ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15−/− macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.     

Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.     

Protein Kinase PKR Mediates the Apoptosis Induction and Growth Restriction Phenotypes of C Protein-Deficient Measles Virus

The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKR^kd cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (C^ko) virus but had no effect on the growth of either wild-type (WT) or V knockout (V^ko) virus. Increased growth of the C^ko virus in PKR^kd cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, V^ko, or especially C^ko virus caused significantly less apoptosis in PKR^kd cells than in PKR-sufficient cells. Although apoptosis induced by C^ko virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of C^ko virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.     

Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus

Introduction

More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.

Methods

Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.

Results

Circulating CD14⁺ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.

Conclusions

Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.     

Endothelial Cell Stimulation Overcomes Restriction and Promotes Productive and Latent HIV-1 Infection of Resting CD4+ T Cells

Highly active antiretroviral therapy (HAART) is able to suppress human immunodeficiency virus type 1 (HIV-1) to undetectable levels in the majority of patients, but eradication has not been achieved because latent viral reservoirs persist, particularly in resting CD4⁺ T lymphocytes. It is generally understood that HIV-1 does not efficiently infect resting CD4⁺ T cells, and latent infection in those cells may arise when infected CD4⁺ T lymphoblasts return to resting state. In this study, we found that stimulation by endothelial cells can render resting CD4⁺ T cells permissible for direct HIV infection, including both productive and latent infection. These stimulated T cells remain largely phenotypically unactivated and show a lower death rate than activated T cells, which promotes the survival of infected cells. The stimulation by endothelial cells does not involve interleukin 7 (IL-7), IL-15, CCL19, or CCL21. Endothelial cells line the lymphatic vessels in the lymphoid tissues and have frequent interactions with T cells in vivo. Our study proposes a new mechanism for infection of resting CD4⁺ T cells in vivo and a new mechanism for latent infection in resting CD4⁺ T cells.     

ADAR1 Facilitates HIV-1 Replication in Primary CD4+ T Cells

Unlike resting CD4⁺ T cells, activated CD4⁺T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4⁺T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4⁺T cells.     

Emerging role of ISG15 in antiviral immunity

Cells express a plethora of interferon-stimulated genes (ISGs) in response to viral infection. Among these is ISG15, a ubiquitin-like protein (UBL) that can be covalently attached to both host and viral proteins. Here we review recent advances toward understanding the role and mechanism of ISG15 modification in antiviral defense.     

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an “immune exhaustion”, with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28⁻CD57⁺CD8⁺ T cells between the groups. However, the frequency of Tim-3⁺CD8⁺ and Tim-3⁺CD4⁺ exhausted T cells, but not PD-1⁺ T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1⁺CD8⁺ T cells were directly associated with T cell immune activation in children. The frequency of Tim-3⁺CD8⁺ T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.     

Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo

The innate immune response, and in particular the alpha/beta interferon (IFN-alpha/beta) system, plays a critical role in the control of viral infections. Interferons alpha and beta exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-alpha/beta. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-alpha/beta receptor (IFN-alpha/betaR)(-/-) mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.     

Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2′-5′-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.     

LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction

LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS ^−/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS ^−/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.     

The IFITM proteins inhibit HIV-1 infection

Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.     

Immune reconstitution in lymphoid tissue following potent antiretroviral therapy

During untreated HIV-1 infection, a chronic state of immune activation and inflammation develops at the lymphoid tissue sites of viral replication. The early effect of potent combination drug therapy is a reduction in peripheral viral burden and a reduction in the production of inflammatory and type 1 cytokines. Further along in treatment there are trends toward normalization in the frequencies of CD8⁸ T-cells, CD4⁺ CD45RA⁺ cells, as well as CD4⁺ CD45R0⁺ cells. Finally, the CD1a⁺ dendritic cell network is re-established and germinal centers are reformed. Although this restoration of the lymphoid dynamic form is coupled to a reconstitution of peripheral blood T-cell function in vitro and by skin testing, sterilizing immunity to HIV-1 does not develop. Furthermore there is no heightened development of cytotoxic CD8⁺ T-cell function at the site of HIV-1 latency. This is evidenced by a massive recrudescence of HIV-1 viral replication within lymphoid tissue when therapy is stopped. The development of supplemental therapies, which reconstitute anti-HIV-1 immunity, will be required. Specific defects in anti-HIV-1 activity which occur in lymphoid tissue during infection include a downregulation of perform expression by cytotoxic T-cells, the down regulation of the TCR signal transducing chain CD3ζ, and inadequate CD4⁺ T-cell help within the tissue compartment of immune regeneration.     

