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Valutazione dell'effetto immunomodulatorio della Glicoproteina vOX2 dell'Herpesvirus umano di tipo 8 su monociti-macrofagi
Full text linkAim of this work is to clarify the human herpervirus 8 vOX2 activity in monocytes-macrophages in order to define its funtional role in Kaposi Sarcoma pathogenesis.
Kaposi’s Sarcoma (KS) is an inflammatory cytokines-mediated angioproliferative disease triggered by human herpesvirus 8 (HHV-8) infection. This virus is unique because of its extensive molecular piracy of host critical cell regulatory and immune modulatory genes encoding proteins that could contribute to viral immune evasion and tumorigenesis. Among them we can find the vOX2 glycoprotein. Cellular homologous OX2 is a member of the immunoglobulin superfamily.
This glycoprotein is expressed by several cell types in vivo and it down-modulates inflammatory response through the interaction with a specific receptor of the myeloid cells, the CD200R. By this mechanism, cellular OX2 prevents autoimmune disease, displaying immune modulatory functions.
This latter feature is also present in the HHV-8 vOX2. Indeed, several reports suggest that the vOX2 has an anti-inflammatory and immunosuppressive activity in basophil and neutrophil cells, but its effect on monocytes-macrophages is still controversial.
The aim of our work is to clarify the vOX2 activity in monocytes-macrophages in order to define its functional role in KS pathogenesis. The relevance of monocytesmacrophages relies on the fact that this cell type is infected by HHV-8 in vivo, it is present in KS lesions and it expresses CD200R that functions as a receptor for vOX2 exactly like it does for cellular OX2.
We decided to express the viral glycoprotein into two different monocyticmacrophagic cell lines (U937 and THP1) and/or into PBMC-derived macrophages (primary MØ) for our study. Compared to chemical and physical methods, the viral transduction has resulted the most efficient system to transfer transgenes into the target cells; based on this finding, the HHV-8 orf K14 encoding vOX2 has been cloned into HIV-1 based lentiviral vector that has been used to transduce the cells.
After verifying the vOX2 expression in the transduced target cells, we evaluated the glycoprotein effect on the transcription level and secretion of two inflammatory cytokines involved in the KS development, TNF? and IL-1?. Our data show that the vOX2 up-regulates both TNF? and IL-1? in U937 and THP1 cell lines and in primary MØ kept in basal conditions. In addition, the TNF? and IL-1? up-regulation was observed in the IFN?-activated U937 cell line. By contrast, in the IFN?-activated THP1 cell line and primary MØ the viral glycoprotein inhibits TNF? and IL-1? gene expression.
Taking into account these controversial data in the different cell types, in order to carry on our research in a model more representative of the physiological condition, we checked the expression of vOX2 receptor on U937 and THP1 cell line and on primary MØ. Since the primary MØ resulted to be the only cell type expressing CD200R, we decided to evaluate the vOX2 activity employing this cellular system.
However, being the level of viral protein expression in primary MØ low compared to the one obtained in the cell lines, we performed coculture between THP1 CD200R ̄ cell line expressing vOX2 and primary MØ CD200R+ in order to confirm the results on inflammatory-cytokines modulation by vOX2. Our data show that the vOX2 promotes TNF? secretion in the primary MØ in basal conditions; on the contrary, in the IFN?-activated cells the viral glycoprotein induces a significant reduction of cytokine production as we observed in monoculture of primary MØ expressing vOX2.
Starting from these data, we next evaluated the vOX2 effect on the transcription level of IL-10, an inhibitory cytokine of the TNF? and IL-1?-mediated inflammatory responses. Our data show that IL-10 expression profile is the opposite with respect to TNF? and IL-1?, as we expected.
Moreover, these results are in agreement with the CD200R mRNA down-modulation that we observed in basal conditions and with its up-regulation in the IFN?-activated cells.
In addition, we were able to show that vOX2 promotes the phagocytosis of the primary MØ in basal conditions while it inhibits this activity in the IFN?-activated cells.
Our results suggest that the immune modulatory activity of vOX2 is tightly dependent on the activation state of the cells.
This conclusion is also supported by the analysis of vOX2 effect on the global gene expression profile in the primary MØ.
Finally, we observed that the antigen presentation to T cells by primary MØ is compromised by vOX2 expression regardless of the activation state of the cells. This effect seems to be related to the down-modulation of HLA expression on cell surface.
Overall, our results lead to the conclusion that vOX2 may be involved in viral immune evasion because of its anti-inflammatory and immunosuppressive effect in the activated monocytes-macrophages. At the same time, in basal conditions, vOX2 can also stimulate monocytes-macrophages contributing to the inflammatory state that is important for the KS development. This finding would imply the presence of at least one unknown receptor that would compete with the “inhibitory receptor” CD200R for the binding to vOX2. An immune modulatory activity of vOX2, linked to cell activation state, could explain the contradictory results reported in literature
Functional activity of KSHV vOX2 glycoprotein in macrophage: immunoactivator or immunosuppressor?
