Dimitrios Lagos

Kaposi sarcoma herpesvirus–induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma

The biology of Kaposi sarcoma is poorly understood because the dominant cell type in Kaposi sarcoma lesions is not known. We show by gene expression microarrays that neoplastic cells of Kaposi sarcoma are closely related to lymphatic endothelial cells (LECs) and that Kaposi sarcoma herpesvirus (KSHV) infects both LECs and blood vascular endothelial cells (BECs) in vitro. The gene expression microarray profiles of infected LECs and BECs show that KSHV induces transcriptional reprogramming of both cell types. The lymphangiogenic molecules VEGF-D and angiopoietin-2 were elevated in the plasma of individuals with acquired immune deficiency syndrome and Kaposi sarcoma. These data show that the gene expression profile of Kaposi sarcoma resembles that of LECs, that KSHV induces a transcriptional drift in both LECs and BECs and that lymphangiogenic molecules are involved in the pathogenesis of Kaposi sarcoma.

miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator

MicroRNAs are small, non-coding RNAs that negatively regulate gene expression. It has been proposed that microRNAs could function in the regulation of innate immunity, but this has not been demonstrated for viral infection. Here we test this hypothesis using the human pathogenic virus Kaposis sarcoma-associated herpesvirus (KSHV) and one of its putative natural cellular targets, primary lymphatic endothelial cells (LECs). We show that an early antiviral microRNA response (6 h post-infection) includes expression of microRNAs that enhance viral gene expression. In particular, the CREB-induced miR-132 microRNA is highly upregulated after infection and has a negative effect on the expression of interferon-stimulated genes, facilitating viral replication. We show a similar function for miR-132 during infection of monocytes with herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). miR-132 regulates innate antiviral immunity by inhibiting expression of the p300 transcriptional co-activator. p300 is downregulated early after KSHV infection, and inhibition of miR-132 induction restores p300 expression. Furthermore, p300 regulates miR-132 levels, revealing a dynamic equilibrium between miR-132 and p300. By targeting p300, rather than a transcription factor or signalling protein, miR-132 has a broad role in the regulation of antiviral immunity.

KSHV-encoded miRNAs target MAF to induce endothelial cell reprogramming

Kaposi sarcoma herpesvirus (KSHV) induces transcriptional reprogramming of endothelial cells. In particular, KSHV-infected lymphatic endothelial cells (LECs) show an up-regulation of genes associated with blood vessel endothelial cells (BECs). Consequently, KSHV-infected tumor cells in Kaposi sarcoma are poorly differentiated endothelial cells, expressing markers of both LECs and BECs. MicroRNAs (miRNAs) are short noncoding RNA molecules that act post-transcriptionally to negatively regulate gene expression. Here we validate expression of the KSHV-encoded miRNAs in Kaposi sarcoma lesions and demonstrate that these miRNAs contribute to viral-induced reprogramming by silencing the cellular transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog). MAF is expressed in LECs but not in BECs. We identify a novel role for MAF as a transcriptional repressor, preventing expression of BEC-specific genes, thereby maintaining the differentiation status of LECs. These findings demonstrate that viral miRNAs could influence the differentiation status of infected cells, and thereby contribute to KSHV-induced oncogenesis.

Short- and long-term effects of highly active antiretroviral therapy on Kaposi sarcoma-associated herpesvirus immune responses and viraemia.

Objective: To investigate the effect of highly active antiretroviral therapy (HAART) on Kaposi sarcoma-associated herpesvirus (KSHV) DNA load, anti-KSHV antibody responses and KSHV-specific CD8 T cell responses in HIV-infected individuals over a 2 year period. Design: Prospective study of 27 HIV-infected antiretroviral therapy-naive individuals, with (n = 4) and without KS (n = 23), before HAART and at 3-month intervals, during treatment with HAART. Methods: Sequential blood samples were collected for anti-KSHV antibody detection, KSHV DNA load in peripheral blood mononuclear cells (PBMC) and plasma, HIV Gag-specific and KSHV-specific CD8 T cell responses, HIV-1 plasma RNA load and CD4 and CD8 T cell counts. Results: KSHV DNA in PBMC and plasma became less detectable over time during HAART, in particular after 12 months. KSHV DNA was undetectable in plasma after 24 months on HAART. Anti-KSHV lytic, but not latent, antibody levels increased within 12 months of treatment. KSHV-specific CD8 T cell responses were absent prior to HAART but became detectable in some patients within 6 months of starting treatment, and continued to increase thereafter. Conclusions: HAART (both protease inhibitor-based and non-nucleoside reverse transcriptase inhibitor-based antiretroviral combinations) is associated with immune reconstitution to KSHV and with undetectable KSHV viraemia. However, this restoration is apparent (in particular) only after a relatively long (> 24 months) period of treatment. These immune responses could contribute to the decreased incidence of KS during HAART, but it is unlikely to be a complete explanation for the often rapid resolution of KS when HAART is started.

