Activation of Toll-Like Receptor 9 Impairs Blood Flow Recovery After Hind-Limb Ischemia

Background: Peripheral artery disease causes significant functional disability and results in impaired quality of life. Ischemic tissue injury releases various endogenous ligands for Toll-like receptors (TLRs), suggesting the involvement of TLRs in blood flow recovery. However, the role of TLR9, which was originally known as a sensor for bacterial DNA, remains unknown. This study investigated the role of TLR9 in blood flow recovery in the ischemic limb using a mouse hind-limb ischemia model.
Methods and Results: Unilateral femoral artery ligation was performed in TLR9-deficient (Tlr9−/−) mice and wild-type mice. In wild-type mice, femoral artery ligation significantly increased mRNA expression of TLR9 in the ischemic limb (P < 0.001) and plasma levels of cell-free DNA (cfDNA) as determined by single-stranded DNA (ssDNA) (P < 0.05) and double-stranded DNA (dsDNA) (P < 0.01), which are endogenous ligands for TLR9, compared with the sham-operated group. Laser Doppler perfusion imaging demonstrated significantly improved ratio of blood flow in the ischemic to non-ischemic limb in Tlr9−/− mice compared with wild-type mice at 2 weeks after ligation (P < 0.05). Tlr9−/− mice showed increased capillary density and reduced macrophage infiltration in ischemic limb. Genetic deletion of TLR9 reduced the expression of TNF-α, and attenuated NF-kB activation in ischemic muscle compared with wild-type mice (P < 0.05, respectively) at 3 days after the surgery. ODN1826, a synthetic agonistic oligonucleotide for TLR9, or plasma obtained from mice with ischemic muscle promoted the expression of TNF-α in wild-type macrophages (P < 0.05), but not in Tlr9−/− macrophages. ODN1826 also activated NF-kB signaling as determined by the degradation of IkBα in wild-type macrophages (P < 0.05), but not in Tlr9−/− macrophages. In vitro experiments using human umbilical vein endothelial cells demonstrated that TNF-α, or conditioned medium obtained from wild-type macrophages treated with ODN1826 accelerated cell death as determined by MTS assay (P < 0.05 and P < 0.01, respectively).
Conclusion: Our results suggest that ischemic muscle releases cfDNA, which activates TLR9 and enhances inflammation, leading to impairment of blood flow recovery in the ischemic limb. cfDNA-TLR9 signaling may serve as a potential therapeutic target in ischemic limb disease.

Copyright © 2018 Nishimoto, Aini, Fukuda, Higashikuni, Tanaka, Hirata, Yagi, Kusunose, Yamada, Soeki, Shimabukuro and Sata.This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)(https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.