INTIMP-03251; No of Pages 10 International Immunopharmacology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

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Article history: Received 20 March 2014 Received in revised form 28 April 2014 Accepted 1 May 2014 Available online xxxx

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Keywords: Affymetrix genechip microarray Bovine mastitis Staphylococcus aureus Lipoteichoic acid Peptidoglycan

Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea School of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea Division of High-risk Pathogen Research, Center for Infectious Diseases, Korean National Institute of Health, Seoul, Republic of Korea

i n f o

a b s t r a c t

A Gram-positive bacterium, Staphylococcus aureus is known to be one of the major pathogenic bacteria responsible for causing bovine mastitis. Among the various cell wall components of S. aureus, lipoteichoic acid (LTA) and peptidoglycan (PGN) are closely associated with inflammatory responses. However, the role of LTA and PGN derived from S. aureus in bovine mastitis has not been clearly elucidated. In this study, we characterized the gene expression profile of a bovine mammary gland epithelial cell line, MAC-T cells, in the presence of LTA and PGN from S. aureus. LTA plus PGN, but not LTA or PGN alone, activated MAC-T cells. The analysis of transcriptional profiles using an Affymetrix genechip microarray showed that stimulation with LTA plus PGN produced a total of 2019 (fold change N 1.2) differentially expressed genes (DEGs), with 801 up-regulated genes and 1218 down-regulated genes. Of the up-regulated genes, 14 inflammatory mediator-related DEGs, 22 intra-cellular signaling molecule-related DEGs, and 15 transcription factor-related DEGs were observed, whereas among the down-regulated DEGs 17 inflammation-related DEGs were found in MAC-T cells. The microarray results were confirmed using real-time RT-PCR of 18 genes with substantial changes in expression (9 each from the upregulated and down-regulated DEGs). These results provide a comprehensive analysis of gene-expression profiles elicited by S. aureus LTA and PGN in MAC-T cells, contributing to an understanding of the pathogenesis for S. aureus-induced bovine mastitis. © 2014 Published by Elsevier B.V.

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Jintaek Im a, Taeheon Lee b, Jun Ho Jeon c, Jung Eun Baik a, Kyoung Whun Kim b, Seok-Seong Kang a, Cheol-Heui Yun b, Heebal Kim b, Seung Hyun Han a,⁎

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Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus

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1. Introduction

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Bovine mastitis is an inflammatory disease caused by intramammary microbial infections in dairy cattle [1], resulting in economic losses through reduced milk yield and quality, along with increased veterinary costs [2]. Once pathogens pass the teat canal, they multiply in the gland cistern and glandular tissue, causing bovine mastitis [3]. Clinical mastitis is mainly caused by Escherichia coli infections, and is often accompanied by severe clinical symptoms including hot and swollen udders, fever, and loss of appetite [4]. Subclinical mastitis, characterized by non-visible clinical signs of illness, is more common than clinical mastitis and also leads to a huge economic loss [1]. Staphylococcus aureus is well recognized as a major pathogen responsible for causing subclinical mastitis in cattle [5]. It colonizes the mammary gland tissues

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⁎ Corresponding author at: Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. Tel.: +82 2 740 8641; fax: +82 2 743 0311. E-mail address: [email protected] (S.H. Han).

by internalizing epithelial and endothelial cells [6]. Subsequently, S. aureus enhances the expression of various pro-inflammatory mediators in the mammary gland epithelial cells [7]. Although the expression of pro-inflammatory mediators is important for eliminating an invasive pathogen, excessive production of these cytokines often leads to a continuous inflammatory response in the mammary glands, possibly resulting in subclinical mastitis [7]. Among the virulence factors of S. aureus, lipoteichoic acid (LTA) and peptidoglycan (PGN) have manifested their pathologic roles in infectious diseases [8]. LTA is regarded as a counterpart of lipopolysaccharide (LPS) of Gram-negative bacteria due to its structural and functional similarities. LTA is an amphiphilic molecule composed of hydrophilic polysaccharides and hydrophobic glycolipids [9]. It is involved in biofilm formation, bacterial adherence to the host, and stimulates the production of various inflammatory mediators [10,11]. Nevertheless, LTA differs from LPS since (i) LTA alone cannot cause sepsis, while LPS alone is sufficient to do so [12]; (ii) LTA is recognized by Toll-like receptor 2 (TLR2), while LPS is sensed mostly by TLR4 [13,14]; and (iii) LTA is secreted from the cell wall during cell growth, while LPS is not [15]. On the other hand, PGN is a common cell wall constituent of both Gram-

http://dx.doi.org/10.1016/j.intimp.2014.05.002 1567-5769/© 2014 Published by Elsevier B.V.

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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Table 1 Primer sequences of genes used for PCR analysis.

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2.1. Bacteria, reagents and chemicals

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S. aureus ATCC 29213 was purchased from the American Type Culture Collection (Manassas, VA, USA) and was grown in tryptic soy broth (BD Biosciences, Franklin, NJ, USA). LPS from E. coli O111:B4 and polymyxin B (PMB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). S. aureus PGN and a synthetic lipopeptide, Pam2CSK4, were obtained from InvivoGen (San Diego, CA, USA).

95 96

2.2. Preparation of S. aureus LTA

101

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t1:1 t1:2

75 76

2. Materials and methods

Highly-pure and structurally-intact LTA from S. aureus was prepared as previously described [20]. The structural intactness of LTA was confirmed by performing high-field nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry as previously described [21]. Any biological contaminants in the purified LTA, including endotoxins, proteins and nucleic

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negative and Gram-positive bacteria. Remarkably, Gram-positive bacteria possess much more PGN than Gram-negative bacteria, it comprises approximately 90% of the Gram-positive bacterial cell wall [16]. PGN is mainly recognized by intracellular pattern recognition receptors, nucleotide-binding oligomerization domains (NODs). NOD1 exclusively recognizes PGN of Gram-negative bacteria, whereas NOD2 recognizes PGN of both Gram-positive and Gram-negative bacteria [17]. Once PGN is recognized by NOD, PGN also triggers signaling pathways for the production of various inflammatory mediators, including cytokines, chemokines, and lipid metabolites [18,19]. LTA alone does not strongly elicit inflammatory responses, whereas the combination of LTA and PGN synergistically induces inflammatory responses and leads to systemic inflammation [8]. Although S. aureus has been considered to be a major pathogenic Gram-positive bacterium, responsible for causing bovine mastitis, the precise role of both cell wall components of S. aureus in bovine mastitis has not been clearly elucidated. Therefore, in order to gain insights into S. aureus-induced mastitis, we investigated the gene-expression profiles in a bovine epithelial cell line, MAC-T cells, in response to LTA and PGN using a DNA microarray analysis.

Genes

GenBank accession no.

