Heart failure (HF) is a leading cause of morbidity and mortality in the western world. Although optimal medical care and treatment is widely available, the prognosis of patients with HF is still poor. Toll-like receptors (TLRs) are important compartments of the innate immunity. Current studies have identified TLRs as critical mediators in cardiovascular diseases. In the present study, we investigated the involvement of TLRs and interferon (IFN) regulatory factors (IRFs) in different experimental HF models including viral myocarditis, myocardial ischemia, diabetes mellitus, and cardiac hypertrophy. In addition, we investigated for the first time comprehensive TLR and IRF gene and protein expression under basal conditions in murine and human cardiac tissue. We found that Tlr4, Tlr9 and Irf7 displayed highest gene expression under basal conditions, indicating their significant role in first-line defense in the murine and human heart. Moreover, induction of TLRs and IRFs clearly differs between the various experimental HF models of diverse etiology and the concomitant inflammatory status. In the HF model of acute viral-induced myocarditis, TLR and IRF activation displayed the uppermost gene expression in comparison to the remaining experimental HF models, indicating the highest amount of myocardial inflammation in myocarditis. In detail, Irf7 displayed by far the highest gene expression during acute viral infection. Interestingly, post myocardial infarction TLR and IRF gene expression was almost exclusively increased in the infarct zone after myocardial ischemia (Tlr2, Tlr3, Tlr6, Tlr7, Tlr9, Irf3, Irf7). With one exception, Irf3 showed a decreased gene expression in the remote zone post infarction. Finally, we identified Irf7 as novel cardiovascular stress-inducible factor in the pathologically stressed heart. These findings on TLR and IRF function in the inflamed heart highlight the complexity of inflammatory immune response and raise more interesting questions for future investigation.

The leishmanin skin test (LST) is an in vivo technique commonly used to evaluate the Leishmania-specific cellular immune response in dogs. However, information regarding the local immune response in LST-positive reactions is scarce. We examined the pattern of toll-like receptor 2 (TLR2), TLR4, TLR7, interleukin- (IL-) 10, interferon gamma (IFN-γ), and (program death ligand) PD-L1 gene expression in LST-positive reactions and paired normal-looking skin of nine infected Ibizan hound dogs. Healthy skin from ten seronegative dogs from a nonendemic area was analysed as a negative control. Immune gene expressions were examined by quantitative PCR (qPCR) analysis. LST-positive reactions presented significant upregulation of TLR2, TLR4, IL-10, IFN-γ, and PD-L1 and downregulation of TLR7 when compared with healthy skin of seronegative control dogs from a nonendemic area. All transcripts but TLR7 were significantly higher in LST-positive reaction than in paired normal-looking skin of Ibizan hound. The expression profile of immune genes in LST-positive reactions was similar to that previously observed in clinically lesioned skin of mildly diseased dogs with papular dermatitis due to Leishmania infantum infection. This data provide additional support for the important role of TLRs in canine leishmaniosis.

During a viral infection, the intracellular RIG-I-like receptors (RLRs) sense viral RNA and signal through the mitochondrial antiviral signaling adaptor MAVS (also known as IPS-1, Cardif and VISA) whose activation triggers a rapid production of type I interferons (IFN) and of pro-inflammatory cytokines through the transcription factors IRF3/IRF7 and NF-κB, respectively. While MAVS is essential for this signaling and known to operate through the scaffold protein NEMO and the protein kinase TBK1 that phosphorylates IRF3, its mechanism of action and regulation remain unclear.

Canine leishmaniosis (CanL) caused by Leishmania infantum can have several dermatological manifestations. The type of immune response elicited against the parasite appears to be at the basis for such clinical variability. Much of the work in CanL has focused on adaptive immune response and there are scarce data on the importance of the innate immune responses. Moreover, few studies have evaluated the immunological response in the cutaneous lesions in dogs naturally infected with L. infantum and with different degrees of disease severity, and no study has compared clinically-lesioned with normal-looking skin.

