IFN Regulatory Factor 3 in Health and Disease

Viral infections usually occur in specific cell types, based on viral attachment proteins and cellular receptors. The interaction between virus and cell is the necessary first step in human viral infectious diseases that have significant morbidity and mortality. However, the immediate innate immune responses of infected cells determine whether viruses succeed or whether hosts avoid morbidity and mortality. To counteract innate immune responses and gain the requisite foothold for replication, viruses evolved evasion strategies that obstruct key innate immune responses. One factor that is the target of viral evasion is the cytosolic protein IFN regulatory factor (IRF) 3 (1). However, unlike other IRFs (2), IRF3 and the closely related IRF7, that arose evolutionarily with the emergence of jawed vertebrates (3), are distinguished as key targets for viral virulence factors that evade innate immune responses. The importance of IRF3 is evident in that it has deciding roles in resistance against many viral infections, including West Nile virus (4), Theiler’s virus, influenza A virus (5), vesicular stomatitis virus (6), and hepatitis C virus (7).

IRFs are a family of proteins that are transcription factors for a wide range of genes associated with innate immune and adaptive immune responses (8). The first two IRFs that were discovered (IRF1 and IRF2) (9) were found to be involved with the expression of IFNs (10) and IFN-stimulated genes (ISGs) during innate antiviral immune responses (11). In these roles, IRF1 was found to induce, whereas IRF2 was found to repress, IFNs and ISGs (12). Later, it was realized that two other proteins, ICSBP and IFN-α–stimulated transcription factor (ISGF) 3γ, already under investigation as antiviral factors (13), were indeed part of the IRF family (14). Thus, ICSBP and ISGF3γ were designated as IRF8 and IRF9, respectively (8, 15). In their respective roles in innate antiviral immunity, IRF8 is either an activator (16) or repressor of IFNs (14), whereas IRF9 associates with STAT1/STAT2 dimers to form the ISGF3 complex as an inducer of IFNs (17). Although the roles for these IRFs in antiviral immune responses in cells are clear, all of them are induced after activation of IRF3.

Unlike other IRFs, IRF3 is constitutively expressed in cells and therein key to rapid antiviral innate responses (2). The discovery of the 427 aa IRF3 (419 aa in mouse) and the understanding of its properties is the basis for the current model for the immediate IFN and ISG expression during viral infections. The details of the model indicate that IRF3 activation is critical to the early establishment of an antiviral state after virus infection of cells. Therefore, in most cells, IRF3 is preformed but latent, so that upon viral encounters, it is rapidly activated by cell signaling pathways linked to membrane-bound and cytosolic pattern recognition receptors (PRRs) that recognize microbial molecular components. It was found that hyperphosphorylation of IRF3 promotes its homodimerization (18), its association with additional cytosolic proteins (1), its nuclear localization, its transcriptional activity at early innate antiviral genes (19), and even its nuclear export (20). Rapid activation of IRF3 impedes virus infection of cells so much that most human viruses have evolved virulence factors that prevent, reduce, or delay the hyperphosphorylation, dimerization, nuclear localization, and/or the transcriptional activity of IRF3 (21).

The critical role for IRF3 in host resistance to virus infection was confirmed in 2000 with the deletion of the IRF3 gene in mice by Taniguchi and colleagues (22). IRF3^−/− mice did not exhibit any impact on T or B cell development but did show significantly increased susceptibility to viral infection that was accompanied by impaired induction of type I IFNs, IRF7, and IRF9. In contrast to development of T and B cells, our group and several others showed that T cells from these IRF3^−/− mice have impaired production of several cytokines, including IL-17, and failed to sustain memory T cells following influenza virus infection (23). The primacy of IRF3 in the immediate antiviral response was also shown in the protein stability of IRF3 compared with IRF7, the latter having a very short half-life (24).

Because IRF3 is constitutively expressed and not induced in cells, only a few investigations have identified IRF3 gene promoter elements. However, various cell types have different levels of constitutive IRF3 expression, suggesting the importance of some transcriptional regulation of IRF3 expression. Those studies showed that some transcription factors positively, whereas others negatively, regulate IRF3 promoter activity. SBF/Staf, Sp1, YY1, Egr2, and Sp3 are positive regulators (25) of IRF3 gene expression, whereas E2F1 is a negative regulator of IRF3 expression (26). Interestingly Egr2 is highly expressed in cells of the nervous system (27) and developing T cells (28), both of which have somewhat higher levels of constitutive IRF3.