Primer orientation

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49 t1:50 t1:51 t1:52 t1:53

Bovine NOD2

NM_001002889

Bovine TLR2

NM_174197

Bovine β-actin

NM_173979.3

Mouse NOD2

NM_145857

Mouse TLR2

NM_011905

Mouse β-actin

NM_001101.3

CTGF

NM_174030

TGF-β2

NM_001113252

PTGS-1

NM_001105323

CCDC-80

NM_001098982

CSRP-2

NM_001038183

WISP-2

NM_001102176

PC-TP

NM_174835

Transcription factor 4

NM_001034621

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

MMP-1 CD36

E

T

C E R

R

O

C

NM_001038065 NM_174112

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GPNMB

NM_001034751

N

LMO-4

D

t1:3

NM_174010

CCR7

NM_001024930

RENBP

NM_001046223

MMP-9

NM_174744

CEACAM1

NM_205788

IL-13RA

XM_583913

LTBP2

NM_174385

GAPDH

U85042.1

R O

73 74

J. Im et al. / International Immunopharmacology xxx (2014) xxx–xxx

Primer sequence (5′ → 3′)

Product size

CCTTGCCGGGTGAGGCCAAG CCCGCAGCCGTGATGTGGTT AGGTGGCAGGCAGCTACGGAC CCTCTGCAGGTCTCTGTTGCCGA CCGGTCGACACCGCAACCAG GACCCCGTCACCGGAGTCCA GAGGAGTCGTGATGGTTGGT CAGTGGAGAGGCAGAGAACC GACTCACAGCAGCCATGAAA TCGCGGATCGACTTTAGACTT GTGGGGCGCCCCAGGCACCA CTCCTTAATGTCACGCACGATTTC GTGGGAGGAGGCCAGTAGAAAGCC GATGGCTGGAGAACGCACATCCG CCCCTCCATCTCGTCGCTCCAA GCAACGTCGTTCCCCAAGTGGAAA CTGGGTGGCCCTCCAGAATGTTGA GCCAGCCACTGTTCTGGATCAGC GATGGCCACCTGAAACCCGCAAA TTCGTTTGCCTTCAAAGGGCCCC CTGCGACCTGTTCTCGAACGCTCA TCGGCGTGGTACACGGTCCTC TCTGTGTCAGCCGCTCTGCAGG GACCAGCTGGCTTGGGAATACGC ATGCTGCTCCTTGAAGTGCGACG GATGTCAGCCACACACGCTCGG TCCGAGGCCATGTACTGCGCAT GAAGGGTAGCCTGGCGAGTCCC TTGAGAGGAGCTCGTGGCCCC GGTCCGCAATCTTGCCCCCG AGATGCCAAGGGTGAGTGAGTCAGA TGAGCCTCGGGGTGGATCATGT GAGACCAACATGCCCAGACTGCC GAAGTTGCTGCTGGGAAGCCGT TCCTGGACCCTGAACACTAGCCTTC TGGGTCTGTGTTTTGCAGGGACAC TCTCCTCAGGCTCTCCACGCTG CCTGGCTGGGAACATGGCTTAGG GCAGCGGACCATCTTCAGCGA GGCTTCGTTCTGGTACCGTGCA CCTTCGACCTCCTGAAGTGCCCT TTCCCTATTGGCAGGGTCCCCC ACCCTGAATGTCCTCTACCCAGTGG ACCACGGGGCCCTCATGTTCT TGGAACCTTCATCCCCTCCAGCA CTGTAGTCACAGCTGGCTGACACG CTTCCCGGTGCCAAAGTGGGT CTCGGAGGGATAGTTCAGCCCCC ATGATTCCACCCACGGCAA ATCACCCCACTTGATGTTGGC

487

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2

415 503 404 451 540 148 103 90 88 110 104 115 137 140 110 80 82 80 91 94 83 80 80 122

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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J. Im et al. / International Immunopharmacology xxx (2014) xxx–xxx

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acids, were not found as determined by Limulus amebocyte lysate assay and silver staining.

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2.3. Cell culture

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A bovine mammary gland epithelial cell line, MAC-T cells [22], was cultured in the complete DMEM (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone),

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3

100 U/mL of penicillin, and 100 μg/mL of streptomycin (HyClone) at 37 °C in a 5% CO2 humidified incubator. A murine macrophage-like cell line, RAW 264.7, and a human embryonic kidney 293 (HEK 293) cell line were also maintained in complete DMEM as described above. NF-κB reporter cell lines co-expressing CD14 together with TLR2 or TLR4, as CHO/CD14/TLR2 or CHO/CD14/TLR4, respectively, were kindly provided by Dr. Douglas Golenbock (Boston Medical Center, Boston, MA, USA) and were cultured in complete Ham's F-12 media (HyClone)

(A) Without PMB With PMB

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* Nitrite (μM)

Nitrite (μM)

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Without PMB With PMB

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30

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PGN (μg/mL)

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30

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LPS (μg/mL)

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1

LPS (μg/mL)

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LTA(μg/mL)

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59.4

-

0.1

66.6

77.8

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30

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-

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LTA ( μg/mL) Pam2CSK4 ( μg/mL)

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-

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30

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LTA ( μg/mL)

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CD25 expression

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N C O

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LPS (μg/mL)

5.9

7.2

52.9

CD25 expression

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2.0

NF- κB activity (Fold increase)

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(D)

1.5 1.0 0.5 0

PGN (μg/mL) Q8

0

10

30

Fig. 1. LTA and PGN prepared from S. aureus retain their functional properties. (A) RAW 264.7 cells (2 × 105 cells/mL) were stimulated with S. aureus LTA (30 μg/mL), S. aureus PGN (30 μg/mL), or a positive control, E. coli LPS (1 μg/mL) in the absence or presence of 25 μg/mL of PMB for 48 h. After stimulation, nitrite accumulations in the culture supernatants were measured using the Griess reagent for the determination of NO production. Values represent the mean ± standard deviation (S.D.) of triplicate samples. (B) CHO/CD14/TLR2 or (C) CHO/ CD14/TLR4 cells at 3 × 105 cells/mL were treated with the indicated concentrations of S. aureus LTA for 20 h. Pam2CSK4 (0.1 μg/mL) and E. coli LPS (1 μg/mL) were used as positive controls for TLR2 and TLR4 stimulation, respectively. After treatment, the cells were stained with anti-human CD25 antibody conjugated with APC, and CD25 expression was measured by flow cytometric analysis. Values in the histogram represent the percentage of positive cells. One of three similar results is shown. (D) HEK 293 cells (5 × 105 cells/mL) were transiently transfected with a NOD2 expression plasmid together with an NF-κB luciferase reporter plasmid and Renilla luciferase plasmid using the TransFectin lipid reagent for 24 h. Next, the cells were stimulated with MDP (1 μg/mL) or S. aureus PGN (0 to 30 μg/mL) for an additional 24 h. After stimulation, the cell lysates were prepared and the dual luciferase activities were measured using a luminometer. Each firefly luciferase activity was normalized to Renilla luciferase activity to correct for the difference in transfection efficiency. Values are the mean ± S.D. of triplicate samples.

