Cytomegalovirus viral interleukin-10 in patients with Aspergillus infection and effects on clinical outcome.

BACKGROUND: Human cytomegalovirus (CMV) is associated with aspergillosis, but the simultaneous presence of CMV viral interleukin-10 (cmvIL-10) and aspergillosis has never been investigated. CmvIL-10 is produced by CMV-infected cells and acts as an immune modulator during CMV infection. The aim of this study was to evaluate cmvIL-10 levels in peripheral blood and its influence on the clinical outcomes of Aspergillus infection. METHODS: Patients who visited or were admitted to the hospital with suspected Aspergillus infection, including invasive aspergillosis (IA) and chronic pulmonary aspergillosis (CPA), were prospectively enrolled. The cmvIL-10, human IL-10 (hIL-10), IL-1B, IL-6, IL-8, IFN-γ, and TNF-α levels in peripheral blood were measured. RESULTS: Patients with Aspergillus infection had a higher level of cmvIL-10 than the control group (158 ± 305 vs 27.9 ± 30.4 pg/ml, p < .05). The level of cmvIL-10 was not correlated with CMV viremia or end-organ disease. The cmvIL-10 but not hIL-10 level was positively correlated with the IFN-γ level (p < .05) and marginally negatively correlated with IL-1B and IL-8 levels (p < .1). In patients with CPA, a high level of cmvIL-10 (≥100 pg/ml) was a poor prognostic factor for long-term survival (p < .05). In contrast, CMV viremia or end-organ disease was associated with poor survival in patients with IA (p = .05). CONCLUSIONS: Aspergillus infection was associated with CMV coinfection with cmvIL-10 in blood. A cmvIL-10 concentration ≥100 pg/ml was a predictor for unfavourable outcome in CPA patients.

Detection of Cytomegalovirus Interleukin 10 (cmvIL-10) by Enzyme-Linked Immunosorbent Assay (ELISA).

Since its introduction in 1971, the enzyme-linked immunosorbent assay (ELISA) has revolutionized medicine by enabling detection of both antigens and antibodies in a variety of samples. We describe here a customized sandwich ELISA developed for the detection of Human Cytomegalovirus interleukin-10 (cmvIL-10). CmvIL-10 is a virally encoded cytokine and ortholog of human interleukin 10 (hIL-10). While cmvIL-10 and hIL-10 are similar in structure and function, overall amino acid sequence identity is only 27%, resulting in antigenically distinct proteins. The cmvIL-10 ELISA is specific and does not detect hIL-10. The assay is sensitive enough to detect cmvIL-10 in both culture supernatants and patient serum. The ability to quantify cmvIL-10 levels during HCMV infection could provide valuable information about immune evasion strategies and viral control of host signaling pathways.

Control of Cytokines in Latent Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) has evolved a number of mechanisms for long-term co-existence within its host. HCMV infects a wide range of cell types, including fibroblasts, epithelial cells, monocytes, macrophages, dendritic cells, and myeloid progenitor cells. Lytic infection, with the production of infectious progeny virions, occurs in differentiated cell types, while undifferentiated myeloid precursor cells are the primary site of latent infection. The outcome of HCMV infection depends partly on the cell type and differentiation state but is also influenced by the composition of the immune environment. In this review, we discuss the role of early interactions between HCMV and the host immune system, particularly cytokine and chemokine networks, that facilitate the establishment of lifelong latent infection. A better understanding of these cytokine signaling pathways could lead to novel therapeutic targets that might prevent latency or eradicate latently infected cells.

Identification of a novel signaling complex containing host chemokine receptor CXCR4, Interleukin-10 receptor, and human cytomegalovirus US27.

Human cytomegalovirus (HCMV) is a widespread herpesvirus that establishes latency in myeloid cells and persists by manipulating immune signaling. Chemokine receptor CXCR4 and its ligand CXCL12 regulate movement of myeloid progenitors into bone marrow and out into peripheral tissues. HCMV amplifies CXCL12-CXCR4 signaling through viral chemokine receptor US27 and cmvIL-10, a viral cytokine that binds the cellular IL-10 receptor (IL-10R), but precisely how these viral proteins influence CXCR4 is unknown. We used the proximity ligation assay (PLA) to examine association of CXCR4, IL-10R, and US27 in both transfected and HCMV-infected cells. CXCR4 and IL-10R colocalized to discrete clusters, and treatment with CXCL12 and cmvIL-10 dramatically increased receptor clustering and calcium flux. US27 was associated with CXCR4 and IL-10R in PLA clusters and further enhanced cluster formation and calcium signaling. These results indicate that CXCR4, IL-10R, and US27 form a novel virus-host signaling complex that enhances CXCL12 signaling during HCMV infection.

