Date Published: October 10, 2013
Publisher: Public Library of Science
Author(s): Emilio Yángüez, Alicia García-Culebras, Aldo Frau, Catalina Llompart, Klaus-Peter Knobeloch, Sylvia Gutierrez-Erlandsson, Adolfo García-Sastre, Mariano Esteban, Amelia Nieto, Susana Guerra, Pinghui Feng.
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.
The host innate immune response represents a critical initial line of defense against invading pathogens, and the magnitude of this early response can influence the course of disease progression. One of the earliest host responses to viral infection is the production of type I interferon (IFN-α and-β) and the subsequent upregulation of IFN-stimulated genes (ISGs) , . These ISGs generate an antiviral state and play an important role in determining the host innate and adaptive immune responses. One of the most highly induced genes in the IFN response is ISG15, which encodes a small UBL protein of 17 kDa that forms covalent conjugates with cellular proteins mediating considerable antiviral responses . During viral infection in mice, ISG15 exists in three different forms: unconjugated within the cell, conjugated to target proteins and released into the serum . When ISG15 is secreted, free ISG15 can function as a cytokine that modulates the immune response. For example, free ISG15 can activate natural killer (NK) and cytotoxic T-cells, stimulate IFN-γ production and induce dendritic cell maturation and neutrophil recruitment , . In addition, antiviral activity associated with protein ISGylation has been described in vitro and/or in vivo for both DNA and RNA viruses, including influenza A and B viruses , Sindbis virus , , hepatitis B virus , herpes simplex type-1 virus , vaccinia virus , vesicular stomatitis virus , , lymphocytic choriomeningitis virus , respiratory syncytial virus , HIV-1  and Ebola virus . In contrast, free ISG15, but not ISGylation, promotes antiviral responses against Chikungunya virus infection .
The host innate immune response, including the production of type-I IFN, represents the primary line of defense against viral pathogens. Of the hundreds of IFN-stimulated genes (ISGs) discovered to date, ISG15 was one of the firstly identified and shown to encode an ubiquitin-like protein modifier . ISG15 knock-out mice are more susceptible to infection by several viruses, pointing out the relevance of this molecule in the antiviral response in animal models . However, the underlying causes of the enhanced susceptibility of ISG15−/− mice and the cell types involved are only weakly defined. In order to clarify these questions, we evaluated the role of ISG15 in VACV replication in different cells types. Both MEFs and dendritic cells (not shown) VACV-infected were unaffected by the lack of ISG15 in the course of infection. In contrast, ISG15−/− peritoneal macrophages showed a clear resistance to VACV-induced cell death in comparison with wild type control macrophages where VACV replication is abortive and infectious progeny is not released . Whereas WT macrophages became apoptotic and die after infection, in ISG15−/− macrophages, in which VACV infection is also abortive, no apoptosis was observed, as evidenced by the absence of PARP cleavage and CPE (Fig. 2). Apoptosis after infection with many types of viruses is generally considered as a self-defense mechanism , as loss of host cell activity should impair virus propagation. For instance, apoptotic cells are engulfed and digested in lysosomes of phagocytes . Moreover, a higher increase in the expression of ISG15 and the protein ISGylation levels were observed in ISG15+/+ infected macrophages when compared to infected MEFs, indicating that the factor involved in the different phenotype among VACV infection in these cells could be modified by ISG15.