Aimee L. Anderson

Toll-like receptor 4–dependent Kupffer cell activation and liver injury in a novel mouse model of parenteral nutrition and intestinal injury†‡

Infants with intestinal failure who are parenteral nutrition (PN)‐dependent may develop cholestatic liver injury and cirrhosis (PN‐associated liver injury: PNALI). The pathogenesis of PNALI remains incompletely understood. We hypothesized that intestinal injury with increased intestinal permeability combined with administration of PN promotes lipopolysaccharide (LPS)–Toll‐like receptor 4 (TLR4) signaling dependent Kupffer cell (KC) activation as an early event in the pathogenesis of PNALI. We developed a mouse model in which intestinal injury and increased permeability were induced by oral treatment for 4 days with dextran sulphate sodium (DSS) followed by continuous infusion of soy lipid‐based PN solution through a central venous catheter for 7 (PN7d/DSS) and 28 (PN28d/DSS) days. Purified KCs were probed for transcription of proinflammatory cytokines. PN7d/DSS mice showed increased intestinal permeability and elevated portal vein LPS levels, evidence of hepatocyte injury and cholestasis (serum aspartate aminotransferase, alanine aminotransferase, bile acids, total bilirubin), and increased KC expression of interleukin‐6 (Il6), tumor necrosis factor α (Tnfα), and transforming growth factor β (Tgfβ). Markers of liver injury remained elevated in PN28d/DSS mice associated with lobular inflammation, hepatocyte apoptosis, peliosis, and KC hypertrophy and hyperplasia. PN infusion without DSS pretreatment or DSS pretreatment alone did not result in liver injury or KC activation, even though portal vein LPS levels were elevated. Suppression of the intestinal microbiota with broad spectrum antibiotics or ablation of TLR4 signaling in Tlr4 mutant mice resulted in significantly reduced KC activation and markedly attenuated liver injury in PN7d/DSS mice. Conclusion: These data suggest that intestinal‐derived LPS activates KC through TLR4 signaling in early stages of PNALI. (HEPATOLOGY 2012)

Phytosterols Promote Liver Injury and Kupffer Cell Activation in Parenteral Nutrition–Associated Liver Disease

Intravenous phytosterols, components of soy lipid emulsions, promote cholestasis and liver injury during parenteral nutrition in a mouse model. Solving the Mysteries of Intravenous Nutrition Parenteral nutrition (PN), an intravenously delivered replacement for food, is a life-saving option for patients who cannot tolerate enteral feeding. These patients’ diagnoses vary widely, ranging from short-term illnesses like pancreatitis to long-term intestinal problems. Unfortunately, the use of PN takes a toll on the liver and can lead to cholestatic liver damage known as PN-associated liver disease (PNALD). The risk of PNALD is particularly high for premature infants and for children with intestinal failure or short bowel syndrome, who are often PN-dependent for months or years. It is well known that the duration of PN use contributes to the risk of PNALD, but other factors that cause this disease are not fully understood. Clinical evidence had suggested that the use of soy lipids in PN increases the risk of PNALD compared to fish oil–based lipids, but an explanation for this difference had been elusive. Now, El Kasmi and colleagues have identified the likely culprit for this side effect of soy lipids, as well as its pathogenic mechanism. The authors designed a mouse model, which they treated with a variety of PN formulations. Some of these included stigmasterol, a phytosterol derived from soy. Only the mice that received stigmasterol, either alone or as a component of soy lipid infusion, developed cholestasis. To clarify the mechanism for this effect, the authors showed that stigmasterol decreased the expression of sterol transporters in hepatocytes, resulting in a buildup of phytosterols in the liver cells. The researchers also found evidence that gut microbiota contribute to the risk of PNALD through stimulation of Toll-like receptor 4 and that antibiotic treatment reduces this risk. Additional work will be needed to investigate the role of other phytosterols and further details of the mechanism, including the effects of specific gut microbes on PNALD. However, the results of this study add to the accumulating evidence of PNALD risk associated with soy-based lipids, clarify the pathogenesis of this disease, and may help promote a shift away from stigmasterol-containing solutions for PN-dependent patients. Parenteral nutrition–associated liver disease (PNALD) is a serious complication of PN in infants who do not tolerate enteral feedings, especially those with acquired or congenital intestinal diseases. Yet, the mechanisms underlying PNALD are poorly understood. It has been suggested that a component of soy oil (SO) lipid emulsions in PN solutions, such as plant sterols (phytosterols), may be responsible for PNALD, and that use of fish oil (FO)–based lipid emulsions may be protective. We used a mouse model of PNALD combining PN infusion with intestinal injury to demonstrate that SO-based PN solution causes liver damage and hepatic macrophage activation and that PN solutions that are FO-based or devoid of all lipids prevent these processes. We have furthermore demonstrated that a factor in the SO lipid emulsions, stigmasterol, promotes cholestasis, liver injury, and liver macrophage activation in this model and that this effect may be mediated through suppression of canalicular bile transporter expression (Abcb11/BSEP, Abcc2/MRP2) via antagonism of the nuclear receptors Fxr and Lxr, and failure of up-regulation of the hepatic sterol exporters (Abcg5/g8/ABCG5/8). This study provides experimental evidence that plant sterols in lipid emulsions are a major factor responsible for PNALD and that the absence or reduction of plant sterols is one of the mechanisms for hepatic protection in infants receiving FO-based PN or lipid minimization PN treatment. Modification of lipid constituents in PN solutions is thus a promising strategy to reduce incidence and severity of PNALD.

