Establishing the role of apoptosis and mtDNA in the pathobiology of lupus
DNA normally remains locked away within cells, and people who don’t have lupus carry little of it in their blood. However, blood levels of DNA are much higher in patients with lupus. One possible source for this DNA is the billions of cells in the body that kill themselves each day. We will test whether dying cells are an important source of blood DNA in lupus by studying mice that lack key proteins that enable cells to commit suicide. We also recently demonstrated that mitochondria, the structures that function as power plants in cells, also release their DNA, which is another potential source. We will test the importance of DNA from mitochondria in the disease by measuring its levels in patients with lupus. We will then ask if patients with more severe lupus carry more mitochondrial DNA. Our studies could enable researchers to design possible lupus treatments to block cell suicide.
What this study means for people with lupus
“The immune system attacks patients’ own DNA in lupus, but researchers haven’t resolved where the large amount of DNA in their blood comes from. We will identify the sources of this DNA by studying mice and blood samples from patients with lupus. Our research could solve one of the long-standing mysteries about lupus and provide important data for studies to identify new drug targets.”
Autoantibodies that bind DNA and associated nucleoproteins are a hallmark of systemic lupus erythematosus (SLE). Thus, loss of tolerance to DNA represents a major mechanism of pathogenesis in lupus. This is underscored by the monogenic forms of SLE and lupus-like disease, a large proportion of which are caused by mutations in genes that encode regulators of nucleic acid repair, sensing and/or degradation. Both nuclear and mitochondrial DNA are found in the serum and plasma of humans; this is known as “circulating cell free” (ccf) DNA. The amount of ccf-DNA in the blood is tightly regulated by Dnase1 and Dnase1l3, secreted enzymes that degrade extracellular DNA. Impairments in their function results in elevated ccf-DNA levels, and mutations in Dnase1 or Dnase1l3 cause lupus in humans and mice. But where does ccf-DNA come from? It has long been held that it originates from the aberrant apoptosis and/or clearance of blood cells. In recent years there has been an explosion of interest in the role of mitochondrial DNA (mtDNA) in autoimmunity. Owing to its unmethlyated CpG motifs and frequent oxidative damage, it is a potent stimulator of major immune pathways including cGAS/STING and TLR9. More than 60 studies have documented increased ccf-mtDNA levels in a myriad of disease settings including lupus. Several groups have reported elevated ccf-mtDNA levels in lupus patients, and in one study the level of anti-mtDNA antibodies correlated with the lupus nephritis activity index better than anti-gDNA antibody levels did. Multiple recent publications have suggested that neutrophil extracellular traps (NETs) in lupus patients are highly enriched for mtDNA, and this promotes autoimmunity. However, the mechanism(s) by which mtDNA is mobilised is unclear.
We recently demonstrated that, during apoptosis, mitochondria undergo herniation. Large macropores form in the outer membrane, allowing the inner membrane to balloon out into the cytoplasm, bringing mtDNA with it. We observed the appearance of single membrane-bound DNA-containing mitochondrial-derived vesicles (dMDVs) which we hypothesise are one mechanism by which mtDNA might find its way to the cell surface and into the circulation. Published in Science in 2018, this is the first documented example of mtDNA escape. Our work raises important questions about the relationship between apoptosis, mtDNA release, and pro-inflammatory signaling in lupus. We propose to 1) Establish the relationship between ccf-mtDNA levels and lupus development in collaboration with the Australian Lupus Registry, 2) Elucidate the role of apoptosis in the generation of ccf-mtDNA using mice deficient for key regulators of apoptosis, and 3) Define mitochondrial dynamics and mtDNA release during neutrophil NETosis using a suite of cutting edge microscopy techniques including lattice light sheet, 3D-SIM and cryoEM.