RESUMEN
MicroRNAs (miRNAs) are important regulators of cell fate decisions in immune responses. They act by coordinate repression of multiple target genes, a property that we exploited to uncover regulatory networks that govern T helper-2 (Th2) cells. A functional screen of individual miRNAs in primary T cells uncovered multiple miRNAs that inhibited Th2 cell differentiation. Among these were miR-24 and miR-27, miRNAs coexpressed from two genomic clusters, which each functioned independently to limit interleukin-4 (IL-4) production. Mice lacking both clusters in T cells displayed increased Th2 cell responses and tissue pathology in a mouse model of asthma. Gene expression and pathway analyses placed miR-27 upstream of genes known to regulate Th2 cells. They also identified targets not previously associated with Th2 cell biology which regulated IL-4 production in unbiased functional testing. Thus, elucidating the biological function and target repertoire of miR-24 and miR-27 reveals regulators of Th2 cell biology.
Asunto(s)
Asma/inmunología , Interleucina-4/biosíntesis , MicroARNs/genética , Células Th2/inmunología , Animales , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Inflamación/inmunología , Interleucina-4/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Noqueados , Familia de Multigenes/genética , Análisis de Secuencia de ARN , Células Th2/citologíaRESUMEN
RBC transfusion therapy is essential for the treatment of anemia. A serious complication of transfusion is the development of non-ABO alloantibodies to polymorphic RBC Ags; yet, mechanisms of alloantibody formation remain unclear. Storage of mouse RBCs before transfusion increases RBC immunogenicity through an unknown mechanism. We previously reported that sterile, stored mouse RBCs activate splenic dendritic cells (DCs), which are required for alloimmunization. Here we transfused mice with allogeneic RBCs to test whether stored RBCs activate pattern recognition receptors (PRRs) on recipient DCs to induce adaptive immunity. TLRs are a class of PRRs that regulate DC activation, which signal through two adapter molecules: MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation and migration, and the subsequent alloantibody response to transfused RBCs require MyD88 but not TRIF, suggesting that a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.
Asunto(s)
Inmunidad Adaptativa , Eritrocitos/inmunología , Eritrocitos/metabolismo , Inmunidad Innata , Factor 88 de Diferenciación Mieloide/metabolismo , Animales , Biomarcadores , Transfusión de Eritrocitos , Técnica del Anticuerpo Fluorescente , Humanos , Isoanticuerpos/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/genéticaRESUMEN
Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.
Asunto(s)
Proteínas Portadoras/fisiología , Movimiento Celular/genética , Células Dendríticas/inmunología , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Factores de Intercambio de Guanina Nucleótido/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Mutación PuntualRESUMEN
B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a "de novo" variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Tolerancia Inmunológica/inmunología , Lupus Eritematoso Sistémico/inmunología , Mutación Missense/inmunología , Receptores Purinérgicos P2Y/inmunología , Animales , Síndrome Antifosfolípido/genética , Síndrome Antifosfolípido/metabolismo , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Tolerancia Inmunológica/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Nefritis Lúpica/genética , Nefritis Lúpica/inmunología , Nefritis Lúpica/metabolismo , Masculino , Ratones Endogámicos C57BL , Mutación Missense/genética , Linaje , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Receptores Purinérgicos P2Y/genética , Receptores Purinérgicos P2Y/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Many skin manifestations of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection reflect activation of cutaneous and systemic immune responses involving effector pathways of both the innate and adaptive arms of the immune system. This article reviews evidence from the recent clinical and scientific literature that informs the current understanding of the consequences of coronavirus disease 2019 (COVID-19)-induced immune cell activation, as relevant to dermatology. Topics include the clinical consequences of autoantibody production in patients with COVID-19, immunologic evidence for chilblains as a manifestation of SARS-CoV-2 infection, and the relationship between type I interferons and COVID-19 disease severity.