Transcription factors for expression of IRF3 engage its gene locus located on human chromosome 19q13.3–13.4 (2). The structural gene is composed of eight exons, which include a 5′UTR. Several laboratories uncovered critical domains of IRF3 that are involved in DNA binding, autoregulation, nuclear localization, nuclear export, and association with other transcription factors (Fig. 1). At first, IRF3 is rapidly phosphorylated after virus infection, and this occurs at its C terminus by kinases that are part of several PRR pathways. IRF3 phosphorylation is linked to PRR pathways that include TLR3 (29), TLR4 (30), retinoic acid–inducible gene I (RIG-I) (31), melanoma differentiation-associated protein 5 (MDA5) (32), and stimulator of IFN genes (STING) (33). Following IRF3 phosphorylation, IRF3 dimerizes and then localizes to the nucleus, where it functions as an integrating transcription factor for promoters of type I IFNs, several cytokines, as well as several antiviral ISGs, such as IFN-induced proteins with tetratricopeptide repeats (IFIT) 2 (Fig. 2). The N-terminal region of IRF3 has a well-conserved DNA binding domain stretching from aa 1 to 120, which is followed by a linker domain with sequences for nuclear localization (34), autoinhibition (inhibitory domain [ID] 1) (18), nuclear export, and IRF association (Fig. 1). The apoptosis activation (35) and signal response domains (SRDs), which include ID2 (18), are positioned near the C terminus. During IRF3 latency, ID1 of the linker and ID2 of the SRD associate together, preventing both IRF3 binding with IRFs and nuclear localization. Phosphorylation at SRD/ID2 dissociates ID1 from ID2, allowing for homo- or heterodimerization with IRFs at the IAD and nuclear localization (36). Thus, IRF3 activation occurs quickly following virus infection but is dependent upon distinct kinases in several PRR pathways, any of which can be targeted by viral evasion.

Activation of IRF3 occurs through well-characterized PPR pathways (Fig. 3). Viral ssRNA and LPS engage TLR3 and TLR4, respectively, to activate TRIF (30, 37). In contrast, 5′ppp dsRNAs bind to RIG-I like receptors (RLRs) (RIG-I/MDA5)– mitochondrial antiviral signaling protein (MAVS) (38, 39), whereas cytosolic DNA activation of IRF3 involves the cyclic GAMP synthase (cGAS)/STING axis (40). cGAS association with dsDNA triggers synthesis of cGAMP, which then binds to STING. Moreover, DNA-dependent activator of IRFs (DAI) is another cytosolic DNA sensor (41). In the end, engagement of TLR3, TLR4, RLR–MAVS, cGAMP/STING, and DAI brings about activation of TBK1/IKKε, resulting in hyperphosphorylation of IRF3 at several serines in the SRD (aa 385, 386, 396, 398, and 402) (42), thereby relieving autoinhibition. In addition to TBK1, the PI3K–Akt pathway was suggested (43) and then shown (44) to be involved in IRF3 activation. In addition, several reports suggest that MAP kinases (45, 46) also phosphorylate IRF3 at serine 171 (173 in human IRF3) within the ID1 domain (47), thereby preparing IRF3 for homodimerization and nuclear localization (Fig. 2). The latter takes place simultaneous with its association to CBP/p300 (48) and β-catenin (49, 50), after which the complex participates in transcriptional activity at the IFN-β enhanceosome.

Besides IRF3, this enhanceosome contains elements for ATF-2, c-Jun, IRF3/7, and NF-κB p50/RelA (1, 51). The specific promoter elements at the enhanceosome to which IRF3 binds are termed IFN-stimulated response elements (ISREs), with the consensus sequence 5′-A/GNNGAAANNGAAA-3′ (42, 52). The ISREs to which IRF3 homodimers bind are also the same for heterodimers of IRF3 with IRF5 or IRF7 (53). Still further, IRF3 associated with NF-κB subunits can bind to ISRE half-sites, whereas the NF-κB half-binds to κB elements (54). The induced IFN-β from this initial IRF3 activity then stimulates expression of several ISGs as well as IRF7 (22). Subsequent activation of IRF7 (55) results in its heterodimerization with IRF3, with enhanced transcriptional activity, and with a heightened second wave of IFN-β and ISGs (56). However, monocytic lineage cells have stronger initial IFN-β expression to viruses because IRF3 in these cells associates and synergizes with IRF8, which is expressed to a greater extent in myeloid cells (57). In the end, augmented second-wave antiviral responses are pivotal to enhanced control of virus infections during innate antiviral immune responses.

