Endotoxin uptake in mouse liver is blocked by endotoxin pretreatment through a suppressor of cytokine signaling-1-dependent mechanism.
The liver is the main organ that clears lipopolysaccharide (LPS) and hepatocytes are a major cell-type involved in LPS uptake. LPS tolerance, or desensitization, is important in negative regulation of responses to LPS, but little is known about its mechanisms in hepatocytes. Primary isolated C57BL/6 hepatocytes, and liver in vivo, internalized fluorescent LPS, and this was dependent on Toll-like receptor 4 (TLR4) at the cell surface but not on TLR4-TIR signaling through MyD88. LPS clearance from plasma was also TLR4-dependent. Pretreatment of C57BL/6 hepatocytes with LPS prevented uptake of LPS 24 hours later and this LPS-mediated suppression was dependent on TLR4 signaling through MyD88. Many regulators of TLR4 signaling have been identified and implicated in LPS desensitization, including suppressor of cytokine signaling 1 (SOCS1). SOCS1 mRNA and protein expression increased after LPS stimulation in hepatocytes and in whole liver. LPS uptake in hepatocytes and liver was significantly reduced following infection with adenoviral vectors overexpressing SOCS1. Similarly, inhibition of SOCS1 using small interfering (si)RNA-mediated knockdown prevented LPS desensitization in hepatocytes. SOCS1 is known to interact with Toll/IL-1 receptor associated protein (TIRAP) and cause TIRAP ubiquitination and degradation, which regulates TLR signaling. We have also shown previously that TIRAP regulates LPS uptake in hepatocytes. SOCS1 coimmunoprecipitated with TIRAP in wild type hepatocyte cell lysates up to 8 hours after LPS stimulation, but not at later times. In the same samples, ubiquitinated TIRAP was detected after 4 hours and up to 8 hours after LPS stimulation, but not at later times.


CONCLUSIONS
These data indicate hepatocytes are desensitized by LPS in a TLR4 signaling-dependent manner. LPS-induced SOCS1 upregulation increases degradation of TIRAP and prevents subsequent LPS uptake. The exploitation of these mechanisms of LPS desensitization in the liver may be important in future sepsis therapies.
Scott
Melanie J
MJ
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Liu
Shubing
S
Shapiro
Richard A
RA
Vodovotz
Yoram
Y
Billiar
Timothy R
TR
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000818:Animals; D003888:Desensitization, Immunologic; D022781:Hepatocytes / Q000276:immunology* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology* / Q000378:metabolism; D008099:Liver / Q000276:immunology* / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D015854:Up-Regulation
D033942:Adaptor Proteins, Vesicular Transport; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D053594:Myeloid Differentiation Factor 88; D017472:Receptors, Interleukin-1; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D051197:Toll-Like Receptor 4; C411728:Ly96 protein, mouse; C507780:Myd88 protein, mouse; C491590:Socs1 protein, mouse; C476286:Ticam1 protein, mouse; C499045:TIRAP protein, mouse; C493487:Tlr4 protein, mouse