Background: Due to common evolutionary origins, mitochondrial DNA (mtDNA) shares many similarities with immunogenic bacterial DNA, and is recognised as a damage-associated molecular pattern (DAMP) that activates pro-inflammatory TLR9 signalling pathways. We hypothesised that mtDNA-TLR9 mediated inflammation is important in IBD, and mtDNA is released during active disease serving as a key pro-inflammatory trigger.
Methods: Between 2015–2016, we collected plasma separated within 2 hours of sampling from 128 prospectively recruited IBD patients comprising 98 ulcerative colitis (UC) and 30 Crohn's disease (CD), and 39 non-IBD controls, with 210 sample points. We assessed plasma mtDNA levels by qPCR using primers amplifying mitochondrial specific ND2 and COXIII genes. In parallel, we investigated plasma mtDNA in vivo, in acute DSS- and chronic spontaneous mdr1a-deficient colitis models; in human IBD: faecal mtDNA levels (n=12 UC vs. 12 healthy controls), electron microscopy (EM) of inflamed colonic mucosa (6 UC vs. 6 healthy controls; intestinal TLR9 protein expression (10 UC, 10 CD and 20 age-matched controls); and acute DSS-colitis in tlr9-deficient mice.
Results: Increased cell-free plasma mtDNA was detected in UC (p<0.0001) and CD (p<0.003), as well as acute DSS- and chronic mdr1a-deficient colitis (both p<0.05). MtDNA levels were higher in active disease compared to those in remission in UC (p<0.001) as measured by the Simple Clinical Colitis Activity Index (SCCAI), and mtDNA levels correlated positively with C-reactive protein (r=0.33, p<0.0001) and negatively with albumin (r=−0.32, p<0.0001). MtDNA levels also correlated with severity of DSS-colitis. MtDNA is significantly higher in faecal samples during active UC vs. controls (>20 fold, p=0.005) indicative of local gut release and supported by the presence of intestinal sub-and epithelial deposits of mitochondrial debris by EM. TLR9 expression is higher in intestinal human IBD epithelium suggesting that a downstream pathway is present. Finally, tlr9-gene deletion in mice resulted in significant attenuation of acute-DSS colitis (colitis score, weight loss, and histological severity) confirming the importance of TLR9 pro-inflammatory signalling in colitis.
Conclusions: For the first time, we show that significant levels of mtDNA are found systemically and locally in human IBD and in mouse models of acute and chronic colitis. These levels are associated with disease severity. TLR9, the target of mtDNA, is highly expressed in the gut and tlr9-deletion is protective in colitis. Collectively, our data suggest that mtDNA-TL9 signalling is important and a targetable pathway, and mtDNA itself represents an attractive functional potential biomarker in IBD.