Human cytomegalovirus IL-10 augments NK cell cytotoxicity.

Human cytomegalovirus (HCMV) persistently infects most of the adult population with periods of productive and latent infection differentially orchestrated by multiple HCMV-encoded gene products. One HCMV gene (UL111a) encodes cmvIL-10, a virokine homologous to human IL (hIL)-10. Although the effects of cmvIL-10 on most human lymphocyte subsets have been extensively studied, its impact on NK cell function was unreported prior to this study. We investigated effects of short-term cmvIL-10 exposure on human NK cells and found it substantially enhanced NK cell cytotoxicity through natural cytotoxicity receptors NKp30 and NKp46 as well as through C-type lectin-like receptors NKG2C and NKG2D. Antibody-dependent cell-mediated cytotoxicity triggered through CD16 also increased significantly with short-term cmvIL-10 exposure. These effects of cmvIL-10 on NK cell cytotoxicity were rapid, dose dependent, neutralized by polyclonal anti-cmvIL-10 or monoclonal anti-IL-10 receptor (IL-10R) antibodies and independent of increased perforin synthesis or up-regulation of activating receptors. A low percentage (0.5-5.4%; n = 12) of NK cells expressed IL-10R and the impact of cmvIL-10 on NK cells degranulation following CD16 stimulation directly correlated with this percentage (P = 0.0218). Short-term exposure of human NK cells to cmvIL-10 did not introduce phenotypic changes reminiscent of NK adaptation to HCMV infection in vivo. Determining how expression of a viral protein that activates NK cells contributes to their function in vivo will increase understanding of HCMV infection and NK cell biology.

Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms.

Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes lifelong infection in the host. Virus persistence is aided by extensive manipulation of the host immune system, particularly cytokine and chemokine signaling pathways. The HCMV UL111A gene encodes cmvIL-10, an ortholog of human interleukin-10 that has many immunomodulatory effects. We found that cmvIL-10 increased signaling outcomes from human CXCR4, a chemokine receptor with essential roles in hematopoiesis and immune cell trafficking, in response to its natural ligand CXCL12. Calcium flux and chemotaxis to CXCL12 were significantly greater in the presence of cmvIL-10 in monocytes, epithelial cells, and fibroblasts that express CXCR4. cmvIL-10 effects on CXCL12/CXCR4 signaling required the IL-10 receptor and Stat3 activation. Heightened signaling occurred both in HCMV-infected cells and in uninfected bystander cells, suggesting that cmvIL-10 may broadly influence chemokine networks by paracrine signaling during infection. Moreover, CXCL12/CXCR4 signaling was amplified in HCMV-infected cells compared to mock-infected cells even in the absence of cmvIL-10. Enhanced CXCL12/CXCR4 outcomes were associated with expression of the virally encoded chemokine receptor US27, and CXCL12/CXCR4 activation was reduced in cells infected with a deletion mutant lacking US27 (TB40/E-mCherry-US27Δ). US27 effects were Stat3 independent but required close proximity to CXCR4 in cell membranes of either HCMV-infected or US27-transfected cells. Thus, HCMV encodes two proteins, cmvIL-10 and US27, that exhibit distinct mechanisms for enhancing CXCR4 signaling. Either individually or in combination, cmvIL-10 and US27 may enable HCMV to exquisitely manipulate CXCR4 signaling to alter host immune responses and modify cell trafficking patterns during infection.IMPORTANCE The human chemokine system plays a central role in host defense, as evidenced by the many strategies devised by viruses for manipulating it. Human cytomegalovirus (HCMV) is widespread in the human population, but infection rarely causes disease except in immunocompromised hosts. We found that two different HCMV proteins, cmvIL-10 and US27, act through distinct mechanisms to upregulate the signaling activity of a cellular chemokine receptor, CXCR4. cmvIL-10 is a secreted viral cytokine that affects CXCR4 signaling in both infected and uninfected cells, while US27 is a component of the virus particle and impacts CXCR4 activity only in infected cells. Both cmvIL-10 and US27 promote increased intracellular calcium signaling and cell migration in response to chemokine CXCL12 binding to CXCR4. Our results demonstrate that HCMV exerts fine control over the CXCL12/CXCR4 pathway, which could lead to enhanced virus dissemination, altered immune cell trafficking, and serious health implications for HCMV patients.

Modulation of the Host Environment by Human Cytomegalovirus with Viral Interleukin 10 in Peripheral Blood.

