RESUMEN
Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.
Asunto(s)
Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Pandemias/prevención & control , Neumonía Viral/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Diseño Universal , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/química , COVID-19 , Vacunas contra la COVID-19 , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Coronavirus del Síndrome Respiratorio de Oriente Medio/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/inmunología , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , SARS-CoV-2 , Células Sf9 , Organismos Libres de Patógenos Específicos , Spodoptera , Transfección , Vacunación/métodos , Células Vero , Vacunas ViralesRESUMEN
The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/aislamiento & purificación , Linfocitos B/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Análisis de la Célula Individual , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , COVID-19 , Convalecencia , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Pandemias , Análisis de Secuencia de ARN , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Exones VDJRESUMEN
Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/patología , Neumonía Viral/patología , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/química , Anticuerpos Antivirales/uso terapéutico , Reacciones Antígeno-Anticuerpo , Sitios de Unión , COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Cricetinae , Microscopía por Crioelectrón , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/inmunología , Femenino , Pulmón/patología , Masculino , Simulación de Dinámica Molecular , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Estructura Cuaternaria de Proteína , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.
Asunto(s)
Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Evaluación Preclínica de Medicamentos/métodos , Pandemias/prevención & control , Neumonía Viral/prevención & control , Vacunas de Productos Inactivados/uso terapéutico , Vacunas Virales/uso terapéutico , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/genética , COVID-19 , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Femenino , Cobayas , Inmunogenicidad Vacunal , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Filogenia , Neumonía Viral/virología , Conejos , Ratas , Ratas Wistar , SARS-CoV-2 , Vacunas de Productos Inactivados/efectos adversos , Células Vero , Vacunas Virales/efectos adversosRESUMEN
Zika virus (ZIKV) persists in the semen of male patients, a first for flavivirus infection. Here, we demonstrate that ZIKV can induce inflammation in the testis and epididymidis, but not in the prostate or seminal vesicle, and can lead to damaged testes after 60 days post-infection in mice. ZIKV induces innate immune responses in Leydig, Sertoli, and epididymal epithelial cells, resulting in the production of pro-inflammatory cytokines/chemokines. However, ZIKV does not induce a rapid and abundant cytokine production in peritubular cell and spermatogonia, suggesting that these cells are vulnerable for ZIKV infection and could be the potential repositories for ZIKV. Our study demonstrates a correlation between ZIKV and testis infection/damage and suggests that ZIKV infection, under certain circumstances, can eventually lead to male infertility.
Asunto(s)
Infertilidad Masculina/virología , Testículo/virología , Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Citocinas/metabolismo , Epidídimo/patología , Epidídimo/virología , Humanos , Infertilidad Masculina/patología , Masculino , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Interferón alfa y beta/genética , Testículo/patología , Internalización del Virus , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/patología , Infección por el Virus Zika/transmisión , Tirosina Quinasa del Receptor AxlRESUMEN
Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Células B de Memoria , SARS-CoV-2 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/uso terapéutico , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/inmunología , Modelos Animales de Enfermedad , Humanos , Células B de Memoria/inmunología , Ratones , Pruebas de Neutralización , SARS-CoV-2/clasificación , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Immune-mediated necrotizing myopathy (IMNM) is an autoimmune disorder associated with the presence of autoantibodies, characterized by severe clinical presentation with rapidly progressive muscular weakness and elevated levels of creatine kinase, while traditional pharmacological approaches possess varying and often limited effects. Considering the pathogenic role of autoantibodies, chimeric antigen receptor (CAR)-T cells targeting B cell maturation antigen (BCMA) have emerged as a promising therapeutic strategy. We reported here a patient with anti-signal recognition particle IMNM refractory to multiple available therapies, who was treated with BCMA-targeting CAR-T cells, exhibited favorable safety profiles, sustained reduction in pathogenic autoantibodies, and persistent clinical improvements over 18 mo. Longitudinal single-cell RNA, B cell receptor, T cell receptor sequencing analysis presented the normalization of immune microenvironment after CAR-T cell infusion, including reconstitution of B cell lineages, replacement of T cell subclusters, and suppression of overactivated immune cells. Analysis on characteristics of CAR-T cells in IMNM demonstrated a more active expansion of CD8+ CAR-T cells, with a dynamic phenotype shifting pattern similar in CD4+ and CD8+ CAR-T cells. A comparison of CD8+ CAR-T cells in patients with IMNM and those with malignancies collected at different timepoints revealed a more NK-like phenotype with enhanced tendency of cell death and neuroinflammation and inhibited proliferating ability of CD8+ CAR-T cells in IMNM while neuroinflammation might be the distinct characteristics. Further studies are warranted to define the molecular features of CAR-T cells in autoimmunity and to seek higher efficiency and longer persistence of CAR-T cells in treating autoimmune disorders.
