Interferon regulatory factors 3 and 7 have distinct roles in the pathogenesis of alphavirus encephalomyelitis.

Interferon (IFN) regulatory factors (IRFs) are important determinants of the innate response to infection. We evaluated the role(s) of combined and individual IRF deficiencies in the outcome of infection of C57BL/6 mice with Sindbis virus, an alphavirus that infects neurons and causes encephalomyelitis. The brain and spinal cord levels of Irf7, but not Irf3 mRNAs, were increased after infection. IRF3/5/7-/- and IRF3/7-/- mice died within 3-4 days with uncontrolled virus replication, similar to IFNα receptor-deficient mice, while all wild-type (WT) mice recovered. IRF3-/- and IRF7-/- mice had brain levels of IFNα that were lower, but brain and spinal cord levels of IFNß and IFN-stimulated gene mRNAs that were similar to or higher than WT mice without detectable serum IFN or increases in Ifna or Ifnb mRNAs in the lymph nodes, indicating that the differences in outcome were not due to deficiencies in the central nervous system (CNS) type I IFN response. IRF3-/- mice developed persistent neurological deficits and had more spinal cord inflammation and higher CNS levels of Il1b and Ifnγ mRNAs than WT mice, but all mice survived. IRF7-/- mice died 5-8 days after infection with rapidly progressive paralysis and differed from both WT and IRF3-/- mice in the induction of higher CNS levels of IFNß, tumour necrosis factor (TNF) α and Cxcl13 mRNA, delayed virus clearance and more extensive cell death. Therefore, fatal disease in IRF7-/- mice is likely due to immune-mediated neurotoxicity associated with failure to regulate the production of inflammatory cytokines such as TNFα in the CNS.

Inflammatory monocytes mediate control of acute alphavirus infection in mice.

Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-transmitted alphaviruses that cause debilitating acute and chronic musculoskeletal disease. Monocytes are implicated in the pathogenesis of these infections; however, their specific roles are not well defined. To investigate the role of inflammatory Ly6ChiCCR2+ monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice, enabling depletion of these cells by administration of diptheria toxin (DT). DT-treated CCR2-DTR mice displayed more severe disease following CHIKV and RRV infection and had fewer Ly6Chi monocytes and NK cells in circulation and muscle tissue compared with DT-treated WT mice. Furthermore, depletion of CCR2+ or Gr1+ cells, but not NK cells or neutrophils alone, restored virulence and increased viral loads in mice infected with an RRV strain encoding attenuating mutations in nsP1 to levels detected in monocyte-depleted mice infected with fully virulent RRV. Disease severity and viral loads also were increased in DT-treated CCR2-DTR+;Rag1-/- mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. Monocytes and macrophages sorted from muscle tissue of RRV-infected mice were viral RNA positive and had elevated expression of Irf7, and co-culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of type I IFN gene expression in monocytes that was Irf3;Irf7 and Mavs-dependent. Consistent with these data, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in Mavs-/- mice. Finally, reconstitution of Irf3-/-;Irf7-/- mice with CCR2-DTR bone marrow rescued mice from severe infection, and this effect was reversed by depletion of CCR2+ cells, indicating that CCR2+ hematopoietic cells are capable of inducing an antiviral response. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1, the viral RNA capping protein, counteract this response.

Novel Lesions of Bones and Joints Associated with Chikungunya Virus Infection in Two Mouse Models of Disease: New Insights into Disease Pathogenesis.

