A 77 Amino Acid Region in the N-Terminal Half of the HSV-1 E3 Ubiquitin Ligase ICP0 Contributes to Counteracting an Established Type 1 Interferon Response.

Herpes simplex virus 1 (HSV-1) is a human pathogen capable of establishing lifelong latent infections that can reactivate under stress conditions. A viral immediate early protein that plays important roles in the HSV-1 lytic and latent infections is the viral E3 ubiquitin ligase, ICP0. ICP0 transactivates all temporal classes of HSV-1 genes and facilitates viral gene expression. ICP0 also impairs the antiviral effects of interferon (IFN)-ß, a component of host innate defenses known to limit viral replication. To begin to understand how ICP0 allows HSV-1 to disarm the IFN-ß response, we performed genetic analyses using a series of ICP0 truncation mutants in the absence and presence of IFN-ß in cell culture. We observed that IFN-ß pretreatment of cells significantly impaired the replication of the ICP0 truncation mutants, n212 and n312, which code for the first 211 and 311 amino acids of ICP0, respectively; this effect of IFN-ß correlated with decreased HSV-1 early and late gene expression. This increased sensitivity to IFN-ß was not as apparent with the ICP0 mutant, n389. Our mapping studies indicate that loss of 77 amino acids from residues 312 to 388 in the N-terminal half of ICP0 resulted in a virus that was significantly more sensitive to cells pre-exposed to IFN-ß. This 77 amino acid region contains a phospho-SUMO-interacting motif or -SIM, which we propose participates in ICP0's ability to counteract the antiviral response established by IFN-ß. IMPORTANCE Interferons (IFNs) are secreted cellular factors that are induced by viral infection and limit replication. HSV-1 is largely refractory to the antiviral effects of type 1 IFNs, which are synthesized shortly after viral infection, in part through the activities of the viral regulatory protein, ICP0. To understand how ICP0 impedes the antiviral effects of type 1 IFNs, we used a series of HSV-1 ICP0 mutants and examined their viral replication and gene expression levels in cells stimulated with IFN-ß (a type 1 IFN). Our mapping data identifies a discrete 77 amino acid region in the N-terminal half of ICP0 that facilitates HSV-1 resistance to IFN-ß. This region of ICP0 is modified by phosphorylation and binds to the posttranslational modification SUMO, suggesting that HSV, and potentially other viruses, may counteract type 1 IFN signaling by altering SUMO and/or SUMO modified cellular proteins.

Interleukin 22 Signaling Regulates Acinar Cell Plasticity to Promote Pancreatic Tumor Development in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that invades surrounding structures and metastasizes rapidly. Although inflammation is associated with tumor formation and progression, little is known about the mechanisms of this connection. We investigate the effects of interleukin (IL) 22 in the development of pancreatic tumors in mice. METHODS: We performed studies with Pdx1-Cre;LSL-KrasG12D;Trp53+/-;Rosa26EYFP/+ (PKCY) mice, which develop pancreatic tumors, and PKCY mice with disruption of IL22 (PKCY Il22-/-mice). Pancreata were collected at different stages of tumor development and analyzed by immunohistochemistry, immunoblotting, real-time polymerase chain reaction, and flow cytometry. Some mice were given cerulean to induce pancreatitis. Pancreatic cancer cell lines (PD2560) were orthotopically injected into C57BL/6 mice or Il22-/-mice, and tumor development was monitored. Pancreatic cells were injected into the tail veins of mice, and lung metastases were quantified. Acini were collected from C57BL/6 mice and resected human pancreata and were cultured. Cell lines and acini cultures were incubated with IL22 and pharmacologic inhibitors, and protein levels were knocked down with small hairpin RNAs. We performed immunohistochemical analyses of 26 PDACs and 5 nonneoplastic pancreas specimens. RESULTS: We observed increased expression of IL22 and the IL22 receptor (IL22R) in the pancreas compared with other tissues in mice; IL22 increased with pancreatitis and tumorigenesis. Flow cytometry indicated that the IL22 was produced primarily by T-helper 22 cells. PKCY Il22-/-mice did not develop precancerous lesions or pancreatic tumors. The addition of IL22 to cultured acinar cells increased their expression of markers of ductal metaplasia; these effects of IL22 were prevented with inhibitors of Janus kinase signaling to signal transducer and activator of transcription (STAT) (ruxolitinib) or mitogen-activated protein kinase kinase (MEK) (trametinib) and with STAT3 knockdown. Pancreatic cells injected into Il22-/- mice formed smaller tumors than those injected into C57BL/6. Incubation of IL22R-expressing PDAC cells with IL22 promoted spheroid formation and invasive activity, resulting in increased expression of stem-associated transcription factors (GATA4, SOX2, SOX17, and NANOG), and increased markers of the epithelial-mesenchymal transition (CDH1, SNAI2, TWIST1, and beta catenin); ruxolitinib blocked these effects. Human PDAC tissues had higher levels of IL22, phosphorylated STAT3, and markers of the epithelial-mesenchymal transition than nonneoplastic tissues. An increased level of STAT3 in IL22R-positive cells was associated with shorter survival times of patients. CONCLUSIONS: We found levels of IL22 to be increased during pancreatitis and pancreatic tumor development and to be required for tumor development and progression in mice. IL22 promotes acinar to ductal metaplasia, stem cell features, and increased expression of markers of the epithelial-mesenchymal transition; inhibitors of STAT3 block these effects. Increased expression of IL22 by PDACs is associated with reduced survival times.

