The central hypothesis is that activation of Toll-like receptor (TLR) 2 contributes to the connected processes of endothelial dysfunction, coagulopathy, and increased vascular permeability in sepsis. TLR2 mediates the inflammatory effects of bacterial lipoproteins. The studies will define mechanisms by which TLR2 agonists modulate coagulation pathways in endothelial cells, and will assess the functional significance of TLR2 activation on endothelial permeability and coagulopathy in sepsis. These studies will provide insights into the mechanisms of coagulopathy, vascular leak, and respiratory dysfunction in sepsis. TLR2 agonists are present in all of the major classes of microorganisms that cause sepsis. Thus if TLR2 is important in sepsis-induced coagulopathy, vascular leak or respiratory failure, then TLR2 signaling pathways could be suitable targets for sepsis therapies.
Specific Aim #1 : Define mechanisms by which TLR2 activation modulates endothelial cell (EC) expression of coagulation pathway factors in vitro. Studies will test the hypotheses that TLR2 agonists alter expression of factors involved in coagulation, anticoagulation, and fibrinolysis: 1) through NF-?B, and 2) through additional mediators, including TGF-?, TNF?, and/or NO. EC will be treated with TLR2 agonists, and expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibitor type 1 (PAI-1) will be quantified. Mechanisms by which TLR2 agonists modulate coagulation pathways will be defined using EC from knockout mice, and using targeted inhibitors with human endothelial cells.
Specific Aim #2 : Assess the effects of TLR2 activation on endothelial permeability in vitro. Studies will test the hypotheses that TLR2 activation increases endothelial leakiness, as assessed by permeability of EC monolayers to albumin.
Specific Aim #3 : Define the functional significance of TLR2 activation on coagulopathy and on lung vascular permeability in sepsis. Sepsis will be induced in mice using peritonitis and pneumonia models. Studies will compare responses of TLR2 knockout mice with those of wild-type mice, and will assess the relative importance of TLR2 in the pathophysiology of Gram- positive versus Gram-negative sepsis. Blood coagulation times will be measured, and levels of factors involved in coagulation and fibrinolysis will be quantified in blood and in lung. Lung vascular leakiness will be assessed using lung wet:dry weight ratios and permeability to albumin. Histologic analyses will assess for microvascular thrombosis, architectural changes, evidence of pulmonary edema, and lung expression of PAI-1 and TF.
Sepsis is a life-threatening consequence of infection that causes shock, abnormalities of coagulation, and organ failure. The persistent high mortality associated with sepsis underscores the need to continue to understand the mechanisms of this highly complex process, to try to define new treatment targets. The proposed studies will evaluate the importance of Toll-like receptor 2 (TLR2), which is involved in the host's immune response to infection, in important processes that occur during sepsis, including respiratory failure and problems with coagulation.
|Lin, Tian; Sammy, Fatima; Yang, Huan et al. (2012) Identification of hemopexin as an anti-inflammatory factor that inhibits synergy of hemoglobin with HMGB1 in sterile and infectious inflammation. J Immunol 189:2017-22|
|Prakash, Arun; Mesa, Kailin R; Wilhelmsen, Kevin et al. (2012) Alveolar macrophages and Toll-like receptor 4 mediate ventilated lung ischemia reperfusion injury in mice. Anesthesiology 117:822-35|
|Wilhelmsen, Kevin; Mesa, Kailin R; Prakash, Arun et al. (2012) Activation of endothelial TLR2 by bacterial lipoprotein upregulates proteins specific for the neutrophil response. Innate Immun 18:602-16|
|Wilhelmsen, Kevin; Mesa, Kailin R; Lucero, Jennifer et al. (2012) ERK5 protein promotes, whereas MEK1 protein differentially regulates, the Toll-like receptor 2 protein-dependent activation of human endothelial cells and monocytes. J Biol Chem 287:26478-94|
|Shin, Hae-Sook; Xu, Fengyun; Bagchi, Aranya et al. (2011) Bacterial lipoprotein TLR2 agonists broadly modulate endothelial function and coagulation pathways in vitro and in vivo. J Immunol 186:1119-30|
|Petersen, Bodil; Bloch, Kenneth D; Ichinose, Fumito et al. (2008) Activation of Toll-like receptor 2 impairs hypoxic pulmonary vasoconstriction in mice. Am J Physiol Lung Cell Mol Physiol 294:L300-8|
|Bagchi, Aranya; Herrup, Elizabeth A; Warren, H Shaw et al. (2007) MyD88-dependent and MyD88-independent pathways in synergy, priming, and tolerance between TLR agonists. J Immunol 178:1164-71|
|Valentine, Catherine H; Hellman, Judith; Beasley-Topliffe, Laura K et al. (2006) Passive immunization to outer membrane proteins MLP and PAL does not protect mice from sepsis. Mol Med 12:252-8|
|Liang, Michael D; Bagchi, Aranya; Warren, H Shaw et al. (2005) Bacterial peptidoglycan-associated lipoprotein: a naturally occurring toll-like receptor 2 agonist that is shed into serum and has synergy with lipopolysaccharide. J Infect Dis 191:939-48|