Chikungunya virus is an arbovirus spread predominantly by Aedes aegypti and Ae. albopictus mosquitoes, and causes debilitating arthralgia and arthritis. While these are common manifestations during acute infection and it has been suggested they can recur in patients chronically, gaps in knowledge regarding the pathogenesis still exist. Two established mouse models were utilized (adult IRF 3/7 -/- -/- and wild-type C57BL/6J mice) to evaluate disease manifestations in bones and joints at various timepoints. Novel lesions in C57BL/6J mice consisted of periostitis (91%) and foci of cartilage of necrosis (50% of mice at 21 DPI). Additionally, at 21 DPI, 50% and 75% of mice exhibited periosteal bone proliferation affecting the metatarsal bones, apparent via histology and µCT, respectively. µCT analysis did not reveal any alterations in trabecular bone volume measurements in C57BL/6J mice. Novel lesions demonstrated in IRF 3/7 -/- -/- mice at 5 DPI included focal regions of cartilage necrosis (20%), periosteal necrosis (66%), and multifocal ischemic bone marrow necrosis (100%). Contralateral feet in 100% of mice of both strains had similar, though milder lesions. Additionally, comparison of control IRF 3/7 -/- -/- and wild-type C57BL/6J mice demonstrated differences in cortical bone. These experiments demonstrate novel manifestations of disease similar to those occurring in humans, adding insight into disease pathogenesis, and representing new potential targets for therapeutic interventions. Additionally, results demonstrate the utility of µCT in studies of bone and joint pathology and illustrate differences in bone dynamics between mouse strains.

Interferon regulatory factor 7 functions as a novel negative regulator of pathological cardiac hypertrophy.

Cardiac hypertrophy is a complex pathological process that involves multiple factors including inflammation and apoptosis. Interferon regulatory factor 7 (IRF7) is a multifunctional regulator that participates in immune regulation, cell differentiation, apoptosis, and oncogenesis. However, the role of IRF7 in cardiac hypertrophy remains unclear. We performed aortic banding in cardiac-specific IRF7 transgenic mice, IRF7 knockout mice, and the wild-type littermates of these mice. Our results demonstrated that IRF7 was downregulated in aortic banding-induced animal hearts and cardiomyocytes that had been treated with angiotensin II or phenylephrine for 48 hours. Accordingly, heart-specific overexpression of IRF7 significantly attenuated pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction, whereas loss of IRF7 led to opposite effects. Moreover, IRF7 protected against angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we identified that IRF7-dependent cardioprotection was mediated through IRF7 binding to inhibitor of κB kinase-ß, and subsequent nuclear factor-κB inactivation. In fact, blocking nuclear factor-κB signaling with cardiac-specific inhibitors of κBα(S32A/S36A) super-repressor transgene counteracted the adverse effect of IRF7 deficiency. Conversely, activation of nuclear factor-κB signaling via a cardiac-specific conditional inhibitor of κB kinase-ß(S177E/S181E) (constitutively active) transgene negated the antihypertrophic effect of IRF7 overexpression. Our data demonstrate that IRF7 acts as a novel negative regulator of pathological cardiac hypertrophy by inhibiting nuclear factor-κB signaling and may constitute a potential therapeutic target for pathological cardiac hypertrophy.

MAVS-dependent IRF3/7 bypass of interferon ß-induction restricts the response to measles infection in CD150Tg mouse bone marrow-derived dendritic cells.

Measles virus (MV) infects CD150Tg/Ifnar (IFN alpha receptor)(-/-) mice but not CD150 (a human MV receptor)-transgenic (Tg) mice. We have shown that bone marrow-derived dendritic cells (BMDCs) from CD150Tg/Ifnar(-/-) mice are permissive to MV in contrast to those from simple CD150Tg mice, which reveals a crucial role of type I interferon (IFN) in natural tropism against MV. Yet, the mechanism whereby BMDCs produce initial type I IFN has not been elucidated in MV infection. RNA virus infection usually allows cells to generate double-stranded RNA and induce activation of IFN regulatory factor (IRF) 3/7 transcription factors, leading to the production of type I IFN through the retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5)-mitochondrial antiviral signaling protein (MAVS) pathway. In mouse experimental BMDCs models, we found CD150Tg/Mavs(-/-)BMDCs, but not CD150Tg/Irf3(-/-)/Irf7(-/-)BMDCs, permissive to MV. IFN-α/ß were not induced in MV-infected CD150Tg/Mavs(-/-)BMDCs, while IFN-ß was subtly induced in CD150Tg/Irf3(-/-)/Irf7(-/-)BMDCs. In vivo systemic infection was therefore established by transfer of MV-infected CD150Tg/Mavs(-/-) BMDCs to CD150Tg/Ifnar(-/-) mice. These data indicate that MAVS-dependent, IRF3/7-independent IFN-ß induction triggers the activation of the IFNAR pathway so as to restrict the spread of MV by infected BMDCs. Hence, MAVS participates in the initial induction of type I IFN in BMDCs and IFNAR protects against MV spreading. We also showed the importance of IL-10-producing CD4(+) T cells induced by MV-infected BMDCs in vitro, which may account for immune modulation due to the functional aberration of DCs.

