Inflammasome activation is gaining recognition as an important mechanism for protection during viral infection. Here, we investigate whether Rift Valley fever virus, a negative-strand RNA virus, can induce inflammasome responses and IL-1β processing in immune cells. We have determined that RVFV induces NLRP3 inflammasome activation in murine dendritic cells, and that this process is dependent upon ASC and caspase-1. Furthermore, absence of the cellular RNA helicase adaptor protein MAVS/IPS-1 significantly reduces extracellular IL-1β during infection. Finally, direct imaging using confocal microscopy shows that the MAVS protein co-localizes with NLRP3 in the cytoplasm of RVFV infected cells.
inflammasome; NLRP3; ASC; caspase-1; Rift Valley fever virus; virus; IL-1β; dendritic cells; murine
Hemozoin (Hz) is the crystalline detoxification product of hemoglobin in plasmodial-infected erythrocytes. We previously proposed that Hz can carry plasmodial DNA into a subcellular compartment accessible to Toll-like receptor 9 (TLR9), inducing an inflammatory signal. Hemozoin also activates the NLRP3 inflammasome in primed cells. We found that Hz appears to co localize with DNA in infected erythrocytes, even prior to RBC rupture or phagolysosomal digestion. Using synthetic Hz coated in vitro with plasmodial genomic DNA (gDNA) or CpG-oligonucleotides, we observed that DNA-complexed Hz induced TLR9 translocation, providing a priming and an activation signal for inflammasomes. After phagocytosis, Hz and DNA dissociate. Hz subsequently induces phagolysosomal destabilization, allowing phagolysosomal contents access to the cytosol where DNA receptors become activated. Similar observations were made with plasmodial-infected RBC. Finally, infected erythrocytes activated both the NLRP3 and AIM2 inflammasomes. These observations suggest that Hz and DNA work together to induce systemic inflammation during malaria.
Autophagy has been implicated as a component of host defense, but the significance of antimicrobial autophagy in vivo and the mechanism by which it is regulated during infection are poorly defined. Here we found that antiviral autophagy was conserved in flies and mammals during infection with Rift Valley fever virus (RVFV), a mosquito-borne virus that causes disease in humans and livestock. In Drosophila, Toll-7 limited RVFV replication and mortality through activation of autophagy. RVFV infection also elicited autophagy in mouse and human cells, and viral replication was increased in the absence of autophagy genes. The mammalian Toll-like receptor adaptor, MyD88, was required for anti-RVFV autophagy, revealing an evolutionarily conserved requirement for pattern-recognition receptors in antiviral autophagy. Pharmacologic activation of autophagy inhibited RVFV infection in mammalian cells, including primary hepatocytes and neurons. Thus, autophagy modulation might be an effective strategy for treating RVFV infection, which lacks approved vaccines and therapeutics.
Loss of function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-Tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces SLE-like disease. While Fas/FasL interactions have been strongly implicated in the activation induced cell death of both lymphocytes and other antigen presenting cells, FasL can also trigger the production of pro-inflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase (MMP) cleavage site in the ectodomain. MMP cleavage inactivates membrane-bound FasL (mFasL) and releases a soluble form, sFasL, reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the pro-apoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation, to produce the ΔCS mouse line. ΔCS mice express higher levels of mFasL than WT mice and fail to release sFasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared to control TMPD-injected BALB/c mice. We found that FasL-deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. By contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in ANA specificity, and markedly increased proteinuria and kidney pathology, compared to controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL pro-inflammatory activity.
Activation of the NLRP3 inflammasome by diverse stimuli requires a priming signal from Toll-like receptors (TLRs) and an activation signal from purinergic receptors or pore-forming toxins. Here we demonstrate through detailed analysis of NLRP3 activation in macrophages deficient in key downstream TLR signaling molecules that MyD88 is required for an immediate early phase, whereas TRIF is required for a subsequent intermediate phase of posttranslational NLRP3 activation. Both IRAK1 and IRAK4 kinases are critical for rapid activation of NLRP3 through the MyD88 pathway, but only IRAK1 is partially required in the TRIF pathway. IRAK1 and IRAK4 are also required for rapid activation of NLRP3 by Listeria monocytogenes as deletion of IRAK1 or IRAK4 led to defective inflammasome activation. These findings define the pathways that lead to rapid NLRP3 activation and identify IRAK1 as a critical mediator of a transcription-independent, inflammasome-dependent early warning response to pathogenic infection.
