Regulation of MRP2/ABCC2 and BSEP/ABCB11 Expression in Sandwich Cultured Human and Rat Hepatocytes Exposed to Inflammatory Cytokines TNF-α, IL-6, and IL-1β

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-α, IL-6, or IL-1β. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1β. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-α and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-α or IL-1β. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.     

Autoregulatory Effect of Prostaglandin E2 on Interleukin 6 Production by Kupffer Cells

The Kupffer cell (KC) is the resident hepatic macrophage, whose functions include local intrahepatic immune responses implicated in tolerance induction, participation in the septic state, and regulation of hepatic regeneration. The ability of the KC to participate In these biologically diverse functions is thought to be due to its release of pleiotropic cytokines, such as interleukin 6 (IL-6), which can act locally in a paracrine fashion or as hormones at distant sites. Many of the KC′s secretory responses are carefully regulated in an autocoid fashion by the eicosanoid prostaglandin E₂ (PGE₂). The degree of regulation depends on the particular cytokine and local environmental factors. In this review, we describe our method for isolating KCs by their adherence to plastic and for testing their IL-6 and PGE₂ secretory responses to lipopolysaccharide. In comparing the responses of KCs from normal and regenerating rat livers, we describe an in vitro KC secretory pattern of eicosanoid inhibition of IL-6, whereas both responses to LPS are augmented in the KC during hepatic regeneration. Such an enhancement is consistent with the shared putative supportive roles of IL-6 and PGE₂ in liver regeneration.     

The role of HGF on invasive properties and repopulation potential of human fetal hepatic progenitor cells

The success of hepatocyte transplantation has been limited by the low efficiency of transplanted cell integration into liver parenchyma. Human fetal hepatic progenitor cells (hepatoblasts) engraft more effectively than adult hepatocytes in mouse livers. However, the signals required for their integration are not yet fully understood. We investigated the role of HGF on the migration and invasive ability of human hepatic progenitors in vitro and in vivo.Hepatoblasts were isolated from the livers of human fetuses between 10 and 12 weeks of gestation. Their invasive ability was assessed in the presence or absence of HGF. These cells were also transplanted into immunodeficient mice and analyzed by immunohistochemistry.In contrast to TNF-alpha, HGF increased the motogenesis and invasiveness of hepatoblasts, but not of human adult hepatocytes, via phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The invasive ability of human hepatoblasts correlated with the expression and secretion of matrix metalloproteinases (MMPs). Hepatoblasts stimulated with HGF prior transplantation into newborn mice migrated from the portal area into the hepatic parenchyma.Conclusions: In contrast to adult hepatocytes, hepatoblasts display invasive ability that can be modulated by HGF in vitro and in vivo.     

Hepatocyte growth factor modulates interleukin-6 production in bone marrow derived macrophages: implications for inflammatory mediated diseases

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.     

Endocrine,paracrine and autocrine actions of prolactin on immune cells

The immune response is regulated by locally released factors, collectively referred to as cytokines. Data on the human immune system have convincingly demonstrated that the hormone prolactin (PRL), in addition to exerting its endocrine control on the immune system, acts as a cytokine in that it is released within the immune system and regulates the lymphocyte response by paracrine and autocrine mechanisms. Both lymphocyte and pituitary PRLs are under the control of immune factors. Synthesis of human PRL by lymphocytes is induced by T-cell stimuli, while increased release of PRL by the pituitary, observed in vivo after immune challenge, may be mediated by cytokines produced by monocyte-macrophages. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain basal immunocompetence. The effects of Cyclosporin (CsA) on IL-2 and PRL gene activation and the analysis of the intracellular signaling events downstream IL-2 and PRL receptors suggest coordinate actions of these two cytokines during T cell activation.     

Bone Marrow Progenitor Cell Therapy-Mediated Paracrine Regulation of Cardiac miRNA-155 Modulates Fibrotic Response in Diabetic Hearts

