Abstract
Purpose :
The current study was designed to examine the role of a carbohydrate-binding protein, galectin 8, (Gal-8) in P. aeruginosa (PA) keratitis pathogenesis, and to characterize the mechanism by which Gal-8 modulates innate immune response in PA keratitis.
Methods :
Two approaches were used. In the first, corneas of C57BL/6 wild-type (WT) or Gal-8 knockout (Gal-8 KO) mice were infected with PA. The severity of bacterial keratitis was graded on day 1 and day 3 post-infection (p.i.) by slit lamp using a scoring system ranging from 0 to 4, and then corneas were harvested for bacterial enumeration. In the second approach, to study the mechanism by which Gal-8 modulates the innate immune response: (i) affinity chromatography experiments were conducted to determine whether specific molecules of the TLR-4 complex bind to Gal-8, and (ii) in vitro experiments were performed to determine whether the addition of exogenous Gal-8 influences the activation of TLR-4 pathway in bone marrow-derived macrophages (BMDMs) stimulated with LPS.
Results :
Gal-8 KO mice exhibited less severe infection in comparison to WT mice as indicated by lower opacity scores and reduced bacterial load. In vitro studies showed that: (i) Gal-8 binds to CD14 but not to TLR4 in a carbohydrate-dependent manner, (ii) the addition of exogenous rGal-8 significantly inhibits the delivery of LPS from CD14 to MD2/TLR4 and impairs activation of TLR4 pathway in BMDMs stimulated with LPS as indicated by a decrease in TNFα, IL-6, MD-2 and MyD88 expression and IkB phosphorylation.
Conclusions :
Gal-8 modulates the pathogenesis of PA keratitis by inhibiting the activation of TLR4 pathway.
This is a 2021 ARVO Annual Meeting abstract.