Federico Cozzolino

Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences.

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser87 and Thr56 as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38α knock-out mice (p38α-/- MEF), whereas they occur within 12 h of serum withdrawal in p38α+/+ MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.

Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

Influenza A virus replication is dependent on an antioxidant pathway that involves GSH and Bcl-2

Growing evidence indicates that viral replication is regulated by the redox state of the host cell. We demonstrate that cells of different origins display differential permissivity for influenza A virus replication, depending on their intracellular redox power as reflected by Bcl‐2 expression and glutathione (GSH) content. Bcl‐2 expressing cells were found to have higher intracellular levels of GSH and to produce lower amounts of virus than Bcl‐2 negative cells. Two different steps in the virus life‐cycle were involved in Bcl‐2/GSH mediated viral inhibition: 1) expression of late viral proteins (in particular hemagglutinin and matrix); and 2) nuclear‐cytoplasmic translocation of viral ribonucleoproteins (vRNPs). Buthionine‐sulfoximine‐induced inhibition of GSH synthesis in Bcl‐2 expressing cells caused an increase in the expression of late viral proteins but did not restore vRNP export to the cytoplasm. Collectively, our findings show that both Bcl‐2 expression and GSH content contribute to the host cells ability to down‐regulate influenza virus replication, although their effects are exerted at different stages of the viral life‐cycle. In certain cell populations, this form of down‐regulation might conceivably favor the establishment of persistent viral infection.

Potential role of interleukin-1 as the trigger for diffuse intravascular coagulation in acute nonlymphoblastic leukemia

Abnormalities of coagulation are common in patients with acute nonlymphoblastic leukemia, although the mechanisms involved are unclear, except in a few cases. To investigate the pathogenesis of this coagulopathy, suspensions of purified leukemic cells were prepared and tested for procoagulant activity. Neither the leukemic cells nor their supernatants directly accelerated the clotting of plasma. Since the leukemic cells did not possess direct procoagulant activity, their ability or inability to elaborate a mediator of cellular coagulant properties, interleukin-1, was studied. Leukemic cells from patients with coagulopathy elaborated interleukin-1, and addition of phytohemagglutinin increased interleukin-1 release. In contrast, no interleukin-1 was released, before or after stimulation with phytohemagglutinin, from leukemic cells from patients without coagulopathy. Leukemic cells from another group of patients with abnormalities of coagulation released interleukin-1 only after phytohemagglutinin treatment. In terms of the coagulation mechanism, interleukin-1 containing supernatants from leukemic cell cultures induced the procoagulant receptor tissue factor, a co-factor in the initiation of coagulation, on the endothelial cell surface. There was coordinate suppression of the anticoagulant endothelial cell receptor thrombomodulin, a co-factor for the antithrombotic protein C pathway. Antibody to interleukin-1 prevented these changes in cellular coagulant properties. Taken together, these changes result in a shift in the balance of endothelial cell coagulant properties to an activated state in which mechanisms promoting procoagulant reactions on the vessel surface predominate. Synthesis and release of the mediator interleukin-1 by leukemic cells thus defines a new mechanism through which malignant cells can potentially activate the coagulation mechanism.

Bcl-2 Expression and p38MAPK Activity in Cells Infected with Influenza A Virus IMPACT ON VIRALLY INDUCED APOPTOSIS AND VIRAL REPLICATION

Previous reports have shown that various steps in the influenza A virus life cycle are impaired in cells expressing the antiapoptotic protein Bcl-2 (Bcl-2+ cells). We demonstrated a direct link between Bcl-2 and the reduced nuclear export of viral ribonucleoprotein (vRNP) complexes in these cells. However, despite its negative impact on viral replication, Bcl-2 did not prevent host cells from undergoing virally triggered apoptosis. The proteins reduced antiapoptotic capacity was related to phosphorylation of its threonine 56 and serine 87 residues by virally activated p38MAPK. In infected Bcl-2+ cells, activated p38MAPK was found predominantly in the cytoplasm, colocalized with Bcl-2, and both Bcl-2 phosphorylation and virally induced apoptosis were diminished by specific inhibition of p38MAPK activity. In contrast, in Bcl-2-negative (Bcl-2−) cells, which are fully permissive to viral infection, p38MAPK activity was predominantly nuclear, and its inhibition decreased vRNP traffic, phosphorylation of viral nucleoprotein, and virus titers in cell supernatants, suggesting that this kinase also contributes to the regulation of vRNP export and viral replication. This could explain why in Bcl-2+ cells, where p38MAPK is active in the cytoplasm, phosphorylating Bcl-2, influenza viral replication is substantially reduced, whereas apoptosis proceeds at rates similar to those observed in Bcl-2− cells. Our findings suggest that the impact of p38MAPK on the influenza virus life cycle and the apoptotic response of host cells to infection depends on whether or not the cells express Bcl-2, highlighting the possibility that the pathological effects of the virus are partly determined by the cell type it targets.

