Curtarello, Matteo (2008) Valutazione dell'effetto immunomodulatorio della Glicoproteina vOX2 dell'Herpesvirus umano di tipo 8 su monociti-macrofagi. [Ph.D. thesis]
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Aim 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.
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