Type I interferons (IFN-I) broadly control innate immunity and are typically transcriptionally induced by Interferon Regulatory Factors (IRFs) following stimulation of pattern recognition receptors within the cytosol of host cells. For bacterial infection, IFN-I signaling can result in widely variant responses, in some cases contributing to the pathogenesis of disease while in others contributing to host defense. In this work, we addressed the role of type I IFN during Yersinia pestis infection in a murine model of septicemic plague. Transcription of IFN-β was induced in vitro and in vivo and contributed to pathogenesis. Mice lacking the IFN-I receptor, Ifnar, were less sensitive to disease and harbored more neutrophils in the later stage of infection which correlated with protection from lethality. In contrast, IRF-3, a transcription factor commonly involved in inducing IFN-β following bacterial infection, was not necessary for IFN production but instead contributed to host defense. In vitro, phagocytosis of Y. pestis by macrophages and neutrophils was more effective in the presence of IRF-3 and was not affected by IFN-β signaling. This activity correlated with limited bacterial growth in vivo in the presence of IRF-3. Together the data demonstrate that IRF-3 is able to activate pathways of innate immunity against bacterial infection that extend beyond regulation of IFN-β production.

Recombinant beta interferons-1 (IFNβ-1) are used as first line therapies in patients with relapsing multiple sclerosis (MS), a chronic inflammatory and neurodegenerative disease of the CNS. IFNβ-1a/b has moderate effects on the prevention of relapses and slowing of disease progression. Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) are known to play a key role in the pathology of MS and its model EAE. To investigate the effects of short-term treatment with s.c. IFNβ-1a versus the combined application of s.c. IFNβ-1a and oligodendrocyte-specific deletion of FGFR1 (Fgfr1ind-/- mice) in MOG35-55-induced EAE. IFNβ-1a (30 mg/kg) was applied s.c. from days 0-7 p.i. of EAE in controls and Fgfr1ind-/- mice. FGFR signaling proteins associated with inflammation/degeneration in MS/EAE were analyzed by western blot in the spinal cord. Further, FGFR1 in Oli-neu oligodendrocytes were inhibited by PD166866 and treated with IFNβ-1a (400 ng/mL). Application of IFNβ-1a over 8 days resulted in less symptoms only at the peak of disease (days 9-11) compared to controls. Application of IFNβ-1a in Fgfr1ind-/- mice resulted in less symptoms primarily in the chronic phase of EAE. Fgfr1ind-/- mice treated with IFNβ-1a showed increased expression of pERK and BDNF. In Oli-neu oligodendrocytes, treatment with PD166866 and IFNβ-1a also showed an increased expression of pERK and BDNF/TrkB. These data suggest that the beneficial effects in the chronic phase of EAE and on signaling molecules associated with ERK and BDNF expression are caused by the modulation of FGFR1 and not by interferon beta-1a. FGFR may be a potential target for therapy in MS.

Severe dengue is caused by host responses to viral infection, but the pathogenesis remains unknown. This is, in part, due to the lack of suitable animal models. Here, we report a non-mouse-adapted low-passage DENV-3 clinical isolate, DV3P12/08, derived from recently infected patients. DV3P12/08 caused a lethal systemic infection in type I and II IFN receptor KO mice (IFN-α/β/γR KO mice), which have the C57/BL6 background. Infection with DV3P12/08 induced a cytokine storm, resulting in severe vascular leakage (mainly in the liver, kidney and intestine) and organ damage, leading to extensive hemorrhage and rapid death. DV3P12/08 infection triggered the release of large amounts of TNF-α, IL-6, and MCP-1. Treatment with a neutralizing anti-TNF-α antibody (Ab) extended survival and reduced liver damage without affecting virus production. Anti-IL-6 neutralizing Ab partly prolonged mouse survival. The anti-TNF-α Ab suppressed IL-6, MCP-1, and IFN-γ levels, suggesting that the severe response to infection was triggered by TNF-α. High levels of TNF-α mRNA were expressed in the liver and kidneys, but not in the small intestine, of infected mice. Conversely, high levels of IL-6 mRNA were expressed in the intestine. Importantly, treatment with Angiopoietin-1, which is known to stabilize blood vessels, prolonged the survival of DV3P12/08-infected mice. Taken together, the results suggest that an increased level of TNF-α together with concomitant upregulation of Tie2/Angiopoietin signaling have critical roles in severe dengue infection.