Fighting mycobacteria through ISGylation

The role of ISGylation in humans has been a long-standing question. A recent groundbreaking study by the Casanova group shows it is essential in the defence against mycobacterial disease but dispensable for other types of infection.Bogunovic et al. (2012). Science, Epub: Aug 2. DOI: 10.1126/science.1224026Our bodies use interferon (IFN) signalling as a central pathway to limit the spread of pathogens, such as viruses, bacteria and parasites. After pathogen exposure, IFN production leads to the activation of immune cells—such as natural killer cells, macrophages and T lymphocytes—which mediate pathogen clearance. There are two main types of IFN signalling, type I and II. In type I signalling, IFNs α and β—produced mainly by leukocytes and fibroblasts, respectively—stimulate macrophages and natural killer (NK) cells to initiate an antiviral response. In type II signalling, IFNγ from activated T cells and NK cells potentiate type I signalling and promote inflammation. During type I signalling IFNα and IFNβ bind to their cognate receptors, which results in the induction of IFN-stimulated genes (ISGs). A key function of ISGs is to interfere with viral replication, hence the name interferon. One of the most strongly induced ISGs is ISG15, a small protein consisting of two ubiquitin folds connected with a hinge spacer, thus resembling di-ubiquitin [1]. It has been proposed that ISGylation is antiviral in mice, but its effects on human virus infection or other functional roles have been a long-standing question in the field. A study by the Casanova group provides important new insights into the role of ISG15 in humans, showing it is essential in the defence against mycobacterial disease but dispensable for other types of infection [2].Similarly to ubiquitin, ISG15 can be conjugated to other proteins and several hundred targets have been suggested from proteomic studies [3]. However, few targets have been carefully evaluated, among them JAK1, STAT1, ERK1/2, PLCγ1, p63, PML-RARα, UBC13, filamin B and several viral proteins. In analogy to the ubiquitin system, ISG15 conjugation is mediated by an enzymatic cascade consisting of an E1 activating enzyme Ube1L, an E2 conjugating enzyme UbcH8 and a HECT-domain containing E3 ligase HERC5 (Fig 1A). Notably, both ISG15 and the E1/E2/E3 cascade are induced by type I IFN signalling.Open in a separate windowFigure 1ISGylation and its function in the antimycobacterial response. (A) The ISG15 conjugation system. ISG15 is activated by an E1 enzyme and conjugated to substrates (green) by the action of E2 and E3 enzymes. HERC5 (HERC6 in mice) is the main ISGylating E3. (B) Mycobacterial infection induces IFNα/β production, which stimulates ISG15 synthesis and secretion in granulocytes. Secreted ISG15 can then activate NK cells to produce IFNγ, which stimulates immune system cells. See text for details. (C) HERC5 has been described to bind to polysomes and to promote cotranslational ISGylation of newly synthesized proteins. EFP, oestrogen-responsive finger protein; HERC5/6, HECT and RLD domain containing E3 ubiquitin protein ligases 5/6; HHARI, human homologue of Drosophila ariadne; IFNα/β/γ;, interferon α/β/γ; IFNαR, interferon α receptor; IL-12, interleukin 12; ISG15, interferon-stimulated gene 15; Lys, lysine; mRNA, messenger RNA; NK, natural killer; TLR, Toll-like receptor; UbcH8, ubiquitin conjugating enzyme H8; Ube1L, ubiquitin activating enzyme E1-like.Knowledge of the biological functions of ISGylation comes mainly from the analysis of knockout mice for ISG15, Ube1L and from in vitro studies. ISG15^−/− mice are more prone to infection by certain viruses, such as Sindbis, influenza A/B and herpes simplex 1 [4]. Ube1L^−/− mice are also sensitized towards Sindbis and influenza infections [5]. Furthermore, ISG15 can inhibit the budding of certain viruses and modify viral proteins, and some viruses have developed strategies to inhibit ISGylation, underscoring the function of ISG15 and ISGylation in the antiviral response [6]. However, other viruses—such as vesicular stomatitis and lymphocytic choriomeningitis virus—have similar effects on ISG15^−/− and wild-type mice [7], suggesting specialized functions of ISGylation after viral infection. In addition, a closer look at the role of ISG15 in regulating human viruses complicates the picture further: ISG15 has been found to stimulate rather than inhibit hepatitis C virus production in vitro, probably by preventing the degradation of viral proteins through competition between ISGylation and ubiquitylation [8]. Considering the limited number of viral infections common to both mice and man, it has been difficult to extrapolate what the in vivo functions of ISGylation might