RESUMEN
Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX3CR1+ cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology.
Asunto(s)
Células de la Médula Ósea , Médula Ósea , Ratones , Animales , Células de la Médula Ósea/metabolismo , Células Madre Hematopoyéticas , Hematopoyesis , Células del EstromaRESUMEN
In neuroinflammatory and neurodegenerative disorders such as multiple sclerosis, mitochondrial damage caused by oxidative stress is believed to contribute to neuroaxonal damage. Previously, we demonstrated that exposure to hydrogen peroxide (H2O2) alters mitochondrial morphology and motility in myelinated axons and that these changes initiate at the nodes of Ranvier, where numerous sodium channels are located. Therefore, we suggested that mitochondrial damage may lead to ATP deficit, thereby affecting the efficiency of the sodium-potassium ATPase and eventually leading to sodium overload in axons. The increased intra-axonal sodium may revert the axonal sodium-calcium exchangers and thus may lead to a pathological calcium overload in the axoplasm and mitochondria. Here, we used the explanted murine ventral spinal roots to investigate whether modulation of sodium or calcium influx may prevent mitochondrial alterations in myelinated axons during exogenous application of H2O2 inducing oxidative stress. For that, tetrodotoxin, an inhibitor of voltage-gated sodium ion channels, and ruthenium 360, an inhibitor of the mitochondrial calcium uniporter, were applied simultaneously with hydrogen peroxide to axons. Mitochondrial shape and motility were analyzed. We showed that inhibition of axonal sodium influx prevented oxidative stress-induced morphological changes (i.e., increase in circularity and area and decrease in length) and preserved mitochondrial membrane potential, which is crucial for ATP production. Blocking mitochondrial calcium uptake prevented decrease in mitochondrial motility and also preserved membrane potential. Our findings indicate that alterations of both mitochondrial morphology and motility in the contexts of oxidative stress can be counterbalanced by modulating intramitochondrial ion concentrations pharmacologically. Moreover, motile mitochondria show preserved membrane potentials, pointing to a close association between mitochondrial motility and functionality.
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Calcio , Peróxido de Hidrógeno , Adenosina Trifosfato/metabolismo , Animales , Axones/fisiología , Calcio/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Ratones , Mitocondrias/metabolismo , Estrés Oxidativo , Sodio/metabolismoRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.
Asunto(s)
COVID-19 , Gripe Humana , Adulto , Humanos , Enzima Convertidora de Angiotensina 2 , Pulmón/patología , Macrófagos Alveolares/metabolismo , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2 , Tropismo ViralRESUMEN
The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZFhi ILCP expressing central LTi molecules in a RORα-dependent fashion. Our data unveil an unexpected role for T-bet and RORα during embryonic ILC function and highlight that RORγt is crucial in counteracting the suppressive effects of T-bet.
Asunto(s)
Diferenciación Celular/inmunología , Inmunidad Innata/inmunología , Ganglios Linfáticos/inmunología , Linfocitos/inmunología , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Proteínas de Dominio T Box/inmunología , Animales , Linaje de la Célula/inmunología , Femenino , Tejido Linfoide/inmunología , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
BACKGROUND: Tissue-resident memory T (TRM) cells are known to be important for the first line of defense in mucosa-associated tissues. However, the composition, localization, effector function, and specificity of TRM cells in the human kidney and their relevance for renal pathology have not been investigated. METHODS: Lymphocytes derived from blood, renal peritumor samples, and tumor samples were phenotypically and functionally assessed by applying flow cytometry and highly advanced histology (multi-epitope ligand cartography) methods. RESULTS: CD69+CD103+CD8+ TRM cells in kidneys display an inflammatory profile reflected by enhanced IL-2, IL-17, and TNFα production, and their frequencies correlate with increasing age and kidney function. We further identified mucosa-associated invariant T and CD56dim and CD56bright natural killer cells likewise expressing CD69 and CD103, the latter significantly enriched in renal tumor tissues. CD8+ TRM cell frequencies were not elevated in kidney tumor tissue, but they coexpressed PD-1 and TOX and produced granzyme B. Tumor-derived CD8+ TRM cells from patients with metastases were functionally impaired. Both CD69+CD103-CD4+ and CD69+CD103-CD8+ TRM cells form distinct clusters in tumor tissues in proximity to antigen-presenting cells. Finally, EBV, CMV, BKV, and influenza antigen-specific CD8+ T cells were enriched in the effector memory T cell population in the kidney. CONCLUSIONS: Our data provide an extensive overview of TRM cells' phenotypes and functions in the human kidney for the first time, pointing toward their potential relevance in kidney transplantation and kidney disease.
