The Role of Endogenous TLR4 Ligand S100A8/A9 in Ocular Inflammation

Abstract

Purpose: The importance of the toll-like receptor 4 (TLR4) in endotoxin-induced uveitis (EIU) from administration of lipopolysaccharide (LPS) is established, but the role of endogenous TLR4 ligands in the activation of this system is not well-characterized. We focus on one endogenous TLR4 ligand, S100A8/A9, and study its potential contribution to the pathogenesis of the EIU mouse model.

Methods: We injected S100A8/A9 (7.8 μg) or saline intravitreally in BALB/c mice (n=6 mice/treatment/timepoint) and monitored ocular inflammation with intravital microscopy at 3 and 6 hours post-injection. Iris tissue explants were stimulated with LPS or increasing concentrations of S100A8/A9, and the IL-6 response was measured by ELISA at 24 hours post-stimulation. We administered intravitreal LPS (250 ng) to BALB/c mice or to S100A9 knockout mice and corresponding C57BL/6 controls (n = 4 mice/genotype/timepoint) and measured inflammation by histology at 6 and 24 hours post-injection. S100A8/A9 levels in whole eye explants from BALB/c mice injected intravitreally with LPS or saline (n = 8 mice/treatment/timepoint) were also measured by ELISA.

Results: Intravital microscopy showed that intravitreal injection of S100A8/A9 resulted in an increase of rolling and adhering cells in the iris vasculature at both 3 and 6 hours post-injection as well as an increase in cells infiltrating surrounding iris tissue at 6 hours compared to saline-injected controls. ELISA of whole eye explants at 2, 6, and 24 hours after intravitreal LPS showed an increase in S100A8/A9 levels in LPS-injected eyes over time. Stimulation of iris tissue explants with S100A8/A9 resulted in an increase in IL-6 production in a dose-dependent manner, although the highest concentration of S100A8/A9 induced less IL-6 production than a relatively low dose of LPS. Unexpectedly, S100A9 knockout mice showed increased levels of infiltrating cells in both posterior and anterior segments after LPS stimulation compared to wild type C57BL/6 mice.

Conclusions: Our data show that S100A8/A9 can induce or enhance ocular inflammation in mice, but paradoxically suggest that its absence, analogously to the absence of TLR2 or NOD2 in some models, can also lead to increased inflammation. The ability of endogenous TLR ligands to induce eye inflammation broadens the importance of the innate immune system in the eye.

Keywords: 746 uveitis-clinical/animal model • 555 immunomodulation/immunoregulation • 557 inflammation