The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo.

Human cytomegalovirus (HCMV) is a betaherpesvirus that is a significant pathogen within newborn and immunocompromised populations. Morbidity associated with HCMV infection is the consequence of viral dissemination. HCMV has evolved to manipulate the host immune system to enhance viral dissemination and ensure long-term survival within the host. The immunomodulatory protein vCXCL-1, a viral chemokine functioning primarily through the CXCR2 chemokine receptor, is hypothesized to attract CXCR2+ neutrophils to infection sites, aiding viral dissemination. Neutrophils harbor HCMV in vivo; however, the interaction between vCXCL-1 and the neutrophil has not been evaluated in vivo Using the mouse model and mouse cytomegalovirus (MCMV) infection, we show that murine neutrophils harbor and transfer infectious MCMV and that virus replication initiates within this cell type. Utilizing recombinant MCMVs expressing vCXCL-1 from the HCMV strain (Toledo), we demonstrated that vCXCL-1 significantly enhances MCMV dissemination kinetics. Through cellular depletion experiments, we observe that neutrophils impact dissemination but that overall dissemination is largely neutrophil independent. This work adds neutrophils to the list of innate cells (i.e., dendritic and macrophages/monocytes) that contribute to MCMV dissemination but refutes the hypothesis that neutrophils are the primary cell responding to vCXCL-1.IMPORTANCE An adequate in vivo analysis of HCMV's viral chemokine vCXCL-1 has been lacking. Here we generate recombinant MCMVs expressing vCXCL-1 to study vCXCL-1 function in vivo using MCMV as a surrogate. We demonstrate that vCXCL-1 increases MCMV dissemination kinetics for both primary and secondary dissemination. Additionally, we provide evidence, that the murine neutrophil is largely a bystander in the mouse's response to vCXCL-1. We confirm the hypothesis that vCXCL-1 is a HCMV virulence factor. Infection of severely immunocompromised mice with MCMVs expressing vCXCL-1 was lethal in more than 50% of infected animals, while all animals infected with parental virus survived during a 12-day period. This work provides needed insights into vCXCL-1 function in vivo.

Anticytomegalovirus Peptides Point to New Insights for CMV Entry Mechanisms and the Limitations of In Vitro Screenings.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that can cause severe disease following in utero exposure, during primary infection, or latent virus reactivation in immunocompromised populations. These complications lead to a 1- to 2-billion-dollar economic burden, making vaccine development and/or alternative treatments a high priority. Current treatments for HCMV include nucleoside analogues such as ganciclovir (GCV), foscarnet, and cidofovir. Recently, letermovir, a terminase complex inhibitor, was approved for prophylaxis after stem cell transplantation. These treatments have unwanted side effects, and HCMV is becoming resistant to them. Therefore, we sought to develop an alternative treatment that targets a different stage in viral infection. Currently, small antiviral peptides are being investigated as anti-influenza and anti-HIV treatments. We have developed heparan sulfate-binding peptides as tools for preventing CMV infections. These peptides are highly effective at stopping infection of fibroblasts with in vitro-derived HCMV and murine cytomegalovirus (MCMV). However, they do not prevent MCMV infection in vivo Interestingly, these peptides inhibit infectivity of in vivo-derived CMVs, albeit not as well as tissue culture-grown CMVs. We further demonstrate that this class of heparan sulfate-binding peptides is incapable of inhibiting MCMV cell-to-cell spread, which is independent of heparan sulfate usage. These data indicate that inhibition of CMV infection can be achieved using synthetic polybasic peptides, but cell-to-cell spread and in vivo-grown CMVs require further investigation to design appropriate anti-CMV peptides.IMPORTANCE In the absence of an effective vaccine to prevent HCMV infections, alternative interventions must be developed. Prevention of viral entry into susceptible cells is an attractive alternative strategy. Here we report that heparan sulfate-binding peptides effectively inhibit entry into fibroblasts of in vitro-derived CMVs and partially inhibit in vivo-derived CMVs. This includes the inhibition of urine-derived HCMV (uCMV), which is highly resistant to antibody neutralization. While these antiviral peptides are highly effective at inhibiting cell-free virus, they do not inhibit MCMV cell-to-cell spread. This underscores the need to understand the mechanism of cell-to-cell spread and differences between in vivo-derived versus in vitro-derived CMV entry to effectively prevent CMV's spread.

The D-form of a novel heparan binding peptide decreases cytomegalovirus infection in vivo and in vitro.

Human cytomegalovirus (HCMV) infection in utero can lead to congenital sensory neural hearing loss and mental retardation. Reactivation or primary infection can increase the morbidity and mortality in immune suppressed transplant recipients and AIDS patients. The current standard of care for HCMV disease is nucleoside analogs, which can be nephrotoxic. In addition resistance to current treatments is becoming increasingly common. In an effort to develop novel CMV treatments, we tested the effectiveness of the D-form of a novel heparan sulfate binding peptide, p5RD, at reducing infection of ganciclovir (GCV) resistant HCMVs in vitro and MCMV in vivo. HCMV infection was reduced by greater than 90% when cells were pretreated with p5RD. Because p5RD acts by a mechanism unrelated to those used by current antivirals, it was effective at reducing GCV resistant HCMVs by 85%. We show that p5RD is resistant to common proteases and serum inactivation, which likely contributed to its ability to significantly reduced infection of peritoneal exudate cells and viral loads in the spleen and the lungs in vivo. The ability of p5RD to reduce HCMV infectivity in vitro including GCV resistant HCMVs and MCMV infection in vivo suggests that this peptide could be a novel anti-CMV therapeutic.

Novel heparan sulfate-binding peptides for blocking herpesvirus entry.

Human cytomegalovirus (HCMV) infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV) infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs), serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function.

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (ß-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.

