Red blood cells (RBCs) are typically considered to be immunologically inert. However, Lam et al. now show that mammalian RBCs bind cell-free DNA, which leads to phagocytosis of RBCs and innate immune activation in pathological settings.

A previous study from the same group detected intracellular expression of the nucleic-acid receptor Toll-like receptor 9 (TLR9) by RBCs and showed a role for RBCs in sequestering potentially harmful cell-free DNA during homeostasis. In this study, Lam et al. used antibodies to the TLR9 ectodomain to show that TLR9 can also be detected on intact, nonpermeabilized RBCs from humans, mice and chimpanzees. Furthermore, RBCs from healthy human donors were shown to bind bacterial genomic DNA from Legionella pneumophila, mitochondrial DNA (mtDNA) from the malaria parasite Plasmodium falciparum and synthetic CpG DNA. As CpG-containing mtDNA is increased in the circulation during sepsis, the authors examined RBCs from critically ill patients with sepsis and showed that both surface TLR9 expression and mtDNA binding are increased compared with RBCs from healthy donors. Levels of mtDNA on RBCs were similarly increased in mouse models of sepsis, bacterial pneumonia and parasite infection.

Credit: S. Bradbrook/Springer Nature Limited

So what is the effect of binding of excess CpG DNA to RBCs? High-dose CpG treatment in vitro led to marked alterations of RBC morphology and changes to the distribution of cytoskeletal and membrane proteins in a TLR9-dependent manner. CpG treatment of RBCs also led to loss of surface expression of the antiphagocytic signal CD47 in a dose-dependent and TLR9-dependent manner.

Consistent with the loss of CD47, mice infused with CpG-treated RBCs had higher levels of erythrophagocytosis by splenic red pulp macrophages than mice infused with control RBCs. Mice infused with CpG-treated RBCs also had neutrophil infiltration of the spleen, increased splenic expression of interferon signalling pathway genes and increased plasma levels of IFNγ and IL-6, which are indicative of both local and systemic immune activation. In critically ill patients with sepsis, those who were anaemic had higher levels of RBC-associated mtDNA than those who were not anaemic, which also supports increased erythrophagocytosis in response to DNA binding by RBCs. In erythrocyte-specific Tlr9-knockout mice compared with wild-type mice, IL-6 levels were attenuated after CpG administration or in a caecal slurry model of sepsis.

Together, the results suggest that when plasma CpG DNA levels increase beyond homeostatic norms, such as during sepsis or infection, TLR9-dependent binding to RBCs results in erythrophagocytosis, with consequent anaemia and innate immune activation. Anaemia and high cytokine levels are common features of multiple inflammatory pathologies, so further investigation of this pathway could have important therapeutic implications. Indeed, this study also reported that in hospitalized patients with COVID-19 pneumonia, the amount of RBC-bound mtDNA correlated with both anaemia and disease severity.