Autophagy regulates hepatocyte identity and epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions promoting Snail degradation.

Epithelial-to-mesenchymal transition (EMT) and the reverse process mesenchymal-to-epithelial transition (MET) are events involved in development, wound healing and stem cell behaviour and contribute pathologically to cancer progression. The identification of the molecular mechanisms underlying these phenotypic conversions in hepatocytes are fundamental to design specific therapeutic strategies aimed at optimising liver repair. The role of autophagy in EMT/MET processes of hepatocytes was investigated in liver-specific autophagy-deficient mice (Alb-Cre;ATG7(fl/fl)) and using the nontumorigenic immortalised hepatocytes cell line MMH. Autophagy deficiency in vivo reduces epithelial markers' expression and increases the levels of mesenchymal markers. These alterations are associated with an increased protein level of the EMT master regulator Snail, without transcriptional induction. Interestingly, we found that autophagy degrades Snail in a p62/SQSTM1 (Sequestosome-1)-dependent manner. Moreover, accordingly to a pro-epithelial function, we observed that autophagy stimulation strongly affects EMT progression, whereas it is necessary for MET. Finally, we found that the EMT induced by TGFß affects the autophagy flux, indicating that these processes regulate each other. Overall, we found that autophagy regulates the phenotype plasticity of hepatocytes promoting their epithelial identity through the inhibition of the mesenchymal programme.

Applying proteomic technology to clinical virology.

Developing antiviral drugs, vaccines and diagnostic markers is still the most ambitious challenge in clinical virology. In the past few decades, data from high-throughput technologies have allowed for the rapid development of new antiviral therapeutic strategies, thus making a profound impact on translational research. Most of the current preclinical studies in virology are aimed at evaluating the dynamic composition and localization of the protein platforms involved in various host-virus interactions. Among the different possible approaches, mass spectrometry-based proteomics is increasingly being used to define the protein composition in subcellular compartments, quantify differential protein expression among samples, characterize protein complexes, and analyse protein post-translational modifications. Here, we review the current knowledge of the most useful proteomic approaches in the study of viral persistence and pathogenicity, with a particular focus on recent advances in hepatitis C research.

Role of STAT3 and PI 3-kinase/Akt in mediating the survival actions of cytokines on sensory neurons.

The binding of cytokines to the gp130 receptor activates the STAT3, MEK/MAPK, and PI3K/Akt signalling pathways. To assess the relative importance of these pathways in promoting the survival of cytokine-dependent neurons, we conditionally inactivated STAT3 in mice and inhibited MEK, PI3K, and Akt in cultured neurons using pharmacological reagents and by expressing specific inhibitory proteins. Inactivation of STAT3 enhanced the death of the cytokine-dependent sensory neurons of the nodose ganglion in vivo and substantially reduced the response of these neurons to CNTF and LIF in vitro. LY294002, an inhibitor of PI3K, but not PD98059, an inhibitor of MEK, markedly reduced the response of these neurons to CNTF, as did dominant-negative PI3K, dominant-negative Akt, and overexpression of Ruk (a natural PI3K inhibitor). These results demonstrate that STAT3 and PI3K/Akt signalling play major roles in mediating the survival response of neurons to cytokines.

Effect of IL-6 on IGF binding protein-3: a study in IL-6 transgenic mice and in patients with systemic juvenile idiopathic arthritis.

