Proinflammatory Cytokine Gene Expression by Equine Epidermal Epithelial Cells Upon Exposure To Bacterial Components in vitro
The host response to bacterial components involves the innate immune system through recognition of, pathogen-associated molecular patterns (PAMPs). These PAMPs are recognized by different pattern recognition receptors (PRRs), such as the toll-like (TLR) and NOD receptors present on the surface (and cytoplasm) of the host cells; subsequent activation of these receptors can result not only in a protective response, but commonly in inflammatory injury to the host cells and tissue. The response to various bacterial ligands has been well documented in human epidermal and visceral (i.e., renal, intestinal) epithelial cells, where inflammatory signaling due to PRR binding plays an important role in both superficial and systemic sepsis. However, little information exists regarding equine epithelial cells and their response to bacterial ligands and toxins. The epidermal epithelial cell response is not only important clinically from the perspective of skin infection or wound healing in the horse, but is also important in laminitis where the laminar epidermal epithelial cells are at the point of failure in laminitis. Therefore, we investigated the response of equine epidermal epithelial cells to bacterial components likely to be present in the septic equine patient at risk of sequelae such as laminitis. Equine epidermal epithelial cells were cultured from skin of horses euthanized for reasons other than systemic illness; cells (passage 3-4) from two horses were exposed to lipopolysaccharide (LPS, TLR4 ligand; 100 ng/ml, 500 ng/ml, and 5 µg/ml), gram-positive peptidoglycan (PGN, NOD2 receptor ligand;100 ng/ml, 500 ng/ml, 5 µg/ml, and 10 µg/ml), lipoteichoic acid (LTA, TLR2 ligand; 1 µg/ml and 10 µg/ml), bacterial DNA (CpG, TLR9 ligand; 1 uM ), gram-negative flagellin (100 ng/ml, TLR5 ligand; 500 ng/ml, and 5 µg/ml) or maintained in media (controls) for 4 and 24 hours. Conditions were performed in triplicate. After incubation, epithelial cells were harvested and RNA isolated to quantify interleukin-1β, interleukin-6 and interleukin-8 mRNA expression via real-time PCR. Significant (P<0.05) increases in IL-1β, IL-6, and IL-8 gene expression were present after stimulation with LPS when compared to controls. Increased expression of both IL-6 and IL-8 were also noted in response to flagellin stimulation when compared to control culture. Overall responses were greatest at 4 hours. No significant increases in cytokine expression in epidermal epithelial cells were present after stimulation with PGN, LTA, or CpG. The cytokine expression present after stimulation of equine epithelial cells by LPS is similar to reports in the human literature. However, the negative response to gram-positive ligands is not consistent with responses of human keratinocytes in culture. These findings are consistent with the observations that, in regards to sepsis-related sequelae such as laminitis, horses are much more sensitive to gram-negative sepsis and bacterial toxin systemically compared to gram-positive sepsis.