Beside its role in expression of IFN-β, IRF3 contributes to expression of other cytokines and chemokines. We have shown that IRF3 binds to the IL-23 p19 promoter region (58), so that without the presence of this IRF3 element, p19 expression is greatly diminished. In addition, expression of RANTES (CCL5) and IP-10 (CXCL10) (59, 60), as well as ISGs, such as IFIT1 and IFIT2 that are involved in antiviral responses (61), are completely dependent on IRF3.

Beyond its role as an inducer of critical antiviral proteins immediately, IRF3 also plays a critical role in transcriptionally dependent and independent initiation of apoptosis (Fig. 2). In this case, activated IRF3 induces Noxa and Puma (62), thereby decreasing activity of Bcl-2 proteins Mcl-1 (63) and Bcl-xL (64) and increasing Bax/Bak activity, cytochrome c from mitochondria, apatosome activation, and programmed cell death (65). Alternatively, activated IRF3 itself associates with Bax at the mitochondrial membrane, thereby directly initiating release of cytochrome c and apoptosis (63). Moreover, ubiquitination of IRF3 plays a role in its induction of apoptosis, whereby lysine 193, 313, or 315 of IRF3 is ubiquitinated by LUBAC ubiquitinase, which is part of a complex of proteins associated with MAVS at mitochondria (66). Thus, the role for IRF3 in innate antiviral responses is through transcriptional activation of key antiviral proteins and cytokines but also induction of apoptosis of the infected cell.

Although activation of IRF3 is well characterized, IRF3 is tightly regulated. One aspect of IRF3 regulation stems from investigations into the effects of EBV on IRF3 activity. In these studies, EBV BGLF4 kinase was shown to inhibit IRF3 activation by phosphorylating three serine/threonine (S123, S173, T180) residues in the linker region (67). This sequence in the linker domain, revealed it to be a suspected targeted of glycogen synthase kinase-3 (GSK-3) (68). Therefore, subsequent studies showed that this linker region of IRF3 is a substrate for GSK-3 phosphorylation (69). Therefore, it appears that this endogenous mechanism to regulate IRF3 activity is exploited by EBV to depress immediate IRF3 activation during its infection of cells. IRF3 activity is also negatively regulated by its proteasomal degradation after polyubiquination. Cellular proteins RAUL (70), TRIM26 (71), and FOXO1 (72), all of which are enhanced by propyl isomerase Pin1 (73), polyubiquitinate IRF3 at K48. In addition, IRF3 acetylation by lysine acetyltransferase 8 (KAT8) was demonstrated to inhibit innate antiviral immunity by acetylation of IRF3 at K359 (74). Cellular protein FLIP-L blocks the interaction between IRF3 and CBP (75), thereby inhibiting IRF3 transcriptional activity. C6orf106 blocks the association of IRF3–CBP/p300 complex with the IFN-β promoter (76). Although many ISGs are induced by IRF3 activity, one such ISG, IFI44L, has a negative feedback effect on IRF3 activity. IFI44L decreases phosphorylation of IRF3, therein decreasing IRF3 activity (77). Finally, certain phosphatases have a negative impact on IRF3 activity. Both protein phosphatase 1 (78) and MAPK phosphatase 5 (79) dephosphorylate IRF3, decreasing its activity prior to its dimerization.

Besides its negative regulation of IRF3, GSK-3 also has a role in augmenting IRF3 activation (68) through its phosphorylation of β-catenin, which is a substrate of GSK-3. In this case, GSK-3 enhances the association of β-catenin with IRF3, thereby facilitating the transcriptional activity of IRF3-CBP/p30-βcatenin. In addition, IRF3 is regulated in a positive manner through methylation and dephosphorylation. NSD3 methyltransferase positively regulates IRF3 activity by methylation of K366 of IRF3 (80). Likewise, PTEN positively regulates IRF3 nuclear localization by dephosphorylating S97 of IRF3 (81). However, Pin1 interaction with IRF3 is blocked by HERC5 that catalyzes conjugation of ISG15 to IRF3, thereby preventing it from interacting with Pin1 (82). Thus, HERC5 has a positive effect on IRF3 activity. Therefore, regulation of IRF3 is equally as complicated as its activation.

