In this study we show that specific induction of DNA damage by gamma irradiation was associated with activation of type I IFN signaling that was required for enhanced antiviral gene expression. Type I IFN signaling in irradiated cells was modified by select DDR proteins, including the DNA damage sensor complex and ATM, a central DDR regulatory kinase. Furthermore, IRF-1, but not IRF-3 and IRF-7, was required for the optimal expression of antiviral ISGs examined here, further differentiating innate immune signaling in irradiated cells from the classical signaling induced by pattern recognition receptors. Importantly, the type I IFN responses induced by DDR were actively blocked in the context of MHV68 infection and conferred an antiviral state that attenuated viral replication. Thus, induction of type I IFN signaling in the context of DNA damage, as shown here, extends the DDR signaling landscape to include innate immune responses.
In spite of many previous publications investigating gene expression changes in response to irradiation, the direct connection between DDR and type I IFN has not been appreciated, since a majority of gene expression studies were conducted in transformed cell lines with altered DDR and IFN responses and/or were limited to well-established DDR effects, such as apoptosis and cell cycle regulation. Several studies have examined changes in gene expression in response to irradiation in vivo
, with the most recent study reporting differential gene expression associated with immune activation (11
). However, it still remains to be determined whether immune activation in vivo
is simply a response to tissue damage induced by irradiation. Irradiation was also shown to stimulate the expression of ligands for NKG2D, an activating NK receptor; however, the involvement of immune signaling pathways in NKG2D ligand expression has not been defined (12
). In a recent study published during the preparation of this manuscript, the Reich group had shown induction of IFN-α and -λ, but not IFN-β, in primary human monocytes treated with etoposide (8
). The differences in the form of type I IFN induction observed between our study and the work of Brzostek-Racine et al., are likely to stem from different experimental approaches (immediate versus delayed responses and a single dose of gamma irradiation versus 24 h of etoposide treatment) and different sources of tissue (human versus mouse).
Based on the studies described here, we propose the following working model of the cross talk between DDR and type I IFN responses (). DNA damage activates transcription of type I IFN. In irradiated primary macrophages, type I IFN signaling downstream of IFN receptor intersects with select DDR components, such as the MRN complex and ATM, and these interactions contribute to regulation of Stat1 phosphorylation. In addition, type I IFN signaling and IRF-1 cooperate to induce the transcription of antiviral ISGs in irradiated cells. One prediction of this model is that viruses uncouple DDR induction from type I IFN activation, a prediction corroborated by the results of our study (). While gamma irradiation is unlikely to be present during viral infection in vivo, low levels of DNA damage may be induced in both infected and bystander cells via the generation of reactive oxygen species and other oxidative moieties released in the context of inflammation. Thus, induction of type I IFN by DDR may reflect an innate immune host defense system that has evolved to confer a protective, antiviral state in vivo that attenuates viral spread prior to the generation of adaptive immune responses. To support this hypothesis, MHV68 spread was attenuated in irradiated macrophage cultures, and this attenuation was in part mediated by type I IFN signaling ().