To trigger type I interferon (IFN) responses, pattern recognition receptors activate signaling cascades that lead to transcription of IFN and IFN-stimulated genes (ISGs). The promyelocytic leukemia (PML) protein has been implicated in these responses, although its role has not been defined. Here, we show that PML isoform II (PML-II) is specifically required for efficient induction of IFN-β transcription and of numerous ISGs, acting at the point of transcriptional complex assembly on target gene promoters. PML-II associated with specific transcription factors NF-κB and STAT1, as well as the coactivator CREB-binding protein (CBP), to facilitate transcriptional complex formation. The absence of PML-II substantially reduced binding of these factors and IFN regulatory factor 3 (IRF3) to IFN-β or ISGs promoters and sharply reduced gene activation. The unique C-terminal domain of PML-II was essential for its activity, while the N-terminal RBCC motif common to all PML isoforms was dispensable. We propose a model in which PML-II contributes to the transcription of multiple genes via the association of its C-terminal domain with relevant transcription complexes, which promotes the stable assembly of these complexes at promoters/enhancers of target genes, and that in this way PML-II plays a significant role in the development of type I IFN responses.

Natural killer (NK) cells are equipped to innately produce the cytokine gamma interferon (IFN-γ) in part because they basally express high levels of the signal transducer and activator of transcription 4 (STAT4). Type 1 interferons (IFNs) have the potential to activate STAT4 and promote IFN-γ expression, but concurrent induction of elevated STAT1 negatively regulates access to the pathway. As a consequence, it has been difficult to detect type 1 IFN stimulation of NK cell IFN-γ during viral infections in the presence of STAT1 and to understand the evolutionary advantage for maintaining the pathway. The studies reported here evaluated NK cell responses following infections with lymphocytic choriomeningitis virus (LCMV) in the compartment handling the earliest events after infection, the peritoneal cavity. The production of type 1 IFNs, both IFN-α and IFN-β, was shown to be early and of short duration, peaking at 30 h after challenge. NK cell IFN-γ expression was detected with overlapping kinetics and required activating signals delivered through type 1 IFN receptors and STAT4. It took place under conditions of high STAT4 levels but preceded elevated STAT1 expression in NK cells. The IFN-γ response reduced viral burdens. Interestingly, increases in STAT1 were delayed in NK cells compared to other peritoneal exudate cell (PEC) populations. Taken together, the studies demonstrate a novel mechanism for stimulating IFN-γ production and elucidate a biological role for type 1 IFN access to STAT4 in NK cells.

Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits.

Interferon-α (IFN-α) and IFN-λ are structurally distinct cytokines that bind to different receptors, but induce expression of similar sets of genes through Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. The difference between IFN-α and IFN-λ signaling remains poorly understood. Here, using the CRISPR/Cas9 system, we examine the role of STAT1 and STAT2 in the inhibition of hepatitis C virus (HCV) replication by IFN-α and IFN-λ. Treatment with IFN-α increases expression of IFN-stimulated genes (ISGs) such as double-stranded RNA-activated protein kinase (PKR) and decreases viral RNA and protein levels in HCV-infected Huh-7.5 human hepatoma cells. These responses are only partially attenuated by knockout of STAT1 but are abolished by knockout of STAT2. In contrast, the inhibition of HCV replication by IFN-λ is abolished by knockout of STAT1 or STAT2. Microarray analysis reveals that IFN-α but not IFN-λ can induce expression of the majority of ISGs in STAT1 knockout cells. These findings suggest that IFN-α can inhibit HCV replication through a STAT2-dependent but STAT1-independent pathway, whereas IFN-λ induces ISG expression and inhibits HCV replication exclusively through a STAT1- and STAT2-dependent pathway.

It is currently believed that type I and III interferons (IFNs) have redundant functions. However, the preferential distribution of type III IFN receptor on epithelial cells suggests functional differences at epithelial surfaces. Here, using human intestinal epithelial cells we could show that although both type I and type III IFNs confer an antiviral state to the cells, they do so with distinct kinetics. Type I IFN signaling is characterized by an acute strong induction of interferon stimulated genes (ISGs) and confers fast antiviral protection. On the contrary, the slow acting type III IFN mediated antiviral protection is characterized by a weaker induction of ISGs in a delayed manner compared to type I IFN. Moreover, while transcript profiling revealed that both IFNs induced a similar set of ISGs, their temporal expression strictly depended on the IFNs, thereby leading to unique antiviral environments. Using a combination of data-driven mathematical modeling and experimental validation, we addressed the molecular reason for this differential kinetic of ISG expression. We could demonstrate that these kinetic differences are intrinsic to each signaling pathway and not due to different expression levels of the corresponding IFN receptors. We report that type III IFN is specifically tailored to act in specific cell types not only due to the restriction of its receptor but also by providing target cells with a distinct antiviral environment compared to type I IFN. We propose that this specific environment is key at surfaces that are often challenged with the extracellular environment.

