RESUMEN
Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.
Asunto(s)
Predisposición Genética a la Enfermedad , Lupus Eritematoso Sistémico , Receptor Toll-Like 7 , Lupus Eritematoso Sistémico/genética , Humanos , Animales , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Ratones , Niño , Femenino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Masculino , Edad de Inicio , Variación Genética , FN-kappa B/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Adolescente , Células THP-1 , Interferón Tipo I/metabolismoRESUMEN
Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic T cell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and T cells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.
Asunto(s)
Atresia Biliar/inmunología , Atresia Biliar/terapia , Hígado/inmunología , Animales , Antígenos CD20/metabolismo , Linfocitos B/inmunología , Atresia Biliar/sangre , Atresia Biliar/tratamiento farmacológico , Biopsia , Receptor 1 de Quimiocinas CX3C/metabolismo , Muerte Celular , Línea Celular , Proliferación Celular , Transdiferenciación Celular , Niño , Preescolar , Estudios de Cohortes , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G/metabolismo , Lactante , Inflamación/patología , Células Asesinas Naturales/inmunología , Macrófagos del Hígado/patología , Hígado/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Depleción Linfocítica , Linfopoyesis , Masculino , Ratones Endogámicos BALB C , Fagocitosis , ARN/metabolismo , Rituximab/administración & dosificación , Rituximab/farmacología , Rituximab/uso terapéutico , Rotavirus/fisiología , Análisis de la Célula Individual , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/terapia , Mucosa Intestinal/patología , Antígenos CD/metabolismo , Apirasa/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Muerte Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Niño , Estudios de Cohortes , Colon/patología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Dipiridamol/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Homeostasis/efectos de los fármacos , Humanos , Inmunoglobulina G/sangre , Memoria Inmunológica , Inflamación/patología , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/genética , Interferón Tipo I/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metilprednisolona/farmacología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismoRESUMEN
Post-translational modifications (PTMs) of proteins are crucial to guarantee the proper biological functions in immune responses. Although protein phosphorylation has been extensively studied, our current knowledge of protein pyrophosphorylation, which occurs based on phosphorylation, is very limited. Protein pyrophosphorylation is originally considered to be a non-enzymatic process, and its function in immune signaling is unknown. Here, we identify a metabolic enzyme, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), as a pyrophosphorylase for protein serine pyrophosphorylation, by catalyzing the pyrophosphorylation of interferon regulatory factor 3 (IRF3) at serine (Ser) 386 to promote robust type I interferon (IFN) responses. Uap1 deficiency significantly impairs the activation of both DNA- and RNA-viruse-induced type I IFN pathways, and the Uap1-deficient mice are highly susceptible to lethal viral infection. Our findings demonstrate the function of protein pyrophosphorylation in the regulation of antiviral responses and provide insights into the crosstalk between metabolism and innate immunity.
Asunto(s)
Factor 3 Regulador del Interferón , Interferón Tipo I , Animales , Ratones , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Fosforilación , Transducción de Señal , Galactosiltransferasas/metabolismoRESUMEN
Microglia and astrocytes are subgroups of brain glia cells that support and protect neurons within the central nervous system (CNS). At early stages of viral infection in the CNS, they are predominant responding cells and lead to recruitment of peripheral immune cells for viral clearance. Inhibitor of nuclear factor κB kinase subunit epsilon (IKKi) is critical for type I interferon signalling and inflammation, which modulate heterogenic immune responses during CNS infection. Balanced autophagy is vital to maintain brain integrity, yet regulation of autophagy and immune activity within brain glia cells is poorly understood. Here we identify SHISA9 as an autophagy cargo receptor that mediates the autophagy-dependent degradation of IKKi during herpes simplex virus type 1 infection. IKKi is recognized by SHISA9 through unanchored K48-linked poly-ubiquitin chains and bridged to autophagosome membrane components GABARAPL1. Single-cell RNA sequencing analysis shows that SHISA9 has temporal characteristics while modulating both antiviral and inflammatory responses in microglia and astrocytes at different stages during viral infection. We found that Shisa9-/- mice are highly susceptible to herpes simplex virus encephalitis, have pathogenic astrocytes and display more severe neuroinflammation compared with wild-type mice. Taken together, our study unravels a critical role of selective autophagy by orchestrating immune heterogeneity of different CNS resident cells through the SHISA9-IKKi axis.
