Dmd062307 335..343

Bacterial sepsis is characterized by a rapid increase in the expression of inflammatory mediators to initiate the acute phase response in liver. Inflammatory mediator release is counterbalanced by the coordinated expression of anti-inflammatory molecules such as interleukin 1 receptor antagonist (IL1-Ra) through time. This study determined whether activation of pregnane X receptor (PXR, NR1I2) alters the lipopolysaccharide (LPS)-inducible gene expression program in primary cultures of hepatocytes (PCHs). Preactivation of PXR for 24 hours in PCHs isolated from wild-type mice suppressed the subsequent LPS-inducible expression of the key inflammatory mediators interleukin 1b (IL-1b), interleukin 6 (IL-6), and tumor necrosis factor a (TNFa) but not in PCHs isolated from Pxr-null (PXR-knockout [KO]) mice. Basal expression of key inflammatory cytokines was elevated in PCHs from PXR-KO mice. Stimulation of PCHs from PXR-KO mice with LPS alone produced enhanced levels of IL-1b when compared with wild-type mice. Experiments performed using PCHs from both humanized-PXR transgenic mice as well as human donors indicate that prolonged activation of PXR produces an increased secretion of IL1-Ra from cells through time. Our data reveal a working model that describes a pivotal role for PXR in both inhibiting as well as in resolving the inflammatory response in hepatocytes. Understanding the molecular details of how PXR is converted from a positive regulator of drug-metabolizing enzymes into a transcriptional suppressor of inflammation in liver will provide new pharmacologic strategies for modulating inflammatory-related diseases in the liver and intestine. Pregnane X receptor (PXR, NR1I2) is a ligand-activated nuclear receptor (NR) superfamily member expressed at high levels within the enterohepatic system of mammals. The biologic function of PXR is mediated together with its obligate partner retinoid X receptor a (Kliewer et al., 1998; Lehmann et al., 1998). To date, the ligands identified for PXR have been numerous, and they are structurally diverse as naturally occurring steroids (Kliewer et al., 1998), antibiotics (Lehmann et al., 1998), bile acids (Staudinger et al., 2001a; Xie et al., 2001; Goodwin et al., 2003), anticancer agents (Desai et al., 2002; Nallani et al., 2004), and the active ingredients in several herbal remedies (Moore et al., 2000; Brobst et al., 2004; Ding and Staudinger, 2005). Ligand-activated PXR positively regulates the drug-inducible expression of genes encoding key drug transporters and drug metabolizing enzymes that function coordinately to increase the uptake, metabolism, excretion, and efflux of xenobiotics from the body. In this way, PXR activation is associated with increased metabolism and clearance of a myriad of potentially toxic compounds, and is classically thought of as a protective response. Clinical treatment with PXR activators can also lead to the repression or attenuation of other biochemical pathways in liver and intestine including both energy metabolism and the inflammatory response (Moreau et al., 2008). For example, it was demonstrated nearly 45 years ago that treatment with rifampicin (Rif), a prototypical ligand of human PXR, leads to a compromised ability to mount an effective immune response in cell-based assays (P aunescu, 1970). In vivo studies in rodents suggest that PXR activation suppresses inflammation and the acute phase response (APR) by attenuating the activity of nuclear factor k-light-chainenhancer of activated B cells (NF-kB) signaling (Shah et al., 2007). More recent studies using rodents indicate that PXR activation regulates intestinal barrier function through an interaction with Toll-like receptor 4 (TLR4), the molecular target of lipopolysaccharide (LPS) stimulation (Venkatesh et al., 2014). It is now widely accepted that the activation of PXR is associated with general suppression of the immune response, particularly in the intestine (Cheng et al., 2012; Dou et al., 2012, 2014). Currently, little is known regarding the molecular mechanism of this phenomenon in the liver or in hepatocytes. Recent investigations indicate that ligand-mediated activation of liverenriched nuclear receptors (NRs), liver receptor homolog-1, and liver X receptor b (LXRb) initiate anti-inflammatory mechanisms and pathways This work was supported by the National Institutes of Health National Institute Diabetes and Digestive and kidney Diseases (NIDDK) [Grant R01 DK090558]. The human hepatocytes used in this study were derived from samples collected and provided by the University of Kansas Medical Center (KUMC) Department of Pharmacology, Toxicology and Therapeutics Hepatocyte Core Laboratory and the