Malaria in pregnancy (MiP) is a distinctive clinical form of Plasmodium infection and is a cause of placental insufficiency leading to poor pregnancy outcomes. Maternal innate immunity responses play a decisive role in the development of placental inflammation, but the action of fetus-derived factors in MiP outcomes has been overlooked. We investigated the role of the Tlr4 and Ifnar1 genes, taking advantage of heterogenic mating strategies to dissect the effects mediated by maternally and fetally derived Toll-like receptor 4 (TLR4) or type I interferon receptor 1 (IFNAR1). Using a mouse infection system displaying severe MiP outcomes, we found that the expressions of TLR4 and IFNAR1 in the maternal compartment take part in deleterious MiP outcomes, but their fetal counterparts patently counteract these effects. We uncovered that fetal TLR4 contributes to the in vitro uptake of infected erythrocytes by trophoblasts and to the innate immune response in the placenta, offering robust protection of fetus viability, but had no sensible impact on the placental parasite burden. In contrast, we observed that the expression of IFNAR1 in the fetal compartment was associated with a reduced placental parasite burden but had little beneficial effect on fetus outcomes. Furthermore, the downregulation of Ifnar1 expression in infected placentas and in trophoblasts exposed to infected erythrocytes indicated that the interferon-IFNAR1 pathway is involved in the trophoblast response to infection. This work unravels that maternal and fetal counterparts of innate immune pathways drive opposing responses in murine placental malaria and implicates the activation of innate receptors in fetal trophoblast cells in the control of placental infection and in the protection of the fetus.

Synthetic double-stranded RNA poly(I:C) is a useful immune adjuvant and exhibits direct antitumor effects against several types of cancers. In this study, we elucidated the mechanisms underlying the effects induced in poly(I:C)-transfected human renal cell carcinoma (RCC) cells.

Neuro-inflammation has long been implicated as a contributor to the progression of Alzheimer's disease in both humans and animal models. Type-1 interferons (IFNs) are pleiotropic cytokines critical in mediating the innate immune pro-inflammatory response. The production of type-1 IFNs following pathogen detection is, in part, through the activation of the toll-like receptors (TLRs) and subsequent signalling through myeloid differentiation factor-88 (Myd88) and interferon regulatory factors (IRFs). We have previously identified that neuronal type-1 IFN signalling, through the type-1 interferon alpha receptor-1 (IFNAR1), is detrimental in models of AD. Using an in vitro approach, this study investigated the TLR network as a potential production pathway for neuronal type-1 IFNs in response to Aβ.

The pathogenesis of systemic lupus erythematosus (SLE) is multifactorial, and the interferon regulatory factors (IRFs) play an important role. Autoantibodies formed in SLE target nuclear antigens, and immune complexes formed by these antibodies contain nucleic acid. These immune complexes can activate antiviral pattern recognition receptors (PRRs), resulting in the downstream activation of IRFs, which can induce type I interferon (IFN-I) and other inflammatory mediators. Genetic variations in IRFs have been associated with susceptibility to SLE, and current evidence supports the idea that these polymorphisms are gain of function in humans. Recent studies suggest that these genetic variations contribute to the break in humoral tolerance that allows for nucleic acid binding autoantibodies, and that the same polymorphisms also augment IFN-I production in the presence of these autoantibody immune complexes, forming a feed-forward loop. In this review, we will outline major features of the PRR/IRF systems and describe the role of the IRFs in human SLE pathogenesis.

The interferon (IFN) response is the major early innate immune response against invading viral pathogens and is even capable of mediating sterilizing antiviral immunity without the support of the adaptive immune system. Cumulative evidence suggests that the gut microbiota can modulate IFN responses, indirectly determining virological outcomes. This review outlines our current knowledge of the interactions between the gut microbiota and IFN responses and dissects the different mechanisms by which the gut microbiota may alter IFN expression to diverse viral infections. This knowledge offers a basis for translating experimental evidence from animal studies into the human context and identifies avenues for leveraging the gut microbiota-IFN-virus axis to improve control of viral infections and performance of viral vaccines.

