Recombinant Soluble Forms of Extracellular TLR4 Domain and MD-2 Inhibit Lipopolysaccharide Binding on Cell Surface and Dampen Lipopolysaccharide-Induced Pulmonary Inflammation in Mice

Abstract

In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-κB activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [3H]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Coinstillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-α levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.

The significance of Toll-like receptor 4 (TLR4) expression in patients with chronic hepatitis B

“…[33][34][35][36][37] Inhibition of TLR4 with a TLR4 antagonist or recombinant soluble forms of extracellular TLR4 domain and MD-2 can attenuate myocardial ischemia-reperfusion injury, dampen lipopolysaccharide-induced pulmonary inflammation in mice and have anti-inflammatory effects in murine models of inflammatory bowel disease. [38][39][40] Our finding of elevated expression of TLR4 in hepatocytes may have a role in the pathogenesis of CHB while little effect on the inhibition of replication of HBV, TLR4 antagonist may be helpful in the treatment of CHB.…”

Pulmonary Surfactant Protein D Inhibits Lipopolysaccharide (LPS)-induced Inflammatory Cell Responses by Altering LPS Binding to Its Receptors

“…Consistently, SP-D attenuates cell surface binding of Alexa-labeled LPS to TLR4/MD-2-expressing cells. We also examined the effect of SP-D on the binding of sMD-2 to LPS, because lipid A avidly binds to sMD-2 but not to sTLR4 (29). The results indicate that SP-D significantly decreases the sMD-2 binding to LPS.…”

Amniotic Fluid Soluble Myeloid Differentiation-2 (sMD-2) as Regulator of Intra-amniotic Inflammation in Infection-induced Preterm Birth

“…Because the sTLR4/sMD-2 complex binds LPS [32], the down-regulatory mechanism could be the consequence of a competitive LPS binding effect between the soluble complex and membrane bound TLR4/MD-2 complex. Because in animal models of sepsis, the combination sTLR4-sMD-2 reduced neutrophil inflammation and TNF-α production it was proposed that this complex can be used therapeutically [33]. If this is the responsible mechanism in humans it remains to be further explored as none of the TLR4 antibodies that are commercially available so far were able to confirm presence of sTLR4 in human AF.…”