Asunto(s)
Enfermedades Autoinmunes , Mieloma Múltiple , Enfermedades Musculares , Receptores Quiméricos de Antígenos , Humanos , Mieloma Múltiple/tratamiento farmacológico , Antígeno de Maduración de Linfocitos B , Enfermedades Neuroinflamatorias , Inmunoterapia Adoptiva , Enfermedades Autoinmunes/terapia , Autoanticuerpos , Enfermedades Musculares/terapia , Análisis de la Célula Individual , Tratamiento Basado en Trasplante de Células y Tejidos , Microambiente TumoralRESUMEN
The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.
Asunto(s)
Anticuerpos Monoclonales , Inmunoglobulina A Secretora , Animales , Ratones , Humanos , Inmunoglobulina G , Inmunoglobulina A , Administración Intranasal , Ratones TransgénicosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19.
Asunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Pulmón/patología , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , Transgenes , Enzima Convertidora de Angiotensina 2 , Animales , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Betacoronavirus/inmunología , Betacoronavirus/metabolismo , Bronquios/patología , Bronquios/virología , COVID-19 , Infecciones por Coronavirus/inmunología , Modelos Animales de Enfermedad , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Humanos , Inmunoglobulina G/inmunología , Pulmón/inmunología , Pulmón/virología , Linfocitos/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/virología , Masculino , Ratones , Ratones Transgénicos , Pandemias , Neumonía Viral/inmunología , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , SARS-CoV-2 , Replicación Viral , Pérdida de PesoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.
Asunto(s)
Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/prevención & control , Modelos Animales de Enfermedad , Pandemias/prevención & control , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/prevención & control , Animales , Betacoronavirus/efectos de los fármacos , Betacoronavirus/inmunología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/inmunología , Hurones/virología , Humanos , Mesocricetus/virología , Ratones , Neumonía Viral/inmunología , Primates/virología , SARS-CoV-2 , Vacunas Virales/inmunologíaRESUMEN
Microglia play a critical role in the clearance of myelin debris, thereby ensuring functional recovery from neural injury. Here, using mouse model of demyelination following two-point LPC injection, we show that the microglial autophagic-lysosomal pathway becomes overactivated in response to severe demyelination, leading to lipid droplet accumulation and a dysfunctional and pro-inflammatory microglial state, and finally failed myelin debris clearance and spatial learning deficits. Data from genetic approaches and pharmacological modulations, via microglial Atg5 deficient mice and intraventricular BAF A1 administration, respectively, demonstrate that staged suppression of excessive autophagic-lysosomal activation in microglia, but not sustained inhibition, results in better myelin debris degradation and exerts protective effects against demyelination. Combined multi-omics results in vitro further showed that enhanced lipid metabolism, especially the activation of the linoleic acid pathway, underlies this protective effect. Supplementation with conjugated linoleic acid (CLA), both in vivo and in vitro, could mimic these effects, including attenuating inflammation and restoring microglial pro-regenerative properties, finally resulting in better recovery from demyelination injuries and improved spatial learning function, by activating the peroxisome proliferator-activated receptor (PPAR-γ) pathway. Therefore, we propose that pharmacological inhibition targeting microglial autophagic-lysosomal overactivation or supplementation with CLA could represent a potential therapeutic strategy in demyelinated disorders.
