Abstract
Purpose:
To determine the effect of MyD88 deficiency on ocular inflammation in a murine model of TLR ligand induced inflammation.
Methods:
Inflammation was induced in seven-week-old MyD88 knockout and wild type littermates by intraperitoneal injection of 50 µg LPS in 0.1 ml PBS, 25 µg of PAM3CSK4 in 0.1 ml water, or 100 µg polyI:C in 0.1 ml PBS. Mice were euthanized 3, 8, or 24 hours after injection. Plasma and right eyes were collected and protein extracts prepared for cytokine and chemokine analysis using a multiplex assay (Aushon Biosystems). The contralateral eyes were fixed in 4% paraformaldehyde and the retinas and posterior eye cups were dissected and prepared for whole flatmount immuno-staining of neutrophils (Gr-1), macrophages (F4/80), and microglia (Iba1). Fluorescent images of five regions of each retina and two regions of each posterior eye cup were captured using an Axiocam MR3 camera on an Axio.ImageM1 microscope. The numbers of neutrophils, macrophages, and microglia were counted with Axiovision software.
Results:
In comparison with wild-type littermates, MyD88 knockout mice had a reduced number of neutrophils and macrophages in the retina after intraperitoneal injection of the TLR2 & TLR4 ligands (PAM3CSK4 and LPS, respectively). Injection of the TLR3 ligand -poly I:C- had no effect on neutrophils and macrophages in the retina. Microglia cells in the posterior eye cup in response to the TLR2, TLR4, and TLR3 ligands in MyD88 knock mice were reduced, in comparison with wild-type littermates, by 100%, 50%, and 0%, respectively. Cytokines in the plasma and eye induced by the TLR2 ligand (PAM3CSK4) was severely reduced, partially inhibited with TLR4 ligand (LPS), and mostly unaffected with TLR3 ligand (poly I:C) in MyD88 deficient mice compared to wild-type littermates.
Conclusions:
MyD88 deficiency impaired TLR2 & TLR4 responses, but not TLR3 responses, as measured by retinal inflammation and cytokine production. These in vivo data support the hypothesis that TLR2 signaling is MyD88 dependent, TLR3 signaling is MyD88 independent, and TLR4 signaling is partially MyD88 dependent.
Keywords: 746 uveitis-clinical/animal model •
557 inflammation •
688 retina