Mycobacterium tuberculosis (Mtb) extracellular DNA (eDNA) gains access to the host cell cytosol via the ESX-1 secretion system. It is puzzling that this eDNA of Mtb does not induce activation of the AIM2-inflammasome since AIM2 recognizes cytosolic DNA. Here we show that non-virulent mycobacteria such as M. smegmatis induce AIM2-inflammasome activation, which is dependent upon their strong induction of IFN-β production. In contrast, Mtb, but not an ESX-1 deficient mutant, inhibits the AIM2-inflammasome activation induced by either M. smegmatis or transfected dsDNA. The inhibition does not involve changes in host cell AIM2 mRNA or protein levels but led to decreased activation of caspase-1. We furthermore demonstrate that Mtb inhibits IFN-β production and signaling, which was partially responsible for the inhibition of AIM2 activation. In conclusion, we report a novel immune evasion mechanism of Mtb that involves the ESX-1-dependent, direct or indirect, suppression of the host cell AIM2-inflammasome activation during infection.
Synthetic oligodeoxynucleotides comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. Here, we demonstrate that such suppressive oligodeoxynucleotides (sup ODN) abrogate activation of cytosolic nucleic acid sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNFα and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and murine cytomegalovirus (MCMV). Inhibition was dependent on A151’s phosphorothioate backbone while substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases.
Measurement of protease activity in living cells or organisms remains a challenging task. We here present a transgene-encoded biosensor that reports the proteolytic activity of caspase-1 in the course of inflammasome activation and that of other proteases in a highly sensitive and specific manner. This protease reporter is based on the biological activity of a pro-interleukin (IL)-1β-Gaussia luciferase (iGLuc) fusion construct, in which pro-IL-1β-dependent formation of protein aggregates renders GLuc enzyme inactive. Cleavage leads to monomerization of this biosensor protein, resulting in a strong gain in luciferase activity. Exchange of the canonical caspase-1 cleavage site in this reporter construct allows the generation of protease biosensors with additional specificities. The high sensitivity, signal-to-background ratio and specificity of the iGLuc system renders it a useful tool to study proteolytic events in mouse and human cells at high throughput and to monitor protease activity in mice in vivo.
Autophagosomes delivers cytoplasmic constituents to lysosomes for degradation while inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. Here we show that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered RalB activation and autophagosome formation. The induction of autophagy did not depend upon ASC or capase-1, but was dependent on the presence of the inflammasome sensor. Blocking autophagy potentiated inflammasome activity while stimulating autophagy limited it. Assembled inflammasomes underwent ubiquitination and recruited the autophagic adaptor p62, which assisted their delivery to autophagosomes. Our data indicate that autophagy accompanies inflammasome activation to temper inflammation by eliminating active inflammasomes.
inflammasome; macrophage; autophagy; signal transduction; ubiquitination
The nucleotidyl transferase cGAS, its second messenger product cGAMP and the cGAMP sensor STING, form the basic mechanism of DNA sensing in the cytoplasm of mammalian cells. Several new reports now uncover key structural features associated with DNA recognition by cGAS and the catalytic mechanisms of cGAMP generation. Concurrent studies also reveal unique phosphodiester linkages in endogenous cGAMP that distinguish it from microbial cGAMP and other cyclic-di-nucleotides. Together, these studies provide a new perspective on DNA recognition in the innate immune system.
Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a novel pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/β receptor–dependent manner. This signaling pathway is critical for immune cell–mediated host resistance to liver-stage Plasmodium infection, which can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.
Obesity is associated with the development of asthma and considerable asthma-related healthcare utilization. To understand the immunological pathways that lead to obesity-associated asthma, we fed mice a high fat diet for 12 weeks, which resulted in obesity and the development of airway hyperreactivity (AHR), a cardinal feature of asthma. This AHR depended on innate immunity, since it occurred in obese Rag−/− mice, and on IL-17A and the NLRP3 inflammasome, since it did not develop in obese Il17−/− or Nlrp3−/− mice. The AHR was also associated with the presence in the lungs of CCR6+ innate lymphoid cells producing IL-17A (ILC3 cells), which could by themselves mediate AHR when adoptively transferred into Rag2−/−
Il2rγ−/− mice. IL-1β played an important role by expanding the ILC3 cells, and treatment to block the function of IL-1β abolished obesity-induced AHR. Since we found ILC3-like cells in the bronchoalveolar lavage fluid of human patients with asthma, we suggest that obesity-associated asthma is facilitated by inflammation mediated by NLRP3, IL-1β and ILC3 cells.
airway hyperreactivity; asthma; obesity; innate lymphoid cells; IL-17; NLRP3; ILC3
Vascular disrupting agents (VDAs) such as DMXAA (5,6-dimethylxanthenone-4-acetic acid) represent a novel approach for cancer treatment. DMXAA has potent anti-tumor activity in mice and, despite significant pre-clinical promise, failed human clinical trials. The anti-tumor activity of DMXAA has been linked to its ability to induce type I interferons in macrophages although the molecular mechanisms involved are poorly understood. Here we identify STING as a direct receptor for DMXAA leading to TBK1 and IRF3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic-dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anti-cancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.
