in endometrial cells. Stars indicated the genes which were

down-regulated in endometrial epithelial cells but up-regulated in endometrial stromal

cells under hyperthermia conditions.

TNF

TNFR1

TRADDTRAF2/3

RIP1

TAB1/2/3

TAK1

NIK IKK NFκB

ASK1

NFκB

MKK

JNK1/2

p38

MEK1 ERK1/2

AP-1

c/EBPβ

CREB

CCL2 CCL5

CXCL1CCL20

CXCL2 CXCL3

CXCL5 CXCL10

IL1β IL6

IL15 Lif

FOS Jun

COX2

C

76

DISCUSSION

In the present study, we found that hyperthermia increased the expression of BiP,

EDEM2, DERL3, HYOU1, and MOGS in endometrial stromal cells (Figure 15B).

Since these genes are involved in the ER stress [106, 107], the present comprehensive

analysis for the effect of hyperthermia on gene expression in endometrial stromal cells

confirmed that hyperthermia induced the ER stress in endometrial stromal cells, as

described in Chapter 3. In endometrial epithelial cells, the present results of RNA-seq

showed that some of genes which were significantly (P<0.05) altered under

hyperthermia conditions were involved in the ER stress pathway (Table 4 and Figure

15A) and this result was contradictory to our results in Chapter 3. However, in

endometrial epithelial cells under hyperthermia conditions, all of the upregulated genes

related in the ER stress in the present study belonged to the HSP families and the gene

expression of downstream of ER stress sensors was not influenced by hyperthermia

(Figure 15A). Therefore, the present results of RNA-seq in endometrial epithelial cells

under hyperthermia conditions also confirmed our results in Chapter 3.

Since hyperthermia had opposite effects on IL-6 production between endometrial

epithelial and stromal cells, we focused on the opposite changed genes (fold change >

1.2) between endometrial epithelial and stromal cells under hyperthermia conditions.

However, the genes which were upregulated in endometrial epithelial cells but

downregulated in endometrial stromal cells under hyperthermia conditions were not

directly involved in the regulation of IL-6 production. In addition, the genes which were

downregulated in endometrial epithelial cells but upregulated in endometrial stromal

cells were annotated in NFκB signal pathway (Figure 16A), Jak-Stat signal pathway

(Figure 16B), and TNF signal pathway (Figure 16C); however altered genes in these

77

signal pathways were mainly cytokines, such as IL6, CXCL8, and CSF2, not main

functional genes for regulation of cytokine production, such as p65, p38, JNK, ERK,

Jak, and STAT. Therefore, we did not decide that the opposite changed genes between

endometrial epithelial and stromal cells under hyperthermia conditions were involved in

the opposite effects of hyperthermia on IL-6 production between endometrial epithelial

and stromal cells. Moreover, p65, p38, JNK, ERK, and STAT are activated by

phosphorylation. Therefore, to reveal whether these factors are involved in the opposite

effects of hyperthermia on IL-6 production between endometrial epithelial and stromal

cells, further studies for protein analysis are necessary.

In the present comprehensive analysis showed that hyperthermia altered 213 genes in

endometrial epithelial cells but 396 genes were altered in endometrial stromal cells

(Table 3). In addition, result of PCA showed that hyperthermia had a little effect on the

gene expression in endometrial epithelial and stromal cells; whereas, the gene

expression was different among each cows cells (Figure 12, Figure 13 A and B). In the

microarray analysis, mammary epithelial cells cultured at 42°C alter approximately

20000 genes (2 fold change) compared with that cultured at 37°C in riverine buffalo

[105]. However, in this report, mammary epithelial cells cultured at 42°C cause the

apoptosis. On the other hand, in our study, culture condition of hyperthermia did not

cause the apoptosis in both types endometrial cells [89]. In addition, Skibiel et al.

reported that the mammary glands from heat stressed heifers altered the 117 genes

compared with that from cooled heifers [14]. Therefore, since the number of the altered

genes under hyperthermia conditions in endometrial epithelial and stromal cells was

same as that in the other type of cells from in vivo, our hyperthermia condition might

mimic the in vivo heat stress conditions. Taken together, several studies about the effect

78

of hyperthermia on cellular functions are conducted under severe conditions, such as

culture at the above 42°C [105, 108]; however, the hyperthermia conditions of our study

was appropriate to reveal the effect of hyperthermia on the cellular functions.