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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J. Im et al. / International Immunopharmacology xxx (2014) xxx–xxx

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2.4. Determination of nitric oxide (NO)

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To determine NO production, nitrite accumulation in the culture supernatants was measured as described previously [23]. Briefly, RAW 264.7 cells (2 × 105 cells/mL) were plated on a 96-well plate and incubated overnight. The cells were then stimulated with 30 μg/mL of S. aureus LTA or PGN in the presence or absence of 25 μg/mL of PMB for 48 h. E. coli LPS (1 μg/mL) was used as a positive control. At the end of stimulation, the culture supernatants were collected, mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2% phosphoric acid) and then incubated for 5 min at room temperature. The absorbance was measured at 540 nm of optical density using a microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA). NO production was determined by comparison with the optical density of a standard curve of NaNO2 concentrations.

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2.5. Measurement of TLR2 and NOD2 activation

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To determine TLR2 or NOD2 expression in MAC-T and RAW 264.7 cells, total RNA from the cells was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Following the synthesis of cDNA using random hexamers (Promega) with the total RNA, a reverse transcription-polymerase chain reaction (RT-PCR) was performed with specific primers for TLR2, NOD2 and β-actin (Table 1), as described previously [24]. The amplified PCR products were electrophoresed and visualized by staining with ethidium bromide. The relative intensity of TLR2 or NOD2 normalized to β-actin was determined through densitometric analysis with Multi Gauge (Fuji Film, Tokyo, Japan). In a separate experiment, CHO/CD14/TLR2 and CHO/CD14/TLR4 cells (3 × 105 cells/mL) were plated on a 24-well plate and incubated overnight. The cells were then stimulated with various doses of S. aureus LTA, Pam2CSK4, or E. coli LPS for 20 h. Since NF-κB transcription factor activates the expression of a reporter gene encoding CD25 in CHO/CD14/TLR2 or CHO/CD14/TLR4 cells [25], the cells were stained with anti-human CD25 antibody conjugated with allophycocyanin (APC) (BD Biosciences). The CD25 expression was measured by a FACSCalibur with CellQuest software (BD Biosciences). Flow cytometric data were analyzed by FlowJo software (Tree Star, San Carlos, CA, USA).

161

2.6. Transfection and luciferase reporter gene assay

162 163

HEK 293 cells (5 × 105 cells/mL) were plated on a 12-well plate and cultured for 16 h. The cells were then transiently transfected with 0.02 μg of NOD2 expression plasmid (InvivoGen), together with 0.2 μg of NF-κB luciferase reporter plasmid (Clontech, Palo Alto, CA, USA) and 0.02 μg of pRL-TK Renilla luciferase plasmid (Promega, Madison, WI, USA) using serum- and antibiotic-free DMEM with TransFectin lipid reagent (Bio-Rad, Hercules, CA, USA) for 24 h. After the transfection, the cells were stimulated with PGN at concentrations ranging from 0 to 30 μg/mL for an additional 24 h. In a separate experiment, MAC-T cells (2 × 105 cells/mL) were plated on a 12-well plate and transiently transfected with 0.2 μg of NF-κB luciferase reporter plasmid (Clontech) together with 0.02 μg of pRL-TK Renilla luciferase plasmid (Promega) using serum- and antibiotic-free DMEM containing Lipofectamine and PLUS reagent (Invitrogen) for 24 h. Next, the cells were stimulated with 30 μg/mL of S. aureus LTA or PGN for an additional 24 h. After stimulation, the cells were lysed with Glo Lysis Buffer (Promega) and the cell lysates were collected by centrifugation at 13,000 ×g for 5 min. The luciferase activity in the cell lysates was measured using a luminometer (Victor 3, Waltham, MA, USA). Firefly

152 153 154 155 156 157 158 159

164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

β -actin

NOD2

P

MAC-T

E

D

RAW 264.7 Relative mRNA (TLR2 or NOD2 toβ-actin)

150 151

(A)

120 100

*

RAW 264.7 MAC-T

80

*

60 40 20 0

TLR2

NOD2

(B) 2.0 NF-κB activity (Fold increase)

148 149

184 185

T

146 147

MAC-T cells (2 × 105 cells/mL) were stimulated with 30 μg/mL of LTA together with 30 μg/mL of PGN from S. aureus for 1 h. Then, total RNA was isolated using an RNeasy® Mini kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer's instruction. DNA microarray analysis was conducted according to the recommended protocols of Affymetrix (Santa Clara, CA, USA). Briefly, complementary DNA (cDNA) was synthesized from 5 μg of total RNA using random hexamers and then purified with a GeneChip® sample cleanup module (Qiagen). An in vitro transcription (IVT) reaction was performed to produce biotin-labeled cRNA from the prepared cDNA using an IVT labeling kit (Ambion, Austin, TX, USA). The biotin-labeled cRNA was fragmented using the GeneChip® sample cleanup module (Qiagen), and 10 μg of

C

144 145

183

E

142 143

2.7. Microarray gene expression profiling

TLR2

R

136 137

R

134 135

O

132 133

C

130 131

N

128 129

U

126 127

luciferase activity was normalized by the Renilla luciferase activity to 181 correct for the difference in the transfection efficiency. 182

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containing 100 μg/mL of G418 (Invitrogen, Grand Island, NY, USA) and 40 μg/mL of hygromycin (Invitrogen).

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R O

4

*

1.5 1.0 0.5 0

LTA (30 μg/mL) PGN (30 μg/mL)

-

+ -

+

+ +

Fig. 2. Co-treatment with LTA and PGN from S. aureus induces NF-κB activation in bovine mammary gland epithelial cells. (A) Total RNA was prepared from MAC-T and RAW 264.7 cells, and the mRNA expression of TLR2 and NOD2 was determined using RT-PCR. The graph represents the percentage of TLR2 or NOD2/β-actin ratio obtained from the intensities of bands. Values are the mean ± S.D. of triplicate samples. (B) MAC-T cells (2 × 105 cells/mL) were co-transfected with an NF-κB luciferase reporter plasmid and a Renilla luciferase plasmid using the Lipofectamine and PLUS reagent for 24 h. Next, the cells were stimulated with 30 μg/mL of LTA, PGN, or LTA plus PGN for an additional 24 h. After stimulation, the cell lysates were prepared and the dual luciferase activities were measured using a luminometer. Each firefly luciferase activity was normalized to the Renilla luciferase activity to correct for the different transfection efficiency. Values are the mean ± S.D. of triplicate samples.