Asunto(s)
COVID-19/fisiopatología , Enfermedades de la Piel/fisiopatología , Eritema Pernio/fisiopatología , Eritema Multiforme/fisiopatología , Exantema/fisiopatología , Humanos , Pitiriasis Rosada/fisiopatología , Piel/fisiopatología , Enfermedades Cutáneas Vesiculoampollosas/fisiopatologíaRESUMEN
P2RY8 promotes the confinement and growth regulation of germinal center (GC) B cells, and loss of human P2RY8 is associated with B cell lymphomagenesis. The metabolite S-geranylgeranyl-l-glutathione (GGG) is a P2RY8 ligand. The mechanisms controlling GGG distribution are poorly understood. Here, we show that gamma-glutamyltransferase-5 (Ggt5) expression in stromal cells was required for GGG catabolism and confinement of P2RY8-expressing cells to GCs. We identified the ATP-binding cassette subfamily C member 1 (Abcc1) as a GGG transporter and showed that Abcc1 expression by hematopoietic cells was necessary for P2RY8-mediated GC confinement. Furthermore, we discovered that P2RY8 and GGG negatively regulated trafficking of B and T cells to the bone marrow (BM). P2RY8 loss-of-function human T cells increased their BM homing. By defining how GGG distribution was determined and identifying sites of P2RY8 activity, this work helps establish how disruptions in P2RY8 function contribute to lymphomagenesis and other disease states.
Asunto(s)
Glutatión , Linfocitos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Receptores Purinérgicos P2Y , gamma-Glutamiltransferasa , Animales , Femenino , Humanos , Masculino , Ratones , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/metabolismo , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Glutatión/metabolismo , Células HEK293 , Activación de Linfocitos , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Receptores Purinérgicos P2Y/genética , Receptores Purinérgicos P2Y/metabolismoRESUMEN
BACKGROUND: Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. METHOD: We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. RESULTS: Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. CONCLUSION: Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.
RESUMEN
Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.
Asunto(s)
Betacoronavirus , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Betacoronavirus/genética , Betacoronavirus/inmunología , Betacoronavirus/aislamiento & purificación , Biotecnología , COVID-19 , Prueba de COVID-19 , Cromatografía de Afinidad , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/epidemiología , Neumonía Viral/inmunología , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Evidence suggests that distinct splenic dendritic cell (DC) subsets activate either CD4+ or CD8+ T cells in vivo. This bias has been partially ascribed to differential antigen presentation; however, all DC subsets can activate both T cell lineages in vitro. Therefore, we tested whether the organization of DC and T cell subsets in the spleen dictated this preference. We discovered that CD4+ and CD8+ T cells segregated within splenic T cell zones prior to immunization. After intravenous immunization, the two major conventional DC populations, distinguished by 33D1 and XCR1 staining, migrated into separate regions of the T cell zone: 33D1+ DCs migrated into the CD4+ T cell area, whereas XCR1+ DCs migrated into the CD8+ T cell area. Thus, the post-immunization location of each DC subset correlated with the T cell lineage it preferentially primes. Preventing this co-localization selectively impaired either CD4+ or CD8+ T cell immunity to blood-borne antigens.
Asunto(s)
Inmunidad Adaptativa , Presentación de Antígeno , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/clasificación , Bazo/inmunología , Animales , Antígenos/administración & dosificación , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Movimiento Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Transfusión de Eritrocitos , Expresión Génica , Inmunización , Inmunofenotipificación , Isoanticuerpos/biosíntesis , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/administración & dosificación , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Bazo/citologíaAsunto(s)
COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/virología , Piel/virología , Adulto , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades de la Piel/inmunología , Enfermedades de la Piel/patología , Adulto JovenRESUMEN
Red blood cell (RBC) transfusion is a life-saving therapeutic tool. However, a major complication in transfusion recipients is the generation of antibodies against non-ABO alloantigens on donor RBCs, potentially resulting in hemolysis and renal failure. Long-lived antibody responses typically require CD4(+) T cell help and, in murine transfusion models, alloimmunization requires a spleen. Yet, it is not known how RBC-derived antigens are presented to naive T cells in the spleen. We sought to answer whether splenic dendritic cells (DCs) were essential for T cell priming to RBC alloantigens. Transient deletion of conventional DCs at the time of transfusion or splenic DC preactivation before RBC transfusion abrogated T and B cell responses to allogeneic RBCs, even though transfused RBCs persisted in the circulation for weeks. Although all splenic DCs phagocytosed RBCs and activated RBC-specific CD4(+) T cells in vitro, only bridging channel 33D1(+) DCs were required for alloimmunization in vivo. In contrast, deletion of XCR1(+)CD8(+) DCs did not alter the immune response to RBCs. Our work suggests that blocking the function of one DC subset during a narrow window of time during RBC transfusion could potentially prevent the detrimental immune response that occurs in patients who require lifelong RBC transfusion support.