Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling.

Silymarin, a characterized extract of the seeds of milk thistle (Silybum marianum), suppresses cellular inflammation. To define how this occurs, transcriptional profiling, metabolomics, and signaling studies were performed in human liver and T cell lines. Cellular stress and metabolic pathways were modulated within 4 h of silymarin treatment: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed silymarin suppression of glycolytic, tricarboxylic acid (TCA) cycle, and amino acid metabolism. Anti-inflammatory effects arose with prolonged (i.e., 24 h) silymarin exposure, with suppression of multiple pro-inflammatory mRNAs and signaling pathways including nuclear factor kappa B (NF-κB) and forkhead box O (FOXO). Studies with murine knock out cells revealed that silymarin inhibition of both mTOR and NF-κB was partially AMPK dependent, whereas silymarin inhibition of mTOR required DDIT4. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Thus, natural products activate stress and repair responses that culminate in an anti-inflammatory cellular phenotype. Natural products like silymarin may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation.

A Plasmodium α/ß-hydrolase modulates the development of invasive stages.

The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/ß-hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a Plasmodium BEM46-like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The Plasmodium BEM46-like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo-erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P. yoelii knockout (Δpblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with Δpblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild-type (WT) parasites. Δpblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although Δpblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with Δpblp sporozoites exhibited a delay in the onset of blood-stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo-erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive-stage morphogenesis throughout the parasite life cycle.

Hepatitis C virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (STAT) 1 protein expression.

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or ß-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.

Direct, interferon-independent activation of the CXCL10 promoter by NF-κB and interferon regulatory factor 3 during hepatitis C virus infection.

Hepatitis C virus (HCV) infection of hepatocytes leads to transcriptional induction of the chemokine CXCL10, which is considered an interferon (IFN)-stimulated gene. However, we have recently shown that IFNs are not required for CXCL10 induction in hepatocytes during acute HCV infection. Since the CXCL10 promoter contains binding sites for several proinflammatory transcription factors, we investigated the contribution of these factors to CXCL10 transcriptional induction during HCV infection in vitro. Wild-type and mutant CXCL10 promoter-luciferase reporter constructs were used to identify critical sites of transcriptional regulation. The proximal IFN-stimulated response element (ISRE) and NF-κB binding sites positively regulated CXCL10 transcription during HCV infection as well as following exposure to poly(I·C) (a Toll-like receptor 3 [TLR3] stimulus) and 5' poly(U) HCV RNA (a retinoic acid-inducible gene I [RIG-I] stimulus) from two viral genotypes. Conversely, binding sites for AP-1 and CCAAT/enhancer-binding protein ß (C/EBP-ß) negatively regulated CXCL10 induction in response to TLR3 and RIG-I stimuli, while only C/EBP-ß negatively regulated CXCL10 during HCV infection. We also demonstrated that interferon-regulatory factor 3 (IRF3) is transiently recruited to the proximal ISRE during HCV infection and localizes to the nucleus in HCV-infected primary human hepatocytes. Furthermore, IRF3 activated the CXCL10 promoter independently of type I or type III IFN signaling. The data indicate that sensing of HCV infection by RIG-I and TLR3 leads to direct recruitment of NF-κB and IRF3 to the CXCL10 promoter. Our study expands upon current knowledge regarding the mechanisms of CXCL10 induction in hepatocytes and lays the foundation for additional mechanistic studies that further elucidate the combinatorial and synergistic aspects of immune signaling pathways.

Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

BACKGROUND & AIMS: The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. METHODS: CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. RESULTS: HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r(2) = 0.88, p ≤ 0.001). CONCLUSIONS: While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures.

Molecular pathways: hepatitis C virus, CXCL10, and the inflammatory road to liver cancer.

An estimated 170 million people worldwide are chronically infected with the hepatitis C virus (HCV), which is characterized histologically by a persistent immune and inflammatory response that fails to clear HCV from hepatocytes. This response is recruited to the liver, in part, by the chemokine CXCL10, the serum and intrahepatic levels of which have been inversely linked to the outcome of interferon-based therapies for hepatitis C. Bystander tissue damage from this ineffective response is thought to lead to increased hepatocyte turnover and the development of fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). However, CXCL10 is traditionally viewed as an orchestrator of the angiostatic and antitumor immune response. In this review, we will explore this duality and the pathways by which CXCL10 is produced by hepatocytes during HCV infection, its effects on resident and infiltrating immune cells, and how deregulation of these cell populations within the liver may lead to chronic liver inflammation. We will also discuss potential host-directed therapies to slow or reverse HCV-induced inflammation that leads to fibrosis, cirrhosis, and HCCs.

In vitro toxicity assessment of amphiphillic polymer-coated CdSe/ZnS quantum dots in two human liver cell models.

Semiconductor quantum dots (Qdots) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. Qdots generally consist of a semiconductor core, capping shell, and surface coating. The semiconductor core of Qdots is often composed of group II and VI metals (e.g., Cd, Se, Te, Hg) that are known to have toxic properties. Various surface coatings have been shown to stabilize Qdots and thus shield cells from the toxic properties of their core elements. In this study, HepG2 cells and primary human liver (PHL) cells were chosen as in vitro tissue culture models of human liver to examine the possible adverse effects of tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) copolymer (TOPO-PMAT)-coated CdSe/ZnS Qdots (TOPO-PMAT Qdots). The TOPO-PMAT coating is desirable for increasing aqueous solubility and ease of conjugation to targeting moieties (e.g., aptamers and peptides). HepG2 cells avidly incorporated these TOPO-PMAT Qdots into subcellular vesicles. However, PHL cells did not efficiently take up TOPO-PMAT Qdots, but nonparenchymal cells did (especially Kupffer cells). No acute toxicity or morphological changes were noted in either system at the exposure levels used (up to 40 nM). However, cellular stress markers and pro-inflammatory cytokines/chemokines were increased in the PHL cell cultures, suggesting that TOPO-PMAT Qdots are not likely to cause acute cytotoxicity in the liver but may elicit inflammation/hepatitis, demonstrating the importance of relevant preclinical safety models. Thus, further in vivo studies are warranted to ensure that TOPO-PMAT-coated Qdots used in biomedical applications do not induce inflammatory responses as a consequence of hepatic uptake.