Post-translational regulation of interleukin 1β secretion

Abstract In view of the key role played by interleukin 1 (IL-1) β in inflammation, its production is likely to be precisely regulated. Previous studies have shown that IL-1β biosynthesis is controlled at the transcriptional and translational levels. We have investigated whether post-translational events also play a role in regulating the production of bioactive IL-1β. IL-1β, which lacks a signal sequence, is released by activated monocytes through a novel pathway of secretion, alternative to the classical endoplasmic reticulum-Golgi route. Secretion of mature 17 kDA IL-1β is increased when pulse-labelled activated monocytes are chased in the presence of heat-aggregated immunoglobulins or of various drugs. Febrile temperatures inhibit secretion of mature IL-1β, but only reduce its synthesis: treatment with cycloheximide restores secretion. Processing of the 33 kDa precursor to the 17 kDa mature molecule is inhibited when the external pH is 8 or higher: under these conditions, release of unprocessed, biologically inactive 33 kDa IL-1β is observed. Thus, secretion of IL-1β is regulated by post-translational mechanisms which operate at the level of both proteolytic processing and extracellular export.

Low molecular weight, non-peptidic agonists of TrkA receptor with NGF-mimetic activity

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

Sex differences in the response to viral infections: TLR8 and TLR9 ligand stimulation induce higher IL10 production in males.

Background Susceptibility to viral infections as well as their severity are higher in men than in women. Heightened antiviral responses typical of women are effective for rapid virus clearance, but if excessively high or prolonged, can result in chronic/inflammatory pathologies. We investigated whether this variability could be in part attributable to differences in the response to the Toll-Like Receptors (TLR) more involved in the virus recognition. Methods Cytokine production by peripheral blood mononuclear cells (PBMCs) from male and female healthy donors after stimulation with Toll-like receptors (TLR) 3, 7, 8, 9 ligands or with viruses (influenza and Herpes-simplex-1) was evaluated. Results Compared to females, PBMCs from males produced not only lower amounts of IFN-α in response to TLR7 ligands but also higher amounts of the immunosuppressive cytokine IL10 after stimulation with TLR8 and TLR9 ligands or viruses. IL10 production after TLR9 ligands or HSV-1 stimulation was significantly related with plasma levels of sex hormones in both groups, whereas no correlation was found in cytokines produced following TLR7 and TLR8 stimulation. Conclusions Given the role of an early production of IL10 by cells of innate immunity in modulating innate and adaptive immune response to viruses, we suggest that sex-related difference in its production following viral nucleic acid stimulation of TLRs may be involved in the sex-related variability in response to viral infections.

Plasmodium falciparum soluble extracts potentiate the suppressive function of polyclonal T regulatory cells through activation of TGFβ‐mediated signals

Increased numbers of T regulatory cells (Tregs), key mediators of immune homeostasis, were reported in human and murine malaria and it is current opinion that these cells play a role in balancing protective immunity and pathogenesis during infection. However, the mechanisms governing their expansion during malaria infection are not completely defined. In this article we show that soluble extracts of Plasmodium falciparum (PfSEs), but not equivalent preparation of uninfected erythrocytes, induce the differentiation of polyclonally activated CD4+ cells in Tregs endowed with strong suppressive activity. PfSEs activate latent TGFβ bound on the membrane of Treg cells, thus allowing the cytokine interaction with TGFβ receptor, and inducing Foxp3 gene expression and TGFβ production. The activation of membrane‐bound latent TGFβ by PfSEs is significantly reduced by a broad‐spectrum metalloproteinases inhibitor with Zn++‐chelating activity, and completely inhibited by the combined action of such inhibitor and antibodies to a P. falciparum thrombospondin‐related adhesive protein (PfTRAP). We conclude that Pf‐Zn++‐dependent proteinases and, to a lesser extent, PfTRAP molecules are involved in the activation of latent TGFβ bound on the membrane of activated Treg cells and suggest that, in malaria infection, this mechanism could contribute to the expansion of Tregs with different antigen specificity.

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4+ Lymphocytes

ABSTRACT The optimal immune response to malaria infection comprises rapid induction of inflammatory responses promptly counteracted by regulatory mechanisms to prevent immunopathology. To evaluate the role of dendritic cells (DC) in the balance of parasite-induced inflammatory/anti-inflammatory mechanisms, we studied the activity of monocyte-derived dendritic cells (MDDC), previously exposed to soluble extracts of Plasmodium falciparum-infected red blood cells (PfSE), in the differentiation of CD4 cells isolated from donors never exposed to malaria infection. We show that MDDC exposed to PfSE are extremely efficient to induce a contemporary differentiation of TH1 effector cells and T regulatory (Treg) cells in CD4 T cells even when exposed to low concentrations of parasitic extracts. Treg cells induced by MDDC infected with PfSE (MDDC-PfSE) produce transforming growth factor beta (TGF-β) and interleukin 10 (IL-10) and are endowed with strong suppressive properties. They also show phenotypical and functional peculiarities, such as the contemporary expression of markers of Treg and TH1 differentiation and higher sensitivity to TLR4 ligands both inducing an increasing production of suppressive cytokines. On the whole, our data indicate that MDDC exposed to PfSE orchestrate a well-balanced immune response with timely differentiation of TH1 and Treg cells in CD4 cells from nonimmune donors and suggest that, during the infection, the role of MDCC could be particularly relevant in low-parasitemia conditions.