Mitochondrial DNA Is a Pro-Inflammatory Damage-Associated Molecular Pattern Released During Active IBD

Abstract

Background: Due to common evolutionary origins, mitochondrial DNA (mtDNA) shares many similarities with immunogenic bacterial DNA. MtDNA is recognized as a pro-inflammatory damage-associated molecular pattern (DAMP) with a pathogenic role in several inflammatory diseases. We hypothesised that mtDNA is released during active disease, serving as a key pro-inflammatory factor in inflammatory bowel disease (IBD).

Methods: Between 2014 and 2015, we collected plasma separated within 2 hours of sampling from 97 prospectively recruited IBD patients (67 ulcerative colitis [UC] and 30 Crohn's disease [CD]) and 40 non-IBD controls. We measured circulating mtDNA using quantitative polymerase chain reaction (amplifying mitochondria COXIII/ND2 genes) and also in mouse colitis induced by dextran sulfate-sodium (DSS). We used a mass spectometry approach to detect free plasma mitochondrial formylated peptides. Furthermore, we examined for mitochondrial damage using electron microscopy (EM) and TLR9 expression, the target for mtDNA, in human intestinal IBD mucosa.

Results: Plasma mtDNA levels were increased in UC and CD (both P < 0.0001) compared with non-IBD controls. These levels were significantly correlated to blood (C-reactive protein, albumin, white cell count), clinical and endoscopic markers of severity, and disease activity. In active UC, we identified 5 mitochondrial formylated peptides (the most abundant being fMMYALF with known chemoattractant function) in plasma. We observed mitochondrial damage in inflamed UC mucosa and significantly higher fecal MtDNA levels (vs non-IBD controls [P < 0.0001]), which supports gut mucosal mitochondrial DAMP release as the primary source. In parallel, plasma mtDNA levels increased during induction of acute DSS colitis and were associated with more severe colitis (P < 0.05). In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC and CD compared with controls (P < 0.05).

Conclusions: We present the first evidence to show that mtDNA is released during active IBD. MtDNA is a potential mechanistic biomarker, and our data point to mtDNA-TLR9 as a therapeutic target in IBD. 10.1093/ibd/izy095_videoizy095.video5776747659001.

Figures

FIGURE 1.

A, Plasma mtDNA (copy/µL) in IBD, HC, and IBS (n = 97, 20, and 20 samples respectively; P ≤ 0.001). B, Plasma mtDNA (copy/µL) in CD, UC, and non-IBD (n = 30, 67, and 40 samples, respectively). Median ± interquartile range.

FIGURE 2.

A, Plasma mtDNA (copy/µL) in UC in clinical remission (ambulatory), active (ambulatory), and severe active (hospitalized; n = 13, 18, and 44 samples, respectively; 8 UC individuals had samples taken at more than 1 time point during active disease and in remission). B, Categorized according to mild, moderate, and severe endoscopic appearances (n = 4, 41, and 23 samples, respectively). C, Plasma mtDNA (copy/µL) in CD, clinical remission (ambulatory), active (ambulatory), and severe disease (hospitalized; n = 10, 5, and 16 samples, respectively; 1 CD individual had samples taken at more than 1 time point during active disease and in remission).

FIGURE 3.

Mitochondrial formylated peptide quantification in 5 UC vs 5 non-IBD controls (*P ≤ 0.01 for fMMYALF, fMTPMRK, fMNPLAQ, fMNFALI, and fMTMHTT). Mean ± standard error of mean.

FIGURE 4.

A, Plasma mtDNA (copies/mL) after acute DSS colitis vs controls (n = 12 and 14; P = 0.01).† B, Plasma mtDNA (copies/mL) stratified to < or >10% weight loss at day 7 of DSS colitis (P = 0.03). Median ± interquartile range.

FIGURE 5.

A, Representative transmission electron microscopy of distal colonic epithelium from active UC vs non-IBD controls (n = 6/group; bar = 5 µm). Annotated image: purple indicates damaged mitochondria (DM), blue indicates healthy mitochondria (HM), and yellow indicates lipid droplets (LD). Black scale bar = 2 µm. Yellow insert indicates damaged and healthy mitochondria from UC and controls, respectively (orange bar = 0.5 µm). B, Fecal mtDNA (copy/nl) in active UC and non-IBD controls (n = 12/group). C, Longitudinal analysis of plasma mtDNA (copy/µL) in UC (n = 8 patients) during active disease and the same patient postcolectomy in clinical remission. All data represent median ± interquartile range.

FIGURE 6.

A, Immunohistochemistry for anti-TLR9 in human IBD colon (UC and CD: n = 7/group; vs non-IBD control: n = 14). Red arrows indicate TLR9-positive cells. Black scale bar = 100 µm. B, TLR9+ cell counts in lamina propria (LP) of human IBD colon per 2 mm2 (UC and CD: n = 7/group; vs non-IBD control: n = 14).‡ Mean ± standard error of mean.