State of the art review
Cell-free DNA beyond a biomarker for rejection: Biological trigger of tissue injury and potential therapeutics

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Cell-free DNA, measured as donor-derived cell-free DNA is developed as a non-specific biomarker for allograft injury and transplant rejection. However, cell-free DNA characteristics are more specific, its fragment length, nucleotide content, and composition, as well as the tissue source of origin, are intrinsically linked to the underlying disease pathogenesis, showing distinct features in acute cellular rejection and antibody-mediated rejection for example. Further, cell-free DNA and cell-free mitochondrial DNA can directly trigger tissue injury as damage-associated molecular patterns through three major intracellular receptors, toll-like receptor 9 , cyclic guanosine monophosphate-adenosine monophosphate synthase, and inflammasomes (i.e., absent in melanoma 2: AIM2). Therefore, in addition to its role as a non-specific marker for allograft injury, cell-free DNA analysis may be used to phenotype transplant rejection, and to non-invasively point the underlying molecular mechanisms with allograft injury. Novel treatment approaches targeting these cell-free DNA pathways may be useful to treat transplant rejection and prevent end-organ dysfunction. In this review, we discuss the link between cell-free DNA characteristics and disease, the role of cell-free DNA as a damage-associated molecular pattern, and novel therapeutics targeting these cell-free DNA molecular pathways and their potential utility to treat transplant rejection.

Section snippets

Cell-free DNA tissue source and characteristics is linked to disease pathogenesis

Advances in genome sequencing now permit the identification of the tissue source of cfDNA. Prior technologies in stem cell transplant used HLA haplotypes differences between donor and recipient and identified hematopoietic cells as a major contributor of cfDNA.6 The novel genome sequencing approaches leverage DNA methylation7,8 or histone footprints,9 markers that are tissue-specific, to map the tissue source of cfDNA in transplant and non-transplant conditions. In our own experiments, the

The potential roles of cell-free DNA in cell/tissue injury

In addition to its diagnostic role discussed above, existing research focusing on the pathogenic roles of cfDNA has proceeded rapidly and in several directions (Figure 2). cfDNA plays a direct role in thrombosis, a complication that is frequent in transplantation.13 cfDNA is also a damage-associated molecular pattern (DAMP) that is recognized by pattern recognition receptors (PRRs), which activate innate immune signaling or cell death pathways that either provoke or prevent disease progression.

How does extracellular DNA interact with intracellular DNA sensors?

Protein binding is often a necessary step to internalize cfDNA. For example, purified naked DNA is endocytosed35 when bound to proteins such as LL37,36 C1q,37 anti-dsDNA antibodies,38 and histones.39 cfDNA complexed with nucleosomes requires binding to HMGB1 with RAGE40 before it is taken up by phagocytic cells,40 or binding to anti-dsDNA for uptake by Fc or other unidentified receptors.41 Nucleosome- HMGB1 complex can also bind TLR2 on macrophages, resulting in direct signaling into the cell (

Sepsis

Circulating cfDNA is associated with poor outcomes in patients with severe sepsis. 48 DNA-sequence analyses show the majority of cfDNA from sepsis patients is from the host and not from the infecting bacteria.48 Circulating cell-free mitochondrial DNA (cf-mtDNA) released with sepsis activates TLR9 and contributes to cytokine production, splenic apoptosis, and kidney injury with overproduction of mitochondrial ROS .49 Inhibition of TLR9 or MyD-88 attenuates septic acute kidney injury (AKI) and

Biological role of cfDNA in Transplantation

The accompanying review (page xx) discusses donor-derived cfDNA (dd-cfDNA) as a potential surrogate marker for heart transplant rejection. Similarly findings have been reported in lung transplantation.79, 80, 81 Nolan et al. suggested a Q-score consisting of six urinary biomarkers, including urinary dd-cfDNA, that could distinguish quiescent no rejection from acute rejection of kidney transplantation, reducing the need for kidney biopsy.82 However, few studies have investigated the role of

Future prospective of cfDNA-targeted therapy

In recent years, various types of cfDNA- targeted drugs have been studied (Table 1). Only recombinant human DNase (rhDNase), dornase alpha (Plumozyme®) is approved by FDA, used as an adjunct therapy in the treatment of cystic fibrosis by inhalation, which digests increased cfDNA in the airway followed by improving lung function and reducing the risk of pulmonary exacerbations.87 However, some clinical trials have shown the benefit of rhDNase in other diseases and some trials using rhDNase for

Conclusion and perspective

Cell-free DNA as a non-specific marker for allograft injury. However, growing evidence in transplant and non-transplant conditions indicates that cfDNA is biologically active. For example, the tissue source and characteristics of cfDNA are intrinsically linked to it the underlying disease pathogenesis. These show distinctive features in antibody-mediated rejection versus acute cellular rejection,12 potentially increasing the specificity of the cfDNA test. This would be welcome news in thoracic

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