Distinct responses of lung and liver macrophages to acute endotoxemia: role of toll-like receptor 4.

Exposure to excessive quantities of bacterial-derived lipopolysaccharide (LPS) is associated with injury to the lung and the liver. Macrophages are thought to play a key role in the pathogenic response to LPS by releasing proinflammatory/cytotoxic mediators. Macrophage responses to LPS are mediated in large part by toll-like receptor 4 (TLR4). In the present studies we used C3H/HeJ mice, which possess a mutated nonfunctional TLR4, to examine its role in lung and liver macrophage responses to acute endotoxemia induced by LPS administration. Treatment of control C3H/HeOuJ mice with LPS (3 mg/ml, i.p.) was associated with a significant increase in the number of macrophages in both the lung and the liver. This was most prominent after 48 h, and was preceded by expression of proliferating cell nuclear antigen (PCNA), suggesting that macrophage proliferation contributes to the response. In liver, but not lung macrophages, LPS administration resulted in a rapid (within 3 h) increase in mRNA expression of Mn superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), key enzymes in antioxidant defense. In contrast, HO-1 protein expression decreased 3 h after LPS administration in liver macrophages, while in lung macrophages it increased. mRNA expression of enzymes mediating the biosynthesis of eicosanoids, including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1), but not 12/15-lipoxygenase (LOX), was upregulated in liver macrophages 3-24 h after LPS, with no effect on lung macrophages. However, COX-2 protein expression increased in both cell types. Loss of functional TLR4 significantly blunted the effects of LPS. Thus, no major changes were observed after LPS administration in the number of lung and liver macrophages recovered from TLR4 mutant mice, or on expression of PCNA. Increases in HO-1, MnSOD, COX-2 and PGES-1 mRNA expression in liver macrophages were also reduced in these mice. Conversely, in lung macrophages, loss of functional TLR4 resulted in increased expression of COX-2 protein and 12/15-LOX mRNA. These results demonstrate distinct lung and liver macrophage responses to acute endotoxemia are mediated, in part, by functional TLR4.

INT-767 improves histopathological features in a diet-induced ob/ob mouse model of biopsy-confirmed non-alcoholic steatohepatitis.

AIM: To characterize the efficacy of the dual FXR/TGR5 receptor agonist INT-767 upon histological endpoints in a rodent model of diet-induced and biopsy-confirmed non-alcoholic steatohepatitis (NASH). METHODS: The effects of INT-767 on histological features of NASH were assessed in two studies using Lepob/ob (ob/ob) NASH mice fed the AMLN diet (high fat with trans-fat, cholesterol and fructose). In a proof-of-concept study, Lepob/ob (ob/ob) NASH mice were first dosed with INT-767 (3 or 10 mg/kg for 8 wk). A second ob/ob NASH study compared INT-767 (3 and 10 mg/kg) to obeticholic acid (OCA) (10 or 30 mg/kg; 16 wk). Primary histological endpoints included qualitative and quantitative assessments of NASH. Other metabolic and plasma endpoints were also assessed. A comparative assessment of INT-767 and OCA effects on drug distribution and hepatic gene expression was performed in C57Bl/6 mice on standard chow. C57Bl/6 mice were orally dosed with INT-767 or OCA (1-30 mg/kg) for 2 wk, and expression levels of candidate genes were assessed by RNA sequencing and tissue drug levels were measured by liquid chromatography tandem-mass spectrometry. RESULTS: INT-767 dose-dependently (3 and 10 mg/kg, PO, QD, 8 wk) improved qualitative morphometric scores on steatohepatitis severity, inflammatory infiltrates and fibrosis stage. Quantitative morphometric analyses revealed that INT-767 reduced parenchymal collagen area, collagen fiber density, inflammation (assessed by Galectin-3 immunohistochemistry) and hepatocyte lipid droplet area following INT-767 treatment. In a comparative study (16 wk), the FXR agonists OCA (10 and 30 mg/kg) and INT-767 (3 and 10 mg/kg) both improved NASH histopathology, with INT-767 exerting greater therapeutic potency and efficacy than OCA. Mechanistic studies suggest that both drugs accumulate similarly within the liver and ileum, however, the effects of INT-767 may be driven by enhanced hepatic, but not ileal, FXR function. CONCLUSION: These findings confirm the potential utility of FXR and dual FXR/TGR5 activation as disease intervention strategies in NASH.

Regulation of cyclooxygenase-2 by nitric oxide in activated hepatic macrophages during acute endotoxemia.