Diabetes is associated with a higher incidence of myocardial infarction (MI) and increased risk for adverse vascular and fibrogenic events post-MI. Bone marrow-derived progenitor cell (BMPC) therapy has been shown to promote neovascularization, decrease infarct area and attenuate left ventricular (LV) dysfunction after MI. Unlike vascular effects, the anti-fibrosis mechanisms of BMPC, specifically under diabetic conditions, are poorly understood. We demonstrated that intramyocardial delivery of BMPCs in infarcted diabetic db/db mice significantly down-regulates profibrotic miRNA-155 in the myocardium and improves LV remodeling and function. Furthermore, inhibition of paracrine factor hepatocyte growth factor (HGF) signaling in vivo suppressed the BMPC-mediated inhibition of miR-155 expression and the associated protective effect on cardiac fibrosis and function. In vitro studies confirmed that the conditioned media of BMPC inhibited miR-155 expression and profibrotic signaling in mouse cardiac fibroblasts under diabetic conditions. However, neutralizing antibodies directed against HGF blocked these effects. Furthermore, miR-155 over-expression in mouse cardiac fibroblasts inhibited antifibrotic Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene, non-Alu-containing (SnoN) signaling and abrogated antifibrogenic response of HGF. Together, our data demonstrates that paracrine regulation of cardiac miRNAs by transplanted BMPCs contributes to the antifibrotic effects of BMPC therapy. BMPCs release HGF, which inhibits miR-155-mediated profibrosis signaling, thereby preventing cardiac fibrosis. These data suggest that targeting miR-155 might serve as a potential therapy against cardiac fibrosis in the diabetic heart.     

Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression

Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.     

CYLD Enhances Severe Listeriosis by Impairing IL-6/STAT3-Dependent Fibrin Production

The facultative intracellular bacterium Listeria monocytogenes (Lm) may cause severe infection in humans and livestock. Control of acute listeriosis is primarily dependent on innate immune responses, which are strongly regulated by NF-κB, and tissue protective factors including fibrin. However, molecular pathways connecting NF-κB and fibrin production are poorly described. Here, we investigated whether the deubiquitinating enzyme CYLD, which is an inhibitor of NF-κB-dependent immune responses, regulated these protective host responses in murine listeriosis. Upon high dose systemic infection, all C57BL/6 Cyld^−/− mice survived, whereas 100% of wildtype mice succumbed due to severe liver pathology with impaired pathogen control and hemorrhage within 6 days. Upon in vitro infection with Lm, CYLD reduced NF-κB-dependent production of reactive oxygen species, interleukin (IL)-6 secretion, and control of bacteria in macrophages. Furthermore, Western blot analyses showed that CYLD impaired STAT3-dependent fibrin production in cultivated hepatocytes. Immunoprecipitation experiments revealed that CYLD interacted with STAT3 in the cytoplasm and strongly reduced K63-ubiquitination of STAT3 in IL-6 stimulated hepatocytes. In addition, CYLD diminished IL-6-induced STAT3 activity by reducing nuclear accumulation of phosphorylated STAT3. In vivo, CYLD also reduced hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 and NOX2 mRNA production as well as fibrin production in murine listeriosis. In vivo neutralization of IL-6 by anti-IL-6 antibody, STAT3 by siRNA, and fibrin by warfarin treatment, respectively, demonstrated that IL-6-induced, STAT3-mediated fibrin production significantly contributed to protection in Cyld^−/− mice. In addition, in vivo Cyld siRNA treatment increased STAT3 phosphorylation, fibrin production, pathogen control and survival of Lm-infected WT mice illustrating that therapeutic inhibition of CYLD augments the protective NF-κB/IL-6/STAT3 pathway and fibrin production.     

Calcineurin mediates the immune response of hemocytes through NF-κB signaling pathway in pearl oyster (Pinctada fucata)

Calcineurin (CN), a multifunctional protein, mediates the immune response through diverse signaling pathways in mammals, while the function of CN in the immune response of molluscan hemocytes still remains unclear. In the present study, we detected the distribution of CN in various tissues and the expression levels of Pf-CNA and Pf-CNB gene in hemocytes of Pinctada fucata. After the preparation of hemocyte monolayers, we checked the response of enzymatic activity of CN, the degradation level of IκBα, the activity of iNOS and the production of NO, and IL-2 to the challenge of lipopolysaccharide (LPS) and cyclosporin A (CsA). CN activity in hemocytes was very sensitive to both the stimulation of LPS and the inhibition of CsA. Most importantly, IκBα degradation in hemocytes was induced by LPS and attenuated by CsA. Consequently, the activity of iNOS was elevated and the production of NO was increased. Additionally, we found that the synthesis of IL-2 was increased by LPS but was apparently weakened by CsA. In vivo bacterial clearance experiments showed that CsA significantly decreased the ability of in vivo bacteria clearance in pearl oyster. All the results revealed, for the first time, that CN mediated the immune response of molluscan hemocytes via activating NF-κB signaling pathway.     