Asunto(s)
Enfermedades Desmielinizantes , Microglía , Ratones , Animales , Microglía/metabolismo , Ácido Linoleico/metabolismo , Autofagia , Enfermedades Desmielinizantes/metabolismo , RegeneraciónRESUMEN
Lymphocyte homing to draining lymph nodes is critical for the initiation of immune responses. Secondary lymphoid organs of germ-free mice are underdeveloped. How gut commensal microbes remotely regulate cellularity and volume of secondary lymphoid organs remains unknown. We report here that, driven by commensal fungi, a wave of CD45(+)CD103(+)RALDH(+) cells migrates to the peripheral lymph nodes after birth. The arrival of these cells introduces high amounts of retinoic acid, mediates the neonatal to adult addressin switch on endothelial cells, and directs the homing of lymphocytes to both gut-associated lymphoid tissues and peripheral lymph nodes. In adult mice, a small number of these RALDH(+) cells might serve to maintain the volume of secondary lymphoid organs. Homing deficiency of these cells was associated with lymph node attrition in vitamin-A-deficient mice, suggesting a perpetual dependence on retinoic acid signaling for structural and functional maintenance of peripheral immune organs.
Asunto(s)
Células Dendríticas/inmunología , Células Endoteliales/inmunología , Isoenzimas/metabolismo , Ganglios Linfáticos/metabolismo , Retinal-Deshidrogenasa/metabolismo , Vitamina A/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Animales Recién Nacidos , Antígenos CD/metabolismo , Antígenos CD18/metabolismo , Procesos de Crecimiento Celular , Movimiento Celular , Femenino , Microbioma Gastrointestinal/inmunología , Cadenas alfa de Integrinas/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Tretinoina/metabolismo , Vitamina A/genéticaRESUMEN
Microglia-mediated neuroinflammation contributes to acute demyelination in neuromyelitis optica spectrum disorders (NMOSD). Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in the CSF has been associated with microglial activation in several neurodegenerative diseases. However, the basis for this immune-mediated attack and the pathophysiological role of sTREM2 in NMOSD remain to be elucidated. Here, we performed Mendelian randomization analysis and identified a genetic association between increased CSF sTREM2 and NMOSD risk. CSF sTREM2 was elevated in patients with NMOSD and was positively correlated with neural injury and other neuroinflammation markers. Single-cell RNA sequencing of human macrophage/microglia-like cells in CSF, a proxy for microglia, showed that increased CSF sTREM2 was positively associated with microglial dysfunction in patients with NMOSD. Furthermore, we demonstrated that sTREM2 is a reliable biomarker of microglial activation in a mouse model of NMOSD. Using unbiased transcriptomic and lipidomic screens, we identified that excessive activation, overwhelmed phagocytosis of myelin debris, suppressed lipid metabolism and enhanced glycolysis underlie sTREM2-mediated microglial dysfunction, possibly through the nuclear factor kappa B (NF-κB) signalling pathway. These molecular and cellular findings provide a mechanistic explanation for the genetic association between CSF sTREM2 and NMOSD risk and indicate that sTREM2 could be a potential biomarker of NMOSD progression and a therapeutic target for microglia-mediated neuroinflammation.