The plethora of virus interactions with host DDR has been recently appraised in a number of excellent reviews (28
). Importantly, several viruses directly induce DDR and subsequently usurp DDR components for efficient viral replication. MHV68-encoded viral kinase initiates DDR in infected macrophages by mediating serine 139 phosphorylation of H2AX, including at the core promoter of gene50
, an immediate-early gene encoding a key viral regulator of the lytic replication cycle (35
). H2AX and ATM are subsequently usurped by MHV68 to facilitate viral gene expression and replication (35
). Furthermore, several herpesvirus kinases target Tip60, an important activator of ATM, to stimulate viral gene expression and replication (24
). Active induction of DDR is not limited to the herpesvirus family, since human papillomavirus E1- and simian virus 40 large T antigen-induced DDR is commandeered for viral replication (13
). Viruses that actively induce DDR are most likely to use several mechanisms to uncouple the connection between DDR and type I IFN signaling. Indeed, the induction of ISGs by irradiation was blocked in MHV68-infected macrophages, likely by using multiple mechanisms, including the expression of orf36 viral kinase ( and ). Intriguingly, our findings may also explain why certain DDR components are inhibited in virus-infected cells. The MRN complex is targeted for degradation or is relocalized to viral replication compartments in adenovirus and Epstein-Barr virus infection, respectively. However, downstream DDR events, such as γH2AX, are induced in infected cells (21
). It is tempting to speculate that these viruses target the MRN complex to attenuate its role in activation of ISG transcription. Indeed, the transcription of GBP-1 and viperin was attenuated in MHV68-infected Nbs1 hypomorphic macrophages ( and ). Other DDR components inhibited in the course of viral infection may also participate in the DDR-immune signaling cross talk. Restoring the DDR type I IFN cross talk in infected cells may constitute a potent antiviral therapy approach, especially for viruses that actively induce DDR.
In addition to its antiviral nature, the induction of type I IFN signaling in the context of DDR may also participate in cancer development and treatment. DDR is activated in a majority of premalignant lesions (5
), and the induction of type I IFN signaling in this context may contribute to the DDR tumor-suppressive effects. Type I IFN has potent antitumor activity, can facilitate the recruitment of immune cells, and is already in wide use as an adjuvant therapy for melanoma, renal cell carcinoma, and lymphoma (58
). Type I IFN is also induced in a mouse model of local radiotherapy, and this induction facilitates the recruitment and activation of immune cells (9
). Thus, harnessing the interaction between a tumor suppressor and immune signaling pathway could improve cancer therapy and prevention approaches.
Because of the novel nature of the DDR-type I IFN signaling cross talk, many questions remain to be addressed in future studies, including the mechanism of type I IFN induction by DDR. The Reich group reported an attenuated IFN-λ and -α transcription in IRF-7-deficient mouse embryonic fibroblasts in response to long-term etoposide treatment (8
). In contrast, we show that the induction of viperin and Mx1 is not attenuated in IRF-7-deficient irradiated macrophages ( and ), suggesting that several IRF factors may collaborate to induce type I IFN-dependent transcriptional responses in irradiated cells. It is also possible that DNA fragments generated by irradiation are recognized by novel DNA sensor proteins with subsequent activation of type I IFN transcription. Recently, IFN-γ-inducible protein 16 (IFI16) was proposed to be a sensor of Kaposi's sarcoma-associated herpesvirus genome in infected nuclei (17
). It is tempting to speculate that IFI16 or a similar nuclear sensor may be involved in sensing large fragments of DNA or DDR-associated changes in chromatin structure (67
) to mediate the induction of type I IFN transcription. These sensors are likely to be distinct from the classical sensors of double-stranded DNA breaks, since in our studies IFN-β was induced in cells deficient in ATM or a functional MRN complex ( and ).
We observed an immediate decrease in Stat1 levels and phosphorylation in response to irradiation, with subsequent upregulation of Stat1 phosphoprotein and total protein levels in irradiated cells. This observation was unexpected since Stat1 is a long-lived protein (51
). The decrease in Stat1 protein levels and phosphorylation following irradiation likely contribute to the delayed induction of ISGs in irradiated macrophages (6 to 8 h postirradiation) in spite of increased IFN-β transcription evident as early as 1 h postirradiation (). An important question to be resolved in future studies is the mechanism by which Stat1 levels and phosphorylation are regulated in irradiated cells and the physiological relevance of such regulation.
It is also likely that other immune signaling pathways are activated by DNA damage. The immune signaling landscape evoked by DNA damage may be further modified by cell type and differentiation stage, since DDR signaling differs in actively proliferating and terminally differentiated cells (39
) and type II IFN signaling evokes differential gene expression in macrophages and mouse embryonic fibroblasts (43
). An unbiased identification of the immune signaling landscape in the context of virus-induced and physiological DDR is an important future undertaking.