The role of nucleophosmin in mediating the LPS-induced lung injury
CCCF ePoster library. Li M. Oct 27, 2015; 117335; P69 Disclosure(s): Canadian Institutes of Health Research Nature Science Foundation of China
Dr. Manshu Li
Dr. Manshu Li
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P69


Topic: Basic/Translational Science


The role of nucleophosmin in mediating the LPS-induced lung injury



Manshu Li, M. Ghazarian, B. Han, A. Luo, A. Grassi, D. Isalm, J. Khang, A. Slutsky, H. Zhang

Critical Care Medicine, The State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China | Institute of Medical Science, University of Toronto, Toronto, Canada | Anaesthesia, Keenan Research Center, St. Michael’s Hospital, Toronto, Canada | Institute of Medical Science, University of Toronto, Toronto, Canada | Deprtment of Anesthesiology and Critical Care Medicine, University of Milano, Milano, Italy | Institute of Medical Science, University of Toronto, Toronto, Canada | Anaesthesia, Keenan Research Center, St. Michael’s Hospital, Toronto, Canada | Critical Care Medicine, Keenan Research Center, St. Michael’s Hospital, Toronto, Canada | Anaesthesia, Keenan Research Center, St. Michael’s Hospital, Tronto, Canada

Introduction: Endotoxins, also known as lipopolysaccharide(LPS), are a major component found in the outer member of G-negative bacteria,and is known to trigger the development of sepsis [1,2]. Various studies have focusedon neutralizing or blocking LPS signaling via its receptor, toll-like receptor4, (TLR4) to treat sepsis [3,4,5,6,7,8,9,10]. Unfortunately, the treatments in human clinicaltrials have all failed, suggesting that alternative approaches are yet to bediscovered in controlling the LPS signaling. We have demonstrated that LPS canbe internalized by lung epithelial cells independent of TLR4 signal pathway. Wealso observed that the LPS internalized sends signal out contributing toongoing inflammtory responses through interaction with nucleophosmin (NPM), anabundant nucleolar phosphoprotein, in human lung epithelial cells. The presentstudy examined the effects of blocking NPM signaling on the LPS-inducedinflammatory response in a mouse model of sepsis.

Objectives:

To determine the role of Nucleophosmin in mediating intracellular LPS signaling in the lung.



Methods:

TLR4-/-male mice (C57BL/10 ScNJ) and littermate controls (WT) (10-12 weeks) wereanesthetized and challenged with LPS (20 mg/kg, orointratracheally, ListBiological) or vehicle control (saline, NS). Mice were monitored for up to 48 hfor survival study or terminated at 4 h, 8 h and 24 h after LPS challenge toexamine the inflammatory responses. Two strategies were used to attenuate theLPS-induced inflammation: 1) administration of Polymyxin B intratracheally 15min after LPS to neutralize LPS, and 2) use of a selective NPM inhibitorNSC348884 intraperioneally 30 and 90 min after LPS challenge to blockinflammatory signaling induced by LPS internalized. Mice were monitored for 8 hafter LPS challenge.



Results:

TLR4-/- mice all survived from LPS challenge while the WT mice had 100% mortality at 48h (Figure 1A). The total cell count in lung lavage fluid was similar between the two strains at 4h but higher in the TLR4-/- mice than in the WT mice at 8 h (Figure 1B). The albumin concentration was lower in the TLR4-/- mice than in the WT mice at 4h after LPS challenge (Figure 1C), and reached similar level thereafter (Figure 1D). Lung immunohistochemistry analysis showed LPS internalization in the alveolar space at 8 h both in the TLR4-/- and WT mice (Figure 2). The administration of Polymyxin B and NPM inhibitor resulted in decreased inflammatory responses induced by LPS in the TLR4-/- mice (Figure 3).



Conclusion:

LPS was internalized in alveolarspace through TLR4-independent signaling pathway. The LPS-induced intracellularinflammatory responses were attenuated by blocking NPM signaling.



References:

1. Jonathan C. Kagan, Immunology. Sensing endotoxins from within., Science, 2013. 341(6151):1184-5

2. Jonathan Cohen, The immunopathogenesis of sepsis., Nature 2002. 420, 885-891

3. Abraham I. Braude, et al., Antibody to Cell Wall Glycolipid of Gram-Negative Bacteria: Induction of Immunity to Bacteremia and Endotoxemia., Oxford Jounals.1977., 136.

4. H. Shaw Warren, e. al., Assessment of Ability of Murine and Human Anti-lipid A Monoclonal Antibodies to Bind and Neutralize Lipopolysaccharide. J. Exp. Med.,1993., 177.

5. Elizabeth J Zigler., et al., Treatment of Gram Negative Bacterial and Shock with Human Antiserum to a Mutant Escherichia Coli.,NEJM,1982.,307

6. Derek C. Angus, e. al., E5Murine Monoclonal Antiendotoxin Antibody in Gram-Negative Sepsis. JAMA,2000., 283(1723-1730).

7. Thierry Rogera,et al., Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4., PNAS, 2009., 106(2348–2352).

8. He, Z., et al., Toll-like receptor 4 monoclonal antibody attenuates lipopolysaccharide-induced acute lung injury in mice. Exp Ther Med,2014., 8(3): 871-876.

9. Mark Tidswell, a. S. P. L. Toll-like receptor-4 antagonist eritoran tetrasodium for severe sepsis. Expert Rev. 2011., 9(5), 507–520

10. Rice, T. W., et al., A randomized, double-blind, placebo-controlled trial of TAK-242 for the treatment of severe sepsis. Crit Care Med, 2010., 38(8): 1685-1694

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