Severe diarrhea in a 10-year-old girl with Aicardi-Goutières syndrome due to IFIH1 gene mutation.

Interferon-induced with helicase C domain 1 (IFIH1) is a cytosolic sensor of dsRNA that induces an anti-viral Type I interferon (IFN) state. A gain-of-function mutation in IFIH1 can cause increased Type I IFN activity and is clinically associated with Aicardi-Goutières syndrome (AGS). AGS is a multisystem disease, characterized as an early-onset progressive encephalopathy with basal ganglia calcification and systemic lupus erythematosus-like features. Gastrointestinal manifestation is rare in AGS patients. We described a 10-year-old female patient with a heterozygous IFIH1 gene mutation who presented with gastrointestinal colitis, cystitis and very severe diarrhea as initial major manifestations of AGS. Proteinuria with high titer of antinuclear antibody and anti-double-stranded DNA was found in this patient. She also had growth retardation and a history of seizures (about two episodes each year) but without attacks until 7 years old. Serum cytokines detected by flow cytometry indicated extremely high level of interleukin 6 (1970.1 pg/ml) and IFN-α (204.1 pg/ml). A contrast-enhanced CT scan of the whole abdomen and an intestinal hydro-MRI indicated that the walls of her stomach, small bowel, colon, and bladder were in various degrees of edema and thickened states. Whole exome sequencing analysis indicated that she harbors an IFIH1 heterozygous mutation (c.2336G > A (p.R779H)) in both blood and intestinal samples. Abundant inflammatory cells infiltration into the intestinal epithelium was observed by immunohistochemical staining. Positive staining of caspase 4 and caspase 5 suggested that the signaling pathway of pyroptosis was involved in the mechanism of intestinal inflammation in AGS. Diarrhea was significantly improved after steroids and intravenous immunoglobulin treatments. Gastrointestinal colitis and cystitis can be rare manifestations of AGS with IFIH1 mutation. Caspase and its related inflammasome pathway may involve in the pathogenesis of AGS.

ADAR1 Regulates Early T Cell Development via MDA5-Dependent and -Independent Pathways.

ADAR1 is an RNA-editing enzyme that is abundant in the thymus. We have previously reported that ADAR1 is required for establishing central tolerance during the late stage of thymocyte development by preventing MDA5 sensing of endogenous dsRNA as nonself. However, the role of ADAR1 during the early developmental stage remains unknown. In this study, we demonstrate that early thymocyte-specific deletion of ADAR1 in mice caused severe thymic atrophy with excessive apoptosis and impaired transition to a late stage of development accompanied by the loss of TCR expression. Concurrent MDA5 deletion ameliorated apoptosis but did not restore impaired transition and TCR expression. In addition, forced TCR expression was insufficient to restore the transition. However, simultaneous TCR expression and MDA5 deletion efficiently ameliorated the impaired transition of ADAR1-deficient thymocytes to the late stage. These findings indicate that RNA-editing-dependent and -independent functions of ADAR1 synergistically regulate early thymocyte development.

CCR5 is a required signaling receptor for macrophage expression of inflammatory genes in response to viral double-stranded RNA.

Double-stranded (ds) RNA, both synthetic and produced during virus replication, rapidly stimulates MAPK and NF-κB signaling that results in expression of the inflammatory genes inducible nitric oxide synthase, cyclooxygenase 2, and IL-1ß by macrophages. Using biochemical and genetic approaches, we have identified the chemokine ligand-binding C-C chemokine receptor type 5 (CCR5) as a cell surface signaling receptor required for macrophage expression of inflammatory genes in response to dsRNA. Activation of macrophages by synthetic dsRNA does not require known dsRNA receptors, as poly(inosinic:cytidylic) acid [poly(I:C)] activates signaling pathways leading to expression of inflammatory genes to similar levels in wild-type and Toll-like receptor 3- or melanoma differentiation antigen 5-deficient macrophages. In contrast, macrophage activation in response to poly(I:C) is attenuated in macrophages isolated from mice lacking CCR5. These findings support a role for CCR5 as a cell surface signaling receptor that participates in activation of inflammatory genes in macrophages in response to the viral dsRNA mimetic poly(inosinic:cytidylic) acid by pathways that are distinct from classical dsRNA receptor-mediated responses.

A Plant-Derived Nucleic Acid Reconciles Type I IFN and a Pyroptotic-like Event in Immunity against Respiratory Viruses.

