Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

Abstract

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS −/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate-a precursor for enteral generation of nitrite and nitric oxide-and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.neonatal inflammation | prematurity | infant formula | neonatal nutrition | sepsis N ecrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in the premature infant and is gradually increasing in frequency (1). The defining pathological feature of NEC is the presence of patchy areas of ischemia and necrosis of the small and large intestine (2). Although prematurity is the leading risk factor for NEC development, breast milk administration has been identified as the most important protective strategy (3). Importantly, the mechanisms that lead to the acute development of intestinal necrosis in the premature intestine and factors within breast milk that may prevent NEC remain largely unexplored.In seeking to understand the underlying biological mechanisms that lead to NEC, we and others have identified a critical role for the innate immune receptor toll-like receptor 4 (TLR4) in NEC pathogenesis, because mice deficient in TLR4 showed reduced mucosal inflammation and reduced intestinal necrosis in experimental NEC (4, 5). Microcirculatory perfusion of the premature intestine is primarily regulated by the vasodilator nitric oxide (NO), which is generated through the activity of endothelial NO synthase (eNOS) (6). ...

Current Research on the Epidemiology, Pathogenesis, and Management of Necrotizing Enterocolitis

“…Recent studies have highlighted such differences, such as those showing that intraepithelial T cell receptor γδ lymphocytes are decreased in surgical NEC specimens compared with appropriate controls [55], as are lamina propria T regulatory cells [56]. A key player in intestinal inflammation and the response to pathogens is TLR4, and recent work has shown that TLR4 signalling is important in the development of NEC [33,57,58,59]. Intriguingly, TLR4 signalling also links to other factors involved in the pathogenesis of NEC, such as the microcirculation [59] (see below).…”

“…A key player in intestinal inflammation and the response to pathogens is TLR4, and recent work has shown that TLR4 signalling is important in the development of NEC [33,57,58,59]. Intriguingly, TLR4 signalling also links to other factors involved in the pathogenesis of NEC, such as the microcirculation [59] (see below).…”

Experimental TLR4 inhibition improves intestinal microcirculation in endotoxemic rats

“…LPS signaling activates leukocytes and vascular endothelial cells, leading to up-regulation of adhesion molecule expression and function, promoting adhesion of leukocytes to endothelial cells (Kim et al, 2011;Yazji et al, 2013;. Activation of leukocytes and endothelial cells also up-regulates production of pro-inflammatory cytokines (Zhang et al, 2011), resulting in recruitment of more leukocytes to sites of infection Therefore, LPS and TLR4 play a major role in the pathogenesis of sepsis-induced multiple organ dysfunction and death.…”

New Frontiers of Necrotizing Enterocolitis: From Pathophysiology to Treatment

“…In the premature birth situation, intestinal TLR4 levels remain elevated and activation of TLR4 on the lining of the premature intestine by the Gram negative bacteria that colonize the premature gut leads to a number of deleterious effects, including increased enterocyte apoptosis, impaired mucosal healing and enhanced proinflammatory cytokine release, which in aggregate lead to the development of NEC [14] [15]. Also, the translocation of Gram-negative bacteria through the gut mucosa leads to activation of TLR4 on the lining of the endothelium of the premature bowel mesentery, resulting in a reduction in blood flow and the development of intestinal ischaemia and necrosis [16]. This explanation for the pathogenesis of NEC termed as "the cross-switching hypothesis" partially explains the reasons for which the premature infant is at risk of NEC development and why the disease occurs upon bacterial colonization.…”