uncontrolled-hemorrhagic-shock-modeled-via-liver-laceration-mice-with

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

University of Pittsburgh

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.

Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53 up-regulated modulator of apoptosis.

Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4(ΔIEC)) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7(ΔIEC)), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7(ΔIEC) mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis.

Extracorporeal membrane oxygenation (ECMO) has been used in patients with pulmonary and/or cardiac disease. In rare circumstances, some patients may have to undergo simultaneous therapeutic plasma exchange (TPE). We sought to characterize simultaneous ECMO and TPE procedures at our institution.

Simultaneous extracorporeal membrane oxygenation and therapeutic plasma exchange procedures are tolerable in both pediatric and adult patients.

Red blood cell transfusions in the setting of trauma are a double-edged sword, as it is a necessary component for life-sustaining treatment in massive hemorrhagic shock, but also associated with increased risk for nosocomial infections and immune suppression. The mechanisms surrounding this immune suppression are unclear. Using supernatant from human packed red blood cell (RBC), we demonstrate that clearance of Escherichia coli by macrophages is inhibited both in vitro and in vivo using a murine model of trauma and hemorrhagic shock. We further explore the mechanism of this inhibition by demonstrating that human-stored RBCs contain soluble high-mobility group box 1 protein (HMGB1) that increases throughout storage. HMGB1 derived from the supernatant of human-stored RBCs was shown to inhibit bacterial clearance, as neutralizing antibodies to HMGB1 restored the ability of macrophages to clear bacteria. These findings demonstrate that extracellular HMGB1 within stored RBCs could be one factor leading to immune suppression following transfusion in the trauma setting.

Article	Year
Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53 up-regulated modulator of apoptosis. The Journal of biological chemistry\| PubMed ID: 22955282	2012
A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 23455503	2013
Simultaneous extracorporeal membrane oxygenation and therapeutic plasma exchange procedures are tolerable in both pediatric and adult patients. Transfusion\| PubMed ID: 24117674	2014
Aged Human Stored Red Blood Cell Supernatant Inhibits Macrophage Phagocytosis in an HMGB1 Dependent Manner After Trauma in a Murine Model. Shock (Augusta, Ga.)\| PubMed ID: 27488090	2017

Mitchell Dyer

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