One interesting regulatory mechanism of IRF3 activity comes through expression of alternate IRF3 isoforms. Karpova et al. (83) first described an isoform of IRF3 termed IRF3a. IRF3a results from alternatively spliced IRF3 RNA, wherein a significant portion of the DNA binding domain of IRF3 is replaced with a short stretch of amino acids encoded by an alternative exon 1. Thus, IRF3a interferes with the transcriptional activity of IRF3. Additional IRF3 isoforms were demonstrated (84), in which a longer, 452-aa C-terminal variant of IRF3 (IRF3CL) was demonstrated, wherein the final 125 aa of IRF3 differ as a result of a 16-bp insert into exon 7 of IRF3 mRNA. Another discovery showed that in C57bl/6ByJ mice splicing of IRF3 RNA is inefficient in this strain such that too many IRF3 mRNAs retain intron 5, resulting in dysfunctional IRF3 (85). Interestingly, upon infection with Listeria monocytogenes, these mice exhibit decreased induced IFN-β but with reduced L. monocytogenes bacterial loads in spleen and liver during this facultative intracellular pathogen infection.

Besides IRF3 isoforms, evidence has accumulated that functionally deficient polymorphisms in IRF3 can enhance susceptibility to viral infections. Several studies showed that IRF3 is required for resistance to HSV and preventing HSV lethal encephalitis (HSE) and specific polymorphisms in the IRF3 gene enhance susceptibility to HSE. In one such case, a child with IRF3 R285Q polymorphism developed HSE (86). Cloning of IRF3 R285Q showed that it failed to become phosphorylated at S386 following infection of cells with HSV, resulting in its transcriptional deficiency. In addition to exogenous viruses like HSV, IRF3 has been shown to play a significant role in keeping endogenous retroviruses (ERV) at bay. ERVs comprise a significant portion of the human genome. Although most remain latent, several ERVs partially or totally reactivate and are then associated with diseases such as cancer. Several reports show that expression of ERV in IRF3^−/− mice is increased (87), suggesting that activation of IRF3 controls reactivation of ERVs and their associated diseases.

Although much is known about the antiviral role for IRF3, its role in antibacterial immunity is beginning to be appreciated. In addition to DNA viruses, cytosolic foreign DNA is a feature of facultative intracellular bacterial infections with Listeria or mycobacteria that persist in macrophage lineage cells (88), which is the key component of infectious diseases with these pathogens. During Listeria infections of macrophage, an abrupt expression of IFN-β occurs within 4 h, and this expression is completely dependent upon IRF3. It is also dependent on listeriolysin O, a key pore-forming virulence factor of Listeria that permits its access to the cytosol from the phagosome (89–92). Moreover, Listeria-induced IFN-β production, although dependent on IRF3, is also independent of MyD88, TRIF, TLR4, TLR9, as well as NOD2.

Like L. monocytogenes, Mycobacterium tuberculosis is a facultative intracellular pathogen that causes disease by chronic infection of macrophage lineage cells. Moreover, M. tuberculosis activation of IRF3 also requires its access to the cytosol. M. tuberculosis–infected macrophages produce IFN-β as well as other ISGs such as IFIT1 and IFIT2 (93, 94). Induction in this case is dependent on IRF3 as well as ESX-1 pore-forming and SecA2 translocase virulence factors of M. tuberculosis. SecA2 translocase transports M. tuberculosis RNA into the lumen of the phagosome, whereas ESX-1 provides the pore in the phagosome for entry of the M. tuberculosis RNA and DNA into the cytosol. Interestingly, M. tuberculosis induction of IFN-β was not dependent on TRIF or MyD88, but was dependent on STING and MAVS. In addition, M. tuberculosis ESX-1 secretion also involves secretion of ESAT-6 protein virulence factor (95) into the lumen of the phagosome, whereby it activates IRF3 and induces IFN-β via the TLR4 pathway. Although there are multiple pathways for activation of IRF3 during M. tuberculosis infection, remarkably, IRF3 deficiency protected mice from M. tuberculosis pathology, morbidity, and mortality. Therefore, the model of L. monocytogenes and M. tuberculosis pathogenesis asserts that phagosomal DNA and RNA of L. monocytogenes and M. tuberculosis gain cytosolic access through LLO and ESX-1, respectively, thereby triggering the STING/TBK1/IRF3 axis that is required for pathogenesis.