Stunted growth is a common complication of childhood diseases characterized by chronic inflammation or infections. We previously demonstrated that NSE/hIL-6 transgenic mice, overexpressing the inflammatory cytokine IL-6 since early phase of life, showed a marked growth defect associated with decreased IGF-I levels, suggesting that IL-6 is one of the factors involved in stunted growth complicating chronic inflammation in childhood. Here we show that NSE/hIL-6 mice have normal liver IGF-I production, decreased levels of IGF binding protein-3 (IGFBP-3) and increased serum IGFBP-3 proteolysis. Reduced IGFBP-3 levels results in a marked decrease in the circulating 150-kDa ternary complex, even in the presence of normally functional acid labile subunit. Pharmacokinetic studies showed that NSE/hIL-6 mice have accelerated IGF-I clearance. Patients with systemic juvenile idiopathic arthritis (s-JIA), a chronic inflammatory disease characterized by prominent IL-6 production and complicated by stunted growth associated with low IGF-I levels, have markedly decreased IGFBP-3 levels, increased serum IGFBP-3 proteolysis and normal acid labile subunit levels. Our data show that chronic overproduction of IL-6 causes decreased IGFBP-3 levels, resulting in a decreased association of IGF-I in the 150-kDa complex. Decreased levels of IGF-I appear to be secondary to increased clearance.

Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver.

We generated mice carrying a STAT3 allele amenable to Cre-mediated deletion and intercrossed them with Mx-Cre transgenic mice, in which the expression of Cre recombinase can be induced by type I interferon. Interferon-induced deletion of STAT3 occurred very efficiently (more than 90%) in the liver and slightly less efficiently (about 70%) in the bone marrow. Analysis of the induction of liver acute-phase genes in response to bacterial lipopolysaccharide unequivocally identifies STAT3 as a fundamental mediator of their induction. The different degrees of defectiveness displayed by the various genes allowed us to differentiate them into three separate groups according to their degree of dependence on STAT3. Induction was totally defective for group I genes, defective at 24 h but almost normal at earlier time points for group II genes, and only slightly defective for group III genes. This division was in good agreement with the known structures of the respective promoters. We also found that the overall induction of the transcription factors C/EBP beta and -delta was only minimally defective in the absence of STAT3. Finally, even though corticosterone levels and action were found to be normal in the conditional-mutant mice, production of both proinflammatory and antiinflammatory cytokines was increased and prolonged, probably as a result of STAT3 deletion in macrophages.

Impaired Stat3 activation following localized inflammatory stimulus in IL-6-deficient mice.

Interleukin 6 (IL-6) and related gp130-signalling cytokines rapidly activate latent cytoplasmic Stat transcription factors and these are believed to play pivotal roles in the expression of downstream cytokine-responsive genes. We have previously shown in IL-6-deficient (-/-) mice that IL-6 is absolutely required for the transcriptional induction of acute phase response (APR) genes in the liver following localized tissue damage caused by subcutaneous injection of turpentine oil, but is not required when the inflammatory stimulus is administered systemically by intraperitoneal injection of bacterial lipopolysaccharide (LPS). In this paper we show that Stat3 is the only Stat factor induced in liver tissue upon localized inflammatory stimuli, and that its activation is virtually absent in IL-6 deficient mice. During LPS-induced inflammation both Stat1 and Stat3 are activated, and only minor kinetic alterations are detected in IL-6-/- mice. These defects are not due to altered intracellular signal transduction, since they could be complemented by injection of recombinant cytokines. These results establish a direct causal relationship in vivo between Stat activation and acute phase gene expression and define unique functions of IL-6 in Stat3 activation upon localized inflammation.

Interleukin 6 is required for the development of collagen-induced arthritis.

Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor alpha (TNF-alpha) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-alpha transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6(-/-) mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.

Induction of interleukin-6 (IL-6) autoantibodies through vaccination with an engineered IL-6 receptor antagonist.

Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.

Differential effects of IL-6 on systemic and central production of TNF: a study with IL-6-deficient mice.