be in humans.The Bogunovic et al [2] study is a clear step forward in our understanding of ISG15 function in infection biology. By analysing patients with the rare paediatric syndrome, Mendelian susceptibility to mycobacterial disease (MSMD), they identified mutations in ISG15 that lead to its loss of expression and suggest that these mutations cause the disease. Importantly, these patients do not suffer from increased sensitivity to viral infections but show severe clinical symptoms when exposed to weakly pathogenic mycobacteria, such as Mycobacterium bovis.As has been shown in ISG15^−/− and Ube1L^−/− mice, Bogunovic et al found that cells from MSMD patients lack ISG15 expression after IFNα/β stimulation, but other ISGs are induced normally, confirming that ISG15 is not essential to elicit an IFN response [2]. Furthermore, patient cell lines are not more susceptible to infection by viruses such as herpes simplex virus, Sindbis virus and vesicular stomatitis virus. Secretion of ISG15 by granulocytes from gelatinase and secretory granules is probably an important process in response to mycobacterial infection that cannot be triggered by, for example, bacterial lipopolysaccharides (Fig 1B). Monocytes and lymphocytes are known to secrete ISG15, and Casanova and colleagues show that even transfected HEK293T cells can do so—suggesting that ISG15 is both an intracellular and a secreted protein—independently of the cellular context. The main function of secreted ISG15 seems to be the triggering of IFNγ release, preferentially from NK cells, but also from T cells. Interestingly, IFNγ secretion is also stimulated by modified ISG15 that can no longer be conjugated to target proteins. This indicates that immune cells either have an ISG15 receptor or that secreted ISG15, which is endocytosed, can induce a response without being conjugated to an intracellular target. Importantly, ISG15 secretion is lost in cells from MSMD patients and, consistently, MSMD leukocytes stimulated with mycobacteria produce greatly reduced amounts of IFNγ, which can be restored by providing recombinant ISG15. In addition, the study also demonstrates that ISG15^−/− mice are more susceptible to mycobacterial infection than their wild-type littermates.Thus, the Bogunovic study identifies a common function of ISG15 in vertebrates: the ability to counteract mycobacterial infections by activating NK cells. The data also suggest that some viruses and bacteria must share pathogen-associated molecular patterns that resemble those of mycobacteria, thus initiating a similar response that involves IFNα/β, which is required for ISG15 induction. The initial activation of this mechanism by mycobacteria remains to be identified and seems to be complex, as treatment of macrophages with IFNα/β during mycobacterial infection has been shown to induce the loss of their mycobacteriostatic properties [9]; partly at odds with the conclusions from this study. These discrepancies notwithstanding, the new insights reveal an essential function for ISG15 in antimycobacterial signalling. Mycobacterial infections are hard to fight and thus the finding that ISG15 is a major effector between granulocytes and NK cells might help develop new treatment strategies. Other cellular mediators, such as the macrophage–T-cell pathway, are a crucial host defence against pathogenic (M. tuberculosis) and nonpathogenic mycobacteria (M. bovis), as well as salmonella, in other variants of MSMD. Hence, Casanova and colleagues speculate that the granulocyte–NK-cell pathway, which involves ISG15 and IFNγ, might constitute a more innate complement to the macrophage–T-cell pathway, which requires IL-12/IFNγ. However, they also report a synergistic effect of a combined treatment of cells with ISG15 and IL-12, which rather argues that the granulocyte–NK-cell and the macrophage–T-cell systems act together. Furthermore, both granulocytes and macrophages express Toll-like receptors (TLRs) that recognize mycobacterial structures—such as TLR2 for the lipomannan of M. tuberculosis.Finally, whether covalent modification of target proteins within cells by ISG15 is important during infection remains unclear. It is difficult to speculate what the main effects would be, as ISG15 modifies targets in diverse cellular pathways. Notably, the E3 ligase for ISG15 conjugation HERC5 has been shown to be associated physically with polysomes, leading to cotranslational ISGylation of newly synthesized proteins, which probably inhibits protein function in general (Fig 1C; [10]). Thus, ISG15 might have two roles in preparing cells to fight pathogens: intracellular proteome remodelling and initiating antimicrobial signalling pathways. It will be interesting to see if and how these functions intersect.