Jieliang Li

Culture of hepatocytes on fructose-modified chitosan scaffolds

Fructose was conjugated onto the inner surface of highly porous chitosan scaffold prepared by lyophilization. The modified scaffold with average pore size 50-200 microm was used to cultivate rat hepatocytes harvested by portal vein collagenase perfusion. The results indicated that while chitosan sponge alone supported cell attachment and growth, the scaffold modified with fructose accommodated a much larger number of hepatocytes due to the specific interaction between seeded hepatocytes and fructose moieties conjugated onto the surface of the scaffold. Hepatocytes exhibited a round cellular morphology with many microvilli evident on the surface of the cells, indicating healthy cells. Metabolic activities in terms of albumin secretion and urea synthesis were evaluated. It was found that hepatocytes cultured within fructose-modified scaffold resulted in much higher activities than within unmodified chitosan sponge. Scanning electron microscopy results showed that fructose-modified porous scaffold promoted the formation of cellular aggregates.

(−)-Epigallocatechin gallate inhibits endotoxin-induced expression of inflammatory cytokines in human cerebral microvascular endothelial cells

Background(−)-Epigallocatechin gallate (EGCG) is a major polyphenol component of green tea that has antioxidant activities. Lipopolysaccharide (LPS) induces inflammatory cytokine production and impairs blood–brain barrier (BBB) integrity. We examined the effect of EGCG on LPS-induced expression of the inflammatory cytokines in human cerebral microvascular endothelial cells (hCMECs) and BBB permeability.MethodsThe expression of TNF-α, IL-1β and monocyte chemotactic protein-1 (MCP-1/CCL2) was determined by quantitative real time PCR (qRT-PCR) and ELISA. Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule (VCAM) in hCMECs were examined by qRT-PCR and Western blotting. Monocytes that adhered to LPS-stimulated endothelial cells were measured by monocyte adhesion assay. Tight junctional factors were detected by qRT-PCR (Claudin 5 and Occludin) and immunofluorescence staining (Claudin 5 and ZO-1). The permeability of the hCMEC monolayer was determined by fluorescence spectrophotometry of transmembrane fluorescin and transendothelial electrical resistance (TEER). NF-kB activation was measured by luciferase assay.ResultsEGCG significantly suppressed the LPS-induced expression of IL-1β and TNF-α in hCMECs. EGCG also inhibited the expression of MCP-1/CCL2, VCAM-1 and ICAM-1. Functional analysis showed that EGCG induced the expression of tight junction proteins (Occludin and Claudin-5) in hCMECs. Investigation of the mechanism showed that EGCG had the ability to inhibit LPS-mediated NF-κB activation. In addition, 67-kD laminin receptor was involved in the anti-inflammatory effect of EGCG.ConclusionsOur results demonstrated that LPS induced inflammatory cytokine production in hCMECs, which could be attenuated by EGCG. These data indicate that EGCG has a therapeutic potential for endotoxin-mediated endothelial inflammation.

Interferon lambda inhibits herpes simplex virus type I infection of human astrocytes and neurons.

Herpes simplex virus Type I (HSV‐1) is a neurotropic virus that is capable of infecting not only neurons, but also microglia and astrocytes and can establish latent infection in the central nervous system (CNS). We investigated whether IFN lambda (IFN‐λ), a newly identified member of IFN family, has the ability to inhibit HSV‐1 infection of primary human astrocytes and neurons. Both astrocytes and neurons were found to be highly susceptible to HSV‐1 infection. However, upon IFN‐λ treatment, HSV‐1 replication in both astrocytes and neurons was significantly suppressed, which was evidenced by the reduced expression of HSV‐1 DNA and proteins. This IFN‐λ‐mediated action on HSV‐1 could be partially neutralized by antibody to IFN‐λ receptor. Investigation of the mechanisms showed that IFN‐λ treatment of astrocytes and neurons resulted in the upregulation of endogenous IFN‐α/β and several IFN‐stimulated genes (ISGs). To block IFN‐α/β receptor by a specific antibody could compromise the IFN‐λ actions on HSV‐1 inhibition and ISG induction. In addition, IFN‐λ treatment induced the expression of IFN regulatory factors (IRFs) in astrocytes and neurons. Furthermore, IFN‐λ treatment of astrocytes and neurons resulted in the suppression of suppressor of cytokine signaling 1 (SOCS‐1), a key negative regulator of IFN pathway. These data suggest that IFN‐λ possesses the anti‐HSV‐1 function by promoting Type I IFN‐mediated innate antiviral immune response in the CNS cells.

TLR3 activation efficiency by high or low molecular mass poly I:C.

