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Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen-associated molecular pattern motifs.
Abstract: Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. Objective: To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen-associated molecular pattern molecules (PAMPs) from both Gram-negative and positive bacteria likely to be present in equine sepsis. Methods: Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), flagellin or maintained in medium (controls) for 4 or 24 h. Real time-quantitative PCR was used to quantify interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and CXCL8 mRNA concentrations. Results: Increases (P<0.05) in IL-1beta, IL-6 and CXCL8 mRNA concentrations were induced by LPS exposure compared to controls. Increased mRNA concentrations of both IL-6 and CXCL8 were also noted (vs. controls) upon exposure to flagellin. Overall, responses were greater at 4 h. No increases (P>0.05) in cytokine expression by keratinocytes were present after LTA, PGN or CpG exposure. Conclusions: Increased proinflammatory cytokine expression in response to LPS and flagellin indicate that equine keratinocytes have functional TLR4 and TLR5 receptor signalling. However, the lack of keratinocyte stimulation by PGN, LTA or CpG provides no evidence for functional TLR2, TLR9 or NOD receptor signalling. These results suggest that equine keratinocytes are more responsive to PAMPs usually associated with Gram-negative sepsis and unresponsive to PAMPs most commonly associated with Gram-positive sepsis. Conclusions: The increased incidence of injury of epidermal structures in clinical cases of Gram-negative (vs. Gram-positive) sepsis in the horse may be due to a lack of functional TLR signalling for Gram-positive PAMPs in the equine keratinocyte.
Publication Date: 2010-06-09 PubMed ID: 20525046DOI: 10.2746/042516409X478523Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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The research paper explores equine keratinocyte responses to different bacterial molecular patterns, with its findings indicating that these keratinocytes respond more to Gram-negative bacterial patterns compared to Gram-positive ones.
Objective
The objective of the study was to investigate how equine keratinocytes react to variations of pathogen-associated molecular pattern molecules (PAMPs) from both Gram-negative and positive bacteria. Furthermore, the study analyses cytokine response to these PAMPs to better understand horse skin diseases.
Methodology
- Keratinocytes, a type of skin cell, were isolated from two horses and cultivated in a controlled environment.
- These cells were then subjected to PAMPs associated with specific types of bacteria, namely lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), bacterial DNA (CpG), and flagellin.
- After exposing the cells to these PAMPs for a duration of 4 or 24 hours, cells were collected for RNA extraction.
- The concentration of three important inflammatory cytokines – interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and CXCL8 – was assessed using real time-quantitative PCR.
Results
- The study reveals that LPS exposure leads to increased concentrations of IL-1beta, IL-6, and CXCL8 mRNA, while flagellin exposure results in elevated IL-6 and CXCL8 mRNA levels compared to the control group.
- The responses were overall more significant after 4 hours of exposure.
- No increases in cytokine expression were observed after exposure to LTA, PGN, or CpG, suggesting an absence of functional TLR2, TLR9, or NOD receptor signaling.
Conclusions
- The research indicates that equine keratinocytes respond more acutely to PAMPs associated with Gram-negative bacteria, compared to those tied to Gram-positive bacteria.
- The findings suggest that this could potentially explain the higher incidence of injury to epidermal structures in cases of Gram-negative sepsis in horses, attributable to a lack of TLR signaling response to Gram-positive PAMPs in the horse’s skin cells.
Cite This Article
APA
Leise BS, Yin C, Pettigrew A, Belknap JK.
(2010).
Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen-associated molecular pattern motifs.
Equine Vet J, 42(4), 294-303.
https://doi.org/10.2746/042516409X478523 Publication
Researcher Affiliations
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA.
MeSH Terms
- Animals
- Bacterial Proteins / pharmacology
- Cells, Cultured
- Cytokines / genetics
- Cytokines / metabolism
- DNA, Bacterial
- Gene Expression Regulation
- Horses
- Keratinocytes / drug effects
- Keratinocytes / metabolism
- Lipopolysaccharides / pharmacology
- RNA, Messenger / metabolism
Citations
This article has been cited 8 times.- Burns TA, Watts MR, Belknap JK, van Eps AW. Digital lamellar inflammatory signaling in an experimental model of equine preferential weight bearing. J Vet Intern Med 2023 Mar;37(2):681-688.
- Reisinger N, Wendner D, Schauerhuber N, Mayer E. Effect of Lipopolysaccharides (LPS) and Lipoteichoic Acid (LTA) on the Inflammatory Response in Rumen Epithelial Cells (REC) and the Impact of LPS on Claw Explants. Animals (Basel) 2021 Jul 9;11(7).
- Watts MR, Hegedus OC, Eades SC, Belknap JK, Burns TA. Association of sustained supraphysiologic hyperinsulinemia and inflammatory signaling within the digital lamellae in light-breed horses. J Vet Intern Med 2019 May;33(3):1483-1492.
- Hellman S, Hjertner B, Morein B, Fossum C. The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC. Vet Res 2018 Oct 22;49(1):108.
- Tarlinton RE, Alder L, Moreton J, Maboni G, Emes RD, Tötemeyer S. RNA expression of TLR10 in normal equine tissues. BMC Res Notes 2016 Jul 19;9:353.
- Schnabel CL, Steinig P, Koy M, Schuberth HJ, Juhls C, Oswald D, Wittig B, Willenbrock S, Murua Escobar H, Pfarrer C, Wagner B, Jaehnig P, Moritz A, Feige K, Cavalleri JM. Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent. BMC Vet Res 2015 Jun 23;11:140.
- Reisinger N, Schaumberger S, Nagl V, Hessenberger S, Schatzmayr G. Milk thistle extract and silymarin inhibit lipopolysaccharide induced lamellar separation of hoof explants in vitro. Toxins (Basel) 2014 Oct 6;6(10):2962-74.
- Leise BS, Watts MR, Roy S, Yilmaz AS, Alder H, Belknap JK. Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis. Equine Vet J 2015 Jul;47(4):478-88.
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