Red Blood Cells Homeostatically Bind Mitochondrial DNA through TLR9 to Maintain Quiescence and to Prevent Lung Injury

Am J Respir Crit Care Med. 2018 Feb 15;197(4):470-480. doi: 10.1164/rccm.201706-1161OC.


Rationale: Potentially hazardous CpG-containing cell-free mitochondrial DNA (cf-mtDNA) is routinely released into the circulation and is associated with morbidity and mortality in critically ill patients. How the body avoids inappropriate innate immune activation by cf-mtDNA remains unknown. Because red blood cells (RBCs) modulate innate immune responses by scavenging chemokines, we hypothesized that RBCs may attenuate CpG-induced lung inflammation through direct scavenging of CpG-containing DNA.

Objectives: To determine the mechanisms of CpG-DNA binding to RBCs and the effects of RBC-mediated DNA scavenging on lung inflammation.

Methods: mtDNA on murine RBCs was measured under basal conditions and after systemic inflammation. mtDNA content on human RBCs from healthy control subjects and trauma patients was measured. Toll-like receptor 9 (TLR9) expression on RBCs and TLR9-dependent binding of CpG-DNA to RBCs were determined. A murine model of RBC transfusion after CpG-DNA-induced lung injury was used to investigate the role of RBC-mediated DNA scavenging in mitigating lung injury in vivo.

Measurements and main results: Under basal conditions, RBCs bind CpG-DNA. The plasma-to-RBC mtDNA ratio is low in naive mice and in healthy volunteers but increases after systemic inflammation, demonstrating that the majority of cf-mtDNA is RBC-bound under homeostatic conditions and that the unbound fraction increases during inflammation. RBCs express TLR9 and bind CpG-DNA through TLR9. Loss of TLR9-dependent RBC-mediated CpG-DNA scavenging increased lung injury in vivo.

Conclusions: RBCs homeostatically bind mtDNA, and RBC-mediated DNA scavenging is essential in mitigating lung injury after CpG-DNA. Our data suggest a role for RBCs in regulating lung inflammation during disease states where cf-mtDNA is elevated, such as sepsis and trauma.

Keywords: CpG-DNA; RBC; Toll-like receptor 9; mitochondrial DNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Animals
  • DNA, Mitochondrial / blood*
  • DNA, Mitochondrial / immunology
  • Disease Models, Animal
  • Erythrocytes / immunology
  • Erythrocytes / physiology*
  • Female
  • Homeostasis
  • Humans
  • Lung Injury / blood
  • Lung Injury / etiology
  • Lung Injury / prevention & control*
  • Male
  • Mice
  • Middle Aged
  • Pneumonia / blood
  • Pneumonia / complications
  • Pneumonia / prevention & control*
  • Reference Values
  • Toll-Like Receptor 9 / blood*
  • Toll-Like Receptor 9 / genetics
  • Toll-Like Receptor 9 / immunology
  • Young Adult


  • DNA, Mitochondrial
  • TLR9 protein, human
  • Tlr9 protein, mouse
  • Toll-Like Receptor 9