Oxidative injury leads to lipid radical accumulation in a human necrotizing enterocolitis enteroid model

Author(s):
Alena Golubkova; Tyler Leiva; Katherine Snyder; Heather Liebe; Camille Schlegel; Jason Hansen; Peter Vitiello; Catherine Hunter

Background:

Necrotizing enterocolitis (NEC) is an infectious and inflammatory intestinal disease that affects premature infants. Oxidative stress has a role in dysregulated cellular responses that follow onset of NEC. Specific targets of peroxidation are yet to be defined in NEC, but intracellular radical accumulation is a potential consequence if cellular detoxification mechanisms are immature, overwhelmed, or dysfunctional. The ability of intestinal epithelial cells to mitigate toxic radicals can push cellular fate towards death versus proliferation, playing a role in its capacity to overcome injury and proceed to intestinal repair.

Hypothesis:

We hypothesized that oxidative injury in a human NEC enteroid model targets fatty acids that are abundant in the cell, leading to lipid peroxidation, and overwhelming cellular antioxidant mechanisms with accumulation of toxic lipid radicals.

Methods:

Human intestinal samples were collected from premature infants undergoing resection for surgical NEC or other clinically indicated reason. Samples underwent processing to propagate primary enteroid lines. Enteroid cultures were grown to maturity and experimental NEC was induced by incubation with LPS (lipopolysaccharide) in hypoxic conditions for 48 hours. Lipid peroxidation was measured in live cultures at the end of the experiment by application of a BODIPY® 581/591 C11-based fluorescent reporter (Invitrogen ^TM) and analyzed with flow cytometry (Stratedigm-3).

Results:

Experimental NEC conditions lead to a significant increase in lipid radicals in treated enteroid cultures (Figure 1). Moreover, human intestinal snap frozen tissue resected for active NEC, reveals significantly decreased protein levels of glutathione peroxidase 4 (Gpx4), a key enzyme in intracellular glutathione (GSH) antioxidant machinery responsible for neutralizing lipid radicals and fighting toxic accumulation.

Conclusions:

Cellular targets of oxidative injury in NEC can play a vital role in influencing cellular fate and ability to fight injury. Accumulation of lipid radicals is a result of exposure to NEC in an enteroid model. If the level of lipid peroxidation overwhelms the ability of intracellular antioxidant machinery to neutralize lipid radicals, there are consequences that favor cellular death. Decreased Gpx4 expression likely contributes to the affected cell’s inability to effectively detoxify.