Asunto(s)
Enfermedad de Alzheimer , Neuromielitis Óptica , Animales , Ratones , Humanos , Microglía/metabolismo , Enfermedad de Alzheimer/metabolismo , Neuromielitis Óptica/genética , Neuromielitis Óptica/metabolismo , Enfermedades Neuroinflamatorias , Biomarcadores/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/genéticaRESUMEN
Chronic cerebral hypoperfusion (CCH), a disease afflicting numerous individuals worldwide, is a primary cause of cognitive deficits, the pathogenesis of which remains poorly understood. Bruton's tyrosine kinase inhibition (BTKi) is considered a promising strategy to regulate inflammatory responses within the brain, a crucial process that is assumed to drive ischemic demyelination progression. However, the potential role of BTKi in CCH has not been investigated so far. In the present study, we elucidated potential therapeutic roles of BTK in both in vitro hypoxia and in vivo ischemic demyelination model. We found that cerebral hypoperfusion induced white matter injury, cognitive impairments, microglial BTK activation, along with a series of microglia responses associated with inflammation, oxidative stress, mitochondrial dysfunction, and ferroptosis. Tolebrutinib treatment suppressed both the activation of microglia and microglial BTK expression. Meanwhile, microglia-related inflammation and ferroptosis processes were attenuated evidently, contributing to lower levels of disease severity. Taken together, BTKi ameliorated white matter injury and cognitive impairments induced by CCH, possibly via skewing microglia polarization towards anti-inflammatory and homeostatic phenotypes, as well as decreasing microglial oxidative stress damage and ferroptosis, which exhibits promising therapeutic potential in chronic cerebral hypoperfusion-induced demyelination.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Isquemia Encefálica , Sustancia Blanca , Animales , Masculino , Ratones , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Isquemia Encefálica/metabolismo , Enfermedad Crónica , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Sustancia Blanca/efectos de los fármacos , Sustancia Blanca/patología , Sustancia Blanca/metabolismoRESUMEN
In brief: The proliferation of the endometrium is regulated by histone methylation. This study shows that decreased NSD2 impairs proliferative-phase endometrial stromal cell proliferation in patients with recurrent implantation failure via epigenetic reprogramming of H3K36me2 methylation on the promoter region of MCM7. Abstract: Recurrent implantation failure (RIF) is a formidable challenge in assisted reproductive technology because of its unclear molecular mechanism. Impaired human endometrial stromal cell (HESC) proliferation disrupts the rhythm of the menstrual cycle, resulting in devastating disorders between the embryo and the endometrium. The molecular function of histone methylation enzymes in modulating HESC proliferation remains largely uncharacterized. Herein, we found that the levels of histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2) and the dimethylation of lysine 36 on histone H3 are decreased significantly in the proliferative-phase endometrium of patients with RIF. Knockdown of NSD2 in an HESC cell line markedly impaired cell proliferation and globally reduced H3K36me2 binding to chromatin, leading to altered expression of many genes. Transcriptomic analyses revealed that cell cycle-related gene sets were downregulated in the endometrium of patients with RIF and in NSD2knockdown HESCs. Furthermore, RNA-sequencing and CUT&Tag sequencing analysis suggested that NSD2 knockdown reduced the binding of H3K36me2 to the promoter region of cell cycle marker gene MCM7 (encoding minichromosome maintenance complex component 7) and downregulated its expression. The interaction of H3K36me2 with the MCM7 promoter was verified using chromatin immunoprecipitation-quantitative real-time PCR. Our results demonstrated a unifying epigenome-scale mechanism by which decreased NSD2 impairs endometrial stromal cell proliferation in the proliferative-phase endometrium of patients with RIF.
Asunto(s)
Endometrio , Histonas , Femenino , Humanos , Proliferación Celular , Cromatina/metabolismo , Endometrio/metabolismo , Histonas/metabolismo , Células del Estroma/metabolismoRESUMEN
The role of microglia in triggering the blood-brain barrier (BBB) impairment and white matter damage after chronic cerebral hypoperfusion is unclear. Here we demonstrated that the vessel-adjacent microglia were specifically activated by the leakage of plasma low-density lipoprotein (LDL), which led to BBB breakdown and ischemic demyelination. Interestingly, we found that LDL stimulation enhanced microglial phagocytosis, causing excessive engulfment of myelin debris and resulting in an overwhelming lipid burden in microglia. Surprisingly, these lipid-laden microglia exhibited a suppressed profile of inflammatory response and compromised pro-regenerative properties. Microglia-specific knockdown of LDLR or systematic medication lowering circulating LDL-C showed protective effects against ischemic demyelination. Overall, our findings demonstrated that LDL-stimulated vessel-adjacent microglia possess a disease-specific molecular signature, characterized by suppressed regenerative properties, which is associated with the propagation of demyelination during ischemic white matter damage.