Legionella and Salmonella are other human intracellular pathogens that, like Listeria and M. tuberculosis, induce IFN-β production from infected cells via the MAVS/IRF3 axis (96) or PKR (97), respectively. In this case, activation of IRF3 was demonstrated to decrease Legionella and Salmonella replication in infected cells.

In contrast to these bacterial pathogens, Staphylococcus aureus and Streptococcus pyogenes cause potentially devastating bacterial infections that are primarily extracellular. Nevertheless, the cytosolic DNA sensor IFI204, which contributes to S. aureus activation of IRF3 and IFN-β production, is demonstrated to be important in resistance against this pathogen (98). In this case, IFI204 and IRF3 contribute to development of macrophage and neutrophil extracellular traps that are pivotal to controlling S. aureus infections. In contrast to S. aureus, the S. pyogenes encounter with macrophages or dendritic cells induces IFN-β in a MyD88- and IRF5- but not in a TLR3- or TLR7-dependent fashion (99). Ultimately, the STING–TBK1–IRF3 pathway is necessary for survival of experimental animals with S. pyogenes infection.

Although IRF3 activation during several bacterial infections is mostly beneficial, its activation during Neisseria gonorrhea infections appears to be detrimental to the host. The Andrade group (100) showed that, during N. gonorrhea infection of human and mouse myeloid cells, type I IFNs are abundantly produced through the GAS/STING and TLR4 pathways. Access of N. gonorrhea DNA to the cytosol from the phagosome is independent of its type IV secretion system. In the end, the IRF3 induced type I IFNs impaired bacterial clearance by myeloid cells through a mechanism that included increasing the intracellular iron pool, which is well-known to be critical to N. gonorrhea growth. Thus, in this case, the pathogen N. gonorrhea uses the cGAS/STING/IRF3/IFN-β pathway for persistence.

IRF3 activation through DNA-sensor PRRs also plays a key role in the response to vaccines with adjuvants. Marichal et al. (101) showed that the mechanism of action for Alum adjuvants essentially damages cells at the injection site, causing release of endogenous DNA, thereby activating IRF3 through DNA PRR sensors such as cGAS/STING and DAI. This activation of IRF3 is crucial to the adjuvancy.

Despite its well-known role in innate antiviral immune responses and lack of a role in the development of T cells, we hypothesized that IRF3 in T cells has a role in mature T cell responses to Ag. We showed that IRF3-deficient mice exhibited impairments in effector cytolytic T cells and memory T cell formation, especially expression of granzyme B (23, 102). Moreover, we showed impairments in IFN-γ, IL-17, and Foxp3 expression in T cells responding to anti-CD3/anti-CD28. Interestingly, Fitzgerald et al. (103) showed that IRF3 deficiency protected mice from development of experimental autoimmune encephalomyelitis (EAE). This protection from EAE was shown to be due to decreased development of Th17 T cells, which are known to play key roles in EAE immunopathology. In polarizing in vitro T cells responses, Th17 subset development was also impaired during IRF3 deficiency. A recent report (104) showed that IRF3 plays a transcriptional role in IL-17 expression and therein Th17 development. In that report, IRF3 bound with RORγT, the signature transcriptional factor of Th17 cells, and this association was critical to optimum development of Th17 polarization. Moreover, we recently demonstrated that IRF3 deficiency impairs cytokine and IFIT2 expression of macrophages in response to IFN-γ. However, we showed that some of the impairment was due to deficient production of IFN-β in response to IFN-γ (61). In contrast to its positive role in Th17 development, IRF3 was shown to regulate development of Tfh and B cell Ab responses and to promote development of Th1 responses during Plasmodium infections in mice (105). Likewise, IRF3 was demonstrated to be important for development of Th2 responses during airway allergy responses to house dust mite allergens (106). However, the contribution of IRF3 in this work was mainly through dendritic cells participating in the development of allergen-specific Th2 (107). Therefore, IRF3 clearly has direct roles in T cell responses.

