A Ferritin Nanoparticle-Based Zika Virus Vaccine Candidate Induces Robust Humoral and Cellular Immune Responses and Protects Mice from Lethal Virus Challenge.

The severe consequences of the Zika virus (ZIKV) infections resulting in congenital Zika syndrome in infants and the autoimmune Guillain-Barre syndrome in adults warrant the development of safe and efficacious vaccines and therapeutics. Currently, there are no approved treatment options for ZIKV infection. Herein, we describe the development of a bacterial ferritin-based nanoparticle vaccine candidate for ZIKV. The viral envelope (E) protein domain III (DIII) was fused in-frame at the amino-terminus of ferritin. The resulting nanoparticle displaying the DIII was examined for its ability to induce immune responses and protect vaccinated animals upon lethal virus challenge. Our results show that immunization of mice with a single dose of the nanoparticle vaccine candidate (zDIII-F) resulted in the robust induction of neutralizing antibody responses that protected the animals from the lethal ZIKV challenge. The antibodies neutralized infectivity of other ZIKV lineages indicating that the zDIII-F can confer heterologous protection. The vaccine candidate also induced a significantly higher frequency of interferon (IFN)-γ positive CD4 T cells and CD8 T cells suggesting that both humoral and cell-mediated immune responses were induced by the vaccine candidate. Although our studies showed that a soluble DIII vaccine candidate could also induce humoral and cell-mediated immunity and protect from lethal ZIKV challenge, the immune responses and protection conferred by the nanoparticle vaccine candidate were superior. Further, passive transfer of neutralizing antibodies from the vaccinated animals to naïve animals protected against lethal ZIKV challenge. Since previous studies have shown that antibodies directed at the DIII region of the E protein do not to induce antibody-dependent enhancement (ADE) of ZIKV or other related flavivirus infections, our studies support the use of the zDIII-F nanoparticle vaccine candidate for safe and enhanced immunological responses against ZIKV.

IL-33 promotes increased replication of Theiler's Murine Encephalomyelitis Virus in RAW264.7 macrophage cells with an IRF3-dependent response.

Interleukin-33 (IL-33), which promotes M2 macrophage development, may influence the control of viruses, such as Theiler's Murine Encephalomyelitis Virus (TMEV) that infect macrophages. Because Interferon Regulatory Factor-3 (IRF3) is also critical to control of TMEV infection in macrophages, information on the relationship between IL-33 and IRF3 is important. Thus, RAW264.7 Lucia murine macrophage lineage cells with an endogenous IRF3-ISRE promoter driving secreted luciferase and IRF3KO RAW Lucia, a subline deficient in IRF3, were challenged with TMEV. After the challenge, considerable TMEV RNA detected at 18 and 24 h in RAW cells was significantly elevated in IRF3KO RAW cells. TMEV induction of ISRE-IRF3 promoter activity, IFN-ß and IL-33 gene expression, and IL-6 and IL-10 protein production, which was strong in RAW cells, was less in IRF3KO RAW cells. In contrast, expression of CD206 and ARG1, classical M2 macrophage markers, was significantly elevated in IRF3KO RAW cells. Moreover, RAW and IRF3KO RAW cells produced extracellular IL-33 prior to and after infection with TMEV and antibody blockade of the IL-33 receptor, ST2, reduced CD206 and ARG1 expression, but increased IL-6 gene expression. Pre-treating both RAW and IRF3KO RAW cells with IL-33 prior to challenge significantly increased TMEV infection, but also increased IL-33, IL-10, IL-6 mRNA expression, and NO production without increasing IFN-ß. Notably, IL-33 induction of IL-33, IRF3-ISRE promoter activity, and IL-10 by TMEV or poly I:C/IFN-γ was significantly dependent upon IRF3. The results show that the expression of IL-33 and the repression of M2 macrophage phenotypic markers are dependent on IRF3 and that IL-33 decreases the ability of macrophages to control infection with macrophage-tropic viruses.

Chlorovirus ATCV-1 Accelerates Motor Deterioration in SOD1-G93A Transgenic Mice and Its SOD1 Augments Induction of Inflammatory Factors From Murine Macrophages.