Moreover, our present results suggested that endometrial stromal cells were more

sensitive to hyperthermia than endometrial epithelial cells. In addition, the present

comprehensive analysis showed that the upregulated genes in endometrial epithelial

cells but downregulated in endometrial stromal cells, such as COL1A2, COL11A2,

ITGA1, ITGA8, ITGB5, MYLK3, MYLK, RELN, and TNXB, are involved in the focal

adhesion (Table 4). The bovine uteri are easily infected by bacteria in the postpartum

period [23, 109] and endometrial epithelial cells act as a barrier against to the infection

of uterine lumen [38, 41, 110]. In the intestinal epithelium, focal adhesion contributes

the restitution of intestinal barrier function after epithelial injury [111]. Taken together,

these results suggest that the endometrial epithelial cells might be more tolerant to

various stresses than endometrial stromal cells to maintain the function of endometrium.

SUMMARY

The present comprehensive analysis could not reveal the opposite effects of

hyperthermia on IL-6 production between endometrial epithelial and stromal cells;

however the relationship between hyperthermia and ER stress in both types of

endometrial cells was confirmed. In addition, the present results of RNA-seq suggested

that endometrial epithelial cells were more resistant to hyperthermia than endometrial

stromal cells.

79

CHAPTER 5

CONCLUSION

The present study investigated the effect of hyperthermia on the chemokine secretion

in bovine endometrial epithelial and stromal cells. The first series of experiments

showed that hyperthermia suppressed the IL-6 production in endometrial epithelial cells

but enhanced the IL-6, MCP1, and IL-8 secretion in endometrial stromal cells. The

second series of experiments showed that hyperthermia was involved in the ER stress in

the endometrial stromal cells but not in endometrial epithelial cells. In addition, the

induction of ER stress under hyperthermia conditions affected the upregulation of IL-6

production in endometrial stromal cells. Whereas, ER stress might not be involved in

the suppression of IL-6 production under hyperthermia conditions in endometrial

epithelial cells. In the third series of experiments, comprehensive analysis of gene

expression patterns was investigated and the results of this experiment suggested that

endometrial epithelial cells were more tolerant to various stresses than endometrial

stromal cells. In addition, it was confirmed that hyperthermia induced the ER stress in

endometrial stromal cells; whereas, any signal pathways which were related to the

regulation of IL-6 production were not identified under hyperthermia conditions in

endometrial epithelial cells. Therefore, to elucidate the mechanism for the suppression

of IL-6 production under hyperthermia conditions in endometrial epithelial cells, further

studies for protein analysis, for example the signal pathway of NFκB, Jak-STAT, and

MAPK, are necessary.

Overall results suggested that hyperthermia enhanced the immune response via

induction of ER stress in endometrial stromal cells; whereas, the immune response was

80

suppressed under hyperthermia conditions in endometrial epithelial cells via different

mechanism from endometrial stromal cells. The alteration of the immune response

under hyperthermia conditions in two types of endometrial cells might cause the

accumulation of macrophages in endometrial stromal layer and not recruit them to

epithelial layer which was main infection site by bacteria. Therefore, the bacteria

infected into uterine lumen might not be eliminated and the symptoms of endometritis

might last longer in summer. The present study will contribute to the livestock industry,

since the prolongation of the endometritis symptom in summer will be improved, if the

immune response of endometrium is appropriately regulated in the postpartum period,

based on our results.