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry.

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Spatial and phenotypic immune profiling of metastatic colon cancer.

Paramount to the efficacy of immune checkpoint inhibitors is proper selection of patients with adequate tumor immunogenicity and a robust but suppressed immune infiltrate. In colon cancer, immune-based therapies are approved for patients with DNA mismatch repair (MMR) deficiencies, in whom accumulation of genetic mutations results in increased neoantigen expression, triggering an immune response that is suppressed by the PD-L1/PD-1 pathway. Here, we report that characterization of the microenvironment of MMR-deficient metastatic colorectal cancer using multiplex fluorescent immunohistochemistry (mfIHC) identified increased infiltration of cytotoxic T lymphocytes (CTLs), which were more often engaged with epithelial cells (ECs) and improved overall survival. A subset of patients with intact MMR but a similar immune microenvironment to MMR-deficient patients was identified and found to universally express high levels of PD-L1, suggesting that they may represent a currently untreated, checkpoint inhibitor-responsive population. Further, PD-L1 expression on antigen-presenting cells (APCs) in the tumor microenvironment (TME) resulted in impaired CTL/EC engagement and enhanced infiltration and engagement of Tregs. Characterization of the TME by mfIHC highlights the interconnection between immunity and immunosuppression in metastatic colon cancer and may better stratify patients for receipt of immunotherapies.

Biological and pathological activities of interleukin-22.

Interleukin (IL)-22, a member of the IL-10 family, is a cytokine secreted by several types of immune cells including IL-22(+)CD4(+) T cells (Th22) and IL-22 expressing innate leukocytes (ILC22). Recent studies have demonstrated that IL-22 is a key component in mucosal barrier defense, tissue repair, epithelial cell survival, and proliferation. Furthermore, accumulating evidence has defined both protective and pathogenic properties of IL-22 in a number of conditions including autoimmune disease, infection, and malignancy. In this review, we summarize the expression and signaling pathway and functional characteristics of the IL-22 and IL-22 receptor axis in physiological and pathological scenarios and discuss the potential to target IL-22 signaling to treat human diseases.

Two overlapping regions within the N-terminal half of the herpes simplex virus 1 E3 ubiquitin ligase ICP0 facilitate the degradation and dissociation of PML and dissociation of Sp100 from ND10.

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in sensory neurons and can reactivate from latency under stress conditions. To promote lytic infection, the virus must interact with specific cellular factors to evade the host's antiviral defenses. The HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), activates transcription of viral genes, in part, by mediating the degradation of certain cellular proteins that play a role in host antiviral mechanisms. One component of the cellular defenses that ICP0 disrupts is the suborganelle, nuclear domain 10 (ND10), by inducing the degradation and dissociation of the major organizer of ND10, a promyelocytic leukemia (PML) and ND10 constituent, Sp100. Because previously identified domains in ICP0 explain only partially how it directs the degradation and dissociation of PML and Sp100, we hypothesized that additional regions within ICP0 may contribute to these activities, which in turn facilitate efficient viral replication. To test this hypothesis, we used a series of ICP0 truncation mutants and examined PML protein levels and PML and Sp100 immunofluorescence staining in human embryonic lung cells. Our results demonstrate that two overlapping regions within the central N-terminal portion of ICP0 (residues 212 to 311) promoted the dissociation and degradation of PML and dissociation of Sp100 (residues 212 to 427). In conclusion, we have identified two additional regions in ICP0 involved in altering ND10 antiviral defenses in a cell culture model of HSV-1 infection.

hTERT extends the life of human fibroblasts without compromising type I interferon signaling.

Primary cells are often used to study viral replication and host-virus interactions as their antiviral pathways have not been altered or inactivated; however, their use is restricted by their short lifespan. Conventional methods to extend the life of primary cultures typically utilize viral oncogenes. Many of these oncogenes, however, perturb or inactivate cellular antiviral pathways, including the interferon (IFN) response. It has been previously shown that expression of the telomerase reverse transcriptase (TERT) gene extends the life of certain cell types. The effect that TERT expression has on the innate antiviral response to RNA- and DNA-containing viruses has not been examined. In the current study, we introduced the human TERT (hTERT) gene into a primary human embryonic lung (HEL-299) cell strain, which is known to respond to the type I IFN, IFN-ß. We show that the resulting HEL-TERT cell line is capable of replicating beyond 100 population doublings without exhibiting signs of senescence. Treatment with IFN-ß resulted in the upregulation of four model IFN stimulated genes (ISGs) in HEL-299 and HEL-TERT cells. Both cell lines supported the replication of herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV) and impaired the replication of both viruses upon IFN-ß pretreatment. Introduction of the viral oncoprotein, simian virus 40 (SV40) large T-antigen, which is frequently used to immortalize cells, largely negated this effect. Taken together, our data indicate that expression of hTERT does not alter type 1 IFN signaling and/or the growth of two viruses, making this cell line a useful reagent for studying viral replication and virus-cell interactions.