BackgroundMacrophage‐mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg‐II) in macrophage function remains elusive. This study characterizes the role of Arg‐II in macrophage inflammatory responses and its impact on obesity‐linked type II diabetes mellitus and atherosclerosis.Methods and ResultsIn human monocytes, silencing Arg‐II decreases the monocytes’ adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low‐density lipoprotein or lipopolysaccharides, as evaluated by real‐time quantitative reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg‐II–deficient (Arg‐II−/−) mice express lower levels of lipopolysaccharide‐induced proinflammatory mediators than do macrophages of wild‐type mice. Importantly, reintroducing Arg‐II cDNA into Arg‐II−/− macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N‐acetylcysteine prevents the Arg‐II–mediated inflammatory responses. Moreover, high‐fat diet–induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg‐II−/− mice. Accordingly, Arg‐II−/− mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)–deficient mice with Arg‐II deficiency (ApoE−/−Arg‐II−/−) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE−/−Arg‐II−/− than ApoE−/−Arg‐II+/+ monocytes infiltrate into the plaque of ApoE−/−Arg‐II+/+ mice. Conversely, recipient ApoE−/−Arg‐II−/− mice accumulate fewer donor monocytes than do recipient ApoE−/−Arg‐II+/+ animals.ConclusionsArg‐II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg‐II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.)

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's , 0.625; < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific. HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

p38 mitogen-activated protein kinase (p38) regulates cellular senescence and senescence-associated secretory phenotype (SASP), i.e., secretion of cytokines and/or chemokines. Previous work showed that augmented arginase-II (Arg-II) and S6K1 interact with each other to promote endothelial senescence through uncoupling of endothelial nitric oxide synthase (eNOS). Here we demonstrate eNOS-uncoupling, augmented expression/secretion of IL-6 and IL-8, elevation of p38 activation and Arg-II levels in senescent endothelial cells. Silencing Arg-II or p38α in senescent cells recouples eNOS and inhibits IL-6 and IL-8 secretion. Overexpression of Arg-II in young endothelial cells causes eNOS-uncoupling and enhances IL-6 and IL-8 expression/secretion, which is prevented by p38 inhibition or by antioxidant. Moreover, p38 activation and expression of IL-6 and KC (the murine IL-8 homologue) are increased in the heart and/or aortas of wild type (WT) old mice, which is abolished in mice with Arg-II gene deficiency (Arg-II−/−). In addition, inhibition of p38 in the old WT mice recouples eNOS function and reduces IL-6 and KC expression in the aortas and heart. Silencing Arg-II or p38α or S6K1 inhibits each other in senescence endothelial cells. Thus, Arg-II, p38, and S6K1 form a positive circuit which regulates endothelial senescence and cardiovascular aging.

MicroRNAs (miRNAs) are endogenously expressed RNAs consisting of 20-24 nucleotides. These molecules are thought to repress protein translation by binding to target mRNAs. However, biological functions have not been assigned to most of the 175 porcine miRNAs registered in miRBase (release 15.0). In an effort to uncover miR-103 important in pigs, we examined the integrative tissue expression profile and gene ontology (GO) term enrichment of predicted target genes to determine the global biological functions of miR-103. Our results demonstrated that miR-103 is involved in various biological processes including brain development, lipid metabolism, adipocyte differentiation, hematopoiesis, and immunity. Moreover, we also experimentally verified effects of miR-103 in porcine preadipocytes. miR-103 levels increased in differentiating adipocytes, and inhibition of miR-103 effectively inhibited preadipocyte differentiation. In addition, mRNA levels of the putative miR-103 target RAI14 were higher in miR-103 inhibitor-treated adipocytes. These results demonstrate that miR-103 is involved in porcine preadipocyte differentiation and may act through the putative target gene RAI14. In a word, our data provide new insights into the global biological role of miR-103.

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

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Human polyomaviruses (HPyVs) encompass more than 10 species infecting 30%-90% of the human population without significant illness. Proven HPyV diseases with documented histopathology affect primarily immunocompromised hosts with manifestations in brain, skin and renourinary tract such as polyomavirus-associated nephropathy (PyVAN), polyomavirus-associated haemorrhagic cystitis (PyVHC), polyomavirus-associated urothelial cancer (PyVUC), progressive multifocal leukoencephalopathy (PML), Merkel cell carcinoma (MCC), Trichodysplasia spinulosa (TS) and pruritic hyperproliferative keratinopathy. Although virus-specific immune control is the eventual goal of therapy and lasting cure, antiviral treatments are urgently needed in order to reduce or prevent HPyV diseases and thereby bridging the time needed to establish virus-specific immunity. However, the small dsDNA genome of only 5 kb of the non-enveloped HPyVs only encodes 5-7 viral proteins. Thus, HPyV replication relies heavily on host cell factors, thereby limiting both, number and type of specific virus-encoded antiviral targets. Lack of cost-effective high-throughput screening systems and relevant small animal models complicates the preclinical development. Current clinical studies are limited by small case

The VG161 represents the first recombinant oncolytic herpes simplex virus type 1 carrying multiple synergistic antitumor immuno‐modulating factors. Here, we report its antitumor mechanisms and thus provide firm theoretical foundation for the upcoming clinical application in pancreatic cancer. Generally, the VG161‐mediated antitumor outcomes were analyzed by a collaboration of techniques, namely the single‐cell sequencing, airflow‐assisted desorption electrospray ionization‐mass spectrometry imaging (AFADSI‐MSI) and nanostring techniques. In vitro, the efficacy of VG161 together with immune checkpoint inhibitors (ICIs) has been successfully shown to grant a long‐term antitumor effect by altering tumor immunity and remodeling tumor microenvironment (TME) metabolisms. Cellular functional pathways and cell subtypes detected from patient samples before and after the treatment had undergone distinctive changes including upregulated CD8+ T and natural killer cells. More importantly, significant antitumor signals have emerged since the administration of VG161 injection. In conclusion, VG161 can systematically activate acquired and innate immunity in pancreatic models, as well as improve the tumor immune microenvironment, indicative of strong antitumor potential. The more robusting antitumor outcome for VG161 monotherapy or in combination with other therapies on pancreatic cancer is worth of being explored in further clinical trials.