Background: Genetically polymorphic Superoxide Dismutase 1 G93A (SOD1-G93A) underlies one form of familial Amyotrophic Lateral Sclerosis (ALS). Exposures from viruses may also contribute to ALS, possibly by stimulating immune factors, such as IL-6, Interferon Stimulated Genes, and Nitric Oxide. Recently, chlorovirus ATCV-1, which encodes a SOD1, was shown to replicate in macrophages and induce inflammatory factors. Objective: This study aimed to determine if ATCV-1 influences development of motor degeneration in an ALS mouse model and to assess whether SOD1 of ATCV-1 influences production of inflammatory factors from macrophages. Methods: Sera from sporadic ALS patients were screened for antibody to ATCV-1. Active or inactivated ATCV-1, saline, or a viral mimetic, polyinosinic:polycytidylic acid (poly I:C) were injected intracranially into transgenic mice expressing human SOD1-G93A- or C57Bl/6 mice. RAW264.7 mouse macrophage cells were transfected with a plasmid vector expressing ATCV-1 SOD1 or an empty vector prior to stimulation with poly I:C with or without Interferon-gamma (IFN-γ). Results: Serum from sporadic ALS patients had significantly more IgG1 antibody directed against ATCV-1 than healthy controls. Infection of SOD1-G93A mice with active ATCV-1 significantly accelerated onset of motor loss, as measured by tail paralysis, hind limb tucking, righting reflex, and latency to fall in a hanging cage-lid test, but did not significantly affect mortality when compared to saline-treated transgenics. By contrast, poly I:C treatment significantly lengthened survival time but only minimally slowed onset of motor loss, while heat-inactivated ATCV-1 did not affect motor loss or survival. ATCV-1 SOD1 significantly increased expression of IL-6, IL-10, ISG promoter activity, and production of Nitric Oxide from RAW264.7 cells. Conclusion: ATCV-1 chlorovirus encoding an endogenous SOD1 accelerates pathogenesis but not mortality, while poly I:C that stimulates antiviral immune responses delays mortality in an ALS mouse model. ATCV-1 SOD1 enhances induction of inflammatory factors from macrophages.

MEK/ERK MAP kinase limits poly I:C-induced antiviral gene expression in RAW264.7 macrophages by reducing interferon-beta expression.

Toll-like receptor 3 (TLR3) recognizes viral double-stranded RNA (or the synthetic dsRNA analog poly I:C) and induces a signal transduction pathway that results in activation of transcription factors that induce expression of antiviral genes including type I interferon (IFN-I). Secreted IFN-I positively feeds back to amplify antiviral gene expression. In this report, we study the role of MEK/ERK MAP kinase in modulating antiviral gene expression downstream of TLR3. We find MEK/ERK is a negative regulator of antiviral gene expression by limiting expression of IFN-ß. However, MEK/ERK does not limit antiviral responses downstream of the type I interferon receptor. These findings provide insights into regulatory mechanisms of antiviral gene expression and reveal potential targets for modulating antiviral immunity.

Epstein-Barr virus-immortalized B lymphocytes exacerbate experimental autoimmune encephalomyelitis in xenograft mice.

Multiple sclerosis (MS) is the most common autoimmune disorder affecting the central nervous system. Epstein-Barr virus (EBV) is a causative agent for infectious mononucleosis (IM) that is associated with MS pathogenesis. However, the exact mechanism by which EBV, specifically in IM, increases the risk for MS remains unknown. EBV immortalizes primary B lymphocytes in vitro and causes excessive B lymphocyte proliferation in IM in vivo. In asymptomatic carriers, EBV-infected B lymphocytes still proliferate to certain degrees, the process of which is tightly controlled by the host immune systems. Experimental autoimmune encephalomyelitis (EAE) mimics key features of MS in humans and is a well-established rodent model for human MS. We have found that xenografts of EBV-immortalized B lymphocytes, which partially resemble the hyperproliferation of EBV-infected cells in IM, exacerbate autoimmune responses in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. After remission, an additional challenge with EBV-immortalized cells induces a relapse in EAE. Moreover, xenografts with EBV-immortalized cells tighten the integrity of the blood-brain barrier (BBB) in the thalamus and hypothalamus areas of the mouse brains. Genomic sequences of prokaryotic 16S ribosomal RNA presented in the feces reveal that EBV-immortalized cells significantly change the diversities of microbial populations. Our data collectively suggest that EBV-mediated proliferation of B lymphocytes may be a risk factor for the exacerbation of MS, which are associated with gut microbiome changes and BBB modulations. Furthermore, multiple xenografts of EBV-immortalized cells into C57BL/6 mice could serve as a useful model for human relapsing-remitting MS with predictable severity and timing.

IFN Regulatory Factor 3 in Health and Disease.