Asunto(s)
Enfermedades Neuroinflamatorias , Virosis , Animales , Ratones , Autofagia , Encéfalo/metabolismo , Sistema Nervioso Central , Enfermedades Neuroinflamatorias/metabolismo , Virosis/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has had a significant impact on healthcare systems and economies worldwide. The continuous emergence of new viral strains presents a major challenge in the development of effective antiviral agents. Strategies that possess broad-spectrum antiviral activities are desirable to control SARS-CoV-2 infection. ACE2, an angiotensin-containing enzyme that prevents the overactivation of the renin angiotensin system, is the receptor for SARS-CoV-2. ACE2 interacts with the spike protein and facilitates viral attachment and entry into host cells. Yet, SARS-CoV-2 infection also promotes ACE2 degradation. Whether restoring ACE2 surface expression has an impact on SARS-CoV-2 infection is yet to be determined. Here, we show that the ACE2-spike complex is endocytosed and degraded via autophagy in a manner that depends on clathrin-mediated endocytosis and PAK1-mediated cytoskeleton rearrangement. In contrast, free cellular spike protein is selectively cleaved into S1 and S2 subunits in a lysosomal-dependent manner. Importantly, we show that the pan-PAK inhibitor FRAX-486 restores ACE2 surface expression and suppresses infection by different SARS-CoV-2 strains. FRAX-486-treated Syrian hamsters exhibit significantly decreased lung viral load and alleviated pulmonary inflammation compared with untreated hamsters. In summary, our findings have identified novel pathways regulating viral entry, as well as therapeutic targets and candidate compounds for controlling the emerging strains of SARS-CoV-2 infection.
Asunto(s)
COVID-19 , SARS-CoV-2 , Internalización del Virus , Quinasas p21 Activadas , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/patología , COVID-19/virología , Citoesqueleto , Quinasas p21 Activadas/metabolismo , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
Mitophagy is a major quality control pathway that removes unwanted or dysfunctional mitochondria and plays an essential role in vascular health. Here we show that MCM8 expression is significantly decreased in children with Kawasaki disease (KD) who developed coronary artery aneurysms. Mechanistically, we discovered that nitric oxide signaling promotes TRIM21-mediated MCM8 ubiquitination, which disrupts its interaction with MCM9 and promotes its cytosolic export. In the cytosol, MCM8 relocates to the mitochondria pore-forming proteins and promotes their ubiquitination by TRIM21. In addition, MCM8 directly recruits LC3 via its LC3-interacting region (LIR) motif and initiates mitophagy. This suppresses mitochondrial DNA-mediated activation of type I interferon via cGAS and STING. Mice that are deficient in Mcm8, Trim21 and Nos2 or reconstituted with the East-Asian-specific MCM8-P276 variant develop more severe coronary artery vasculopathy in the Lactobacillus casei extract-induced KD model. Collectively, the data suggest that MCM8 protects vascular health in the KD setting.
Asunto(s)
Modelos Animales de Enfermedad , Mitofagia , Síndrome Mucocutáneo Linfonodular , Óxido Nítrico , Transducción de Señal , Animales , Síndrome Mucocutáneo Linfonodular/metabolismo , Síndrome Mucocutáneo Linfonodular/genética , Síndrome Mucocutáneo Linfonodular/patología , Humanos , Óxido Nítrico/metabolismo , Ratones Noqueados , Ratones , Masculino , Ubiquitinación , Ratones Endogámicos C57BL , Femenino , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genéticaRESUMEN
Ubiquitination plays a crucial role in retinoic acid-inducible gene I (RIG-I)-induced antiviral responses. However, the precise regulatory mechanisms of RIG-I activity mediated by conjugated and unanchored ubiquitin chains remain to be determined. In this study, we discovered that T55 of RIG-I was required for its binding ability for the unanchored ubiquitin chains. Experimental and mathematical analysis showed that unanchored ubiquitin chains associated with RIG-I were essential for sustained activation of type I interferon (IFN) signaling. Transcriptomics study revealed that the binding of RIG-I with unanchored ubiquitin chains additionally regulated the expression of a subset of metabolic and cell fate decision genes. Moreover, we found that ubiquitin-specific peptidase 21 (USP21) and USP3 deubiquitinate conjugated and unanchored ubiquitin chains on RIG-I respectively. Taken together, characterization of the regulation mode and functions of conjugated ubiquitination and the unconjugated ubiquitin chain-binding of RIG-I may provide means to fine-tune RIG-I-mediated type I IFN signaling.