Kaposi's Sarcoma–Associated Herpesvirus-Encoded Interleukin-6 and G-Protein–Coupled Receptor Regulate Angiopoietin-2 Expression in Lymphatic Endothelial Cells

Kaposis sarcoma (KS) is caused by Kaposis sarcoma-associated herpesvirus (KSHV) and consists of proliferating spindle cells, which are related to lymphatic endothelial cells (LEC). Angiopoietin-2 (Ang2) is a secreted proangiogenic and lymphangiogenic molecule. Here, we show the expression of Ang2 protein in KS and confirm that KSHV infection up-regulates Ang2 in LEC. We show that a paracrine mechanism contributes to this up-regulation. A lentiviral library of individual KSHV-encoding genes, comprising the majority of known latent genes and a selection of lytic viral genes, was constructed to investigate the underlying mechanism of this up-regulation. Two lytic genes, viral interleukin-6 (vIL6) and viral G-protein-coupled receptor (vGPCR), up-regulated Ang2 expression in LEC. Both vIL6 and vGPCR are expressed in KSHV-infected LEC and caused up-regulation of Ang2 in a paracrine manner. KSHV, vIL6, and vGPCR up-regulated Ang2 through the mitogen-activated protein kinase (MAPK) pathway. Gene expression microarray analysis identified several other angiogenic molecules affected by KSHV, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis, which is also affected by vIL6 and vGPCR in LEC, and matrix metalloproteinases, which could act in concert with Ang2 to contribute to KS development. These findings support the paracrine and autocrine roles of the lytic KSHV-encoded proteins, vIL6 and vGPCR, in KS pathogenesis and identify Ang2 as a potential therapeutic target for this neoplasm.

Associations between Burkitt Lymphoma among Children in Malawi and Infection with HIV, EBV and Malaria: Results from a Case-Control Study

Background Burkitt lymphoma, a childhood cancer common in parts of sub-Saharan Africa, has been associated with Epstein Barr Virus (EBV) and malaria, but its association with human immunodeficiency virus (HIV) is not clear. Methodology/Principal Findings We conducted a case-control study of Burkitt lymphoma among children (aged ≤15 years) admitted to the pediatric oncology unit in Blantyre, Malawi between July 2005 and July 2006. Cases were 148 children diagnosed with Burkitt lymphoma and controls were 104 children admitted with non-malignant conditions or cancers other than hematological malignancies and Kaposi sarcoma. Interviews were conducted and serological samples tested for antibodies against HIV, EBV and malaria. Odds ratios for Burkitt lymphoma were estimated using unconditional logistic regression adjusting for sex, age, and residential district. Cases had a mean age of 7.1 years and 60% were male. Cases were more likely than controls to be HIV positive (Odds ratio (OR))  = 12.4, 95% Confidence Interval (CI) 1.3 to 116.2, p = 0.03). ORs for Burkitt lymphoma increased with increasing antibody titers against EBV (p = 0.001) and malaria (p = 0.01). Among HIV negative participants, cases were thirteen times more likely than controls to have raised levels of both EBV and malaria antibodies (OR = 13.2; 95% CI 3.8 to 46.6; p = 0.001). Reported use of mosquito nets was associated with a lower risk of Burkitt lymphoma (OR =  0.2, 95% CI, 0.03 to 0.9, p = 0.04). Conclusions Our findings support prior evidence that EBV and malaria act jointly in the pathogenesis of Burkitt lymphoma, suggesting that malaria prevention may decrease the risk of Burkitt lymphoma. HIV may also play a role in the etiology of this childhood tumor.

Toll-like Receptor 4 Mediates Innate Immunity to Kaposi Sarcoma Herpesvirus

The involvement of Toll-like receptor 4 (TLR4) in immunity against human herpesviruses has not been previously demonstrated. We show that infection of endothelial cells with Kaposi sarcoma herpesvirus (KSHV), a human oncogenic virus, leads to rapid suppression of TLR4 expression. This is a mechanism of immune escape as TLR4 mediates innate immunity against KSHV. In vitro, cells lacking TLR4 are more susceptible to KSHV infection, whereas activation of TLR4 protects cells from infection. In vivo, HIV-1-infected individuals carrying a mutant TLR4 allele appear more likely to have multicentric Castlemans disease, a lymphoproliferation associated with enhanced KSHV replication. ERK activation by KSHV structural proteins and the KSHV-encoded vGPCR plays a key role in the TLR4 downregulation, whereas the KSHV vIRF1 also contributes to this effect. Our findings reveal a role for TLR4 in innate immunity against herpesviruses and suggest the potential use of TLR4 agonists for the treatment of KSHV-related neoplasms.

Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>1010 M−1) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.

KSHV Manipulates Notch Signaling by DLL4 and JAG1 to Alter Cell Cycle Genes in Lymphatic Endothelia

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFκB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.

The CD8 and CD4 T-Cell Response against Kaposi's Sarcoma-Associated Herpesvirus Is Skewed Towards Early and Late Lytic Antigens

Kaposis sarcoma-associated herpesvirus (KSHV) is causally related to Kaposis sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs). We used these to transduce monocyte-derived dendritic cells (moDCs) isolated from 14 KSHV-seropositive (12 HIV-positive) and 7 KSHV-seronegative (4 HIV-positive) individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle). Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group) was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1]) were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37]) was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1]) was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.