Immunity to viruses requires an array of critical cellular proteins that include IFN regulatory factor 3 (IRF3). Consequently, most viruses that infect vertebrates encode proteins that interfere with IRF3 activation. This review describes the cellular pathways linked to IRF3 activation and where those pathways are targeted by human viral pathogens. Moreover, key regulatory pathways that control IRF3 are discussed. Besides viral infections, IRF3 is also involved in resistance to some bacterial infections, in anticancer immunity, and in anticancer therapies involving DNA damage agents. A recent finding shows that IRF3 is needed for T cell effector functions that are involved in anticancer immunity and also in T cell autoimmune diseases. In contrast, unregulated IRF3 activity is clearly not beneficial, considering it is implicated in certain interferonopathies, in which heightened IRF3 activity leads to IFN-ß-induced disease. Therefore, IRF3 is involved largely in maintaining health but sometimes contributing to disease.

Amyloid precursor protein is a restriction factor that protects against Zika virus infection in mammalian brains.

Zika virus (ZIKV) is a neurotropic flavivirus that causes several diseases including birth defects such as microcephaly. Intrinsic immunity is known to be a frontline defense against viruses through host anti-viral restriction factors. Limited knowledge is available on intrinsic immunity against ZIKV in brains. Amyloid precursor protein (APP) is predominantly expressed in brains and implicated in the pathogenesis of Alzheimer's diseases. We have found that ZIKV interacts with APP, and viral infection increases APP expression via enhancing protein stability. Moreover, we identified the viral peptide, HGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGL, which is capable of en-hancing APP expression. We observed that aging brain tissues with APP had protective effects on ZIKV infection by reducing the availability of the viruses. Also, knockdown of APP expression or blocking ZIKV-APP interactions enhanced ZIKV replication in human neural progenitor/stem cells. Finally, intracranial infection of ZIKV in APP-null neonatal mice resulted in higher mortality and viral yields. Taken together, these findings suggest that APP is a restriction factor that protects against ZIKV by serving as a decoy receptor, and plays a protective role in ZIKV-mediated brain injuries.

An Attenuated Zika Virus Encoding Non-Glycosylated Envelope (E) and Non-Structural Protein 1 (NS1) Confers Complete Protection against Lethal Challenge in a Mouse Model.

Zika virus (ZIKV), a mosquito-transmitted flavivirus, emerged in the last decade causing serious human diseases, including congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Although many vaccine platforms are at various stages of development, no licensed vaccines are currently available. Previously, we described a mutant MR766 ZIKV (m2MR) bearing an E protein mutation (N154A) that prevented its glycosylation, resulting in attenuation and defective neuroinvasion. To further attenuate m2MR for its potential use as a live viral vaccine, we incorporated additional mutations into m2MR by substituting the asparagine residues in the glycosylation sites (N130 and N207) of NS1 with alanine residues. Examination of pathogenic properties revealed that the virus (m5MR) carrying mutations in E (N154A) and NS1 (N130A and N207A) was fully attenuated with no disease signs in infected mice, inducing high levels of humoral and cell-mediated immune responses, and protecting mice from subsequent lethal virus challenge. Furthermore, passive transfer of sera from m5MR-infected mice into naïve animals resulted in complete protection from lethal challenge. The immune sera from m5MR-infected animals neutralized both African and Asian lineage viruses equally well, suggesting that m5MR virus could be developed as a potentially broad live virus vaccine candidate.

Interferon regulatory factor 3 plays a role in macrophage responses to interferon-γ.

IFN-γ produced during viral infections activates the IFN-γ receptor (IFNGR) complex for STAT1 transcriptional activity leading to expression of Interferon Regulatory Factors (IRF). Simultaneous activation of TBK/IKKε via TLR3 during viral infections activates the transcription factor IRF3. Together these transcription factors contributes to expression of intracellular proteins (e.g. ISG49, ISG54) and secreted proteins (e.g. IFN-ß, IP-10, IL-15) that are essential to innate antiviral immunity. Here we examined the role of IRF3 in expression of innate anti-viral proteins produced in response to IFN-γ plus TLR3 agonist. Wild-type (WT) and IRF3KO RAW264.7 cells, each with ISG54-promoter-luciferase reporter vectors, were stimulated with IFN-γ, poly I:C, or both together. ISG54 promoter activity was significantly reduced in IRF3KO RAW264.7 cells responding to IFN-γ, poly I:C, or IFN-γ plus poly I:C, compared with WT RAW264.7 cells. These data were confirmed with western blot and qRT-PCR. Primary macrophages and dendritic cells (DCs) from IRF3KO mice also showed decreased ISG54 in response to IFN-γ, poly I:C, or IFN-γ plus poly I:C compared with those from WT mice. Moreover, pharmacological inhibition of TBK/IKKε significantly reduced ISG54 promoter activity in response to IFN-γ, poly I:C, or IFN-γ plus poly I:C. Similarly, expression of ISG49 and IL-15, but not IP-10, was impaired in IRF3KO RAW264.7 cells responding to IFN-γ or poly I:C, which also had impaired STAT1 phosphorylation and IRF1 expression. These data show that IRF3 contributes to IFN-γ/IFNGR signaling for expression of innate anti-viral proteins in macrophages.