Eicosanoids generated via cyclooxygenase-2 (COX-2) and nitric oxide produced from inducible nitric oxide synthase (NOSII) have been implicated in endotoxin-induced tissue injury. In the present studies, we characterized COX-2 and NOSII activity in rat hepatic macrophages and their interaction during acute endotoxemia. Kupffer cells from control animals were found to constitutively express COX-2 and NOSII mRNA and protein. Whereas treatment of the cells with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma) had no major effect on COX-2, NOSII expression increased. Induction of acute endotoxemia resulted in a rapid and transient increase in constitutive COX-2 expression and prostaglandin E2 (PGE2) production by liver macrophages as well as NOSII expression and nitric oxide release. Cells from endotoxin-treated rats were also sensitized to generate more nitric oxide and express increased NOSII in response to LPS and IFN-gamma. Inhibition of NOSII with aminoguanidine reduced COX-2 mRNA and protein expression as well as PGE2 production by activated macrophages from endotoxemic, but not control animals. In contrast, SC236, a specific COX-2 inhibitor, had no effect on NOSII mRNA or protein levels or on nitric oxide production by hepatic macrophages, even after endotoxin administration. These data suggest that activation of COX-2 may be important in the pathophysiological response of hepatic macrophages to endotoxin. Moreover, nitric oxide is involved in regulating COX-2 in activated liver macrophages during acute endotoxemia.

Identification of an Adamantyl Azaquinolone JNK Selective Inhibitor.

3-[4-((1S,2S,3R,5S,7S)-5-Hydroxyadamantan-2-ylcarbamoyl)benzyl]-4-oxo-1-phenyl-1,4-dihydro-[1,8]naphthyridine-2-carboxylic acid methyl ester (4) was identified as a novel, druglike and selective quinolone pan JNK inhibitor. In this communication, some of the structure-activity relationship of the azaquinolone analogues leading to 4 is discussed. The focus is on how changes at the amide functionality affected the biochemical potency, cellular potency, metabolic properties, and solubility of this class of JNK inhibitors. Optimization of these properties led to the identification of the adamantyl analogue, 4. 4 achieved proof of mechanism in both rat and mouse TNF-α challenge models.

Regulation of TREM expression in hepatic macrophages and endothelial cells during acute endotoxemia.

Triggering receptor expressed on myeloid cells (TREM) regulates inflammatory responses to lipopolysaccharide (LPS). In these studies, we analyzed the expression of TREM in hepatic macrophages and endothelial cells which play a central role in LPS clearance. LPS administration to C3H/HeOuJ mice resulted in a rapid induction of TREM-1 and TREM-3, but a decrease in TREM-2 in liver macrophages and endothelial cells. The observation that TREM family members are detectable in endothelial cells is novel and demonstrates that their expression is not limited to myeloid cells. LPS-induced alterations in TREM expression were not evident in cells from C3H/HeJ TLR-4 mutant mice, indicating that the response is dependent on TLR-4. IL-1beta and TNFalpha upregulated TREM-1 and TREM-3 expression and suppressed TREM-2 expression in macrophages and endothelial cells. This activity involved PI3-kinase and p38 MAP kinase signaling. Interestingly, no significant differences were noted in TREM expression between wild-type and TNFR1-/- mice treated with LPS. Treatment of macrophages and endothelial cells with LPS upregulated expression of nitric oxide synthase-2 (NOS-2). This was blocked by TREM-1 Fc/fusion protein, indicating that TREM-1 mediates LPS-induced NOS-2 expression. These results suggest that TREM proteins are important in the inflammatory response of hepatic macrophages and endothelial cells to acute endotoxemia.

Role of TLR-4 in liver macrophage and endothelial cell responsiveness during acute endotoxemia.

Liver macrophages and endothelial cells have been implicated in hepatotoxicity induced by endotoxin (ETX). In these studies, we analyzed the role of toll-like receptor 4 (TLR-4) in the response of these cells to acute endotoxemia. Treatment of control C3H/HeOuJ mice with ETX (3 mg/kg, i.p.) resulted in increased numbers of activated macrophages in the liver. This was associated with morphological changes in the cells and a rapid (within 3 h) induction of nitric oxide synthase-2, cyclooxygenase-2, microsomal PGE synthase-1, interleukin-1 beta and tumor necrosis factor alpha gene expression. In endothelial cells, acute endotoxemia led to increased expression of these genes, as well as 5-lipoxygenase. In contrast, liver sinusoidal cells from C3H/HeJ TLR-4 mutant mice were relatively unresponsive to ETX. Treatment of C3H/HeOuJ, but not C3H/HeJ mice with ETX, resulted in activation of transcription factors AP-1 and NF-kappaB in liver sinusoidal cells, which was evident within 3 h. Whereas in macrophages, transcription factor activation was transient, in endothelial cells, it persisted for 24 h. In C3H/HeOuJ mice treated with ETX, activation of p38 MAP kinase was also evident in macrophages and endothelial cells, and JNK kinase in macrophages. In contrast, reduced protein kinase B (AKT) was noted in macrophages. In C3H/HeJ mice, ETX administration also led to activation of p38 MAP kinase in macrophages with no effects on JNK, p44/42 MAP kinase or AKT. These studies demonstrate that liver macrophages and endothelial cells are highly responsive to acute endotoxemia. Moreover, this activity is largely dependent on TLR-4.