The Concentration and Duration of Lipopolysaccharide Stimulation Produce Different Cytokine Responses in an Ex Vivo Whole Blood Model in Horses.
Abstract: Lipopolysaccharide (LPS) is frequently used in equine research to model clinical endotoxemia; however, there is no standardized protocol for inducing cytokine production in equine whole blood. To address this knowledge gap, the goal of this study was to compare the cytokine responses elicited by three different LPS stimulation protocols. Whole blood was collected from six healthy horses (aged 5-30 years; mixed breeds and genders) from the North Carolina State University teaching herd (IACUC #23-412). Sixty milliliters of heparinized blood were aseptically drawn and divided into 15 mL aliquots. Samples were stimulated with LPS at concentrations of 100 ng/mL, 1000 ng/mL, or using a two-hit model (500 ng/mL initially and again at 1.5 h). Incubation occurred at 37 °C on an orbital shaker for time points ranging from 1.5 h to 24 h. Cytokine concentrations were measured using the Cornell Equine Cytokine and Chemokine Panel and were compared to non-stimulated controls. LPS stimulation induced the production of TNF-α, IL-1β, IL-10, CCL5, and CCL11 in a time- and concentration-dependent manner. Notably, reliable and robust cytokine responses were observed only after 12 h of stimulation with either 1000 ng/mL or the two-hit 500/500 ng/mL protocol. These findings suggest that both the concentration and duration of LPS exposure significantly influence cytokine expression in equine whole blood. Therefore, the optimal stimulation protocol may vary depending on the specific cytokine of interest, and careful consideration of these variables is essential for designing reproducible and physiologically relevant ex vivo models of equine endotoxemia.
Publication Date: 2025-11-16 PubMed ID: 41295728PubMed Central: PMC12656798DOI: 10.3390/vetsci12111090Google Scholar: Lookup
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- Journal Article
Summary
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Overview
- This study investigated how different concentrations and exposure times of lipopolysaccharide (LPS) affect cytokine production in horse whole blood samples.
- The goal was to identify optimal stimulation protocols for reliably inducing cytokine responses in an ex vivo model of equine endotoxemia.
Background
- LPS is a component of the outer membrane of Gram-negative bacteria and commonly used to simulate endotoxemia (a systemic inflammatory response) in horse research.
- Endotoxemia in horses can cause severe inflammatory responses, often characterized by increased production of cytokines, which are signaling proteins involved in immune regulation.
- Despite widespread use, there was no standardized protocol for how best to stimulate equine whole blood with LPS to induce cytokine production.
- This lack of standardization leads to variability in research outcomes, complicating comparisons and reproducibility.
Research Objectives
- To compare cytokine responses in equine whole blood when stimulated with different LPS concentrations and exposure durations.
- To determine which LPS stimulation protocols produce reliable and robust cytokine production ex vivo.
Methodology
- Blood samples were collected from six healthy horses of mixed breed, age, and gender from a university teaching herd.
- Blood was anticoagulated with heparin and divided into 15 mL aliquots for stimulation.
- Three LPS stimulation protocols were tested:
- Single exposure at 100 ng/mL LPS
- Single exposure at 1000 ng/mL LPS
- Two-hit model with 500 ng/mL LPS applied initially and repeated at 1.5 hours
- Samples were incubated at 37 °C on an orbital shaker to simulate physiological conditions and ensure mixing, for times ranging from 1.5 to 24 hours.
- Cytokine concentrations were measured using a specialized equine cytokine panel that detects TNF-α, IL-1β, IL-10, CCL5, and CCL11.
- Results were compared against non-stimulated control samples to determine the effect of LPS stimulation.
Results
- LPS stimulation triggered production of several key cytokines associated with inflammation and immune response:
- Tumor necrosis factor-alpha (TNF-α)
- Interleukin-1 beta (IL-1β)
- Interleukin-10 (IL-10), an anti-inflammatory cytokine
- Chemokines CCL5 and CCL11, which attract immune cells
- The cytokine production depended on both the concentration of LPS and the duration of stimulation:
- Lower concentration (100 ng/mL) or shorter stimulation times (under 12 hours) generated inconsistent or weak cytokine responses.
- Higher concentration (1000 ng/mL) or the two-hit protocol (500 ng/mL twice) induced robust and reliable cytokine responses, particularly after 12 hours.
- This indicates that both the amount of LPS and length of incubation critically affect the responsiveness of equine blood immune cells.
Conclusions and Implications
- Optimal stimulation protocols for measuring cytokines ex vivo in equine whole blood require careful consideration of both LPS concentration and incubation time.
- For strong and reproducible cytokine expression, stimulating with either 1000 ng/mL LPS or a two-hit 500/500 ng/mL protocol for at least 12 hours is recommended.
- The choice of protocol may vary based on which cytokine(s) researchers are interested in, meaning protocols should be tailored to specific research questions.
- These findings help standardize ex vivo models of equine endotoxemia, enhancing the consistency and translational relevance of future inflammatory and immunological studies in horses.
- Ultimately, this contributes to better understanding, diagnosis, and treatment of endotoxemia-related diseases in horses by improving experimental design in this field.
Cite This Article
APA
Mitlyng N, Hobbs KJ, Cooper BL, Sheats MK.
(2025).
The Concentration and Duration of Lipopolysaccharide Stimulation Produce Different Cytokine Responses in an Ex Vivo Whole Blood Model in Horses.
Vet Sci, 12(11), 1090.
https://doi.org/10.3390/vetsci12111090 Publication
Researcher Affiliations
- Department of Clinical Sciences, North Carolina State University, Raleigh, NC 27695, USA.
- Department of Clinical Sciences, North Carolina State University, Raleigh, NC 27695, USA.
- Department of Clinical Sciences, Texas A&M University, College Station, TX 77845, USA.
- Department of Clinical Sciences, North Carolina State University, Raleigh, NC 27695, USA.
- Department of Clinical Sciences, North Carolina State University, Raleigh, NC 27695, USA.
Grant Funding
- Unknown / National Institute for Health and Care Research
Conflict of Interest Statement
The author declare no conflicts of Interest.
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