Asunto(s)
Barrera Hematoencefálica , Isquemia Encefálica , Lipoproteínas LDL , Microglía , Sustancia Blanca , Microglía/metabolismo , Animales , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Ratones , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Isquemia Encefálica/metabolismo , Barrera Hematoencefálica/metabolismo , Masculino , Ratones Endogámicos C57BL , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Fagocitosis/fisiología , Vaina de Mielina/metabolismoRESUMEN
Neutrophils play a crucial role in the immune system within tumor microenvironment. At present, numerous studies have explored the changes of neutrophils' automatic killing effect and cellular communication with other immune cells under pathological conditions through single-cell sequencing. However, there remains a lack of definite conclusion about the identification criteria of neutrophil subgroups. Here, we collected tumor and para-carcinoma tissues, pre- and postoperative blood from patients with non-small cell lung cancer (NSCLC), and performed single-cell RNA (scRNA) sequencing to evaluate the distribution of neutrophil subgroups. We have developed a computational method of over expression rate (OER) to evaluate the specificity of neutrophil subgroups, in order to target gene panels with potential clinical application value. In addition, OER was used to evaluate specificity of neutrophil subsets in healthy people and patients with various diseases to further validate the feasibility of this evaluation system. As a result, we found the specificity of Neu_ c1_ IL1B and Neu_ c2_ cxcr4 (low) in postoperative blood has increased, while that of IL-7R + neutrophils has decreased, indicating that these groups of cells possibly differentiated or migrated to other subgroups in the state of lung cancer. In addition, seven gene panels (Neu_c3_CST7, RSAD2_Neu, S100A2/Pabpc1_Neu, ISG15/Ifit3_Neu, CD74_Neu, PTGS2/Actg1_Neu, SPP1_Neu) were high specific in all the four NSCLC-associated samples, meaning that changes in the percentage of these cell populations would have a high degree of confidence in assessing changes of disease status. In conclusion, combined consideration of the distribution characteristics of neutrophil subgroups could help evaluate the diagnosis and prognosis of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Microambiente Tumoral , Neutrófilos , PulmónRESUMEN
BACKGROUND: Microphthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome. RESULTS: Query of the International Mouse Phenotyping Consortium (IMPC) database (data release 17.0, August 01, 2022) identified 74 unique knockout lines (genes) with genetically associated eye defects in mouse embryos. The vast majority of eye abnormalities were small or absent eyes, findings most relevant to MAC spectrum disease in humans. A literature search showed that 27 of the 74 lines had previously published knockout mouse models, of which only 15 had ocular defects identified in the original publications. These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice. Of these 59, we identified 19 genes with a reported human eye phenotype. Overall, mining of the IMPC data yielded 40 previously unimplicated genes linked to mammalian eye development. Bioinformatic analysis showed that several of the IMPC genes colocalized to several protein anabolic and pluripotency pathways in early eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation. CONCLUSIONS: Using genome-wide phenotype screening of single-gene knockout mouse lines, STRING analysis, and bioinformatic methods, this study identified genes heretofore unassociated with MAC phenotypes providing models to research novel molecular and cellular mechanisms involved in eye development. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.
Asunto(s)
Anoftalmos , Coloboma , Anomalías del Ojo , Microftalmía , Humanos , Ratones , Animales , Anomalías del Ojo/genética , Anoftalmos/genética , Microftalmía/genética , Coloboma/genética , Ratones Noqueados , Desarrollo Embrionario/genética , Fenotipo , Ojo , MamíferosRESUMEN
OBJECTIVE: Cephalic Index (CI), the ratio of head width to length, is one of the indexes reflecting cranial morphological characteristics. Current norms were established by European and American countries. The purpose of the study was to study anthropometry of cranial parameters using computed tomography scans to establish the CI of the sampled Chinese Children. METHODS: The cross-sectional study was carried out on patients of age younger than 14 years old at Shanghai Children's Medical Center. The measurement of maximum cranial breadth and maximum cranial length were taken on a computed tomography scan machine and recorded for analysis. Cephalic Index was calculated for each age and sex group and compared with previously established norms. RESULTS: Five hundred eighteen patients met the inclusion criteria, including 301 males and 217 females. The means for boys and girls were 87.1 (SD: 4.3) and 85.8 (SD: 4.3), respectively. There was a significant difference between boys and girls (P < 0.01). Cephalic Index in different ages and on applying the 1-way analysis of variance association was statistically insignificant (P = 0.19). CONCLUSIONS: Chinese head shape was brachycephalic. A statistically significant correlation was seen between the CI and sex, while not age.