81

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91

ABSTRACT IN JAPANEASE

Alteration of immune responses in bovine endometrial cells

under hyperthermia conditions

(暑熱環境下におけるウシ子宮内膜細胞の免疫応答変化)

酒井 駿介

地球温暖化に伴う気温上昇は畜産業に莫大な損害を与えるため、家畜に対する暑

熱ストレスの影響は世界中において緊急の課題として扱われている。特にウシにおい

て、暑熱ストレスによって受胎率および乳生産が低下することが報告されている。暑熱

環境下におけるウシ受胎率低下に関して、生体レベルに対する暑熱ストレスの影響は

多く研究される一方で、各器官に対する局所的な暑熱ストレスの影響に関する報告は

少ない。これまでに我々はウシ子宮内膜の内分泌機能に着目し、暑熱ストレスが子宮

内膜細胞に直接作用することで Prostaglandin 産生を乱し、受胎率低下の一因となる

可能性を報告した。

また、近年、受胎率低下と共に分娩後の空胎期間の延長が畜産経営を逼迫させて

いる問題として注目されている。分娩後の子宮内細菌感染による子宮内膜炎は空胎

期間延長の一因であるが、夏季の暑熱環境下において子宮内膜炎を発症するリスク

が高いこと、発症した場合、症状が長期化することが報告されている。子宮内膜炎を治

癒するためにはウシ子宮内膜細胞が細菌を認識し、MCP1, IL-6 および IL-8 などの

Chemokine を分泌、さらに主に細菌が存在する子宮内膜上皮層まで免疫細胞を誘引

することが必要である。我々は子宮内膜細胞によるこの一連の免疫反応に暑熱ストレ

スが直接影響することで、細菌除去が適切に行われず夏季の子宮内膜炎症状の長期

化につながると仮説を立てた。この仮説を証明するために、単離したウシ子宮内膜上

皮細胞および間質細胞を暑熱環境下で培養し、細胞機能の変化を検討した。

1. ウシ子宮内膜上皮および間質細胞における Chemokine 産生に対する暑熱スト

レスの影響

ウシ子宮内膜上皮および間質細胞の Chemokine 産生に暑熱ストレスが関与する

かどうか明らかにするために、単離したウシ子宮内膜上皮および間質細胞に

lipopolysaccharide (LPS: 1 µg/ml) を添加し、38.5°C (通常のウシ体温) もしくは

40.5°C (暑熱環境下のウシ体温) で培養した。培養後、MCP1, IL6 および CXCL8

遺伝子発現および LPS 認識に関与する TLR4 関連因子の遺伝子発現を定量的

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RT-PCR によって測定するとともに、MCP1, IL-6 および IL-8 濃度を EIA によって