Background: Human cytomegalovirus (HCMV) is a herpesvirus with both lytic and latent life cycles. Human cytomegalovirus encodes 2 viral cytokines that are orthologs of human cellular interleukin 10 (cIL-10). Both cytomegalovirus interleukin 10 (cmvIL-10) and Latency-associated cytomegalovirus interleukin 10 (LAcmvIL-10) (collectively vIL-10) are expressed during lytic infection and cause immunosuppressive effects that impede virus clearance. LAcmvIL-10 is also expressed during latent infection of myeloid progenitor cells and monocytes and facilitates persistence. Here, we investigated whether vIL-10 could be detected during natural infection. Methods: Plasma from healthy blood donors was tested by enzyme-linked immunosorbent assay for anti-HCMV immunoglobulin G and immunoglobulin M and for cIL-10 and vIL-10 levels using a novel vIL-10 assay that detects cmvIL-10 and LAcmvIL-10, with no cross-reactivity to cIL-10. Results: vIL-10 was evident in HCMV+ donors (n = 19 of 26), at levels ranging 31-547 pg/mL. By comparison, cIL-10 was detected at lower levels ranging 3-69 pg/mL. There was a strong correlation between vIL-10 and cIL-10 levels (P = .01). Antibodies against vIL-10 were also detected and neutralized vIL-10 activity. Conclusions: vIL-10 was detected in peripheral blood of healthy blood donors. These findings suggest that vIL-10 may play a key role in sensing or modifying the host environment during latency and, therefore, may be a potential target for intervention strategies.

Human cytomegalovirus interleukin-10 enhances matrigel invasion of MDA-MB-231 breast cancer cells.

BACKGROUND: While some risk factors for breast cancer are well-known, the influence of other factors, particularly virus infection, remains unclear. Human cytomegalovirus (HCMV) is widespread in the general population, and both molecular and epidemiological evidence has indicated links between HCMV and breast cancer. The HCMV protein cmvIL-10 is a potent suppressor of immune function that has also been shown to promote proliferation and migration of breast cancer cells. In this study, the impact of cmvIL-10 on tumor cell invasion through a simulated basement membrane was investigated. RESULTS: MDA-MB-231 breast cancer cells exhibited invasion through a matrigel layer that was significantly enhanced in the presence of either purified cmvIL-10 or supernatants from HCMV-infected cells containing secreted cmvIL-10. Transcriptional profiling revealed that cmvIL-10 altered expression of several genes implicated in metastasis. Exposure to cmvIL-10 resulted in higher MMP-3 mRNA levels, greater protein expression, and increased enzymatic activity. Treatment with cmvIL-10 also increased expression of both urokinase plasminogen receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), which can stimulate MMP-3 activity and have previously been identified as poor prognostic markers in breast cancer patients. Finally, MDA-MB-231 cells treated with cmvIL-10 showed significant downregulation of metastasis suppressor 1 (MTSS1), a scaffolding protein that regulates cytoskeletal rearrangements and is frequently lost in metastatic tumors. CONCLUSIONS: HCMV, and in particular the secreted viral cytokine, cmvIL-10, can induce cellular changes that facilitate cell migration and invasion. These findings indicate that HCMV may be associated with promoting the malignant spread of breast cancer cells and suggest that antiviral treatment may be a useful complement to chemotherapy in some patients.

Human cytomegalovirus latent infection alters the expression of cellular and viral microRNA.

BACKGROUND: MicroRNAs (miRNAs) play important roles in regulating gene expression of plants, animals and viruses. Comprehensive characterization of host and viral miRNA will help uncover the molecular mechanisms that underlie the progression of human cytomegalovirus (HCMV) latent infection. To investigate the miRNA expression profile of HCMV and host cells during latent infection, we performed deep-sequencing analysis of the small RNAs isolated from HCMV-infected and mock-infected human monocytic leukemia cell line, THP-1. RESULTS: We established a HCMV latent infection cell model using the THP-1 cells. High-throughput sequencing technology was used to sequence small RNA libraries of the HCMV-infected and mock-infected THP-1 and to investigate their small RNA transcriptomes. We found eight miRNAs including miR-US25-1, miR-US25-2-5p and miR-UL112 that were expressed by HCMV during latent infection. The expressions of the host miRNAs were also affected by HCMV latent infection. At least 49 cellular miRNAs were differentially expressed: 39 were up-regulated and 10 were down-regulated upon HCMV latent infection. The expression of the human miRNA hsa-miR-124-3p was significantly up-regulated in the HCMV latent infection library. In addition, we found 14 cellular novel miRNAs in the HCMV-infected and mock-infected THP-1 libraries. Functional annotation of the target genes of the differentially expressed miRNAs suggested that the majority of the genes are involved in melanogenesis, pathways in cancer, endocytosis and wnt signaling pathway. CONCLUSIONS: The small RNA transcriptomes obtained in this study demonstrate the usefulness of the deep-sequencing combined with bioinformatics approach in understanding of the expression and function of host and viral small RNAs in HCMV latent infection. This approach can also be applied to the study of other kinds of viruses.