Differential sensitivity of equine leukocyte populations to toll-like receptor ligands

Abstract

Systemic inflammatory responses to microbial molecules contribute to disease pathogenesis and the development of life-threatening complications such as sepsis. Toll-like receptors function as sentinels for the innate immune system, detecting microbial ligands during infection and inflammation. In naturally occurring infections, multiple pathogenassociated receptors are likely to be activated during the initiation of the host response to infection. Therefore, it is important to understand the regulation of TLR signaling and interactions between the TLRs and their ligands. In the first study in this dissertation, we investigated the effects of ligands recognized by TLR 2, TLR 3 or TLR 4 upon expression of the genes encoding these receptor proteins by equine monocytes. We determined that incubation of monocytes with TLR 2 and 4 ligands, which signal through the intracellular adaptor protein MyD88, induces expression of the TLR 2 and 4 genes, but not the TLR 3 gene. Conversely, incubation with a TLR 3 ligand, which recruits the TRIF adaptor protein, selectively induces expression of the TLR 3 gene, but not TLR 2 or 4 genes. Furthermore, incubation of these cells with TNF-α, the pro-inflammatory cytokine that is a hallmark of TLR activation, does not affect expression of any of the three TLR genes. In the second study, we investigated the responses of equine leukocytes to bacterial flagellin, the ligand recognized by TLR 5. Although equine neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for pro-inflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. While both neutrophils and monocytes expressed mRNA for TLR 5, the message appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR 5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR 4 ligand, LPS. Furthermore, equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR 5 and do not respond to flagellin. Finally, we validated in-house ELISA assays to detect flagellin and antibodies directed against flagellin in serum samples obtained from hospitalized horses and foals. We determined that flagellin enters the circulation of horses with gastrointestinal diseases and septic foals, most likely due to intestinal barrier dysfunction. Furthermore, adaptive immune responses to flagellin were detected in horses with gastrointestinal disease that were reflected by increased levels of antibody directed against flagellin during hospitalization.