The roles of IRF-3 and IRF-7 in innate antiviral immunity against dengue virus.

We investigated the roles of IFN regulatory factor (IRF)-3 and IRF-7 in innate antiviral immunity against dengue virus (DENV). Double-deficient Irf-3(-/-)7(-/-) mice infected with the DENV2 strain S221 possessed 1,000-150,000 fold higher levels of viral RNA than wild-type and single-deficient mice 24 h postinfection (hpi); however, they remained resistant to lethal infection. IFN-α/ß was induced similarly in wild-type and Irf-3(-/-) mice post-DENV infection, whereas in the Irf-7(-/-) and Irf-3(-/-)7(-/-) mice, significantly low levels of IFN-α/ß expression was observed within 24 hpi. IFN-stimulated gene induction was also delayed in Irf-3(-/-)7(-/-) mice relative to wild-type and single-deficient mice. In particular, Cxcl10 and Ifnα2 were rapidly induced independently of both IRF-3 and IRF-7 in the Irf-3(-/-)7(-/-) mice with DENV infection. Higher levels of serum IFN-γ, IL-6, CXCL10, IL-8, IL-12 p70, and TNF were also observed in Irf-3(-/-)7(-/-) mice 24 hpi, at which time point viral titers peaked and started to be cleared. Ab-mediated blockade experiments revealed that IFN-γ, CXCL10, and CXCR3 function to restrict DENV replication in Irf-3(-/-)7(-/-) mice. Additionally, the IFN-stimulated genes Cxcl10, Ifit1, Ifit3, and Mx2 can be induced via an IRF-3- and IRF-7-independent pathway that does not involve IFN-γ signaling for protection against DENV. Collectively, these results demonstrate that IRF-3 and IRF-7 are redundant, albeit IRF-7 plays a more important role than IRF-3 in inducing the initial IFN-α/ß response; only the combined actions of IRF-3 and IRF-7 are necessary for efficient control of early DENV infection; and the late, IRF-3- and IRF-7-independent pathway contributes to anti-DENV immunity.

A FOXO3-IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses.

Antiviral responses must be tightly regulated to defend rapidly against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses and their transcription is regulated by a variety of transcription factors; principal among these is the family of interferon regulatory factors (IRFs). The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.

Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock.

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7(-/-)) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/ß) in serum, ∼50- and ∼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/ß receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/ß induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/ß responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.

Toll-like receptor 7 preconditioning induces robust neuroprotection against stroke by a novel type I interferon-mediated mechanism.

BACKGROUND AND PURPOSE: Systemic administration of Toll-like receptor (TLR) 4 and TLR9 agonists before cerebral ischemia have been shown to reduce ischemic injury by reprogramming the response of the brain to stroke. Our goal was to explore the mechanism of TLR-induced neuroprotection by determining whether a TLR7 agonist also protects against stroke injury. METHODS: C57Bl/6, TNF(-/-), interferon (IFN) regulatory factor 7(-/-), or type I IFN receptor (IFNAR)(-/-) mice were subcutaneously administered the TLR7 agonist Gardiquimod (GDQ) 72 hours before middle cerebral artery occlusion. Infarct volume and functional outcome were determined after reperfusion. Plasma cytokine responses and induction of mRNA for IFN-related genes in the brain were measured. IFNAR(-/-) mice also were treated with the TLR4 agonist (lipopolysaccharide) or the TLR9 agonist before middle cerebral artery occlusion and infarct volumes measured. RESULTS: The results show that GDQ reduces infarct volume as well as functional deficits in mice. GDQ pretreatment provided robust neuroprotection in TNF(-/-) mice, indicating that TNF was not essential. GDQ induced a significant increase in plasma IFNα levels and both IRF7(-/-) and IFNAR(-/-) mice failed to be protected, implicating a role for IFN signaling in TLR7-mediated protection. CONCLUSIONS: Our studies provide the first evidence that TLR7 preconditioning can mediate neuroprotection against ischemic injury. Moreover, we show that the mechanism of protection is unique from other TLR preconditioning ligands in that it is independent of TNF and dependent on IFNAR.