Interleukin 6 (IL-6) is known to inhibit the synthesis of tumour necrosis factor (TNF) in vitro and in vivo. In this study we investigated the possible role of IL-6 as an endogenous inhibitor of TNF production in the brain or in the periphery using IL-6-deficient mice or administering recombinant human IL-6 (rhIL-6). When IL-6-deficient mice were injected intracerebroventricularly (i.c.v.) with lipopolysaccaride (LPS), no differences were observed in the production of TNF in the brain, while in the periphery (serum or spleen) TNF levels were markedly increased (about four-fold). When normal mice were injected i.c.v. with a combination of LPS and rhIL-6, inhibition of TNF production was only slight (about 20%), while IL-6 had a stronger effect (> 80% inhibition) in the periphery. Co-administration of soluble IL-6 receptor (sIL-6R) did not enhance the effect of IL-6 on brain TNF, so this refractoriness cannot be attributed to a lack of IL-6 receptors. Interestingly, IL-6 potently inhibited LPS-induced TNF production by macrophagic cells but not by a microglial cell clone, suggesting that the defective response to IL-6 of the brain lies within the responsiveness TNF producing cells to IL-6. It thus appears that the TNF-inhibitory role of IL-6 is confined to the periphery.

Interleukin 6 causes growth impairment in transgenic mice through a decrease in insulin-like growth factor-I. A model for stunted growth in children with chronic inflammation.

Stunted growth is a major complication of chronic inflammation and recurrent infections in children. Systemic juvenile rheumatoid arthritis is a chronic inflammatory disorder characterized by markedly elevated circulating levels of IL-6 and stunted growth. In this study we found that NSE/hIL-6 transgenic mouse lines expressing high levels of circulating IL-6 since early after birth presented a reduced growth rate that led to mice 50-70% the size of nontransgenic littermates. Administration of a monoclonal antibody to the murine IL-6 receptor partially reverted the growth defect. In NSE/hIL-6 transgenic mice, circulating IGF-I levels were significantly lower than those of nontransgenic littermates; on the contrary, the distribution of growth hormone pituitary cells, as well as circulating growth hormone levels, were normal. Treatment of nontransgenic mice of the same strain with IL-6 resulted in a significant decrease in IGF-I levels. Moreover, in patients with systemic juvenile rheumatoid arthritis, circulating IL-6 levels were negatively correlated with IGF-I levels. Our findings suggest that IL-6-mediated decrease in IGF-I production represents a major mechanism by which chronic inflammation affects growth.

Interleukin-6 and CAAT/enhancer binding protein beta-deficient mice act as tools to dissect the IL-6 signalling pathway and IL-6 regulation.

Interleukin-6 (IL-6) is a pleiotropic cytokine playing important roles in immunity, hemopoiesis and inflammation. IL-6 signalling is known to involve the activation of two independent transcription factors: Stat3 (through phosphorylation by Jak kinases) and C/EBP beta (through activation of the ras pathway). In addition, C/EBP beta is believed to act as a transcriptional activator of the IL-6 gene itself. Making use of IL-6-deficient mice, we have recently demonstrated that IL-6 is essential for the induction of acute phase mRNAs in the liver upon localized tissue damage, but not upon systemically induced inflammation. Here we show that the defective mRNA induction is paralleled by a defective activation of Stat3, thus establishing a direct relationship between IL-6 function, Stat3 activation and acute phase genes induction. On the other hand, making use of C/EBP beta-deficient mice, we show that the induction of IL-6 by a variety of stimuli does not require C/EBP beta activity. In contrast to the predicted activating role of C/EBP beta, IL-6 levels are increased in the C/EBP beta-deficient mice, suggesting that C/EBP beta may act as a down-modulator of the IL-6 gene. Through the generation of C/EBP beta, IL-6 double mutant mice we show that IL-6 hyperproduction is responsible for the development of the Castleman's like lymphoproliferative disease described in the C/EBP beta-deficient mice, since the disorder is completely blocked by inactivating the IL-6 gene.

Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production.