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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The quality of the array image was assessed according to the method described by the GeneChip Expression Analysis Technical Manual (Affymetrix). All arrays were processed by the robust multi-array (RMA) of the affy software package of the R statistical package (version 2.5) for background subtraction, quantile normalization among arrays, and median polish among genes [26]. The data were then subjected to linear model fitting and empirical Bayes functions to generate a p-value using the linear models for microarray data (LIMMA) software package of the R statistical package [27]. After false discovery rate (FDR) correction, the FDR-adjusted p-value was used to identify differentially expressed genes (DEGs) [28]. Heatmaps were drawn using a heatmap software package for the R statistical package [29]. The DEGs of the 3rd level of the biological process term of gene ontology (GO) were counted using the Affymetrix annotated data, NetAffx [30]. The GO enrichment test of DEGs was performed using Fisher's exact test, and p-values used in this test were adjusted with the FDR correction. To characterize the up-regulated DEGs associated

(A) Down-regulated

Up-regulated

E

218

T

216 217

C

214 215

227 228

E

212 213

To validate the microarray data, MAC-T cells (3 mL of 2 × 105 cells/mL) were stimulated with 30 μg/mL of LTA and PGN from S. aureus for 1 h. Total RNA was prepared with TRIzol reagent (Invitrogen) and 3 μg of total RNA was used to synthesize cDNA using random hexamers (Promega). Real-time RT-PCR was performed with SYBR Premix Ex Taq (Takara Bio Inc.) using the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The reaction conditions were as follows; denaturation at 95 °C for 10 s and amplification by cycling 40 times at 95 °C for 5 s and 60 °C for 31 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize the copy numbers of the target gene by the 2− ΔΔCt method. After normalization, the relative target gene mRNA levels of the experimental group were expressed as fold increases compared to those of the control group. The sequences of primers specific for each target gene are listed in Table 1.

R

210 211

226

R

208 209

N C O

206 207

NT

LTA + PGN

(B)

U

204 205

2.9. Real-time RT-PCR

F

202 203

O

2.8. Analysis of microarray data

219 220

R O

201

198 199

with an inflammatory response elicited by LTA plus PGN stimulation, DEGs were categorized as inflammatory mediators, intra-cellular signaling molecules, and transcription factors for up-regulated DEGs having a fold change (FC) N 1.2 and p b 0.05. Categorization of down-regulated DEGs associated with an inflammatory response elicited by LTA plus PGN stimulation was also performed for all downregulated DEGs having a FC N 1.2 and p b 0.05.

P

200

fragmented cRNA was hybridized to a GeneChip® Bovine Genome Array (Affymetrix) at 45 °C and 60 rpm for 16 h. The genechips were stained with a streptavidin–phycoerythrin complex and washed in a GeneChip Fluidics Station 450 (Affymetrix) and then scanned using a GeneChip Scanner 3000 7G (Affymetrix).

D

196 197

5

Gene expression and metabolism

Cell cycle and growth

UP-regulated DEGs Down-regulated DEGs Biosynthetic process Gene expression Regulation of cellular process Regulation of border follicle cell delamination Regulation of biological process Regulation of homeostatic process Regulation of metabolic process Cell cycle Cellular localization Embryonic development Anatomic structure formation Positive regulation of growth 0

10

20

30

40

50

60

Percent (%)

Fig. 3. The combination of LTA and PGN from S. aureus shows different gene-expression profiles in MAC-T cells. (A) MAC-T cells (2 × 105 cells/mL) were stimulated with a combination of LTA (30 μg/mL) and PGN (30 μg/mL) from S. aureus for 1 h. After stimulation, total RNA was isolated and subjected to microarray analysis. The red color represents the up-regulated expression of genes, while the green color represents down-regulated expression of genes. NT denotes no treatment. (B) The DEGs of the 3rd level biological process term of GO were counted using Affymetrix annotated data. The GO enrichment test of the DEGs was performed using Fisher's exact test and the p-values were adjusted with the FDR correction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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229 230 231 232 233 234 235 236 237 238 239 240 241

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3. Results

248 249 250

(A)

Up-regulated

O

R

R

E

C

T

E

Down-regulated

(B)

NT

LTA + PGN

C

261 262

271 272

Real-time RT-PCR

5

N

259 260

Since TLR2 and NOD2 sense LTA and PGN, respectively, we examined whether MAC-T cells expressed TLR2 and NOD2. Fig. 2A indicates that TLR2 and NOD2 mRNAs were observed in MAC-T cells, even though the expression in MAC-T cells was not as much as was observed in RAW 264.7 cells. Next, we examined the stimulatory effect of LTA and PGN on the activation of bovine mammary gland epithelial cells using MAC-T cells. Since NF-κB activity is regulated by microbial-sensing proteins such as TLRs and NODs and controls the induction of inflammatory cytokines and chemokines [32], we measured NF-κB activity in MAC-T cells in response to LTA and/or PGN. Although stimulation with LTA or PGN alone did not induce NF-κB activation, co-stimulation with LTA and PGN significantly enhanced NF-κB activation (Fig. 2B). These results suggest that LTA in the presence of PGN, but not LTA or PGN alone, could activate the MAC-T cells.

Fold change

257 258

First, we examined whether LTA and PGN prepared from S. aureus were biologically active and verified that they were not contaminated with endotoxin using a macrophage cell line RAW 264.7 and transfectants with the NF-κB reporter gene. When the cells were stimulated with 30 μg/mL of LTA or PGN for 48 h, the NO production was significantly (p b 0.05) increased by either LTA or PGN. LTA- or PGNinduced NO production was not affected in the presence of the LPS antagonist, PMB (Fig. 1A). Since LTA-induced immune responses are mediated by TLR2 activation [31], the ability of LTA to activate TLR2 was measured using CHO/CD14/TLR2 cells. When CHO/CD14/TLR2 cells were treated with LTA (0, 10, or 30 μg/mL) or a representative TLR2 ligand, Pam2CSK4 (0.1 μg/mL), CD25 expression was markedly increased (Fig. 1B). In contrast, LTA did not induce CD25 expression in CHO/CD14/ TLR4 cells (Fig. 1C). In addition, since it has been reported that the

U

255 256

3.2. NF-κB activity was induced by LTA plus PGN in MAC-T, a bovine mammary gland epithelial cell line

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3.1. LTA and PGN from S. aureus are immunologically active

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247

O

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The mean ± standard deviation (S.D.) was determined from triplicate samples of each experimental group. All statistical significance was measured using Student's t-test and differences were marked by an asterisk (*) when p b 0.05.

253 254

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immuno-stimulating effect of PGN is mediated by NOD2 [17], we examined the ability of PGN to activate NF-κB through NOD2 in HEK 293 cells co-transfected with NOD2 expression plasmid and NF-κB reporter plasmid. As shown in Fig. 1D, PGN significantly increased NF-κB activity through NOD2 signaling pathways. These results indicate that LTA and PGN were biologically active and were not contaminated.