IFN-γ synergism with poly I:C reduces growth of murine and human cancer cells with simultaneous changes in cell cycle and immune checkpoint proteins.

Previously, we reported that IFN-γ and poly I:C, a TLR3 Pattern Recognition Receptor (PRR) agonist, reduces growth of and induces Cleaved-Caspase-3, ISG54 and p27Kip in B16 melanoma cells. Here, analysis of IFN-γ/PRR synergism was expanded with UM-SCC1 and UM-SCC38 human squamous carcinoma cells and other PRR agonists. As in B16 cells, poly I:C plus IFN-γ synergism reduced UM-SCC1 and UM-SCC38 growth, and no more than 24 h was needed for significant growth reduction. IFN-γ synergism to stem B16 growth also occurred with TLR7, TLR9, TLR4, and STING agonists, but not TLR2 agonist. IFN-γ synergized with TLR3 and TLR4 agonists reducing UM-SCC1 growth, and with TLR7 and TLR3 agonists reducing UM-SCC38 growth. IFN-γ plus poly I:C, which had the most pronounced effect, decreased cyclin-D1, increased G1 cell cycle arrest, and increased Cleaved caspase-3 in B16 cells, as well as RAW264.7, a virus-transformed murine macrophage cell line. Finally, IFN-γ plus poly I:C modulated total but not cell surface expression of immune checkpoint protein PD-L1, as well as cell cycle checkpoint proteins in B16 cells. Thus IFN-γ plus poly I:C, and other PRR agonists, may well be effective adjuvants to cancer immunotherapy against several tumor cell types.

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion.

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

Activation of IRF3 contributes to IFN-γ and ISG54 expression during the immune responses to B16F10 tumor growth.

Interferon Regulatory Factor (IRF-3) has been shown to contribute to immune control of B16 melanoma tumor growth. We have shown previously that IRF-3 has a role in IFN-γ-induced expression of pro-apoptotic interferon stimulated gene 54 (ISG54) in macrophages and IFN-γ in T cells. To investigate the IRF3-IFN-γ-ISG54 nexus, we injected C57Bl/6 (B6) and IRF3KO mice s.c. with luciferase-producing B16-F10 tumor cells. Tumor growth as measured by luciferase levels was similar between B6 and IRF3KO mice at days 2 and 6, but was significantly greater at day 9 in IRF3KO mice compared with B6 mice. Transcription factor assays on splenic protein extracts after tumor inoculation revealed peak activation of IRF3 and IRF7 at day 6 in B6 tumor-bearing mice but not in IRF3KO tumor-bearing mice. Likewise, significant induction of IFN-γ occurred in spleens and tumors in B6 mice from days 6-9 but failed to occur in tumor-bearing IRF3KO mice. Previous reports from other labs showed that the anti-tumor properties of IFN-γ are the result of cell cycle arrest. Using B16F1 cells or B16F1 cells deficient in IFN-γ receptor (B16-IRFGRKO), we found that IFN-γ alone and in synergy with the TLR3/IRF3 agonists, poly I:C, decreased B16F1 cell growth in significant correlation with increased ISG54 expression. Moreover, IFN-γ alone increased expression of the cell cycle inhibitor, p27Kip while IFN-γ plus poly I:C increased cleaved Caspase-3 in B16 cells. Thus, it is likely that an IFN-γ/IRF3/ISG54 nexus can significantly contribute to tumor cell control during anti-tumor immune responses.

Significant role for IRF3 in both T cell and APC effector functions during T cell responses.