RESUMEN
DDX58/RIG-I, is a critical pattern recognition receptor for viral RNA, which plays an essential role in antiviral immunity. Its posttranslational modifications and stability are tightly regulated to mediate the moderate production of type I IFN to maintain the immune homeostasis. Recently, we reported that macroautophagy/autophagy balances type I IFN signaling through selective degradation of ISG15-associated DDX58 via LRRC25. However, the regulatory mechanism about the autophagic degradation of DDX58 remains largely undefined. Here, we identified LRRC59 as a vital positive regulator of DDX58-mediated type I IFN signaling. Upon virus infection, LRRC59 specifically interacted with ISG15-associated DDX58 and blocked its association with LRRC25, the secondary receptor to deliver DDX58 to autophagosomes for SQSTM1/p62-dependent degradation, leading to the stronger antiviral immune responses. Thus, our study reveals a novel regulatory role of selective autophagy in innate antiviral responses mediated by the cross-regulation of LRRC family members. These data further provide insights into the crosstalk between autophagy and innate immune responses.Abbreviations: ATG: Autophagy-related; Baf A1: Bafilomycin A1; DDX58/RIG-I: DEAD [Asp-Glu-Ala-Asp] box polypeptide 58; EV: Empty vector; IC poly[I:C]: Intracellular polyriboinosinic polyribocytidylic acid; IFIH1/MDA5: Interferon induced with helicase C domain 1; IFN: Interferon; ISG15: ISG15 ubiquitin like modifier; IKBKE: Inhibitor of nuclear factor kappa B kinase subunit epsilon; IRF3: Interferon regulatory factor 3; KO: Knockout; LRRC: Leucine rich repeat containing; MAVS: Mitochondrial antiviral signaling protein; CGAS/MB21D1: Cyclic GMP-AMP synthase; SeV: Sendai virus; siRNA: small interfering RNA; SQSTM1/p62: Sequestosome 1; TBK1: TANK binding kinase 1; TLR: Toll like receptor; TMEM173/STING: Transmembrane protein 173; VSV: Vesicular stomatitis virus; WT: Wild type.
Asunto(s)
Autofagia , Proteína 58 DEAD Box/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Proteolisis , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Antivirales/metabolismo , Línea Celular , Citocinas/metabolismo , Proteína 58 DEAD Box/química , Humanos , Modelos Biológicos , Unión Proteica , Estabilidad Proteica , Receptores Inmunológicos , Ubiquitinas/metabolismo , Virosis/metabolismoRESUMEN
The class III phosphoinositide 3-kinase vacuolar protein sorting 34 (VPS34) is a core protein of autophagy initiation, yet the regulatory mechanisms responsible for its stringent control remain poorly understood. Here, we report that the E3 ubiquitin ligase NEDD4-1 promotes the autophagy flux by targeting VPS34. NEDD4-1 undergoes lysine 29 (K29)-linked auto-ubiquitination at K1279 and serves as a scaffold for recruiting the ubiquitin-specific protease 13 (USP13) to form an NEDD4-1-USP13 deubiquitination complex, which subsequently stabilizes VPS34 to promote autophagy through removing the K48-linked poly-ubiquitin chains from VPS34 at K419. Knockout of either NEDD4-1 or USP13 increased K48-linked ubiquitination and degradation of VPS34, thus attenuating the formation of the autophagosome. Our results identify an essential role for NEDD4-1 in regulating autophagy, which provides molecular insights into the mechanisms by which ubiquitination regulates autophagy flux.