Toll-like receptor 3 (TLR3) plays a critical role in initiating type I IFN-mediated innate immunity against viral infections. TLR3 recognizes various forms of double stranded (ds) RNA, including viral dsRNA and a synthetic mimic of dsRNA, poly I:C, which has been used extensively as a TLR3 ligand to induce antiviral immunity. The activation efficiency of TLR3 by poly I:C is influenced by various factors, including size of the ligands, delivery methods and cell types. In this study, we examined the stimulatory effect of two commercially-available poly I:Cs [high molecular mass (HMM) and low molecular mass (LMM)] on TLR3 activation in various human cell types by determining the induction of type I and type III IFNs, as well as the antiviral effect. We demonstrated that the direct addition of both HMM- and LMM-poly I:C to the cultures of primary macrophages or a neuroplastoma cell line could activate TLR3. However, the transfection of poly I:C was necessary to induce TLR3 activation in other cell types studied. In all the cell lines tested, the efficiency of TLR3 activation by HMM-poly I:C was significantly higher than that by LMM-poly I:C. These observations indicate the importance and necessity of developing effective TLR3 ligands for antiviral therapy.

Immune activation of human brain microvascular endothelial cells inhibits HIV replication in macrophages.

There is limited information about the role of blood-brain barrier (BBB) endothelial cells (ECs) in the central nervous system (CNS) and their innate immunity against HIV. We examined whether brain ECs can be immunologically activated to produce antiviral factors that inhibit HIV replication in macrophages. Human brain microvascular ECs expressed functional toll-like receptor 3 (TLR3) that could be activated by polyinosinic-polycytidylic acid (PolyI:C), resulting in the induction of endogenous interferon-β (IFN-β) and IFN-λ. The TLR3 activation of ECs also induced the phosphorylation of interferon regulatory transcription factor 3 (IRF3) and IRF7, the key regulators of IFN signaling pathway. When supernatant (SN) of PolyI:C-activated EC cultures was applied to infected macrophage cultures, HIV replication was significantly suppressed. This SN action of ECs on HIV was mediated through both IFN-β and IFN-λ because antibodies to their receptors could neutralize the SN-mediated anti-HIV effect. The role of IFNs in EC-mediated anti-HIV activity is further supported by the observation that treatment with SN from EC cultures induced the expression of IFN-stimulated genes (ISGs: ISG56, OAS-1, and MxA) in macrophages. These observations indicate that brain microvascular ECs may be a key regulatory bystander, playing a crucial role in the BBB innate immunity against HIV infection.

RIG-I activation inhibits HIV replication in macrophages

The RIG‐I signaling pathway is critical in the activation of the type I IFN‐dependent antiviral innate‐immune response. We thus examined whether RIG‐I activation can inhibit HIV replication in macrophages. We showed that the stimulation of monocyte‐derived macrophages with 5′ppp‐dsRNA, a synthetic ligand for RIG‐I, induced the expression of RIG‐I, IFN‐α/β, and several IRFs, key regulators of the IFN signaling pathway. In addition, RIG‐I activation induced the expression of multiple intracellular HIV‐restriction factors, including ISGs, several members of the APOBEC3 family, tetherin and CC chemokines, the ligands for HIV entry coreceptor (CCR5). The inductions of these factors were associated with the inhibition of HIV replication in macrophages stimulated by 5′ppp‐dsRNA. These observations highlight the importance of RIG‐I signaling in macrophage innate immunity against HIV, which can be beneficial for the treatment of HIV disease, where intracellular immune defense is compromised by the virus.

Induction of interferon-λ contributes to toll-like receptor 3-mediated herpes simplex virus type 1 inhibition in astrocytes

Toll‐like receptor 3 (TLR3) recognizes double‐stranded RNA and induces type I interferon (IFN)‐mediated antiviral immunity against a number of viral infections. Type III IFN (IFN‐λ) is a newly identified antiviral cytokine that has biological functions similar to those of type I IFNs. We thus investigated the role of IFN‐λ in TLR3 activation‐mediated inhibition of herpes simplex virus type 1 (HSV‐1) in human primary astrocytes. Human astrocytes express endogenous IFN‐λ1 and IFN‐λ receptor complex, interleukin‐28 receptor α subunit (IL‐28Rα), and IL‐10Rβ. The activation of TLR3 by poly‐I:C treatment significantly induced the expression of IFN‐λ1 and IFN‐λ2/3 in astrocytes. The induction of IFN‐λ contributed to TLR3 activation‐mediated HSV‐1 inhibition in astrocytes. Investigation of the mechanisms showed that treatment of astrocytes with specific antibody against IFN‐λ receptor attenuated the anti‐HSV‐1 activity of poly‐I:C, indicating that endogenous IFN‐λ contributes to the anti‐HSV‐1 effect of TLR3 activation. The anti‐HSV‐1 effect of endogenous IFN‐λ was also confirmed by the finding that recombinant IFN‐λ treatment inhibited HSV‐1 infection of astrocytes. These results provide direct and compelling evidence that endogenous IFN‐λ participates in TLR3‐mediated antiviral activity, which may have important implications in host cell innate immunity against HSV‐1 infection in the CNS.

