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.