Interferon Regulatory Factor (IRF)3 is a crucial transcription factor during innate immune responses. Here we show IRF3 also has a role in adaptive T cell immune responses. Expression of IFN-γ, IL-17, and Granzyme B (GrB) during in vitro T cell responses was impaired when either dendritic cells (DCs) or T cells were derived from IRF3KO mice. Unexpectedly, IRF3-dependent NK-activating molecule (INAM), which is an NK cell activating factor of the DC innate immune response, was induced during the T cell response. Additionally, supernatants from responding T cells induced ISG54 in the RAW264.7 macrophage cell line in an IRF3 dependent manner. Moreover, addition of anti-IFN-γ prevented supernatant induction of ISG54 and recombinant IFN-γ stimulated ISG54 expression. Thus, IRF3 in APCs and T cells is required for optimal T-cell effector function and the ability of T cells to influence innate immune function of APCs.

Effect of Chlorovirus ATCV-1 infection on behavior of C57Bl/6 mice.

Neuroinflammation induced during immune responses to viral infections in the brain affect behavior. Unexpected evidence that oral gavage of an algal virus in its host algal cells could alter cognition was further examined by directly injecting purified algal virus ATCV-1 intracranially into C57BL/6 mice. After 4weeks, the ATCV-1 infection impaired delayed location recognition memory, and also reduced and anxiety. Corresponding to these effects, heightened ATCV-1, IL-6, iNOS, IFN-γ, and CD11b expression in brains was observed 3-days and/or 8-weeks post infection compared with control mice. These results imply that ATCV-1 infection damages the hippocampus via induction of inflammatory factors.

Response of Mammalian Macrophages to Challenge with the Chlorovirus Acanthocystis turfacea Chlorella Virus 1.

UNLABELLED: It was recently reported that 44% of the oropharyngeal samples from the healthy humans in a study cohort had DNA sequences similar to that of the chlorovirus ATCV-1 (Acanthocystis turfacea chlorella virus 1, family Phycodnaviridae) and that these study subjects had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened interleukin-6 (IL-6), nitric oxide (NO), and ERK mitogen-activated protein (MAP) kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral PFU counts in murine RAW264.7 cells and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after a virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h, and the percentage increased 2- to 3-fold over 72 h. Immunoblot assays with rabbit anti-ATCV-1 antibody detected a 55-kDa protein consistent with the viral capsid protein from 1 to 72 h and increasing de novo synthesis of a previously unidentified 17-kDa protein beginning at 24 h. Emergence of the 17-kDa protein did not occur and persistence of the 55-kDa protein declined over time when cells were exposed to heat-inactivated ATCV-1. Moreover, starting at 24 h, RAW264.7 cells exhibited cytopathic effects, annexin V staining, and cleaved caspase 3. Activation of ERK MAP kinases occurred in these cells by 30 min postchallenge, which preceded the expression of IL-6 and NO. Therefore, ATCV-1 persistence in and induction of inflammatory factors by these macrophages may contribute to declines in the cognitive abilities of mice and humans. IMPORTANCE: Virus infections that persist in and stimulate inflammatory factors in macrophages contribute to pathologies in humans. A previous study showed that DNA sequences homologous to the chlorovirus ATCV-1 were found in a significant fraction of oropharyngeal samples from a healthy human cohort. We show here that ATCV-1, whose only known host is a eukaryotic green alga (Chlorella heliozoae) that is an endosymbiont of the heliozoon Acanthocystis turfacea, can unexpectedly persist within murine macrophages and trigger inflammatory responses including factors that contribute to immunopathologies. The inflammatory factors that are produced in response to ATCV-1 include IL-6 and NO, whose induction is preceded by the activation of ERK MAP kinases. Other responses of ATCV-1-challenged macrophages include an apoptotic cytopathic effect, an innate antiviral response, and a metabolic shift toward aerobic glycolysis. Therefore, mammalian encounters with chloroviruses may contribute to chronic inflammatory responses from macrophages.

Candida albicans Quorum Sensing Molecules Stimulate Mouse Macrophage Migration.

The polymorphic commensal fungus Candida albicans causes life-threatening disease via bloodstream and intra-abdominal infections in immunocompromised and transplant patients. Although host immune evasion is a common strategy used by successful human fungal pathogens, C. albicans provokes recognition by host immune cells less capable of destroying it. To accomplish this, C. albicans white cells secrete a low-molecular-weight chemoattractive stimulant(s) of macrophages, a phagocyte that they are able to survive within and eventually escape from. C. albicans opaque cells do not secrete this chemoattractive stimulant(s). We report here a physiological mechanism that contributes to the differences in the interaction of C. albicans white and opaque cells with macrophages. E,E-Farnesol, which is secreted by white cells only, is a potent stimulator of macrophage chemokinesis, whose activity is enhanced by yeast cell wall components and aromatic alcohols. E,E-farnesol results in up to an 8.5-fold increase in macrophage migration in vitro and promotes a 3-fold increase in the peritoneal infiltration of macrophages in vivo. Therefore, modulation of farnesol secretion to stimulate host immune recognition by macrophages may help explain why this commensal is such a successful pathogen.