Nucleotide-binding oligomerization domain-like receptors (NLRs) detect pathogens and danger-associated signals within the cell. Salmonella Typhimurium, an intracellular pathogen, activates caspase-1 required for the processing of the pro-inflammatory cytokines, pro-IL-1β and pro-IL-18, and pyroptosis. Here we show that Salmonella infection induces the formation of an ASC–Caspase-8–Caspase-1 inflammasome in macrophages. Caspase-8 and caspase-1 are recruited to the ASC focus independently of one other. Salmonella infection initiates caspase-8 proteolysis in a manner dependent on NLRC4 and ASC, but not NLRP3, caspase-1 or caspase-11. Caspase-8 primarily mediates the synthesis of pro-IL-1β, but is dispensable for Salmonella-induced cell death. Overall, our findings highlight that the ASC inflammasome can recruit different members of the caspase family to induce distinct effector functions in response to Salmonella infection.
The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9, and that non-immune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, like macrophages and conventional dendritic cells, detect infections with herpesviruses. Here we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFI16 is important for induction of IFN-β in human macrophages after infection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as IFI16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.
Recognition of microbial nucleic acids is one strategy by which mammalian hosts respond to infectious agents. Intracellular DNA which is introduced into cells during infection elicits potent inflammatory responses by triggering the induction of antiviral type I interferons and the maturation and secretion of inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin (IL)-1β and IL-18. In addition, if nucleases such as DNase II or DNase III (Trex1) fail to clear self-DNA accumulated DNA gains access to intracellular compartments where it drives inflammatory responses leading to autoimmune disease. In this review, we discuss a rapidly evolving view of how cytosolic DNA sensing machineries coordinate antimicrobial immunity and if unchecked lead to autoimmune disease.
Great progress has been made in understanding how immune cells detect microbial pathogens. An area that has received particular attention is nucleic acid sensing where RNA and DNA sensing machineries have been uncovered. For DNA, TLR9 in endosomes and numerous cytoplasmic DNA binding proteins have been identified. Several of these have been proposed to couple DNA recognition to induction of type I IFNs, pro-inflammatory cytokines and/or caspase-1 activation. Given the ubiquitous expression of many of these DNA binding proteins and the significant potential for endogenous DNA to engage these molecules, it is important that DNA recognition is tightly regulated. A better understanding of DNA recognition pathways can provide new insights into infectious, inflammatory and autoimmune diseases.
Particulate ligands including cholesterol crystals and amyloid fibrils induce NLRP3-dependent production of interleukin-1β (IL-1β) in atherosclerosis, Alzheimer's disease and diabetes. Soluble endogenous ligands including oxidized-LDL, amyloid-β and amylin peptides accumulate in these diseases. Here we identify a CD36-mediated endocytic pathway that coordinates the intracellular conversion of these soluble ligands to crystals or fibrils, resulting in lysosomal disruption and NLRP3-inflammasome activation. Consequently, macrophages lacking CD36 failed to elicit IL-1β production in response to these ligands and targeting CD36 in atherosclerotic mice reduced serum IL-1β and plaque cholesterol crystal accumulation. Collectively, these findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation.
Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14+CD16−Caspase-1+ and CD14dimCD16+Caspase-1+ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.
Together Plasmodium falciparum and P. vivax infect approximately 250 million individuals, reaping life of near one million children every year. Extensive research on malaria pathogenesis has funneled into the consensus that the clinical manifestations are often a consequence of the systemic inflammation. Importantly, secondary bacterial and viral infections potentiate this inflammatory reaction being important co-factors for the development of severe disease. One of the hallmarks of malaria syndrome is the paroxysm, which is characterized by high fever associated with peak of parasitemia. In this study we dissected the mechanisms of induction and the importance of the pyrogenic cytokine, IL-1β in the pathogenesis of malaria. Our results demonstrate the critical role of the innate immune receptors named Toll-Like Receptors and inflammasome on induction, processing and release of active form of IL-1β during malaria. Importantly, we provide evidences that bacterial superinfection further potentiates the Plasmodium-induced systemic inflammation, leading to the release of bulk amounts of IL-1β and severe disease. Hence, this study uncovers new checkpoints that could be targeted for preventing systemic inflammation and severe malaria.
