RESUMEN
Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on measurement of vitamin B12 in the blood, which may not accurately reflect the concentration in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro by depleting its target from the cell surface. Despite a normal serum concentration, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased B12 in the CSF and clinical improvement. Optofluidic screening enabled isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier (BBB). Autoantibodies targeting the same epitope of CD320 were identified in seven other patients with neurologic deficits of unknown etiology, 6% of healthy controls, and 21.4% of a cohort of patients with neuropsychiatric lupus. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we found that the low-density lipoprotein receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.
Asunto(s)
Autoanticuerpos , Deficiencia de Vitamina B 12 , Vitamina B 12 , Humanos , Deficiencia de Vitamina B 12/inmunología , Vitamina B 12/sangre , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Femenino , Receptores de Superficie Celular/metabolismo , Antígenos CD/metabolismo , Persona de Mediana Edad , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/sangre , Barrera Hematoencefálica/metabolismo , MasculinoRESUMEN
Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood-borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood-brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Encéfalo/metabolismo , Encéfalo/microbiología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/microbiología , Infecciones Estreptocócicas/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Streptococcus agalactiaeRESUMEN
Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs after bacteria interact with and penetrate the blood-brain barrier (BBB). The BBB is comprised of highly specialized brain microvascular endothelial cells (BMECs) that function to separate the circulation from the CNS and act as a formidable barrier for toxins and pathogens. Certain bacteria, such as Streptococcus agalactiae (group B Streptococcus [GBS]), possess the ability to interact with and penetrate the BBB to cause meningitis. Modeling bacterial interaction with the BBB in vitro has been limited to primary and immortalized BMEC culture. While useful, these cells often do not retain BBB-like properties, and human primary cells have limited availability. Recently, a human induced pluripotent stem cell (iPSC)-derived BMEC model has been established that is readily renewable and retains key BBB phenotypes. Here, we sought to evaluate whether the iPSC-derived BMECs were appropriate for modeling bacterial interaction with the BBB. Using GBS as a model meningeal pathogen, we demonstrate that wild-type GBS adhered to, invaded, and activated the iPSC-derived BMECs, while GBS mutants known to have diminished BBB interaction were attenuated in the iPSC-derived model. Furthermore, bacterial infection resulted in the disruption of tight junction components ZO-1, occludin, and claudin-5. Thus, we show for the first time that the iPSC-derived BBB model can be utilized to study BBB interaction with a bacterial CNS pathogen. IMPORTANCE Here for the first time, human iPSC-derived BMECs were used to model bacterial interaction with the BBB. Unlike models previously used to study these interactions, iPSC-derived BMECs possess robust BBB properties, such as the expression of complex tight junctions that are key components for the investigation of bacterial effects on the BBB. Here, we demonstrated that GBS interacts with the iPSC-derived BMECs and specifically disrupts these tight junctions. Thus, using this BBB model may allow researchers to uncover novel mechanisms of BBB disruption during meningitis that are inaccessible to immortalized or primary cell models that lack substantial tight junctions.