CCL20 is elevated during obesity and differentially regulated by NF-κB subunits in pancreatic ß-cells.

Enhanced leukocytic infiltration into pancreatic islets contributes to inflammation-based diminutions in functional ß-cell mass. Insulitis (aka islet inflammation), which can be present in both T1DM and T2DM, is one factor influencing pancreatic ß-cell death and dysfunction. IL-1ß, an inflammatory mediator in both T1DM and T2DM, acutely (within 1h) induced expression of the CCL20 gene in rat and human islets and clonal ß-cell lines. Transcriptional induction of CCL20 required the p65 subunit of NF-κB to replace the p50 subunit at two functional κB sites within the CCL20 proximal gene promoter. The NF-κB p50 subunit prevents CCL20 gene expression during unstimulated conditions and overexpression of p50 reduces CCL20, but enhances cyclooxygenase-2 (COX-2), transcript accumulation after exposure to IL-1ß. We also identified differential recruitment of specific co-activator molecules to the CCL20 gene promoter, when compared with the CCL2 and COX2 genes, revealing distinct transcriptional requirements for individual NF-κB responsive genes. Moreover, IL-1ß, TNF-α and IFN-γ individually increased the expression of CCR6, the receptor for CCL20, on the surface of human neutrophils. We further found that the chemokine CCL20 is elevated in serum from both genetically obese db/db mice and in C57BL6/J mice fed a high-fat diet. Taken together, these results are consistent with a possible activation of the CCL20-CCR6 axis in diseases with inflammatory components. Thus, interfering with this signaling pathway, either at the level of NF-κB-mediated chemokine production, or downstream receptor activation, could be a potential therapeutic target to offset inflammation-associated tissue dysfunction in obesity and diabetes.

Transcription of the gene encoding TNF-α is increased by IL-1ß in rat and human islets and ß-cell lines.

Synthesis and secretion of immunomodulatory proteins, such as cytokines and chemokines, controls the inflammatory response within pancreatic islets. When this inflammation does not resolve, destruction of pancreatic islet ß-cells leads to diabetes mellitus. Production of the soluble mediators of inflammation, such as TNF-α and IL-1ß, from resident and invading immune cells, as well as directly from islet ß-cells, is also associated with suboptimal islet transplantation outcomes. In this study, we found that IL-1ß induces rapid increases in TNF-α mRNA in rat and human islets and the 832/13 clonal ß-cell line. The surge in transcription of the TNF-α gene required the inhibitor of kappa B kinase beta (IκKß), the p65 subunit of the NF-κB and a signal-specific recruitment of RNA polymerase II to the gene promoter. Of note was the increased intracellular production of TNF-α protein in a manner consistent with mRNA accumulation in response to IL-1ß, but no detectable secretion of TNF-α into the media. Additionally, TNF-α specifically induces expression of CD11b, but not CD11c, on neutrophils, which could contribute to the inflammatory milieu and diabetes progression. We conclude that activation of the NF-κB pathway in pancreatic ß-cells leads to rapid intracellular production of the pro-inflammatory TNF-α protein through a combination of specific histone covalent modifications and NF-κB signaling pathways.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription.

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing ß-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1ß markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and ß-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1ß to promote transcription. In addition, IL-1ß induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1ß induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1ß-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic ß-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

Screening for novel constitutively active CXCR2 mutants and their cellular effects.

Chemokines play an important role in inflammatory, developmental, and homeostatic processes. Deregulation of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the "on" position. This can lead to cellular transformation/tumorigenesis. The CXC chemokine receptor 2 (CXCR2) is a G-protein-coupled receptor (GPCR) expressed on neutrophils, some monocytes, endothelial cells, and some epithelial cells. CXCR2 activation with CXC chemokines induces leukocyte migration, trafficking, leukocyte degranulation, cellular differentiation, and angiogenesis. Activation of CXCR2 can lead to cellular transformation. We hypothesized that CAM CXCR2s may play a role in cancer development. In order to identify CXCR2 CAMs, potential mutant CXCR2 receptors were screened using a modified Saccharomyces cerevisiae high-throughput system. S. cerevisiae has been used successfully to identify GPCR/G-protein interactions and autocrine selection for peptide agonists. The CXCR2 CAMs identified from this screen were characterized in mammalian cells. Their ability to transform cells in vitro was shown using foci formation, soft-agar growth, impedance measurement assays, and in vivo tumor growth following hind flank inoculation into mice. Signaling pathways contributing to cellular transformation were identified using luciferase reporter assays. Studying constitutively active GPCRs is an approach to "capturing" pluridimensional GPCRs in a "locked" activation state. In order to address the residues necessary for CXCR2 activation, we used S. cerevisiae for screening novel CAMs and characterized them using mammalian reporter assays.

Electrical impedance measurements predict cellular transformation.

Cellular transformation is the first step in cancer development. Two features of cellular transformation are proliferation in reduced serum and loss of contact inhibition. Electronic Cell-Substrate Impedance Sensing (ECIS) measurements have been used to measure cellular proliferation, cytotoxicity, apoptosis, and attachment. We have used impedance measurements to distinguish normal cells from cells transformed with a constitutively active chemokine receptor, CXCR2. CXCR2, a member of the G-protein coupled receptor (GPCR) family, is normally involved in cellular activation and migration, but a single amino acid substitution leads to constitutive activity. NIH3T3 cells were transformed with a constitutively active CXCR2 (D143V_CXCR2) and growth in reduced serum and foci formation were measured using established biological assays and compared to data from ECIS. The results of this study show that impedance measurements provide a quick and reliable way of measuring cellular transformation and provide real time assessment of transformed cellular parameters. Use of the ECIS system could allow a rapid screening of anti-cancer drugs that alter cellular transformation.