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2.10. Statistical analysis

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Microarray

4

3 2

1 0 CTGF

TGF-β2

PTGS-1 CCDC-80 CSRP-2 WISP-2

PC-TP

TF-4

LMO-4

Fig. 4. The combination of LTA and PGN up-regulates various inflammation-related genes. (A) The heatmap shows the expression levels of 51 up-regulated DEGs, which were categorized as inflammatory mediators, intra-cellular signaling factors, or transcription factors, in MAC-T cells in the presence or absence of LTA plus PGN from S. aureus. The red color represents the up-regulated expression of genes, while the green color represents down-regulated expression of genes. NT denotes no treatment. (B) MAC-T cells (2 × 105 cells/mL) were stimulated with 0 or 30 μg/mL of LTA and PGN from S. aureus for 1 h. After stimulation, total RNA was isolated and subjected to real-time RT-PCR analysis for 9 randomly selected DEGs with FC N2 among the DEGs listed in Tables 2–4, as described in the Materials and methods. Values are the mean of the fold increases to the non-treatment group ± S.D. of triplicate samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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In order to examine gene-expression profiling in MAC-T cells stimulated with LTA plus PGN, a microarray analysis was performed using a GeneChip® Bovine Genome Array containing 24,027 probe sets representing over 23,000 transcripts. To identify DEGs by LTA plus PGN stimulation, the acquired microarray data were statistically analyzed. Of 2019 DEGs, 801 DEGs were up-regulated whereas 1218 DEGs were down-regulated in MAC-T cells following exposure to LTA plus PGN (Fig. 3A). It was noted that most of them (401 up-regulated DEGs and 371 down-regulated DEGs) appeared to be related to gene expression and metabolism, including biosynthetic processes, gene expression, regulation of cellular processes, regulation of border follicle cell delamination, and regulation of biological processes (Fig. 3B).

306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326

t2:1 t2:2 t2:3

3.5. The combination of LTA and PGN down-regulates various immune response-related genes in MAC-T cells In order to identify down-regulated DEGs associated with immune responses induced in MAC-T cells stimulated with LTA plus PGN, we screened down-regulated DEGs with FC N1.2 and p b 0.05, which yielded 17 down-regulated DEGs (Table 5). The heatmap of the down-

Table 2 List of up-regulated DEGs categorized as inflammatory mediator-related DEGs in bovine mammary gland epithelial cells stimulated with LTA plus PGN. Gene

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18

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304 305

To characterize up-regulated DEGs, which are related to inflammatory responses induced in MAC-T cells in response to LTA plus PGN, up-regulated DEGs with FC N1.2 and p b 0.05 were screened. Next, the DEGs were categorized into three groups, including inflammatory mediators, intra-cellular signaling molecules, and transcription factorrelated groups. Fig. 4A shows the heatmap of the up-regulated DEGs in relation to inflammatory responses in MAC-T cells stimulated with LTA plus PGN for 1 h. Table 2 presents a list of 14 inflammatory mediator-related DEGs. Among them, gene expression of endothelin-1 (ET-1), connective tissue growth factor (CTGF), and transforming growth factor-β2 (TGF-β2) was markedly higher (FC N3) than that of the others. In addition, intra-cellular signaling molecule-related DEGs (n = 22) and transcription factor-related DEGs (n = 16) were listed in Tables 3 and 4, respectively. In order to confirm the microarray results for up-regulated DEGs, we performed real-time RT-PCR with 9 randomly selected genes from the up-regulated DEGs with FC N 2, including CTGF, TGF-β2, PTGS-1, CCDC-80, CSRP-2, WISP-2, PC-TP, TF-4, and LMO-4. As shown in Fig. 4B, the expression of the 9 genes was upregulated, as determined by both real-time RT-PCR and the microarray results (Fig. 4B).

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303

3.4. Co-treatment with LTA and PGN up-regulates various inflammation-related genes in MAC-T cells

D

301 302

Coiled-coil domain containing-80 Cysteine and glycine-rich protein-2 Wnt1 inducible signaling pathway protein-2 Phosphatidylcholine transfer protein Rap1 GTPase-GDP dissociation stimulator 1 Alkaline phosphodiesterase I Peptidyl arginine deiminase Phosphoserine phosphatase NADH dehydrogenase Ras homolog gene family, member B TNF receptor-associated factor 6 Ras-GTPase-activating protein SH3-domain-binding protein Proto-oncogene serine/threonineprotein kinase-1 Lysyl oxidase MAP/microtubule affinityregulating kinase 3 GTP-binding nuclear protein Ran Serine/threonine protein kinase Serine/threonine–protein kinase 12 Protein kinase C alpha type Serine/threonine–protein phosphatase PP1-gamma catalytic subunit Serine–threonine kinase receptorassociated protein Protein kinase B

Endothelin-1 Connective tissue growth factor Transforming growth factor β2 Prostaglandin-endoperoxide synthase-1 Neuregulin-1 Endothelin-2 Stromal interaction molecule-1 High mobility group box-1 Heat shock 70 kDa protein 5 Fibroblast growth factor-binding protein 1 High-mobility group box-2 Monocyte chemotactic protein-2 Chondroitin sulfate proteoglycan-6 Hepatoma-derived growth factor

GenBank accession no.

Foldchange

Adjusted p-value

NM_181010 NM_174030 NM_001113252 NM_001105323 NM_174128 NM_175714 NM_001035409 NM_176612 NM_001075148 NM_174337 NM_001037616 NM_174007 NM_174295 NM_175832

3.49 3.36 3.34 2.71 2.56 1.55 1.40 1.37 1.34 1.32 1.30 1.27 1.25 1.21

0.0010 0.0038 0.0146 0.0075 0.0410 0.0048 0.0114 0.0187 0.0132 0.0278 0.0465 0.0351 0.0498 0.0459

t3:1 t3:2 t3:3

GenBank accession no.

Foldchange

Adjusted p-value

t3:4

NM_001098982 NM_001038183 NM_001102176

3.47 2.15 2.09

0.0337 0.0096 0.0142

t3:5 t3:6 t3:7

NM_174835 NM_174666 NM_001075144 XR_042986 NM_001046355 NM_175829 NM_001077922 XM_592305 NM_001037611

2.01 1.88 1.77 1.59 1.58 1.56 1.54 1.54 1.52

0.0010 0.0129 0.0265 0.0233 0.0095 0.0339 0.0264 0.0381 0.0108

t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16

NM_174144

1.46

0.0139

t3:17

NM_173932 XM_001788800

1.43 1.39

0.0477 0.0220

t3:18 t3:19

XR_027380 XM_589151 NM_183084 NM_174435 NM_174581

1.37 1.30 1.29 1.27 1.24

0.0249 0.0250 0.0277 0.0394 0.0277

t3:20 t3:21 t3:22 t3:23 t3:24

NM_001015567

1.23

0.0365

t3:25

NM_173986

1.21

0.0440

t3:26

E

300

Gene

T

299

C

297 298

E

295 296

R

293 294

R

291 292

N C O

290

U

288 289

Table 3 List of up-regulated DEGs categorized as intra-cellular signaling molecule-related DEGs in bovine mammary gland epithelial cells stimulated with LTA and PGN.