Study on the activity of the signaling pathways regulating hepatocytes from G0 phase into G1 phase during rat liver regeneration

Under normal physiological conditions, the majority of hepatocytes are in the functional state (G0 phase). After injury or liver partial hepatectomy (PH), hepatocytes are rapidly activated to divide. To understand the mechanism underlying hepatocyte G0/G1 transition during rat liver regeneration, we used the Rat Genome 230 2.0 Array to determine the expression changes of genes, then searched the GO and NCBI databases for genes associated with the G0/G1 transition, and QIAGEN and KEGG databases for the G0/G1 transition signaling pathways. We used expression profile function (E _t ) to calculate the activity level of the known G0/G1 transition signal pathways, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the interactions among these signaling pathways. The results of our study show that the activity of the signaling pathways of HGF, IL-10 mediated by p38MAPK, IL-6 mediated by STAT3, and JAK/STAT mediated by Ras/ERK and STAT3 are significantly increased during the priming phase (2–6 h after PH) of rat liver regeneration. This leads us to conclude that during rat liver regeneration, the HGF, IL-10, IL-6 and JAK/STAT signaling pathways play a major role in promoting hepatocyte G0/G1 transition in the regenerating liver.     

Mitochondrial Damage-Associated Molecular Patterns (MTDs) Are Released during Hepatic Ischemia Reperfusion and Induce Inflammatory Responses

Ischemia / reperfusion injury (IRI) during the course of liver transplantation enhances the immunogenicity of allografts and thus impacts overall graft outcome. This sterile inflammatory insult is known to activate innate immunity and propagate organ damage through the recognition of damage-associate molecular pattern (DAMP) molecules. The purpose of the present study was to investigate the role of mitochondrial DAMPs (MTDs) in the pathogenesis of hepatic IRI. Using in vitro models we observed that levels of MTDs were significantly higher in both transplantation-associated and warm IR, and that co-culture of MTDs with human and rat hepatocytes significantly increased cell death. MTDs were also released in an in vivo rat model of hepatic IRI and associated with increased secretion of inflammatory cytokines (TNF-α, IL-6, and IL-10) and increased liver injury compared to the sham group. Our results suggest that hepatic IR results in a significant increase of MTDs both in vitro and in vivo suggesting that MTDs may serve as a novel marker in hepatic IRI. Co-culture of MTDs with hepatocytes showed a decrease in cell viability in a concentration dependent manner, which indicates that MTDs is a toxic mediator participating in the pathogenesis of liver IR injury.     

Human bone marrow mesenchymal stem cells are resistant to HBV infection during differentiation into hepatocytes in vivo and in vitro

Hepatocyte-like cells induced from bone marrow mesenchymal stem cells (BMSCs) recover liver function in animal models with liver failure. Our initial findings revealed that human BMSCs improved liver function in hepatitis B patients with end stage liver disease. However, the susceptibility of BMSCs to HBV infection during induction toward hepatocytes remains unknown. We have assessed whether BMSCs-derived hepatocyte-like cells can function like liver cells and be infected by HBV. A new and efficient way to direct the differentiation of BMSCs into functional hepatocytes was developed. BMSCs obtained from hepatitis B patients were induced to differentiate into hepatocytes through exposure to HGF, FGF-4, and EGF. After 6 days of exposure, BMSCs-derived hepatocyte-like cells that expressed a subset of hepatic genes and showed hepatic functions were obtained. HBV was used to infect the differentiated cells, and subsequently these cells were assayed for the presence of HBeAg, HBsAg, and HBV DNA. BMSCs proved resistant to HBV infection, both in vitro and during differentiation into hepatocytes in vitro. This demonstrates that BMSCs are resistant to HBV infection. BMSCs are viable for transplantation and should facilitate further research exploring the in vivo HBV-resistance of the hepatocytes derived from BMSCs after transplantation, a characteristic that could form the basis for hepatocyte transplantation.     

Quorum sensing governs collective dendritic cell activation in vivo

Dendritic cell (DC) activation by viral RNA sensors such as TLR3 and MDA‐5 is critical for initiating antiviral immunity. Optimal DC activation is promoted by type I interferon (IFN) signaling which is believed to occur in either autocrine or paracrine fashion. Here, we show that neither autocrine nor paracrine type I IFN signaling can fully account for DC activation by poly(I:C) in vitro and in vivo. By controlling the density of type I IFN‐producing cells in vivo, we establish that instead a quorum of type I IFN‐producing cells is required for optimal DC activation and that this process proceeds at the level of an entire lymph node. This collective behavior, governed by type I IFN diffusion, is favored by the requirement for prolonged cytokine exposure to achieve DC activation. Furthermore, collective DC activation was found essential for the development of innate and adaptive immunity in lymph nodes. Our results establish how collective rather than cell‐autonomous processes can govern the initiation of immune responses.     

