RESUMEN
Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells1,2. Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections3,4. Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD226,7, which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life.
Asunto(s)
Inmunidad Materno-Adquirida , Inmunoglobulina G , Espacio Intracelular , Listeria monocytogenes , Madres , Embarazo , Acetilesterasa , Animales , Animales Recién Nacidos , Linfocitos B , Femenino , Inmunidad Materno-Adquirida/inmunología , Inmunoglobulina G/inmunología , Interleucina-10/biosíntesis , Espacio Intracelular/inmunología , Espacio Intracelular/microbiología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/prevención & control , Ratones , Ácido N-Acetilneuramínico/metabolismo , Embarazo/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Linfocitos TRESUMEN
The world is currently in a pandemic of COVID-19 (Coronavirus disease-2019) caused by a novel positive-sense, single-stranded RNA ß-coronavirus referred to as SARS-CoV-2. Here we investigated rates of SARS-CoV-2 infection in the greater Cincinnati, Ohio, USA metropolitan area from August 13 to December 8, 2020, just prior to initiation of the national vaccination program. Examination of 9,550 adult blood donor volunteers for serum IgG antibody positivity against the SARS-CoV-2 Spike protein showed an overall prevalence of 8.40%, measured as 7.56% in the first 58 days and 9.24% in the last 58 days, and 12.86% in December 2020, which we extrapolated to ~20% as of March, 2021. Males and females showed similar rates of past infection, and rates among Hispanic or Latinos, African Americans and Whites were also investigated. Donors under 30 years of age had the highest rates of past infection, while those over 60 had the lowest. Geographic analysis showed higher rates of infectivity on the West side of Cincinnati compared with the East side (split by I-75) and the lowest rates in the adjoining region of Kentucky (across the Ohio river). These results in regional seroprevalence will help inform efforts to best achieve herd immunity in conjunction with the national vaccination campaign.
Asunto(s)
Anticuerpos Antivirales/sangre , Donantes de Sangre/estadística & datos numéricos , COVID-19/epidemiología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Ohio/etnología , Pandemias , Estudios Seroepidemiológicos , Adulto JovenRESUMEN
Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that signal in response to collagen. We had previously shown that collagen binding leads to clustering of DDR1b, a process partly mediated by its extracellular domain (ECD). In this study, we investigated (i) the impact of the oligomeric state of DDR2 ECD on collagen binding and fibrillogenesis, (ii) the effect of collagen binding on DDR2 clustering, and (iii) the spatial distribution and phosphorylation status of DDR1b and DDR2 after collagen stimulation. Studies were conducted using purified recombinant DDR2 ECD proteins in monomeric, dimeric or oligomeric state, and MC3T3-E1 cells expressing full-length DDR2-GFP or DDR1b-YFP. We show that the oligomeric form of DDR2 ECD displayed enhanced binding to collagen and inhibition of fibrillogenesis. Using atomic force and fluorescence microscopy, we demonstrate that unlike DDR1b, DDR2 ECD and DDR2-GFP do not undergo collagen-induced receptor clustering. However, after prolonged collagen stimulation, both DDR1b-YFP and DDR2-GFP formed filamentous structures consistent with spatial re-distribution of DDRs in cells. Immunocytochemistry revealed that while DDR1b clusters co-localized with non-fibrillar collagen, DDR1b/DDR2 filamentous structures associated with collagen fibrils. Antibodies against a tyrosine phosphorylation site in the intracellular juxtamembrane region of DDR1b displayed positive signals in both DDR1b clusters and filamentous structures. However, only the filamentous structures of both DDR1b and DDR2 co-localized with antibodies directed against tyrosine phosphorylation sites within the receptor kinase domain. Our results uncover key differences and similarities in the clustering abilities and spatial distribution of DDR1b and DDR2 and their impact on receptor phosphorylation.
Asunto(s)
Colágeno Tipo I/metabolismo , Receptor con Dominio Discoidina 1/metabolismo , Receptor con Dominio Discoidina 2/metabolismo , Fosforilación/fisiología , Células 3T3 , Animales , Sitios de Unión/fisiología , Línea Celular , Membrana Celular/metabolismo , Análisis por Conglomerados , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Ratones , Unión Proteica/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Tirosina/metabolismoRESUMEN
Fibrin has recently been shown to activate platelets through the immunoglobulin receptor glycoprotein VI (GPVI). In the present study, we show that spreading of human platelets on fibrin is abolished in patients deficient in GPVI, confirming that fibrin activates human platelets through the immunoglobulin receptor. Using a series of proteolytic fragments, we show that D-dimer, but not the E fragment of fibrin, binds to GPVI and that immobilized D-dimer induces platelet spreading through activation of Src and Syk tyrosine kinases. In contrast, when platelets are activated in suspension, soluble D-dimer inhibits platelet aggregation induced by fibrin and collagen, but not by a collagen-related peptide composed of a repeat GPO sequence or by thrombin. Using surface plasmon resonance, we demonstrate that fibrin binds selectively to monomeric GPVI with a KD of 302 nM, in contrast to collagen, which binds primarily to dimeric GPVI. These results establish GPVI as the major signaling receptor for fibrin in human platelets and provide evidence that fibrin binds to a distinct configuration of GPVI. This indicates that it may be possible to develop agents that selectively block the interaction of fibrin but not collagen with the immunoglobulin receptor. Such agents are required to establish whether selective targeting of either interaction has the potential to lead to development of an antithrombotic agent with a reduced effect on bleeding relative to current antiplatelet drugs.