測定した。さらに夏季および冬季のウシ子宮内膜におけるマクロファージおよび好中

球の局在を免疫染色およびギムザ染色によってそれぞれ検討した。

ウシ子宮内膜上皮細胞において MCP1 および IL6 遺伝子発現は LPS によっ

て有意に増加したが、この増加は暑熱ストレスによって有意に抑制された。子宮内膜

上皮細胞において LPS によって増加した CXCL8 遺伝子発現に暑熱ストレスは影

響を及ぼさなかった。また、子宮内膜上皮細胞において IL-6 産生は LPS によって

増加したが、この増加は暑熱ストレスによって検出限界以下まで抑制された。LPS は

IL-8 産生を有意に増加させ、この増加に暑熱ストレスは影響を及ぼさなかった。また、

上皮細胞における MCP1 産生量は LPS 存在下でも検出限界以下だった。一方、

子宮内膜間質細胞において、MCP1, IL-6 および IL-8 産生および遺伝子発現は

LPS によって有意に増加し、この増加は暑熱ストレスによってさらに増強された。さら

に子宮内膜上皮細胞および間質細胞において TLR4 遺伝子発現は暑熱ストレスに

よって増加したが、LPS 認識のために TLR4 と複合体を形成する CD14 および

MD-2 遺伝子発現に暑熱ストレスは影響を及ぼさなかった。また、夏季および冬季の

ウシ子宮内膜組織において好中球の局在に変化はなかった一方、冬季の子宮内膜

組織に比べ夏季の子宮内膜組織においてマクロファージは上皮層直下まで誘引され

ていないことが示された。

以上のことから、暑熱ストレスはウシ子宮内膜上皮細胞の免疫反応を抑制する一方、

間質細胞の免疫反応を増強することで免疫細胞が子宮内膜間質層に蓄積し、上皮層

まで誘引されないため、夏季の子宮内膜炎症状の長期化に関与する可能性が示され

た。しかし、暑熱ストレスによる子宮内膜上皮および間質細胞それぞれの変化に

TLR4 による細菌認識は関与しない可能性が示唆された。

2. 暑熱ストレスによるウシ子宮内膜上皮および間質細胞の IL-6 産生変化に対す

る小胞体ストレスの影響

暑熱ストレスによる子宮内膜上皮および間質細胞の IL-6 産生変化に小胞体ストレ

スが関与するかどうか明らかにするために、単離した子宮内膜上皮および間質細胞に

LPS を添加し、38.5°C もしくは 40.5°C で培養した。培養後、小胞体ストレスマーカ

ー (BiP, sXBP1 および ATF4) 遺伝子発現を定量的 RT-PCR によって測定すると

ともに、BiP タンパク質発現量を Western Blot によって検討した。また、子宮内膜上

皮および間質細胞に LPS と同時に小胞体ストレス阻害剤 (Toyocamycin, KIRA6

および Salubrinal) を添加し、IL6 遺伝子発現の変化を定量的 RT-PCR によって測

定した。同様に、子宮内膜間質細胞に各小胞体ストレス阻害剤を添加し暑熱環境下

で培養後、IL-6 産生量の変化を EIA によって測定した。また、子宮内膜間質細胞

に小胞体ストレス促進剤 (NaF) を添加し、IL6 遺伝子発現および IL-6 産生量の変

93

化を定量的 RT-PCR および EIA によって測定した。

ウシ子宮内膜上皮細胞において BiP, sXBP1 および ATF4 遺伝子発現に暑熱ス

トレスは影響を及ぼさなかった。また、子宮内膜上皮細胞における BiP タンパク質発

現に暑熱ストレスは影響しなかった。一方、子宮内膜間質細胞において、BiP および

sXBP1 遺伝子発現は暑熱ストレスによって増加したが、ATF4 遺伝子発現に暑熱スト

レスは影響を及ぼさなかった。さらに子宮内膜間質細胞における BiP タンパク質発

現は暑熱ストレスによって有意に増加した。各小胞体ストレス阻害剤を添加した子宮内

膜上皮細胞において、暑熱による IL6 遺伝子発現の抑制は回復されなかったが、各

小胞体ストレス阻害剤添加によって、暑熱による子宮内膜間質細胞の IL6 遺伝子発

現および IL-6 産生の増加は有意に抑制された。また、NaF 添加によって、暑熱環

境下の子宮内膜間質細胞における IL6 遺伝子発現および IL-6 産生に変化は見ら

れなかった一方、38.5°C で培養した子宮内膜間質細胞において IL6 遺伝子発現お

よび IL-6 産生の有意な増加が確認された。

以上のことから、暑熱ストレスはウシ子宮内膜上皮細胞の小胞体ストレスに関与しな

い一方、子宮内膜間質細胞において小胞体ストレスを引き起こす可能性が示唆され

た。さらに、子宮内膜間質細胞において暑熱による小胞体ストレスの誘起が、IL-6 産