Lipopolysaccharide Does Not Alter Small Airway Reactivity in Mouse Lung Slices

The bacterial endotoxin, lipopolysaccharide (LPS) has been associated with occupational airway diseases with asthma-like symptoms and in acute exacerbations of COPD. The direct and indirect effects of LPS on small airway reactivity have not been fully elucidated. We tested the hypothesis that both in vitro and in vivo LPS treatment would increase contraction and impair relaxation of mouse small airways. Lung slices were prepared from naïve Balb/C mice and cultured in the absence or presence of LPS (10 μg/ml) for up to 48 h for measurement of TNFα levels in conditioned media. Alternatively, mice were challenged with PBS or LPS in vivo once a day for 4 days for preparation of lung slices or for harvest of lungs for Q-PCR analysis of gene expression of pro-inflammatory cytokines and receptors involved in airway contraction. Reactivity of small airways to contractile agonists, methacholine and serotonin, and bronchodilator agents, salbutamol, isoprenaline and rosiglitazone, were assessed using phase-contrast microscopy. In vitro LPS treatment of slices increased TNFα release 6-fold but did not alter contraction or relaxation to any agonists tested. In vivo LPS treatment increased lung gene expression of TNFα, IL-1β and ryanodine receptor isoform 2 more than 5-fold. However there were no changes in reactivity in lung slices from these mice, even when also incubated with LPS ex vivo. Despite evidence of LPS-induced inflammation, neither airway hyperresponsiveness or impaired dilator reactivity were evident. The increase in ryanodine receptor isoform 2, known to regulate calcium signaling in vascular smooth muscle, warrants investigation. Since LPS failed to elicit changes in small airway reactivity in mouse lung slices following in vitro or in vivo treatment, alternative approaches are required to define the potential contribution of this endotoxin to altered small airway reactivity in human lung diseases.     

Necro-inflammatory response of pancreatic acinar cells in the pathogenesis of acute alcoholic pancreatitis

The role of pancreatic acinar cells in initiating necro-inflammatory responses during the early onset of alcoholic acute pancreatitis (AP) has not been fully evaluated. We investigated the ability of acinar cells to generate pro- and anti-inflammatory mediators, including inflammasome-associated IL-18/caspase-1, and evaluated acinar cell necrosis in an animal model of AP and human samples. Rats were fed either an ethanol-containing or control diet for 14 weeks and killed 3 or 24 h after a single lipopolysaccharide (LPS) injection. Inflammasome components and necro-inflammation were evaluated in acinar cells by immunofluorescence (IF), histology, and biochemical approaches. Alcohol exposure enhanced acinar cell-specific production of TNFα, IL-6, MCP-1 and IL-10, as early as 3 h after LPS, whereas IL-18 and caspase-1 were evident 24 h later. Alcohol enhanced LPS-induced TNFα expression, whereas blockade of LPS signaling diminished TNFα production in vitro, indicating that the response of pancreatic acinar cells to LPS is similar to that of immune cells. Similar results were observed from acinar cells in samples from patients with acute/recurrent pancreatitis. Although morphologic examination of sub-clinical AP showed no visible signs of necrosis, early loss of pancreatic HMGB1 and increased systemic levels of HMGB1 and LDH were observed, indicating that this strong systemic inflammatory response is associated with little pancreatic necrosis. These results suggest that TLR-4-positive acinar cells respond to LPS by activating the inflammasome and producing pro- and anti-inflammatory mediators during the development of mild, sub-clinical AP, and that these effects are exacerbated by alcohol injury.     

GCN2-Dependent Metabolic Stress Is Essential for Endotoxemic Cytokine Induction and Pathology

Activated inflammatory macrophages can express indoleamine 2,3-dioxygenase (IDO) and thus actively deplete their own tryptophan supply; however, it is not clear how amino acid depletion influences macrophage behavior in inflammatory environments. In this report, we demonstrate that the stress response kinase GCN2 promotes macrophage inflammation and mortality in a mouse model of septicemia. In vitro, enzymatic amino acid consumption enhanced sensitivity of macrophages to the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) with significantly increased interleukin 6 (IL-6) production. Tryptophan withdrawal induced the stress response proteins ATF4 and CHOP/GADD153; however, LPS stimulation rapidly enhanced expression of both proteins. Moreover, LPS-driven cytokine production under amino acid-deficient conditions was dependent on GCN2, as GCN2 knockout (GCN2KO) macrophages had a significant reduction of cytokine gene expression after LPS stimulation. To test the in vivo relevance of these findings, monocytic-lineage-specific GCN2KO mice were challenged with a lethal dose of LPS intraperitoneally (i.p.). The GCN2KO mice showed reduced inflammatory responses, with decreased IL-6 and IL-12 expression correlating with significant reduction in animal mortality. Thus, the data show that amino acid depletion stress signals (via GCN2) synergize with proinflammatory signals to potently increase innate immune responsiveness.     

miR-146a Ameliorates Liver Ischemia/Reperfusion Injury by Suppressing IRAK1 and TRAF6

A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor– associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-κB P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.