RESUMEN
The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 µM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.
Asunto(s)
Colágeno/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Colágeno/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores Inmunológicos/metabolismoRESUMEN
Platelets are activated by a range of stimuli that share little or no resemblance in structure to each other or to recognized ligands, including diesel exhaust particles (DEP), small peptides [4N1-1, Champs (computed helical anti-membrane proteins), LSARLAF (Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides (fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation of human and mouse platelets through the glycoprotein VI (GPVI)-FcR γ-chain complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using platelets from mice deficient in either or both of these receptors. In addition, all of these ligands stimulate tyrosine phosphorylation in GPVI/CLEC-2-double-deficient platelets, indicating that they bind to additional surface receptors, although only in the case of dextran sulfate does this lead to activation. DEP, fucoidan and dextran sulfate, but not the other agonists, activate GPVI and CLEC-2 in transfected cell lines as shown using a sensitive reporter assay confirming a direct interaction with the two receptors. We conclude that this miscellaneous group of ligands bind to multiple proteins on the cell surface including GPVI and/or CLEC-2, inducing activation. These results have pathophysiological significance in a variety of conditions that involve exposure to activating charged/hydrophobic agents.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Antígenos CD36/química , Coagulantes/farmacología , Lectinas Tipo C/agonistas , Lectinas Tipo C/química , Glicoproteínas de Membrana/agonistas , Activación Plaquetaria/efectos de los fármacos , Emisiones de Vehículos/toxicidad , Contaminantes Atmosféricos/química , Contaminantes Atmosféricos/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Línea Celular , Pollos , Coagulantes/antagonistas & inhibidores , Coagulantes/química , Coagulantes/metabolismo , Cruzamientos Genéticos , Genes Reporteros/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Células Jurkat , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones Transgénicos , Conformación Molecular , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Ingeniería de Proteínas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Members of the cytomegalovirus family each encode two or more genes with significant homology to G-protein coupled receptors (GPCRs). In rodent models of pathogenesis, these viral encoded GPCRs play functionally significant roles, as their deletion results in crippled viruses that cannot traffic properly and/or replicate in virally important target cells. Of the four HCMV encoded GPCRs, US28 has garnered the most attention due to the fact that it exhibits both agonist-independent and agonist-dependent signaling activity and has been demonstrated to promote cellular migration and proliferation. Thus, it appears that the CMV GPCRs play important roles in viral replication in vivo as well as promote the development of virus-associated pathology. In the current study we have utilized a series of HCMV/US28 recombinants to investigate the expression profile and signaling activities of US28 in a number of cell types relevant to HCMV infection including smooth muscle cells, endothelial cells and cells derived from glioblastoma multiforme (GBM) tumors. The results indicate that US28 is expressed and exhibits constitutive agonist-independent signaling activity through PLC-ß in all cell types tested. Moreover, while CCL5/RANTES and CX3CL1/Fractalkine both promote US28-dependent Ca(++) release in smooth muscle cells, this agonist-dependent effect appears to be cell-specific as we fail to detect US28 driven Ca(++) release in the GBM cells. We have also investigated the effects of US28 on signaling via endogenous GPCRs including those in the LPA receptor family. Our data indicate that US28 can enhance signaling via endogenous LPA receptors. Taken together, our results indicate that US28 induces a variety of signaling events in all cell types tested suggesting that US28 signaling likely plays a significant role during HCMV infection and dissemination in vivo.