Viral RNA is sensed by TLR 7 and 8 or by the RNA helicases LGP2, MDA5 and RIG-I to trigger antiviral responses. Much less is known about sensors for DNA. Here we identify a novel DNA sensing pathway involving RNA polymerase III and RIG-I. AT-rich dsDNA serve as a template for RNA polymerase III, which is transcribed into dsRNA harboring a 5′ triphosphate moiety which signals via RIG-I to activate type I IFN gene transcription and NF-κB. This pathway is also important in sensing Epstein-Barr virus encoded small RNAs, which are transcribed by RNA polymerase III and then trigger RIG-I activation. Thus, RNA Pol III and RIG-I play a pivotal role in coordinating anti-viral defenses in the innate immune response.
Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages, the type I, II and III lineages. However, in South America these strains are rarely isolated and instead a great variety of other strains are found. T. gondii strains differ widely in a number of phenotypes in mice, such as virulence, persistence, oral infectivity, migratory capacity, induction of cytokine expression and modulation of host gene expression. The outcome of toxoplasmosis in patients is also variable and we hypothesize that, besides host and environmental factors, the genotype of the parasite strain plays a major role. The molecular basis for these differences in pathogenesis, especially in strains other than the clonal lineages, remains largely unexplored. Macrophages play an essential role in the early immune response against T. gondii and are also the cell type preferentially infected in vivo. To determine if non-canonical Toxoplasma strains have unique interactions with the host cell, we infected murine macrophages with 29 different Toxoplasma strains, representing global diversity, and used RNA-sequencing to determine host and parasite transcriptomes. We identified large differences between strains in the expression level of known parasite effectors and large chromosomal structural variation in some strains. We also identified novel strain-specifically regulated host pathways, including the regulation of the type I interferon response by some atypical strains. IFNβ production by infected cells was associated with parasite killing, independent of interferon gamma activation, and dependent on endosomal Toll-like receptors in macrophages and the cytoplasmic receptor retinoic acid-inducible gene 1 (RIG-I) in fibroblasts.
Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii. The parasite is found throughout the world. When humans are infected, few have symptoms because a healthy immune system usually prevents the parasite from causing illness. Nevertheless, cases of severe disease in otherwise healthy individuals have been observed. These cases are usually a result of infection with less common atypical strains of Toxoplasma. Factors associated with virulence in the atypical strains are not well understood. Here, we infected host cells with 29 different strains of Toxoplasma, and performed high-throughput RNA sequencing of both host cells and parasites. We found significant differences in gene expression profiles between strains. Host cell transcriptional response also varied substantially depending on the infecting strain. Specifically, we found that a small group of atypical strains are able to induce production of type I interferons, which are immunomodulatory cytokines. Interferon production is a result of the elimination of internalized parasites through a novel killing mechanism. The dataset we generated is a valuable tool for identification of host cell targets of Toxoplasma secreted effectors and can contribute to our understanding of why certain Toxoplasma strains are more prone to cause severe disease in humans.
Fas, a tumor necrosis factor family receptor, is activated by the membrane protein Fas ligand (FasL) expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas induced cytokines, the IL-1β family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1β cytokines via caspase-1. The mechanisms, by which Fas regulates IL-1β activation, however, remain unresolved. Here, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells leading to the maturation of IL-1β and IL-18 independently of inflammasomes or Rip3. Hence, Fas controls a novel non-canonical IL-1β activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance and control of infectious diseases.
Prion diseases are fatal transmissible neurodegenerative diseases, characterized by aggregation of the pathological form of prion protein, spongiform degeneration, neuronal loss and activation of astrocytes and microglia. Microglia can clear prion plaques but on the other hand cause neuronal death via release of neurotoxic species. Elevated expression of the proinflammatory cytokine IL-1β has been observed in brains affected by several prion diseases and IL-1R-deficiency significantly prolonged the onset of the neurodegeneration in mice. We show that microglial cells stimulated by prion protein (PrP) fibrils induced neuronal toxicity. Microglia and macrophages release IL-1β upon stimulation by PrP fibrils, which depends on the NLRP3 inflammasome. Activation of NLRP3 inflammasome by PrP fibrils requires depletion of intracellular K+ and requires phagocytosis of PrP fibrils and consecutive lysosome destabilization. Among the well-defined molecular forms of PrP the strongest NLRP3 activation was observed by fibrils, followed by aggregates, while neither native monomeric nor oligomeric PrP were able to activate the NLRP3 inflammasome. Our results together with previous studies on IL-1R-deficient mice suggest the IL-1 signaling pathway as the perspective target for the therapy of prion disease.
prions; amyloid; inflammasome; NLRP3; IL-1β; neuroinflammation
Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense.
Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.