Adventitial Fibroblasts Induce a Distinct Proinflammatory/Profibrotic Macrophage Phenotype in Pulmonary Hypertension

Macrophage accumulation is not only a characteristic hallmark but is also a critical component of pulmonary artery remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Using multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, and primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive pulmonary arteries (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL-6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL-4/IL-13–STAT6 and TLR–MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation, complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, and deficiency in C/EBPβ or HIF1 attenuated fibroblast-driven macrophage activation. These findings challenge the current paradigm of IL-4/IL-13–STAT6–mediated alternative macrophage activation as the sole driver of vascular remodeling in PH, and uncover a cross-talk between adventitial fibroblasts and macrophages in which paracrine IL-6–activated STAT3, HIF1α, and C/EBPβ signaling are critical for macrophage activation and polarization. Thus, targeting IL-6 signaling in macrophages by completely inhibiting C/EBPβ or HIF1α or by partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL-6 and absent IL-4/IL-13 signaling.

Specific Microbiome Changes in a Mouse Model of Parenteral Nutrition Associated Liver Injury and Intestinal Inflammation

Background Parenteral nutrition (PN) has been a life-saving treatment in infants intolerant of enteral feedings. However, PN is associated with liver injury (PN Associated Liver Injury: PNALI) in a significant number of PN-dependent infants. We have previously reported a novel PNALI mouse model in which PN infusion combined with intestinal injury results in liver injury. In this model, lipopolysaccharide activation of toll-like receptor 4 signaling, soy oil-derived plant sterols, and pro-inflammatory activation of Kupffer cells (KCs) played key roles. The objective of this study was to explore changes in the intestinal microbiome associated with PNALI. Methodology and Principal Findings Microbiome analysis in the PNALI mouse identified specific alterations within colonic microbiota associated with PNALI and further association of these communities with the lipid composition of the PN solution. Intestinal inflammation or soy oil-based PN infusion alone (in the absence of enteral feeds) caused shifts within the gut microbiota. However, the combination resulted in accumulation of a specific taxon, Erysipelotrichaceae (23.8% vs. 1.7% in saline infused controls), in PNALI mice. Moreover, PNALI was markedly attenuated by enteral antibiotic treatment, which also was associated with significant reduction of Erysipelotrichaceae (0.6%) and a Gram-negative constituent, the S24-7 lineage of Bacteroidetes (53.5% in PNALI vs. 0.8%). Importantly, removal of soy oil based-lipid emulsion from the PN solution resulted in significant reduction of Erysipelotrichaceae as well as attenuation of PNALI. Finally, addition of soy-derived plant sterol (stigmasterol) to fish oil-based PN restored Erysipelotrichaceae abundance and PNALI. Conclusions Soy oil-derived plant sterols and the associated specific bacterial groups in the colonic microbiota are associated with PNALI. Products from these bacteria may directly trigger activation of KCs and promote PNALI. Furthermore, the results indicate that lipid modification of PN solutions may alter specific intestinal bacterial species associated with PNALI, and thus suggest strategies for management of PNALI.

Endotoxemia Induces IκBβ/NF-κB–Dependent Endothelin-1 Expression in Hepatic Macrophages

Elevated serum concentrations of the vasoactive protein endothelin-1 (ET-1) occur in the setting of systemic inflammatory response syndrome and contribute to distal organ hypoperfusion and pulmonary hypertension. Thus, understanding the cellular source and transcriptional regulation of systemic inflammatory stress-induced ET-1 expression may reveal therapeutic targets. Using a murine model of LPS-induced septic shock, we demonstrate that the hepatic macrophage is the primary source of elevated circulating ET-1, rather than the endothelium as previously proposed. Using pharmacologic inhibitors, ET-1 promoter luciferase assays, and by silencing and overexpressing NF-κB inhibitory protein IκB expression, we demonstrate that LPS-induced ET-1 expression occurs via an NF-κB–dependent pathway. Finally, the specific role of the cRel/p65 inhibitory protein IκBβ was evaluated. Although cytoplasmic IκBβ inhibits activity of cRel-containing NF-κB dimers, nuclear IκBβ stabilizes NF-κB/DNA binding and enhances gene expression. Using targeted pharmacologic therapies to specifically prevent IκBβ/NF-κB signaling, as well as mice genetically modified to overexpress IκBβ, we show that nuclear IκBβ is both necessary and sufficient to drive LPS-induced ET-1 expression. Together, these results mechanistically link the innate immune response mediated by IκBβ/NF-κB to ET-1 expression and potentially reveal therapeutic targets for patients with Gram-negative septic shock.