Production of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6), in the brain is increased in various diseases. To investigate the relationships between the effect of overproduction of IL-6 in the brain on central and peripheral production of TNF, IL-1 beta and IL-6 itself, we used transgenic mice (NSE-hIL-6) where neuronal human IL-6 expression under the control of the neuronal specific enolase promoter results in astrocytosis and gliosis. These mice had higher cerebral endogenous IL-6 (12-fold), IL-1 beta (12-fold) and TNF (4-fold) production measured in brain homogenates after intracerebroventricular (i.c.v.) injection of 2.5 micrograms LPS, lipopolysaccharide (LPS) than wild-type mice (no TNF or IL-1 were detectable in saline-injected NSE or control mice). Cerebral cytokines production was also increased in NSE-hIL-6 mice treated i.p. with LPS doses that do not normally induce cytokines in the brain. The induction of peripheral (serum or spleen) TNF, IL-1 beta or IL-6 was the same in all these experiments in NSE-hIL-6 and wild-type mice. Furthermore, using microglial cell clone pretreated in vitro with IL-6, we noted an increase in LPS-induced TNF and IL-6 production and proliferation of pretreated cells than control. This study indicates that overproduction of IL-6 in the central nervous system (CNS) may ultimately result in increased central production of inflammatory cytokines, probably due to increased proliferation and activation of the cells which produce cytokine in the CNS.

C/EBPbeta is required for the late phases of acute phase genes induction in the liver and for tumour necrosis factor-alpha, but not Interleukin-6, regulation.

C/EBPbeta is a leucine-zipper transcription factor believed to play an important role in the control of liver functions, and in particular in the transcriptional regulation of acute phase genes in response to several inflammatory stimuli and to recombinant cytokines. Moreover, this factor has been proposed as an important activator of several cytokine genes. We recently described the generation of mice in which the C/EBPbeta gene has been inactivated by gene targeting, showing that they are viable, but present specific defects in the myeloid and lymphoid compartments. Here we demonstrate that C/EBPbeta does indeed play a role in the transcriptional induction of some, but not all, liver acute phase genes. Activation is in particular defective in C/EBPbeta-deficient mice in the later phases of induction, suggesting that the early phases may be triggered by factors other than C/EBPs. Moreover, IL-6 activation is normal and TNFalpha activation is defective in the mutant mice, indicating a differential role of C/EBPbeta in the control of these two cytokines' production.

IL-6 expression in neurons of transgenic mice causes reactive astrocytosis and increase in ramified microglial cells but no neuronal damage.

Growing evidence suggests that aberrant production of inflammatory cytokines within the central nervous system (CNS) contributes to the development of pathological conditions. To test the cause-effect relationship between the overproduction of interleukin-6 (IL-6) in the CNS and the onset of neuropathological changes, we have generated transgenic mice in which human IL-6 expression has been targeted to the neurons by using the rat neuron-specific enolase promoter. These mice develop reactive astrocytosis and an increase in ramified microglial cells but do not show histological or behavioural signs of neuron damage at the light microscope level. We thus conclude that a constant release of human IL-6 by neuronal subpopulations in mice is sufficient to activate cells potentially capable of modulating the local immune response, but at the same time is compatible with normal neuron functions.

Generation and characterization of two human alpha/beta T cell clones. Recognizing autologous breast tumor cells through an HLA- and TCR/CD3-independent pathway.

Cell-mediated immune response to breast tumor has only been marginally investigated. To gain insight into this issue we have developed two clones of distinct phenotype, CD3+ alpha/beta, CD4+, CD8-, CD16-, and CD3+ alpha/beta, CD4-, CD8+, CD16-, respectively, from peripheral blood lymphocytes (PBL) of a breast cancer patient. These effectors, selected on the basis of their cytolytic activity against autologous tumor cells and lack of lysis on NK-sensitive cell lines, preferentially recognize autologous tumor cells. The two clones' cytotoxic activity, while inhibited by anti-LFA-1 mAb, could not be abolished by mAbs to CD3, to class I and class II MHC molecules, and by mAbs to molecules involved in T cell function (i.e., CD4, CD8, CD2). The molecular structure of the alpha and beta T cell receptor chains of the two effector cells, confirmed their clonality and showed that, despite an overlapping killing pattern, they possess distinct TCR alpha and beta chains. These findings demonstrate that breast tumor-specific CTL clones can be generated through current technology and that a alpha/beta effector cell population operating through a HLA-unrestricted and TCR/CD3-independent pathway may be involved in the identification and killing of this tumor.