生増強に関与する可能性が示唆された。一方、子宮内膜上皮細胞において暑熱スト

レスによる IL-6 産生抑制のメカニズムには小胞体ストレス以外の経路が関与する可

能性が示された。

3. 子宮内膜上皮および間質細胞において暑熱ストレスによって変動する遺伝子の

網羅的解析

子宮内膜間質細胞において暑熱ストレスによる IL-6 産生増加に小胞体ストレスが

関与する可能性が示された一方、子宮内膜上皮細胞における暑熱による IL-6 産生

抑制に関わるメカニズムは明らかになっていない。そこで、暑熱環境下における IL-6

産生制御に関与する因子を発見することを目的として、LPS 添加後、38.5°C もしくは

40.5°C で培養した子宮内膜上皮および間質細胞における遺伝子発現の変動を

RNA-seq を用いて網羅的に解析した。

暑熱環境下で培養された子宮内膜上皮細胞において 112 個の遺伝子発現が増

加し、101 個の遺伝子発現が抑制された。また、暑熱環境下で培養された子宮内膜

間質細胞において 169 個の遺伝子発現か増加し、227 個の遺伝子発現が抑制され

た。これにより、子宮内膜上皮細胞に比べ子宮内膜間質細胞は暑熱ストレスに対する

感受性が高い可能性が示された。子宮内膜上皮細胞において発現変化が見られた

遺伝子は小胞体ストレスに関与することが Pathway analysis によって明らかになった

が、Heat shock protein family に属する因子のみが変動しており、小胞体ストレスセン

サー下流の因子は変化が見られなかった。また、Pathway analysis の結果、暑熱環境

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下の子宮内膜上皮細胞において IL-6 産生を直接制御する可能性のある Pathway

は発見できなかった。一方、子宮内膜間質細胞において暑熱ストレスは小胞体ストレ

ス経路を活性化することが示された。さらに、暑熱環境下の子宮内膜上皮細胞で発現

増加し、間質細胞において発現抑制される因子を選抜し Pathway analysis を行った

結果、IL-6 産生制御に関与する可能性のある Pathway は発見できなかった。また、

暑熱環境下の子宮内膜上皮細胞において発現が抑制され、間質細胞において発現

増加する因子を選抜し、Pathway analysis を行った結果、IL-6 産生に関与する

Jak-STAT signal pathway, TNF signaling pathway および NFκB signaling pathway

が変化することが示された。しかし、この経路において変動した遺伝子は IL-6,

CXCL8 および CSF2 などの Cytokine および Chemokine のみで、p65, p38, JNK,

ERK, Jak および STAT などの Cytokine 発現制御に関わる因子の遺伝子発現に

変化は見られなかった。

以上のことから、実験 2 において示したように、暑熱ストレスは子宮内膜上皮細胞

の小胞体ストレスに関与しない可能性がある一方、子宮内膜間質細胞において小胞

体ストレスを誘導する可能性が再確認された。さらに、子宮内膜間質細胞は上皮細胞

に比べ暑熱ストレスに対する感受性が強い可能性が示唆された。また、上述した

Cytokine 発現制御に関与する転写因子やキナーゼは核内移行やリン酸化によって

その機能が活性化される。そのため、暑熱ストレスによる子宮内膜上皮細胞の IL-6

産生抑制に関わるメカニズムを解明するためには、遺伝子レベルだけでなくタンパク

質レベルで各因子の活性化状態を詳細に検討する必要性が示唆された。

本研究から暑熱ストレスは小胞体ストレスを介してウシ子宮内膜間質細胞の免疫反

応を増強する一方、子宮内膜間質細胞とは異なる経路を介して子宮内膜上皮細胞の

免疫反応を抑制する可能性が示された。暑熱ストレスによる子宮内膜上皮細胞および

間質細胞の免疫反応の変化によって、マクロファージが子宮内膜間質層に蓄積し、主

に細菌が存在する子宮内膜上皮層直下まで誘引されない可能性が示された。これに

よって子宮内に感染した細菌の除去が適切に行われず、夏季の子宮内膜炎症状の

長期化に関与する可能性が考えられる。本研究から得られた知見をもとに、夏季に分

娩したウシの子宮に直接アプローチし、子宮内免疫機能を適切に制御することで、夏

季の子宮内膜炎症状の長期化を改善できると考える。これにより、長期的に暑熱ストレ

スに対応することなく、分娩時期に短期間で効率的に暑熱ストレスに対応することで空

胎期間の短縮および夏季の受胎率改善に貢献できると考える。