Platelet-Activating Factor Induces TLR4 Expression in Intestinal Epithelial Cells: Implication for the Pathogenesis of Necrotizing Enterocolitis

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

PAF-induced TLR4 expression in intestinal epithelial cells.

<p>IEC-6 (a and b) and Caco-2 (c) cells were grown in Petri dishes and treated with increasing concentrations of cPAF for for the indicated periods of time (a) or 6 h with 2 µM cPAF (b and c). cDNA copy numbers for TLR4 and GAPDH were quantified using QRT-PCR and TLR4 mRNA levels were expressed as %GAPDH. Treatment with PAF resulted in a dose- and time-dependent increase of TLR4 mRNA. * p<0.01 vs. untreated controls.</p

PAF-induced STAT3 translocation to nuclei in IEC-6 cells and in COS-7 cells transfected with the human PAFR.

<p>IEC-6 cells were treated without (a,b,c), or with (d,e,f) 2 µM PAF for 1 h then fixed and processed for STAT3 immunohistochemistry (a,d; c and f red) and nuclei were counterstained with Hoechst dye (b,e; c and f blue). COS-7 cells were first incubated with AD-PAFR for 24 hrs then without (g,h), or with (i,j) 1 µM PAF for 1 h. Triple label immunohistochemistry was performed with anti STAT3 (red), anti PAFR (green) and the DNA binding dye Hoechst (blue). Rat small intestinal cells (IEC-6) were treated as indicated, then Western blots were performed on cytoplasmic and nuclear fractions using antibodies against STAT3 and phorphorylated STAT3 (p-STAT3).</p

Inhibition of PAF-induced TLR4 expression by STAT3 and NFκB inhibitors.

<p>IEC-6 cells were untreated, treated with PAF (2 µM 4 h), or with PAF (2 µM 4 h) following pretreatment for 1 h with: a) DMSO (vehicle; 1∶1000; all inhibitors were dissolved in DMSO at a 1000× stock), b) Tyrophostin AG490 (25 µM), c) Bay 11–7082 (10 µM), or PSI-II (1 µM). Subsequently, total RNA was isolated and TLR4 mRNA expression was determined using GAPDH reference and quantitative real time PCR. Data shown are mean ±S.E.M. of at least n = 6 in each group. * depicts statistical significance compared to vehicle treated control of p<0.01 and # depicts statistical significance compared to PAF+DMSO of p<0.05 using ANOVA and Tukey's post hoc analysis.</p

Induction of TLR4 expression by intraluminal PAF.

<p>Adult rat ileal loops were perfused with saline (sham), or with saline +10 µM carbamyl PAF for 4 hrs as indicated. Total RNA was isolated from mucosal scrapings and QRT-PCR was performed to quantify TLR4 and GAPDH mRNA copy numbers. TLR4 mRNA level is shown as % GAPDH. L indicates ileal loop mucosa, NL indicates non loop mucosa. TLR4 mRNA was significantly increased only in the mucosa that was in direct contact with PAF. * p<0.05.</p