Cutting edge: independent roles for IRF-3 and IRF-7 in hematopoietic and nonhematopoietic cells during host response to Chikungunya infection.

The host response to Chikungunya virus is dependent on the direct action of type I IFN on infected nonhematopoietic cells. Prior studies have demonstrated that multiple host sensors coordinate an antiviral response; however, the tissue source(s) and signaling pathways for IFN production remain unknown. In this study, we demonstrate that IRF-3 and IRF-7 are functionally redundant, but lack of both factors results in lethal infection in adult mice. Reciprocal bone marrow chimeras indicated that IRF-3 or IRF-7 expression in either hematopoietic or nonhemotopoietic cell compartments was capable of inducing an antiviral response. Interestingly, redundancy of IRF-3 and IRF-7 was age dependent, as neonatal animals lacking either factor succumbed to infection. We further demonstrate that IPS-1 is essential in nonhematopoietic cells and preferentially required during early life. These results highlight the interplay between nonimmune and immune cells during Chikungunya virus infection and suggest an important role for nonhematopoietic cells as a critical source of IFN-α/ß.

The DNA damage response induces IFN.

This study reveals a new complexity in the cellular response to DNA damage: activation of IFN signaling. The DNA damage response involves the rapid recruitment of repair enzymes and the activation of signal transducers that regulate cell-cycle checkpoints and cell survival. To understand the link between DNA damage and the innate cellular defense that occurs in response to many viral infections, we evaluated the effects of agents such as etoposide that promote dsDNA breaks. Treatment of human cells with etoposide led to the induction of IFN-stimulated genes and the IFN-α and IFN-λ genes. NF-κB, known to be activated in response to DNA damage, was shown to be a key regulator of this IFN gene induction. Expression of an NF-κB subunit, p65/RelA, was sufficient for induction of the human IFN-λ1 gene. In addition, NF-κB was required for the induction of IFN regulatory factor-1 and -7 that are able to stimulate expression of the IFN-α and IFN-λ genes. Cells that lack the NF-κB essential modulator lack the ability to induce the IFN genes following DNA damage. Breaks in DNA are generated during normal physiological processes of replication, transcription, and recombination, as well as by external genotoxic agents or infectious agents. The significant finding of IFN production as a stress response to DNA damage provides a new perspective on the role of IFN signaling.

Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production.

Dendritic cell (DC) activation by nucleic acid-containing IgG complexes is implicated in systemic lupus erythematosus (SLE) pathogenesis. However, it has been difficult to definitively examine the receptors and signaling pathways by which this activation is mediated. Because mouse FcgammaRs recognize human IgG, we hypothesized that IgG from lupus patients might stimulate mouse DCs, thereby facilitating this analysis. In this study, we show that sera and purified IgG from lupus patients activate mouse DCs to produce IFN-alpha, IFN-beta, and IL-6 and up-regulate costimulatory molecules in a FcgammaR-dependent manner. This activation is only seen in sera with reactivity against ribonucleoproteins and is completely dependent on TLR7 and the presence of RNA. As anticipated, IFN regulatory factor (IRF)7 is required for IFN-alpha and IFN-beta production. Unexpectedly, however, IRF5 plays a critical role in IFN-alpha and IFN-beta production induced not only by RNA-containing immune complexes but also by conventional TLR7 and TLR9 ligands. Moreover, DC production of IL-6 induced by these stimuli is dependent on a functional type I IFNR, indicating the need for a type I IFN-dependent feedback loop in the production of inflammatory cytokines. This system may also prove useful for the study of receptors and signaling pathways used by immune complexes in other human diseases.