Because of its role in antiviral immunity, it is expected that IRF3 would play some beneficial role in anticancer immunity. Indeed, IRF3 activity appears to contribute to beneficial outcomes in glioblastoma (108, 109) and melanoma (110, 111). Moreover, we demonstrated that IRF3 contributes significantly to NK cell activity (111). However, the greatest contribution of IRF3 during cancer may be in its role during DNA damage responses (DDRs) (112). It was found that activation of IRF3 through the cGAS/STING pathway occurs during chemotherapies using DDR agents (113) and immunotherapies involving checkpoint blockade (114). IRF3 activation in response to DDR was shown to involve its role in expression of RAE1 (115), which is the ligand on tumor cells for NKG2D on NK cells. Binding of RAE1 with NKG2D stimulates NK cell effector function. Moreover, overexpression of IRF3 decreased tumor cell growth in vitro by increasing p53 activity (116). Interestingly glioma cells, which are highly proliferative and metastatic, express low levels of IRF3. Proliferation, migration, and invasion of glioma cells was dramatically decreased with overexpression of IRF3 (109). In contrast, IRF3 appears to also be involved in regulation of STING activity, wherein TBK1 and IRF3 together are required for the p62/SQSTM1–mediated degradation of STING (117). In addition to its regulation of STING, IRF3 also appears to regulate the activity of IKKβ/NF-κB complex that is associated with obesity-related inflammation in the liver (118). Therefore, in addition to the role for IRF3 in immune cells responding to cancer cells, IRF3 plays a beneficial role in tumor cells themselves and in regulating innate responses from tumor cells.

The DDR plays a pivotal role in resistance against DNA damaging anticancer agents. Therefore, DDR inhibitors increase the efficacy of chemotherapeutics, some of which leads to IRF3 activation. Moreover, DDR inhibitors during chemotherapy of tumor cells increased immune checkpoint protein PD-L1. In this case, increased PD-L1 expression with DDR inhibitors was mostly IRF3 dependent (114), and tumor growth inhibition with DDR inhibitors used in conjunction with immune checkpoint blockade was entirely dependent on the cGAS/STING/IRF3 axis. This investigation went on to show that additional benefit in activation of the cGAS/STING/IRF3 axis was due to T cell infiltration into the tumor resulting from increased expression of CXCL10 and CCL5 chemokines.

It is quite clear that most viruses target some aspect of the IRF3 activation pathway. Influenza NS1 proteins of the 2009 H1N1 and H5N1 block IRF3 activation (119). HSV 2 ICP27 protein and ICP0 protein reduce IRF3 activity by binding to IRF3, thereby promoting its degradation (120). Varicella zoster ORF61 protein promotes degradation of IRF3 (121). Hepatitis C virus NS3/4A proteins targets TRIF and MAVS, thereby decreasing IRF3 activity (7). West Nile Virus NS1 protein blocks translocation of IRF3, thereby abrogating its transcriptional activity (122). Porcine reproductive and respiratory syndrome virus NSP1 protein inhibits IRF3 phosphorylation (123). HIV vpu protein decreases IRF3 activity by stimulating its degradation (124, 125). Hepatitis A virus (126) and mouse Theiler virus (127), both picornaviruses, encode leader proteins that block IRF3 dimerization, thereby preventing its complete activation. In contrast, encephalomyocarditis virus 3C protein disrupts the TBK–IKKε–IRF3 complex (128). One mechanism of immune evasion by some viruses is through decoy mimics of cytokines or other factors critical to antiviral immune responses. HHV8 blocks activity of cellular IRF3 by encoding a viral mimic of IRF3 (129). Zika virus NS5 protein, which localizes to the nucleus of infected cells, inhibits nuclear-localized IRF3, thereby preventing its transcriptional activity at the IFN-β enhanceosome (130), whereas dengue virus NS2A and NS4B proteins inhibit TBK1 activation through the RIG-I and MDA5 pathway (131). EBV Rta protein has been shown to decrease IFN-β expression by diminishing production of IRF3 and IRF7 protein levels (132). As mentioned above, EBV BGLF4 kinase phosphorylates several serine and threonine residues of the linker region of IRF3, thereby suppressing IRF3 activation (67).

