Transcriptome Analysis of The Inflammatory Responses of Bovine Mammary Epithelial Cells: Exploring Immunomodulatory Target Genes for Bovine Mastitis

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Abstract

Bovine mastitis is the inflammatory reaction of the mammary gland and is commonly caused by bacterial infections in high-yielding dairy cows. The detailed investigation of the immunotranscriptomic response of bovine mammary epithelial (BME) cells to pattern recognition receptors (PRRs) activation by microbial-associated molecular patterns (MAMPs) can be of great importance for understanding the innate immune defense mechanisms, and for exploring the immunomodulatory candidate genes. In this work, we investigated the transcriptome modifications of BME cells after the in vitro stimulation with Escherichia coli derived lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus JE2 and S. aureus SA003. In addition, the effect of Pam3CSK4 (a synthetic triacylated lipopeptide that activates Toll-like receptor 2 (TLR2)), and the intracellular chemotactic protein cyclophilin A (CyPA), which is secreted by BME cells during mastitis, in the expression changes of selected cytokines and chemokines were evaluated by qPCR. Microarray analysis identified 447, 465 and 520 differentially expressed genes (DEGs) in the BME cells after LPS, S. aureus JE2 and S. aureus SA003 stimulation, respectively. A major differential response in the inflammatory gene expression was noticed between the stimulation of LPS and S. aureus strains. Unlike the S. aureus strains, LPS stimulation resulted in significant upregulation of CCL2, CXCL2, CXCL3, CXCL8, IL1α and IL1β, which were confirmed by qPCR analysis. Pam3CSK4 was not able to induce significant changes in the expression of cytokines and chemokines in challenged BME cells. The exogenous CyPA administration was able to upregulate CXCL2, CXCL3, CXCL8, IL1α and IL1β expression in BME cells indicating its ability to promote inflammation. The identification of transcriptional markers of mastitis specific for individual inflammatory factors such as LPS, Pam3CSK4 or CyPA, which can be evaluated in vitro in BME cells, may enable the development of novel diagnostics and/or immunomodulatory treatments, providing new tools for the effective management of mastitis in dairy cows. The results of this work are an advance in this regard.

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Most cited references 28

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Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved.

Nicolas W J Schröder, Siegfried Morath, Christian Alexander … (2003)

Lipoteichoic acid (LTA) derived from Streptococcus pneumoniae, purified employing a chloroform/methanol protocol, and from Staphylococcus aureus, prepared by the recently described butanol extraction procedure, was investigated regarding its interaction with lipopolysaccharide (LPS)-binding protein (LBP), CD14, Toll-like receptors (TLRs)-2 and -4, and MD-2. LTA from both organisms induced cytokine synthesis in human mononuclear phagocytes. Activation was LBP- and CD14-dependent, and formation of complexes of LTA with LBP and soluble CD14 as well as catalytic transfer of LTA to CD14 by LBP was verified by PhastGel(TM) native gel electrophoresis. Human embryonic kidney (HEK) 293/CD14 cells and Chinese hamster ovary (CHO) cells were responsive to LTA only after transfection with TLR-2. Additional transfection with MD-2 did not affect stimulation of these cells by LTA. Our data suggest that innate immune recognition of LTA via LBP, CD14, and TLR-2 represents an important mechanism in the pathogenesis of systemic complications in the course of infectious diseases brought about by the clinically most important Gram-positive pathogens. However, the involvement of TLR-4 and MD-2 in this process was ruled out.

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Cyclophilin: a specific cytosolic binding protein for cyclosporin A

J. Rice, D. Speicher, R. Handschumacher … (1984)

Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 X 10(-7) moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH2-terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56 degrees C and at pH 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism.

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Cyclophilin A enhances vascular oxidative stress and development of angiotensin II-induced aortic aneurysms

Kimio Satoh, Patrizia Nigro, Tetsuya Matoba … (2009)

Inflammation and oxidative stress are pathogenic mediators of many diseases, but therapeutic targets remain elusive. In the vasculature, abdominal aortic aneurysm (AAA) formation critically involves inflammaton and matrix degradation. Cyclophilin A (CyPA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMC), is secreted in response to reactive oxygen species (ROS), and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe −/− mice, we show that Apoe −/− Ppia −/− mice were completely protected from AngII–induced AAA formation, in contrast to Apoe −/− Ppia +/+ mice. Apoe −/− Ppia −/− mice showed decreased inflammatory cytokine expression, elastic lamina degradation, and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia +/+ mice. Mechanistic studies demonstrated that VSMC-derived intracellular and extracellular CyPA were required for ROS generation and matrix metalloproteinase-2 activation. These data define a novel role for CyPA in AAA formation and suggest CyPA is a new target for cardiovascular therapies.

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Author and article information

Journal

Journal ID (nlm-ta): Pathogens

Journal ID (iso-abbrev): Pathogens

Journal ID (publisher-id): pathogens

Title: Pathogens

Publisher: MDPI

ISSN (Electronic): 2076-0817

Publication date (Electronic): 09 March 2020

Publication date Collection: March 2020

Volume: 9

Issue: 3

Electronic Location Identifier: 200

Affiliations

[1 ]Food and Feed Immunology Group, Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan; aminul.vmed@ 123456bau.edu.bd (M.A.I.); takagimichihiro@ 123456gmail.com (M.T.); fukuyama.k.mc0511@ 123456gmail.com (K.F.); dondonassima26@ 123456gmail.com (R.K.); leonardogenius5@ 123456gmail.com (L.A.); wakako.ohtsubo.a7@ 123456tohoku.ac.jp (W.I.-O.); jcvillena@ 123456cerela.org.ar (J.V.)

[2 ]Livestock Immunology Unit, International Education and Research Center for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan

[3 ]Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

[4 ]Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli, (CERELA-CONICET), Tucuman 980-0845, Argentina

[5 ]Scientific Computing Laboratory, Computer Science Department, Faculty of Exact Sciences and Technology, National University of Tucuman, Tucuman 980-0845, Argentina

[6 ]Infection Immunity Unit, International Education and Research Center for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan; tomonori.nochi.a5@ 123456tohoku.ac.jp

[7 ]Cell Biology Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan

[8 ]Graduate School of Food, Agriculture and Environment, Miyagi University, Sendai 980-8572, Japan; suda@ 123456myu.ac.jp

[9 ]Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands; v.rutten@ 123456uu.nl (V.R.); W.vanEden@ 123456uu.nl (W.v.E.)

[10 ]Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private bag X20, Hatfield 0028, South Africa

Author notes

[* ]Correspondence: asosan@ 123456tohoku.ac.jp (H.A.); haruki.kitazawa.c7@ 123456tohoku.ac.jp (H.K.)

[†]

These authors have contributed equally to this work.

Author information

Md. Aminul Islam https://orcid.org/0000-0003-1979-9652

Leonardo Albarracin https://orcid.org/0000-0001-8216-2190

Julio Villena https://orcid.org/0000-0003-4941-4960

Article

Publisher ID: pathogens-09-00200

DOI: 10.3390/pathogens9030200

PMC ID: 7157600

PubMed ID: 32182886

SO-VID: 3bd47503-3cad-4fb9-b070-44785ee200a9

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Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

Transcriptome Analysis of The Inflammatory Responses of Bovine Mammary Epithelial Cells: Exploring Immunomodulatory Target Genes for Bovine Mastitis

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Biomarkers for Inflammatory Lung Disease

Most cited references 28

Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved.

Cyclophilin: a specific cytosolic binding protein for cyclosporin A

Cyclophilin A enhances vascular oxidative stress and development of angiotensin II-induced aortic aneurysms

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