Asunto(s)
Infecciones por Citomegalovirus/enzimología , Infecciones por Citomegalovirus/metabolismo , Receptores de Quimiocina/metabolismo , Fosfolipasas de Tipo C/metabolismo , Proteínas Virales/metabolismo , Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Quimiocina CCL5/farmacología , Quimiocina CX3CL1/farmacología , Citomegalovirus/enzimología , Citomegalovirus/patogenicidad , Humanos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismoRESUMEN
The human cytomegalovirus (HCMV)-encoded G-protein-coupled receptor (GPCR) US28 is a potent activator of a number of signaling pathways in HCMV-infected cells. The intracellular carboxy-terminal domain of US28 contains residues critical for the regulation of US28 signaling in heterologous expression systems; however, the role that this domain plays during HCMV infection remains unknown. For this study, we constructed an HCMV recombinant virus encoding a carboxy-terminal domain truncation mutant of US28, FLAG-US28/1-314, to investigate the role that this domain plays in US28 signaling. We demonstrate that US28/1-314 exhibits a more potent phospholipase C-beta (PLC-beta) signal than does wild-type US28, indicating that the carboxy-terminal domain plays an important role in regulating agonist-independent signaling in infected cells. Moreover, HMCV-infected cells expressing the US28/1-314 mutant exhibit a prolonged calcium signal in response to CCL5, indicating that the US28 carboxy-terminal domain also regulates agonist-dependent signaling. Finally, while the chemokine CX3CL1 behaves as an inverse agonist or inhibitor of constitutive US28 signaling to PLC-beta, we demonstrate that CX3CL1 functions as an agonist with regard to US28-stimulated calcium release. This study is the first to demonstrate that the carboxy terminus of US28 controls US28 signaling in the context of HCMV infection and indicates that chemokines such as CX3CL1 can decrease constitutive US28 signals and yet simultaneously promote nonconstitutive US28 signals.
Asunto(s)
Citomegalovirus/metabolismo , Fibroblastos/virología , Receptores de Quimiocina/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Separación Celular , Quimiocina CX3CL1/metabolismo , Cromosomas Artificiales Bacterianos/metabolismo , Citometría de Flujo , Humanos , Fosfatos de Inositol/química , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric G(q/11) proteins and not through promiscuous coupling of M33 to the G(s) pathway. Using inhibitors of signaling molecules downstream of G(q/11), we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-beta and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38alpha and transcription factor NF-kappaB, occurs in the absence of G(q/11) and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-beta and PKC plays a central role in MCMV pathogenesis in vivo.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Muromegalovirus/fisiología , Fosfolipasa C beta/metabolismo , Proteína Quinasa C/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Animales , Línea Celular , Femenino , Infecciones por Herpesviridae/enzimología , Infecciones por Herpesviridae/virología , Humanos , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/genética , Fosfolipasa C beta/genética , Proteína Quinasa C/genética , Receptores Acoplados a Proteínas G/genética , Glándulas Salivales/enzimología , Glándulas Salivales/metabolismo , Glándulas Salivales/virología , Proteínas Virales/genética , Replicación ViralRESUMEN
Specific detection of the pathogenic prion protein, PrP(Sc), is essential for determining the prion clearance capacity of purification processes for therapeutic proteins. Use of a previously described indirect (two-antibody) Western blot assay sometimes resulted in the appearance of non-specific protein bands that interfered with the detection of small amounts of PrP(Sc)-specific signal, limiting the amount of clearance that could be determined for steps so affected. It is shown that these non-specific signals are due to the interaction between immunoglobulin fragments in the sample and the secondary antibody used in the assay. To circumvent this problem, a direct Western blot assay using a prion-specific primary antibody conjugated to the reporter enzyme alkaline phosphatase was developed. Application of the direct Western blot assay resulted in a significant reduction of non-specific signal while retaining the detection sensitivity for PrP(Sc)-specific signal. Therefore, the direct Western blot assay format is an improved tool for determining prion clearance capacity, particularly for immunoglobulin-rich samples.
Asunto(s)
Proteínas Sanguíneas/efectos adversos , Western Blotting/métodos , Proteínas PrPSc/farmacocinética , Enfermedades por Prión/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Bioensayo , Proteínas Sanguíneas/aislamiento & purificación , Cricetinae , Proteínas PrPSc/sangre , Proteínas PrPSc/inmunología , Enfermedades por Prión/transmisiónRESUMEN
The misfolded isoform of the prion protein (PrP(Sc)) possesses many unusual physiochemical properties. Previously, we and others reported on the differential partitioning of PrP(Sc) from plasma derived therapeutic proteins during their purification processes. To understand the driving force behind these partitioning differences, we investigated the effects of various solvent conditions on the precipitation of PrP(Sc). In a physiological buffer, PrP(Sc) remained in the supernatant after low speed centrifugation. At pH 5, PrP(Sc) precipitation was nearly complete regardless of the salt content. PrP(Sc) could also be precipitated at pH 8 by adding ethanol, but this precipitation was salt dependent. Based on these observations, an empirical mathematical model was constructed in which the PrP(Sc) precipitation trends were fully described as a function of solvent pH, salt, and ethanol concentration. This model consistently predicted PrP(Sc) partitioning during cold ethanol precipitation steps used in plasma protein purification processes, as shown by experimentally determined distributions of PrP(Sc) and transmissible spongiform encephalopathy (TSE) infectivity. These results indicate that pH, salt, and ethanol content are the major solvent factors determining the precipitation of the infectious PrP(Sc) in these processes and may provide a useful tool for assessing the differential partitioning of PrP(Sc) in a given solvent environment.