IRF3 deficiency impacts granzyme B expression and maintenance of memory T cell function in response to viral infection.

The role of interferon regulatory factor 3 (IRF3) in the innate immune response to infection has been well studied. However, less is known about IRF3 signaling in shaping the adaptive T cell response. To determine the role of IRF3 in the generation and maintenance of effective anti-viral T cell responses, mice deficient in IRF3 were infected with a potentially persistent virus, Theiler's murine encephalomyelitis virus (TMEV) or with a model acute infection, influenza A virus (IAV). IRF3 was required to prevent TMEV persistence and induce robust TMEV specific effector T cell responses at the site of infection. This defect was more pronounced in the memory phase with an apparent lack of TMEV-specific memory T cells expressing granzyme B (GrB) in IRF3 deficient mice. In contrast, IRF3 had no effect on antigen specific T cell responses at the effector stage during IAV infection. However, memory T cell responses to IAV were also impaired in IRF3 deficient mice. Furthermore, addition of cytokines during peptide restimulation could not restore GrB expression in IRF3 deficient memory T cells. Taken together, IRF3 plays an important role in the maintenance of effective anti-viral T cell memory responses.

Interferon response factor 3 is crucial to poly-I:C induced NK cell activity and control of B16 melanoma growth.

Interferon Response Factor 3 (IRF3) induces several NK-cell activating factors, is activated by poly-I:C, an experimental cancer therapeutic, but is suppressed during many viral infections. IRF3 Knockout (KO) mice exhibited enhanced B16 melanoma growth, impaired intratumoral NK cell infiltration, but not an impaired poly-I:C therapeutic effect due to direct suppression of B16 growth. IRF3 was responsible for poly-I:C decrease in TIM-3 expression by intratumoral dendritic cells, induction of NK-cell Granzyme B and IFN-γ, and induction of macrophage IL-12, IL-15, IL-6, and IRF3-dependent NK-activating molecule (INAM). Thus, IRF3 is a key factor controlling melanoma growth through NK-cell activities, especially during poly-I:C therapy.

IRF3 helps control acute TMEV infection through IL-6 expression but contributes to acute hippocampus damage following TMEV infection.

IRF3 is an innate anti-viral factor whose role in limiting Theiler's murine encephalomyelitis virus (TMEV) infection and preventing TMEV-induced disease is unclear. Acute disease and innate immune responses of macrophages were examined in IRF3 knockout mice compared with C57Bl/6 mice following in vitro or intracranial infection with either TMEV GDVII or DA. IRF3 deficiency augmented viral infection, as well as morbidity and mortality following intracranial infection with neurovirulent TMEV GDVII. In contrast, IRF3 deficiency prevented hippocampal injury following intracranial infection with persistent TMEV DA. The extent of TMEV infection in macrophages from C57Bl/6 mice was significantly less than that in IRF3 deficient macrophages, which was associated with poor IFN-ß and IL-6 expression in response to TMEV. Reestablishing IRF3 expression in IRF3 deficient macrophages increased control of TMEV replication and increased expression of IFN-ß and IL-6. In addition, IRF3 deficient macrophages failed to exhibit IL-6 antiviral effects, which was associated with inability to sustain IL-6-induced STAT1 activation compared with C57BL/6 macrophages. Altogether, IRF3 contributes to early control of TMEV replication through induction of IL-6 and IFN-ß and support of IL-6 antiviral effects, but contributes to TMEV-induced hippocampal injury.

IRF3 and ERK MAP-kinases control nitric oxide production from macrophages in response to poly-I:C.

Understanding nitric oxide (NO) in innate anti-viral immunity and immune-mediated pathology is hampered by incomplete details of its transcriptional and signaling factors. We found in macrophages that IRF3, ERK MAP-kinases, and PKR are essential to NO production in response to RNA-virus mimic, poly I:C, a TLR3 agonist. ERK's role in NO induction may be through phosphorylation of serine-171 of IRF3 and expression of NO-inducing cytokines, IL-6 and IFN-ß. However, these cytokines induced less NO in IRF3 knockout or knockdown macrophages. These findings show that ERK and IRF3 coordinate induction of NO by macrophages in response to stimulation of TLR3.