Molecular mechanism of endotoxin tolerance^†

Scott et al.1 are to be congratulated on the delineation of a pathway by which a primary endotoxin challenge causes insensitivity to a secondary challenge because a better understanding of this phenomenon termed endotoxin tolerance is required to target intracellular signaling intermediates responsible for regulating endotoxin signaling leading to inadequate proinflammatory cytokine production in various liver diseases. In hepatocytes isolated from mice and mouse livers, using fluorescent microscopy and imaging software after the administration of fluorescent lipopolysaccharide (LPS), the authors revealed that the initial hepatic uptake of LPS and the clearance of LPS from the systemic circulation were dependent on the expression of toll-like receptor 4 (TLR4), the secondary uptake of LPS decreased with LPS pretreatment, and the suppression of uptake was associated with attenuated TLR4 signaling via a myeloid differentiation protein 88 (MyD88)–dependent pathway induced by the first endotoxin challenge. Moreover, the authors investigated which regulatory proteins are involved in decreased TLR4 signaling associated with LPS uptake desensitization, and they found that only the suppressor of cytokine signaling 1 (SOCS1) expression was up-regulated early after the primary LPS challenge and that the expression of SOCS1 was dependent on TLR4 signaling through MyD88. In the following experiments using hepatocytes infected with an SOCS1-expressing adenovirus and SOCS1 knockout cells, the authors confirmed that SOCS1 plays a central role in the mechanism of desensitization of LPS uptake in hepatocytes. Finally, the authors demonstrated that SOCS1 could inactivate toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) and that the expression of SOCS1 was inversely correlated with that of TIRAP; this suggests that down-regulation of TIRAP induced by up-regulation of SOCS1 is the main cause of desensitization of LPS uptake in murine hepatocytes.

The DNA binding activity of nuclear factor kappa B (NFkB) in response to a second endotoxin challenge after the exposure of 100 ng/mL LPS or control media for 60 minutes was measured to assess the effect of LPS pretreatment on the intensity of TLR4 signaling, but the time course for NFkB activation after LPS treatment was not shown in this study. I would like to ask the authors whether a quantitative relationship between the hepatic uptake of LPS and intrahepatic or extrahepatic TLR4 signaling has been previously examined. As shown in this article, delayed hepatic clearance of endotoxin, which may be associated with endotoxin tolerance in hepatocytes, is likely to lead to prolonged endotoxemia, resulting in sustained activation of Kupffer cells in the liver and immune cells in extrahepatic tissue. Although the authors do not mention the role of myeloid differentiation-2 (MD2) in the desensitization of LPS uptake, MD2 expression was decreased after 4 hours of endotoxin treatment in their study. MD2 is associated with the extracellular domain of TLR4, and this soluble protein is indispensable for LPS recognition and subsequent TLR4 signaling. Moreover, I would like to ask the authors whether an early reduction of MD2 expression with LPS pretreatment triggered the desensitization in hepatocytes. TIRAP appears to be associated with the MyD88-dependent pathway that induces tumor necrosis factor production and also with the MyD88-independent and toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon beta–dependent pathway that induces interferon production. In contrast to this study, SOCS1 regulated interferon-dependent pathways but had no regulatory effect on the activation of NFkB in human monocytes and macrophages infected with an SOCS1-expressing adenovirus.2 I think that the pathways of LPS-induced TLR4 signaling and the mechanism of endotoxin tolerance should be examined in different cells from animals and humans. Furthermore, a study using TIRAP-deficient animals, such as a study showing that expression of TIRAP is not required for bacterial clearance,3 would be necessary.