Nucleic acids carrying pathogen-associated molecular patterns trigger innate immune responses and are used to activate host immunity. Although synthetic nucleic acids have been used for that purpose, they have shown limitations for in vivo and clinical applications. To address this issue, we tested a naturally occurring dsRNA extracted from rice bran (rb-dsRNA) and characterized it as a potent ligand of TLR3 and MDA5. In this study, intranasal administration of rb-dsRNA induced production of type I IFNs by alveolar macrophages and protected mice from morbidity and mortality resulting from respiratory virus infection, such as influenza A virus. This protection was completely absent in mice lacking both TRIF and MDA5, indicating the essential role of TLR3- and MDA5-dependent pathways. Interestingly, IFNAR1-deficient mice retained residual antiviral protection, which was abolished by pharmacological inhibition of caspase 1, but not IL-1ß signaling. In fact, rb-dsRNA activated caspase 1 via TRIF, resulting in the release of IL-1ß and LDH. In addition to the direct antiviral activity, rb-dsRNA modulated the immune cell population in the lungs by repopulating virus-depleted alveolar macrophages. Our data demonstrate that rb-dsRNA orchestrates IFN-dependent and -independent direct antiviral protection and that it is a potent immune stimulator modulating antiviral immunity in the lungs. These findings open doors to a range of precise immune-modulating studies and therapeutic options.

Recurrent rhinovirus infections in a child with inherited MDA5 deficiency.

MDA5 is a cytosolic sensor of double-stranded RNA (ds)RNA including viral byproducts and intermediates. We studied a child with life-threatening, recurrent respiratory tract infections, caused by viruses including human rhinovirus (HRV), influenza virus, and respiratory syncytial virus (RSV). We identified in her a homozygous missense mutation in IFIH1 that encodes MDA5. Mutant MDA5 was expressed but did not recognize the synthetic MDA5 agonist/(ds)RNA mimic polyinosinic-polycytidylic acid. When overexpressed, mutant MDA5 failed to drive luciferase activity from the IFNB1 promoter or promoters containing ISRE or NF-κB sequence motifs. In respiratory epithelial cells or fibroblasts, wild-type but not knockdown of MDA5 restricted HRV infection while increasing IFN-stimulated gene expression and IFN-ß/λ. However, wild-type MDA5 did not restrict influenza virus or RSV replication. Moreover, nasal epithelial cells from the patient, or fibroblasts gene-edited to express mutant MDA5, showed increased replication of HRV but not influenza or RSV. Thus, human MDA5 deficiency is a novel inborn error of innate and/or intrinsic immunity that causes impaired (ds)RNA sensing, reduced IFN induction, and susceptibility to the common cold virus.

RIG-I-like Receptor Triggering by Dengue Virus Drives Dendritic Cell Immune Activation and TH1 Differentiation.

Dengue virus (DENV) causes 400 million infections annually and is one of several viruses that can cause viral hemorrhagic fever, which is characterized by uncontrolled immune activation resulting in high fever and internal bleeding. Although the underlying mechanisms are unknown, massive cytokine secretion is thought to be involved. Dendritic cells (DCs) are the main target cells of DENV, and we investigated their role in DENV-induced cytokine production and adaptive immune responses. DENV infection induced DC maturation and secretion of IL-1ß, IL-6, and TNF. Inhibition of DENV RNA replication abrogated these responses. Notably, silencing of RNA sensors RIG-I or MDA5 abrogated DC maturation, as well as cytokine responses by DENV-infected DCs. DC maturation was induced by type I IFN responses because inhibition of IFN-α/ß receptor signaling abrogated DENV-induced DC maturation. Moreover, DENV infection of DCs resulted in CCL2, CCL3, and CCL4 expression, which was abrogated after RIG-I and MDA5 silencing. DCs play an essential role in TH cell differentiation, and we show that RIG-I and MDA5 triggering by DENV leads to TH1 polarization, which is characterized by high levels of IFN-γ. Notably, cytokines IL-6, TNF, and IFN-γ and chemokines CCL2, CCL3, and CCL4 have been associated with disease severity, endothelial dysfunction, and vasodilation. Therefore, we identified RIG-I and MDA5 as critical players in innate and adaptive immune responses against DENV, and targeting these receptors has the potential to decrease hemorrhagic fever in patients.

MDA-5 activation by cytoplasmic double-stranded RNA impairs endothelial function and aggravates atherosclerosis.

Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA-5) belongs to the intracellular retinoic acid inducible gene-I like receptors and its activation promotes pro-inflammatory mechanisms. Here, we studied the effect of MDA-5 stimulation in vascular biology. To gain insights into MDA-5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA-5 agonist polyIC (long double-stranded RNA). Human coronary endothelial cell expressed MDA-5 and reacted with receptor up-regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro-inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA-5 stimulation. To test these effects in vivo, wild-type mice were transfected with 32.5 µg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC-treated wild-type mice, endothelium-dependent vasodilation and re-endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA-5 deficiency in vivo. Finally, chronic MDA-5 stimulation in Apolipoprotein E/toll-like receptor 3 (TLR3) double(-) deficient (ApoE(-/-) /TLR3(-/-) ) mice-enhanced atherosclerotic plaque formation. This study demonstrates that MDA-5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA-5.