L.M. Bode1, C. Tragesser1, J. Duess1, K. Tsuboi1, H. Moore1, H. Jang1, D. Scheese1, J. Young1, M. Sampah1, S. William-McLeod1, T. Prindle1, W. Fulton1, S. Wang1, M. Wang1, C.P. Sodhi1, D.J. Hackam1 1Johns Hopkins University School Of Medicine, Pediatric Surgery, Baltimore, MD, USA
Introduction:
Toll-like receptor 4 (TLR4) plays a critical role in the pathogenesis of necrotizing enterocolitis (NEC), a severe gastrointestinal disease of premature infants. As part of the innate immune system, TLR4 detects microbial components like lipopolysaccharide (LPS) from Gram-negative bacteria. Activated TLR4 leads to mucosal disruption and impaired intestinal perfusion resulting in the intestinal ischemia and necrosis seen in NEC. Ischemia contributes to mitochondrial dysfunction upon which mitochondria can release damage-associated molecular patterns (DAMPs) and excessive production of reactive oxygen species. DAMPs, sharing structural similarities with LPS, activate TLR4 and amplify the inflammatory response vice versa. We hypothesized that a depletion of mitochondrial DNA would affect TLR4 signaling. We conducted an in vitro model of lipopolysaccharide (LPS)-induced injury in rat intestinal epithelial cells (IEC6) to examine a potential complementary enhancement.
Methods:
Rat intestinal epithelial cells (IEC-6) were cultured under physiologic conditions i.e. 37 °C and 5% CO2 v/v in complete media containing 1% L-glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum. Enterocytes were pre-treated for 2-6 weeks with ethidium bromide 100nM to deplete mitochondria. Sufficient mitochondrial depletion was proven both quantitatively by decreased mRNA levels of cytochrome B, ATP synthase and mitochondrial transcription factor A and qualitatively by ATP assay. To examine the sensitivity of TLR4, experimental wells were exposed to the bacterial ligand LPS (50 ug/mL) for 6 hours in healthy and mitochondrial depleted conditions. To measure gene expression changes, cells underwent total RNA isolation followed by cDNA synthesis and qPCR. For immunohistochemical analyses, cells were washed in cold phosphate-buffered saline and fixed in 4% PFA prior to subsequent antibody staining.
Results:
Mitochondria were most significantly diminished with a treatment concentration of 100nM ethidium bromide for at least 2 weeks (mtcytB 6772.6 vs. 466.1; p=0.0001). LPS treatment resulted in significantly increased pro-inflammatory (IL-1, TNF, iNOS) gene expression and cytokine release (IL-6) close to a level seen upon LPS stimulation. Strikingly, mitochondrial depletion doubled this effect.
Conclusion:
The interaction between mitochondrial DAMPs and TLR4 can create a feedback loop, where inflammation leads to further mitochondrial damage, resulting in increased DAMP release and severe inflammation.Therapeutic approaches such as inhibitors of TLR4 or mitochondrial enforcing treatments to protect mitochondrial function might help mitigate the severity of NEC.