F

3.3. Co-treatment with LTA and PGN regulates expression of various genes in MAC-T cells

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285 286

7

regulated DEGs is shown in Fig. 5A. In order to validate the microarray results of down-regulated DEGs, 9 down-regulated DEGs having a FC N3, including GPNMB, MMP-1, CD36, CCR7, RENBP, MMP-9, CEACAM 1, IL-13RA, and LTBP 2, were selected and their mRNA expression was analyzed by real-time RT-PCR. As shown in Fig. 5B, the expression of 9 genes was up-regulated, as determined by both real-time RT-PCR and the microarray results (Fig. 5B).

327

4. Discussion

334

In this study, we characterized the gene-expression profile of MAC-T cells co-stimulated with LTA and PGN using a DNA microarray analysis. The results of the microarray analysis showed 2019 DEGs induced by the stimulation with LTA and PGN. Among them, 801 DEGs were up-regulated and 1218 DEGs were down-regulated. Through the

335 336

Table 4 List of up-regulated DEGs categorized as transcription factor-related DEGs in bovine mammary gland epithelial cells stimulated with LTA and PGN.

328 329 330 331 332 333

337 338 339 t4:1 t4:2 t4:3

Gene

GenBank accession no.

Foldchange

Adjusted p-value

t4:4

Transcription factor 4 LIM domain only-4 Jun oncogene GATA-6 Zinc finger protein 297B CCAAT/enhancer binding protein alpha Nuclear factor 1 A-type Zinc finger protein 330 Nucleosome assembly protein 1-like 1 Myb-related protein B Transforming growth factor beta 1 induced transcript 1 Nuclear factor I/C Zinc finger protein 262 X-box binding protein 1 CCAAT/enhancer-binding protein-gamma

NM_001034621 NM_001034751 NM_001077827 XM_001253596 NM_001024503 NM_176784 NM_001038209 NM_001038157 NM_001099215 NM_001075448 NM_001035313

2.24 2.18 1.84 1.69 1.64 1.51 1.51 1.46 1.46 1.43 1.43

0.0337 0.0193 0.0079 0.0377 0.0218 0.0299 0.0335 0.0215 0.0207 0.0126 0.0312

t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15

NM_001024574 XM_001790260 NM_001034727 NM_001034801

1.42 1.35 1.31 1.31

0.0402 0.0494 0.0197 0.0192

t4:16 t4:17 t4:18 t4:19

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

Table 5 List of down-regulated DEGs categorized as inflammation-related DEGs in bovine mammary gland epithelial cells stimulated with LTA and PGN. GenBank accession no.

Foldchange

Adjusted p-value

Glycoprotein non-metastatic melanoma protein B Matrix metalloproteinase-1 Cluster of differentiation 36 C–C chemokine receptor type 7 Renin binding protein Matrix metalloproteinase-9 Carcinoembryonic antigen-related cell adhesion molecule 1 Interleukin-13 receptor alpha-1 Latent TGF-β-binding protein 2 Suppressor of cytokine signaling-2 Cyclooxygenase-2 Chemokine (C–X–C motif) ligand-1 Aryl hydrocarbon receptor Early growth response protein 1 Vascular endothelial growth factor Cluster of differentiation 87 Cluster of differentiation 46

NM_001038065

15.37

0.0001

NM_174112 NM_174010 NM_001024930 NM_001046223 NM_174744 NM_205788

10.84 9.71 4.32 4.29 3.45 3.42

0.0041 0.0219 0.0017 0.0002 0.0011 0.0287

XM_583913 NM_174385 NM_177523 NM_174445 NM_001048165 XM_612996 NM_001045875 NM_174216 NM_174423 NM_183080

3.09 3.00 2.52 2.15 2.10 1.99 1.87 1.84 1.31 1.24

0.0060 0.0020 0.0140 0.0070 0.0305 0.0042 0.0020 0.0011 0.0413 0.0262

O

(A)

Up-regulated

R

R

E

C

T

E

Down-regulated

O

342

categorization of DEGs having a FC N 1.2 and p b 0.05, we found 14 inflammatory mediator-related DEGs, 22 intra-cellular signaling molecule-related DEGs, and 15 transcription factor-related DEGs

C

340 341

(B)

N

t5:12 t5:13 t5:14 t5:15 t5:16 t5:17 t5:18 t5:19 t5:20 t5:21

Fold change

t5:6 t5:7 t5:8 t5:9 t5:10 t5:11

U

t5:5

R O

t5:4

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Gene

among the up-regulated DEGs, and 17 down-regulated DEGs associated with inflammatory responses by stimulation with LTA and PGN. Among 801 up-regulated DEGs, 14 DEGs were associated with inflammatory mediator gene expression including ET-1 and CTGF, which were most significantly up-regulated by the co-treatment with LTA and PGN of S. aureus. Even though the inflammatory mediators have not been clearly elucidated for the pathogenesis of bovine mastitis [33–35], accumulating results have demonstrated that ET-1 increased a chemokine, macrophage inflammatory protein-1β, in human peripheral blood monocytes [33] and enhanced the infiltration of tissue macrophages and lymphocytes, as well as the expression of inducible nitric oxide synthase [34]. An increased level of ET-1 is often found in chronic diseases, such as pulmonary hypertension and congestive heart failure [36], as well as in chronic inflammatory bowel diseases [37]. The inflammatory potential of CTGF has previously been reported to be involved with inflammatory cell infiltration, and pro-inflammatory cytokines such as IL-6 and chemokines including monocyte chemoattractant protein 1 (MCP-1) [35,38]. Furthermore, up-regulated CTGF is strongly associated with the progression of many chronic inflammatory diseases [39]. Therefore, our findings suggest that the up-regulation of ET-1 and CTGF, stimulated by LTA and PGN of S. aureus, might contribute to the development of chronic mastitis. On the contrary, we also observed the up-regulation of DEGs that are associated with anti-inflammatory mediators, such as TGF-β2 and NRG-1. TGF-β2 has been shown to suppress the expression of pro-

D

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J. Im et al. / International Immunopharmacology xxx (2014) xxx–xxx

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GPNMB

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MMP-1

CD36

CCR7

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MMP-9 CEACAM 1 IL-13RA LTBP 2