With the devastating pandemic of SARS-CoV-2 (COVID-19) occurring in 2020, evasion of IRF3 activation in infected lung epithelial cells is likely to occur with this viral family. Earlier reports, however, indicate that SARS-CoV-1 and MERS-CoV inhibit IFN-β production and activation of IRF3 through viral M protein (133, 134). SARS-CoV-1 and MERS-CoV M protein localize to Golgi membrane in infected cells. Overexpression of M protein prevented the phosphorylation of IRF3 by sequestering RIG-I, TBK1, IKKε, and TRAF3, thereby preventing IRF3 activation. In contrast to M protein, SARS-CoV-1 (nucleocapsid) N protein inhibits IRF3 transcriptional activity by preventing dimerization of IRF3 (135). Because SARS-CoV–infected cells produce viral polyproteins during viral replication, the virus encodes a protease to cleave the polyprotein into individual mature viral proteins. This protease has been termed papain-like protease (PLpro). PLpro also impairs IRF3 activation during viral infection by disturbing TBK1 activation of IRF3 (136). It remains to be seen whether SARS-CoV-2 M, PLpro, or N proteins have similar impacts on IRF3 activation.

Although type I IFNs are critical to antiviral immunity, unregulated or persistently elevated levels of these IFNs can themselves lead to certain diseases termed interferonopathies (137). Although most interferonopathies do not involve IRF3, some do. STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory interferonopathy (138). In SAVI, the TMEM173 gene encoding STING is mutated (N153S) so that mononuclear cells, fibroblasts, and endothelial cells exhibit heightened constitutive activation of STING (139, 140). This hyperactivated STING gives rise to heightened activation of IRF3 and production of IFN-β. The manifestations of SAVI include fever, skin vasculopathy, and interstitial lung disease, followed by respiratory failure. These manifestations are mostly due to the highly activated STING and IRF3 in SAVI endothelial cells that trigger apoptosis in response to cGAMP. More than likely, the role of IRF3 in triggering apoptosis through association with Bax contributes to SAVI-associated endothelial apoptosis. However, despite high activation of IRF3 in SAVI, at least in a mouse model, knockout of IRF3 failed to protect from highly activated STING.

Like SAVI, Aicardi–Goutières syndrome (AGS) is a rare genetic encephalopathic disease manifesting shortly after birth with microcephaly as well as neurologic and physical impairments (141). Notably, IFN-β levels are elevated in cerebral spinal fluid with AGS. Autosomal recessive mutations in TREX1, RNASEH2, ADAR, or SAMHD1 play pivotal roles in AGS (142, 143). However, in SAMHD1, AGS-heightened IFN-β expression is highly dependent on heightened IRF3 activation. Research into mechanisms for heightened IRF3 activity in SAMHD1–AGS reveal that Akt protein kinase is responsible. In this case, a mutational deficiency of SAMHD1 results in failure to regulate Akt, leaving it with high constitutive activity, thereby hyperactivating IRF3.

Recently, IRF3 has been shown to play a role in poor outcomes following mycocardial infarction (MI) (144). During MI ischemia, which is triggered by accumulation of inflammatory lipid-laden macrophages at heart vasculature, IRF3 is activated. MI-activated IRF3 results from ischemic cell death, release of dying cell DNA, and subsequent activation of the cGAS/STING/IRF3 axis. Activated IRF3 in this instance increases expression of inflammatory factors, such as CXCL10, and ISGs, such as IFIT1 and IFIT2, in damaged heart vasculature. More importantly, deletion of the IRF3 gene in an MI mouse model prevents most of the MI-induced expression of inflammatory factors and ISGs in MI heart tissue, thereby preventing post-MI mortality and increasing post-MI heart function. Therefore, pharmaceuticals that reduce IRF3 activation may be a future treatment post-MI.

Since its discovery, IRF3 was shown to have a pivotal role in antiviral immunity. Newer data shows that it also plays role in adaptive immunity, bacterial infections, obesity, metabolism, and interferonopathies. Therefore, this ancient protein that evolved with jawed vertebrates plays a role in other aspects of human health and disease.

I thank the University of Nebraska Medical Center Department of Oral Biology for support.

The author has no financial conflicts of interest.