Abstract
Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in neonatal intensive care units, however its pathogenesis is not completely understood. We have previously shown that platelet activating factor (PAF), bacteria and TLR4 are all important factors in the development of NEC. Given that Toll-like receptors (TLRs) are expressed at low levels in enterocytes of the mature gastrointestinal tract, but were shown to be aberrantly over-expressed in enterocytes in experimental NEC, we examined the regulation of TLR4 expression and signaling by PAF in intestinal epithelial cells using human and mouse in vitro cell lines, and the ex vivo rat intestinal loop model. In intestinal epithelial cell (IEC) lines, PAF stimulation yielded upregulation of both TLR4 mRNA and protein expression and led to increased IL-8 secretion following stimulation with LPS (in an otherwise LPS minimally responsive cell line). PAF stimulation resulted in increased human TLR4 promoter activation in a dose dependent manner. Western blotting and immunohistochemical analysis showed PAF induced STAT3 phosphorylation and nuclear translocation in IEC, and PAF-induced TLR4 expression was inhibited by STAT3 and NFκB Inhibitors. Our findings provide evidence for a mechanism by which PAF augments inflammation in the intestinal epithelium through abnormal TLR4 upregulation, thereby contributing to the intestinal injury of NEC.
Highlights
Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease primarily affecting premature infants, and despite recent advances in neonatal intensive care, it remains a leading cause of morbidity and mortality in this high-risk population.The etiology of NEC remains elusive, but inflammation is thought to be central to its pathogenesis, leading to intestinal injury and associated morbidities [1,2,3,4]
Our earlier studies have shown that in rodent models of necrotizing enterocolitis where Platelet activating factor (PAF) plays an important role, TLR4 expression is aberrantly elevated in the intestinal epithelium, implicating PAF in the regulation of TLR4 expression
In order to test whether PAF can regulate TLR4 expression, we developed a perfused ileal loop model, where the effect of intraluminal perfusion of PAF on gene expression can be directly evaluated
Summary
Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease primarily affecting premature infants, and despite recent advances in neonatal intensive care, it remains a leading cause of morbidity and mortality in this high-risk population. The etiology of NEC remains elusive, but inflammation is thought to be central to its pathogenesis, leading to intestinal injury and associated morbidities [1,2,3,4]. We and others have shown that platelet activating factor plays a key role in NEC in humans and in animals [5,6,7,8,9,10,11,12]. Platelet activating factor (PAF) is a potent endogenous, extracellular and intracellular phospholipid mediator, produced by a two-step enzymatic process where phosphatidyl choline is the de novo precursor and phospholipase A2 (PLA2-II) is the rate-limiting enzyme. Activation of its G protein-coupled receptor, PAF-receptor (PAFR), leads to activation of several signal transduction pathways including the signal transducers and activators of transcription (STATs) and NFkB [13], culminating in physiological or pathological changes including vasoconstriction and/or vasodilatation, leukocyte stimulation and migration, synthesis and activation of cell adhesion molecules, increased capillary permeability, production of reactive oxygen and nitrogen species, and alterations in intestinal mucosal permeability [14,15]
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