Asunto(s)
Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Ubiquitinación , Línea Celular , Estabilidad de Enzimas , Humanos , Lisina/metabolismo , Unión ProteicaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause acute respiratory distress syndrome, hypercoagulability, hypertension, and multiorgan dysfunction. Effective antivirals with safe clinical profile are urgently needed to improve the overall prognosis. In an analysis of a randomly collected cohort of 124 patients with COVID-19, we found that hypercoagulability as indicated by elevated concentrations of D-dimers was associated with disease severity. By virtual screening of a U.S. FDA approved drug library, we identified an anticoagulation agent dipyridamole (DIP) in silico, which suppressed SARS-CoV-2 replication in vitro. In a proof-of-concept trial involving 31 patients with COVID-19, DIP supplementation was associated with significantly decreased concentrations of D-dimers (P < 0.05), increased lymphocyte and platelet recovery in the circulation, and markedly improved clinical outcomes in comparison to the control patients. In particular, all 8 of the DIP-treated severely ill patients showed remarkable improvement: 7 patients (87.5%) achieved clinical cure and were discharged from the hospitals while the remaining 1 patient (12.5%) was in clinical remission.
RESUMEN
The activation of retinoic acid-inducible gene I (RIG-I), an indispensable viral RNA sensor in mammals, is subtly regulated by ubiquitination. Although multiple ubiquitination sites at the amino terminus of RIG-I have been identified, their functional allocations in RIG-I activation remain elusive. We identified a stratified model for RIG-I amino-terminal ubiquitination, in which initiation at either Lys164 or Lys172 allows subsequent ubiquitination at other lysines, to trigger and amplify RIG-I activation. Experimental and mathematical modeling showed that multisite ubiquitination provides robustness in RIG-I-mediated type I interferon (IFN) signaling. Furthermore, the flexibly controlled ultrasensitivity and IFN activation intensity determine the specificity of the IFN-stimulated gene transcription and manipulate cell fate in antiviral immune response. Our work demonstrates that tunable type I IFN signaling can be regulated through multisite RIG-I ubiquitination and elucidates a new paradigm for dynamic regulation in RIG-I-mediated antiviral signaling.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Regulación de la Expresión Génica , Inmunidad , Línea Celular , Proteína 58 DEAD Box/química , Proteína 58 DEAD Box/genética , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Interferón Tipo I/metabolismo , Modelos Biológicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Inmunológicos , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Virosis/etiología , Virosis/metabolismoRESUMEN
RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Ubiquitinación , Virus/inmunología , Relación Dosis-Respuesta Inmunológica , Células HEK293 , Humanos , Modelos Teóricos , Receptores InmunológicosRESUMEN
Nuclear factor κB (NF-κB) is a key transcription factor in inflammatory immune responses and cell survival. Multiple types of ubiquitination play critical roles in the activation of NF-κB signaling, yet the molecular mechanisms responsible for their reversible deubiquitination are still poorly understood. In this study, we identified a member of the deubiquitinases family, ubiquitin-specific protease 18 (USP18), as a novel negative regulator in Toll-like receptor (TLR)-mediated NF-κB activation in human macrophages. USP18 is an interferon inducible gene, which is also upregulated by various TLR ligands in human monocytes and macrophages. Knockdown of USP18 enhanced the phosphorylation of IKK, the degradation of IκB, and augmented the expression of pro-inflammatory cytokines. Furthermore, USP18 interacted with TAK1-TAB1 complex and IKKα/ß-NEMO complex, respectively. USP18 cleaved the K63-linked polyubiquitin chains attached to TAK1 in a protease-dependent manner. Moreover, USP18 targeted the IKK complex through the regulatory subunit NEMO of IKK, and specifically inhibited K63-linked ubiquitination of NEMO. Mutation analysis revealed direct binding of USP18 to the UBAN motif of NEMO. Our study has identified a previously unrecognized role for USP18 in the negative regulation of NF-κB activation by inhibiting K63-linked ubiquitination of TAK1 and NEMO through distinct mechanisms.