Alcohol impairs interferon signaling and enhances full cycle hepatitis C virus JFH-1 infection of human hepatocytes

Alcohol drinking and hepatitis C virus (HCV) infection frequently coexist in patients with chronic liver disease. There is limited information, however, about the impact of alcohol on host cell innate immunity and full cycle replication of HCV. This study investigated whether alcohol impairs the intracellular innate immunity in human hepatocytes, promoting HCV infection and replication. Alcohol treatment of human hepatocytes before, during and after viral infection significantly enhanced full cycle HCV replication. Alcohol suppressed intracellular expression of type I interferons (IFN-α/β) in human hepatocytes. Investigation of the mechanisms responsible for the alcohol action revealed that alcohol inhibited the expression of the IFN regulatory factors (IRF-5 and IRF-7), and signal transducer and activator of transcription (STAT-1 and STAT-2), the key positive regulators in type I IFN signaling pathway. In addition, alcohol induced the expression of suppressors of cytokine signaling (SOCS-2 and SOCS-3), the key negative regulators of IFN-α/β expression. These in vitro findings suggest that alcohol, through modulating the expression of key regulators in IFN signaling pathway, inhibits type I IFN-based intracellular innate immunity in hepatocytes, which may contribute to the chronicity of HCV infection and the poor efficacy of IFN-α-based therapy.

Mycophenolate mofetil inhibits hepatitis C virus replication in human hepatic cells

Hepatitis C virus (HCV) infection is the most common indication for liver transplantation and the major cause of graft failure. A widely used immunosuppressant, cyclosporine A (CsA), for people who receive organ transplantation, has been recognized to have the ability to inhibit HCV replication both in vivo and in vitro. In this study, we investigated the effects of several other immunosuppressants, including mycophenolate mofetil (MMF), rapamycin and FK506, on HCV replication in human hepatic cells. MMF treatment of hepatic cells before or during HCV infection significantly suppressed full cycle viral replication, as evidenced by decreased expression of HCV RNA, protein and production of infectious virus. In contrast, rapamycin and FK506 had little effect on HCV replication. Investigation of the mechanism(s) disclosed that the inhibition of HCV replication by MMF was mainly due to its depletion of guanosine, a purine nucleoside crucial for synthesis of guanosine triphosphate, which is required for HCV RNA replication. The supplement of exogenous guanosine could reverse most of anti-HCV effect of mycophenolate mofetil. These data indicate that MMF, through the depletion of guanosine, inhibits full cycle HCV JFH-1 replication in human hepatic cells. It is of interest to further determine whether MMF is indeed beneficial for HCV-infected transplant recipients in future clinical studies.

Morphine suppresses IFN signaling pathway and enhances AIDS virus infection.

Background Opioids exert a profound influence on immunomodulation and enhance HIV infection and replication. However, the mechanism(s) of their action remains to be determined. We thus investigated the impact of morphine on the intracellular innate antiviral immunity. Methodology/Principal Findings Seven-day-cultured macrophages were infected with equal amounts of cell-free HIV Bal or SIV DeltaB670 for 2 h at 37°C after 24 h of treatment with or without morphine. Effect of morphine on HIV/SIV infection and replication was evaluated by HIV/SIV RT activity assay and indirect immunofluorescence for HIV p24 or SIV p28 antigen. The mRNA expression of cellular factors suppressed or induced by morphine treatment was analyzed by the real-time RT-PCR. We demonstrated that morphine treatment of human blood monocyte-derived macrophages significantly inhibited the expression of interferons (IFN-α, IFN-β and IFN-λ) and IFN-inducible genes (APOBEC3C/3F/3G and 3H). The further experiments showed that morphine suppressed the expression of several key elements (RIG-I and IRF-7) in IFN signaling pathway. In addition, morphine treatment induced the expression of suppressor of cytokine signaling protein-1, 2, 3 (SOCS-1, 2, 3) and protein inhibitors of activated STAT-1, 3, X, Y (PIAS-1, 3, X, Y), the key negative regulators of IFN signaling pathway. Conclusions These findings indicate that morphine impairs intracellular innate antiviral mechanism(s) in macrophages, contributing to cell susceptibility to AIDS virus infection.