0

-2 -4 -6 -8

-10 -12 -14 -16

Real-time RT-PCR Microarray

Fig. 5. LTA and PGN down-regulate various immune response-related genes. (A) The heatmap shows the expression levels of down-regulated 17 DEGs, which were associated with inflammation, in MAC-T cells treated with LTA plus PGN from S. aureus. Red color represents high expression, while green color represents low expression. NT denotes no treatment. (B) MAC-T cells (2 × 105 cells/mL) were stimulated with 0 or 30 μg/mL of LTA and PGN from S. aureus for 1 h. After stimulation, total RNA was isolated and subjected to real-time RTPCR analysis for the 9 DEGs having FC N3 among the DEGs shown in Table 5, as described in the Materials and methods. Values are the mean of the fold increases to the LTA plus PGN treatment group ± S.D. of triplicate samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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References

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C

389 390

E

387 388

R

385 386

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383 384

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381 382

U

379 380

[7]

[8]

[9]

[10]

[11] [12]

[13] [14] [15]

[1] Zhao X, Lacasse P. Mammary tissue damage during bovine mastitis: causes and control. J Anim Sci 2008;86:57–65. [2] Yang W, Zerbe H, Petzl W, Brunner RM, Gunther J, Draing C, et al. Bovine TLR2 and TLR4 properly transduce signals from Staphylococcus aureus and E. coli, but S. aureus fails to both activate NF-kappaB in mammary epithelial cells and to quickly induce

F

424

377 378

[6]

O

423 Q4

This work was supported by grants from the Biogreen 21 Program, Rural Development Administration (PJ007066), the National Research Foundation of Korea funded by the Korean Ministry of Education, Science, and Technology (2010-0029116 and 2008-0062421), and the R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

375 376

[5]

R O

418

374

[4]

P

Acknowledgments

372 373

[3]

TNFalpha and interleukin-8 (CXCL8) expression in the udder. Mol Immunol 2008;45:1385–97. Bannerman DD, Paape MJ, Lee JW, Zhao X, Hope JC, Rainard P. Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clin Diagn Lab Immunol 2004;11:463–72. Wellenberg GJ, van der Poel WH, Van Oirschot JT. Viral infections and bovine mastitis: a review. Vet Microbiol 2002;88:27–45. Oviedo-Boyso J, Valdez-Alarcon JJ, Cajero-Juarez M, Ochoa-Zarzosa A, Lopez-Meza JE, Bravo-Patino A, et al. Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis. J Infect 2007;54:399–409. Anaya-Lopez JL, Contreras-Guzman OE, Carabez-Trejo A, Baizabal-Aguirre VM, Lopez-Meza JE, Valdez-Alarcon JJ, et al. Invasive potential of bacterial isolates associated with subclinical bovine mastitis. Res Vet Sci 2006;81:358–61. Wesson CA, Deringer J, Liou LE, Bayles KW, Bohach GA, Trumble WR. Apoptosis induced by Staphylococcus aureus in epithelial cells utilizes a mechanism involving caspases 8 and 3. Infect Immun 2000;68:2998–3001. De Kimpe SJ, Kengatharan M, Thiemermann C, Vane JR. The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure. Proc Natl Acad Sci U S A 1995;92:10359–63. Hermann C, Spreitzer I, Schroder NW, Morath S, Lehner MD, Fischer W, et al. Cytokine induction by purified lipoteichoic acids from various bacterial species— role of LBP, sCD14, CD14 and failure to induce IL-12 and subsequent IFN-gamma release. Eur J Immunol 2002;32:541–51. Fabretti F, Theilacker C, Baldassarri L, Kaczynski Z, Kropec A, Holst O, et al. Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infect Immun 2006;74:4164–71. Ginsburg I. Role of lipoteichoic acid in infection and inflammation. Lancet Infect Dis 2002;2:171–9. Fischer W. Molecular analysis of lipid macroamphiphiles by hydrophobic interaction chromatography, exemplified with lipoteichoic acids. Anal Biochem 1993;208: 49–56. Buckley JM, Wang JH, Redmond HP. Cellular reprogramming by Gram-positive bacterial components: a review. J Leukoc Biol 2006;80:731–41. Morrison DC, Ryan JL. Endotoxins and disease mechanisms. Annu Rev Med 1987;38:417–32. Alkan ML, Beachey EH. Excretion of lipoteichoic acid by group A streptococci. Influence of penicillin on excretion and loss of ability to adhere to human oral mucosal cells. J Clin Invest 1978;61:671–7. Palaniyar N, Nadesalingam J, Reid KB. Pulmonary innate immune proteins and receptors that interact with Gram-positive bacterial ligands. Immunobiology 2002;205:575–94. Strober W, Murray PJ, Kitani A, Watanabe T. Signalling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol 2006;6:9–20. Kumar A, Zhang J, Yu FS. Innate immune response of corneal epithelial cells to Staphylococcus aureus infection: role of peptidoglycan in stimulating proinflammatory cytokine secretion. Invest Ophthalmol Vis Sci 2004;45:3513–22. Wang JE, Dahle MK, McDonald M, Foster SJ, Aasen AO, Thiemermann C. Peptidoglycan and lipoteichoic acid in Gram-positive bacterial sepsis: receptors, signal transduction, biological effects, and synergism. Shock 2003;20:402–14. Ryu YH, Baik JE, Yang JS, Kang SS, Im J, Yun CH, et al. Differential immunostimulatory effects of Gram-positive bacteria due to their lipoteichoic acids. Int Immunopharmacol 2009;9:127–33. Jang KS, Baik JE, Han SH, Chung DK, Kim BG. Multi-spectrometric analyses of lipoteichoic acids isolated from Lactobacillus plantarum. Biochem Biophys Res Commun 2011;407:823–30. Huynh HT, Robitaille G, Turner JD. Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp Cell Res 1991;197:191–9. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131–8. Im J, Jeon JH, Cho MK, Woo SS, Kang SS, Yun CH, et al. Induction of IL-8 expression by bacterial flagellin is mediated through lipid raft formation and intracellular TLR5 activation in A549 cells. Mol Immunol 2009;47:614–22. Medvedev AE, Henneke P, Schromm A, Lien E, Ingalls R, Fenton MJ, et al. Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4. J Immunol 2001;167:2257–67. Gautier L, Cope L, Bolstad BM, Irizarry RA. affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 2004;20:307–15. Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004:3 [Article3]. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B (Stat Methodol) 1995: 289–300. Warnes G, Bolker B, Lumley T. gplots: various R programming tools for plotting data. URL http://cran r-project org/web/packages/gplots/index html. Ashburner M, Ball C, Blake J, Botstein D, Butler H, Cherry J, et al. Gene ontology: tool for the unification of biology. Nat Genet 2000;25:25–9. Akira S. TLR signaling. Curr Top Microbiol Immunol 2006;311:1–16. Kawai T, Akira S. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med 2007;13:460–9. Gonsalves C, Kalra VK. Endothelin-1-induced macrophage inflammatory protein1beta expression in monocytic cells involves hypoxia-inducible factor-1alpha and AP-1 and is negatively regulated by microRNA-195. J Immunol 185:6253–64. Hocher B, Schwarz A, Slowinski T, Bachmann S, Pfeilschifter J, Neumayer HH, et al. In-vivo interaction of nitric oxide and endothelin. J Hypertens 2004;22:111–9.

