Platelet Toll-like receptor 4 expression in POAG: The innate immune system

Abstract

Purpose: Toll-like receptor 4 (TLR4) is a critical player in the first line of defense for the innate immune system. TLRs are transmembrane proteins that recognize invading pathogen-associated molecular patterns (PAMPs), such as microbes, as well as damage-associated molecular patterns (DAMPs), such as low molecular weight hyaluronic acid (LMW-HA) generated by hyaluronidase-2 (HYAL2). PAMPs and DAMPs activate signaling pathways that launch immune and inflammatory responses to counter the invaders. The purpose of this study was to determine the expression of TLR4 in the platelets of primary open-angle glaucoma (POAG) patients.

Methods: Platelets from POAG patients (n=3) and age-matched control subjects (n=3) were isolated from whole blood. Platelet-rich plasma was obtained by low speed centrifugation and followed by high speed centrifugation to isolate the platelets. The platelet lysates were prepared with a lysis buffer (Cell Signaling Technologies; Danvers, MA) in the presence of a protease inhibitor cocktail (Sigma-Aldrich; St. Louis, MO). Each platelet lysate sample was analyzed for protein content. Equal amounts of proteins were resolved by SDS polyacrylamide gel electrophoresis and immunoblotted with anti-TLR4 antibody (R&D Systems; Minneapolis, MN; 1:1000 dilution) or anti-HYAL2 antibody (Abcam; Cambridge, MA; 1:1000 dilution). This was followed by a goat HRP conjugated anti-mouse secondary antibody (Genetex; Irvine, CA; 1:3000) or a goat HRP conjugated anti-rabbit secondary antibody (Bio-Rad; Hercules, CA; 1:3000). Blots were visualized using the Clarity Western ECL Substrate (Bio-Rad), analyzed via densitometry (GE Healthcare; Pittsburgh, PA), and normalized using beta-actin as the loading control.

Results: Western blot analysis indicated that platelet TLR4 was decreased two-fold in patients with POAG compared to age-matched controls (p<0.01). In contrast, HYAL2 was increased two-fold in POAG platelets; however, the difference was not statistically significant.

Conclusions: Platelets are sentinel cells of the innate immune response. POAG platelets have decreased TLR4 expression and increased amounts of LMW-HA. Thus, it is possible that as an individual ages or as a disease progresses, the first line of defense (TLR4) can be breached and overwhelmed, and the upregulation of HYAL2 results in generation of the TLR4 agonist LMW-HA. The net result is the individual is confronted with a progressive inflammatory response.

Keywords: 555 immunomodulation/immunoregulation • 557 inflammation