Chicken MDA5 (chMDA5), the essential accepted pattern recognition receptors for detecting cytoplasmic viral RNA in chicken, initiates interferon β (IFN-β) generation. However, there is an incomplete elucidation of regulating chMDA5-mediated IFN-β production. NEMO-related protein, optineurin, was identified as inhibitors of virus triggered IFN-β induction in human or mice. In this study, full length of chicken optineurin (chOPTN) was cloned from chicken embryo fibroblast, and its role in inhibiting IFN-β signaling pathway was further explored. Full-length chOPTN encodes 547 amino acids residues and contains unique LC3 interaction region and ubiquitin binding domain. Chicken optineurin mRNA and protein are widely expressed in different tissues, especially the heart, kidney, and bursal fabricius (BF). Overexpressed chOPTN not only inhibits poly I:C or homos-induced human IFN-β promoter activation in 293T cells but also suppresses poly I:C, infectious bursal disease virus (IBDV) genome double-strand RNA (dsRNA), and chMDA5-induced chicken IFN-β (chIFN-β) promoter activation. In addition, we first revealed that chOPTN negatively regulates chIFN-β production via inhibiting ubiquitination of chicken TBK1, which is dependent on the ubiquitin-binding domain of chOPTN. Moreover, chIFN-β stimulus, poly I:C, and IBDV genome dsRNA improve chOPTN expression. Endogenous chOPTN expression is also upregulated by IBDV infection in 293T, DF-1 cells, as well as in BF. Therefore, our results suggested that chOPTN plays an inhibition role of chMDA5-mediated chIFN-β signaling pathway in chicken cells.

Viruses are detected by different classes of pattern recognition receptors that lead to the activation of interferon regulatory factors (IRF) and consequently to the induction of alpha/beta interferon (IFN-α/β). In turn, efficient viral strategies to escape the type I IFN-induced antiviral mechanisms have evolved. Previous studies established that pestivirus N(pro) antagonizes the early innate immune response by targeting the transcription factor IRF3 for proteasomal degradation. Here, we report that N(pro) of classical swine fever virus (CSFV) interacts also with IRF7, another mediator of type I IFN induction. We demonstrate that the Zn-binding domain of N(pro) is essential for the interaction of N(pro) with IRF7. For IRF3 and IRF7, the DNA-binding domain, the central region, and most of the regulatory domain are required for the interaction with N(pro). Importantly, the induction of IRF7-dependent type I IFN responses in plasmacytoid dendritic cells (pDC) is reduced after wild-type CSFV infection compared with infection with virus mutants unable to interact with IRF7. This is associated with lower levels of IRF7 in pDC. Consequently, wild-type but not N(pro) mutant CSFV-infected pDC show reduced responses to other stimuli. Taken together, the results of this study show that CSFV N(pro) is capable of manipulating the function of IRF7 in pDC and provides the virus with an additional strategy to circumvent the innate defense.

Depression is one of the major side effects of interferon alpha (IFN-α) treatment, but the molecular mechanism underlying IFN-α-induced depression remains unclear. Several studies have shown that the serotonin receptors 5-HTR1b and 5-HTR4 play key roles in the anti-depression effects associated with p11 (S100A10). We investigated the effects of IFN-α on the regulation of p11, 5-HTR1b and 5-HTR4 in mice and human neuroblastoma cells (SH-sy5y). We found that intraperitoneal injection with IFN-α in Balb/c mice resulted in an increased immobility in FST and TST, and potently lowered the protein levels of p11, 5-HTR1b and 5-HTR4 in the hippocampus or cingulate gyrus. IFN-α significantly down-regulated the protein levels of p11, 5-HTR1b and 5-HTR4 in SH-sy5y cells, in a time- and dose-dependent manner. Our study revealed that over-expression of p11 could prevent the IFN-α-induced down-regulation of 5-HTR1b and 5-HTR4. The results indicated that IFN-α treatment resulted in p11 down-regulation, which subsequently decreased 5-HTR1b and 5-HTR4 in vitro or in vivo. Our findings suggested that p11 might be a potential regulator on 5-HTR1b and 5-HTR4 as well as a predictor of or a therapeutic target for IFN-α-induced depression.