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inflammatory cytokines and chemokines including IL-6, IL-8, and MCP1 in human intestinal epithelial cells [40]. A previous report has also demonstrated that NRG-1 attenuated the activation and nitrite release in murine microglial cells [41]. Moreover, a mutation to NRG-1 in lymphoblastoid cells enhanced the expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-8 [42]. Although both E. coli and S. aureus cause acute inflammation of the mammary glands, they confer different kinetics of inflammatory responses. Stimulation of mammary epithelial cells with E. coli LPS elicited the sustained induction of proinflammatory mediators, such as TNF-α and IL-8 [43]. In contrast, S. aureus-induced pro-inflammatory mediators rapidly increased at early time points, after which the pro-inflammatory responses dramatically declined to a basal level at 24 h in mammary epithelial cells [44]. Indeed, S. aureus-induced mastitis is associated with moderate inflammatory responses, resulting in chronic and subclinical mastitis [5]. Therefore, the lack of sustained pro-inflammatory responses could favor persistent infection by S. aureus, leading to chronic mastitis. We also observed several down-regulated DEGs, including GPNMB, MMPs and CD36, related to inflammation in MAC-T cells in response to LTA and PGN. GPNMB is a type I transmembrane protein, which is associated with cell differentiation, inflammation, tissue regeneration, and tumor progression [45]. The overexpression of GPNMB results in enhanced expression of MMPs, such as MMP-3 and MMP-9 [46]. We found a significant decrease of GPNMB in MAC-T cells stimulated with LTA and PGN. Concomitantly, down-regulated expression of MMPs such as MMP-1 and MMP-9 was observed in MAC-T cells in response to LTA and PGN, indicating that the decreased expression of MMP-1 and MMP-9 may be accompanied by down-regulated GPNMB. More importantly, MMPs play an important role in inflammation by regulating cytokine and chemokine activity [47]. For example, MMP-9 has been known to contribute to enhanced IL-8 activity [48]. It has been proposed that CD36 contributes to the concentration of ligands for TLR2 recognition, implicating its involvement in inflammatory responses [49]. The persistent inflammation of mastitis caused by S. aureus could occur as a result of loss of enhancement of pro-inflammatory responses initiated by TLR2 recognition with the down-regulated CD36 expression. S. aureus is regarded as a major pathogenic Gram-positive bacterium responsible for causing bovine mastitis. Moreover, S. aureus infection often causes chronic inflammation in bovine mammary glands for the entire life of dairy cattle, sometimes without visible signs of disease. Despite the pathologic importance of S. aureus in bovine mastitis, most pathologic studies have focused on bovine mastitis caused by E. coli infection and the role of its virulence factor, LPS. However, little is known about the roles of the major virulence factors of S. aureus, LTA and PGN, in bovine mastitis. In this study, we conducted gene-expression profiling in bovine mammary gland epithelial cells treated with LTA and PGN of S. aureus, and our findings might contribute to a better understanding of the pathogenesis of chronic mastitis induced by S. aureus infections.

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[17]

[18]

[19]

[20]

[21]

[22] [23]

[24]

[25]

[26] [27] [28]

[29] [30] [31] [32] [33]

[34]

Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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[43] Strandberg Y, Gray C, Vuocolo T, Donaldson L, Broadway M, Tellam R. Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine 2005;31:72–86. [44] Lahouassa H, Moussay E, Rainard P, Riollet C. Differential cytokine and chemokine responses of bovine mammary epithelial cells to Staphylococcus aureus and Escherichia coli. Cytokine 2007;38:12–21. [45] Rose AA, Siegel PM. Emerging therapeutic targets in breast cancer bone metastasis. Future Oncol 2010;6:55–74. [46] Rich JN, Shi Q, Hjelmeland M, Cummings TJ, Kuan CT, Bigner DD, et al. Bone-related genes expressed in advanced malignancies induce invasion and metastasis in a genetically defined human cancer model. J Biol Chem 2003;278:15951–7. [47] Parks WC, Wilson CL, Lopez-Boado YS. Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 2004;4:617–29. [48] Van den Steen PE, Proost P, Wuyts A, Van Damme J, Opdenakker G. Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact. Blood 2000;96:2673–81. [49] Jimenez-Dalmaroni MJ, Xiao N, Corper AL, Verdino P, Ainge GD, Larsen DS, et al. Soluble CD36 ectodomain binds negatively charged diacylglycerol ligands and acts as a co-receptor for TLR2. PLoS One 2009;4:e7411.

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[35] Sanchez-Lopez E, Rayego S, Rodrigues-Diez R, Rodriguez JS, Rodriguez-Vita J, Carvajal G, et al. CTGF promotes inflammatory cell infiltration of the renal interstitium by activating NF-kappaB. J Am Soc Nephrol 2009;20:1513–26. [36] Cody RJ, Haas GJ, Binkley PF, Capers Q, Kelley R. Plasma endothelin correlates with the extent of pulmonary hypertension in patients with chronic congestive heart failure. Circulation 1992;85:504–9. [37] Murch SH, Braegger CP, Sessa WC, MacDonald TT. High endothelin-1 immunoreactivity in Crohn's disease and ulcerative colitis. Lancet 1992;339:381–5. [38] Wu SH, Wu XH, Lu C, Dong L, Zhou GP, Chen ZQ. Lipoxin A4 inhibits connective tissue growth factor-induced production of chemokines in rat mesangial cells. Kidney Int 2006;69:248–56. [39] Gressner OA, Fang M, Li H, Lu LG, Gressner AM, Gao CF. Connective tissue growth factor (CTGF/CCN2) in serum is an indicator of fibrogenic progression and malignant transformation in patients with chronic hepatitis B infection. Clin Chim Acta 2013;421:126–31. [40] Rautava S, Nanthakumar NN, Dubert-Ferrandon A, Lu L, Rautava J, Walker WA. Breast milk-transforming growth factor-beta(2) specifically attenuates IL-1betainduced inflammatory responses in the immature human intestine via an SMAD6and ERK-dependent mechanism. Neonatology 99:192–201. [41] Dimayuga FO, Ding Q, Keller JN, Marchionni MA, Seroogy KB, Bruce-Keller AJ. The neuregulin GGF2 attenuates free radical release from activated microglial cells. J Neuroimmunol 2003;136:67–74. [42] Marballi K, Quinones MP, Jimenez F, Escamilla MA, Raventos H, Soto-Bernardini MC, et al. In vivo and in vitro genetic evidence of involvement of neuregulin 1 in immune system dysregulation. J Mol Med 88:1133–41.

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Please cite this article as: Im J, et al, Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.05.002

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