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HIV-1-mediated delivery of a short hairpin RNA targeting vascular endothelial growth factor in human retinal pigment epithelium cells
No full textBackground: Vascular endothelial growth factor (VEGF) has been shown to play a major role in the pathological neovascularisation that occurs in degenerative retinal diseases like age-related macular degeneration (AMD). Although several approaches to attenuate VEGF show significant promise, repeated treatments are required to achieve therapeutic benefits. As lentiviruses efficiently and stably infect resting cells, a human immunodeficiency virus type 1 (HIV-1)-based vector was used for the delivery and long-term endogenous expression of a short hairpin RNA (shRNA) specific for VEGF in postmitotic human retinal pigment epithelium (RPE) cells.
Methods: An HIV-1 vector expressing a shRNA targeting VEGF was developed and adopted to transduce RPE cell cultures, in both normoxic and hypoxic conditions in vitro. Intracellular VEGF expression was analysed by western blotting, and the release of VEGF in culture supernatants was determined by ELISA.
Results: At least 90% of RPE cells were successfully transduced by HIV-1 virions. Inhibition of VEGF expression and reduction by 95% of VEGF release in transduced cells were achieved. Moreover, shRNA-VEGF effectively and specifically prevented hypoxia-induced VEGF upregulation.
Conclusion: HIV-1-mediated delivery of a shRNA-VEGF leading to gene expression knockdown could represent a novel therapeutic strategy against neovascularisation-related eye diseases
Increased expression of LGI1 gene triggers growth inhibition and apoptosis of neuroblastoma cells.
No full textThe LGI1 gene has been implicated in the malignant progression of glioblastoma and it has also been genetically linked to a form of partial epilepsy (ADLTE). In this study, we investigated the relevance of LGI1 expression for neuroblastoma cells. The analysis of two cell lines (SH-SY5Y and SK-N-BE) revealed unpredictably low levels of LGI1 and stable cell transfection with LGI1 cDNA yielded moderate increases of LGI1 expression. Neuroblastoma cell clones exhibited impaired cell growth and survival ability in relation to LGI1 levels. The process of growth inhibition could be discerned under experimental conditions of low cell density, since conditions of elevated cell density, which enhance the requirement for survival stimuli, resulted in massive cellular death. At high cell density, spontaneous apoptosis of LGI1 cells was clearly shown by the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria and by phosphatydil serine exposure and nuclear fragmentation. Activation of apoptotic effectors caspase-3/7 also occurred, however, the broad caspase inhibitor Z-VAD-FMK substantially failed to block cell death. Thus the possibility that LGI1-triggered apoptosis may involve initiator caspases linked to activation of death receptors, appears unlikely. The decreased ratio of Bcl-2 to Bax suggests that apoptosis is initiated by the intrinsic mitochondrial pathway through the release of caspase-dependent and -independent apoptogenic molecules. This study provides the first evidence that LGI1 controls neuronal cell survival, suggesting its role in the development of the nervous system in relation to the pathogenesis of neuroblastoma and ADLTE
Patterns of gene expression exhibited by the vOX2 (orfK14) protein of human herpesvirus 8 in PBMC-derived macrophages
No full textvOX2 glycoprotein of human herpesvirus 8 modulates human primary macrophages activity
No full textHuman herpesvirus 8 (HHV-8) is a lymphotropic herpesvirus linked to several disorders such as Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Several HHV-8 proteins regulate host innate and adaptive immune response; in particular, orfK14 is expressed as an immediate early gene during the viral lytic cycle and encodes a surface glycoprotein (vOX2), significantly homologous to the cellular OX2, which delivers inhibitory signals to macrophages. Although it has been suggested that vOX2 may down-regulate basophil and neutrophil functions, its role in macrophages, a cell type lytically infected by HHV-8 in vivo, is still controversial. Therefore, we investigated the effect of vOX2 expression in human primary monocyte-derived macrophages (MDMs). In this report, we demonstrate that vOX2-expressing MDMs in basal conditions are induced to produce inflammatory cytokines and display higher phagocytic activity with respect to mock cells. By contrast, an opposite effect is exhibited by vOX2 in MDMs undergoing IFN-gamma-activation, with a down-modulation of the cytokine production and phagocytic activity. Moreover, we observed that, when MDMs are co-cultured with vOX2-expressing cells, the inflammatory cytokine release is increased, independently from the MDM activation state. Interestingly, we could correlate our results with the mRNA transcript level of the vOX2 cellular CD200R receptor. Finally, we demonstrate a down-regulation of the MHC class I and class II molecules on the cell surface of vOX2-transduced MDMs. Our results provide new insights into the immunomodulatory effects of HHV-8 vOX2 protein
Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients
Full text linkDNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett's esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring
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