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Antígenos de Diferenciación de Linfocitos T/metabolismo , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Linfocitos T/fisiología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Femenino , Alemania , Humanos , Neoplasias Renales/cirugía , Masculino , Persona de Mediana Edad , Nefrectomía , FenotipoRESUMEN
The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-ß, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-ß. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-ß, and is distracted from itself.
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Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Factor de Crecimiento Transformador beta/inmunología , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/virología , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Interleucinas/inmunología , Masculino , Persona de Mediana Edad , Células Plasmáticas/inmunología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Germinal centers (GCs) are the sites where B cells undergo affinity maturation. The regulation of cellular output from the GC is not well understood. Here, we show that from the earliest stages of the GC response, plasmablasts emerge at the GC-T zone interface (GTI). We define two main factors that regulate this process: Tfh-derived IL-21, which supports production of plasmablasts from the GC, and TNFSF13 (APRIL), which is produced by a population of podoplanin+ CD157high fibroblastic reticular cells located in the GTI that are also rich in message for IL-6 and chemokines CXCL12, CCL19, and CCL21. Plasmablasts in the GTI express the APRIL receptor TNFRSF13B (TACI), and blocking TACI interactions specifically reduces the numbers of plasmablasts appearing in the GTI. Plasma cells generated in the GTI may provide an early source of affinity-matured antibodies that may neutralize pathogens or provide feedback regulating GC B cell selection.
Asunto(s)
Centro Germinal/citología , Células Plasmáticas/metabolismo , Transducción de Señal , Células del Estroma/citología , Linfocitos T Colaboradores-Inductores/citología , Animales , Antígenos/metabolismo , Diferenciación Celular , Movimiento Celular , Quimiocinas/metabolismo , Regulación de la Expresión Génica , Inmunidad , Factores Reguladores del Interferón/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Ligandos , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células del Estroma/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
Splenic marginal zone B cells (MZB) shuttle between the blood-filled marginal zone for antigen collection and the follicle for antigen delivery. However, it is unclear how MZBs migrate directionally from the marginal zone to the follicle. Here, we show that murine MZBs migrate up shear flow via the LFA-1 (αLß2) integrin ligand ICAM-1, but adhere or migrate down the flow via the VLA-4 integrin (α4ß1) ligand VCAM-1. MZBs lacking Arhgef6 (Pak-interacting exchange factor (αPIX)) or functional LFA-1 are impaired in shuttling due to mislocalization toward the VCAM-1-rich red pulp. Sphingosine-1-phosphate (S1P) signaling through the S1PR3 receptor inhibits MZB migration up the flow, and deletion of S1pr3 in Arhgef6 -/- mice rescues mislocalized MZBs. These findings establish shear flow as a directional cue for MZB migration to the follicle, and define S1PR3 and VCAM-1 as counteracting forces that inhibit this migration.
Asunto(s)
Linfocitos B/fisiología , Movimiento Celular/fisiología , Molécula 1 de Adhesión Intercelular/metabolismo , Lisofosfolípidos/metabolismo , Receptores de Lisoesfingolípidos/genética , Esfingosina/análogos & derivados , Estrés Mecánico , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Linfocitos B/metabolismo , Integrina alfa4beta1/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Noqueados , Receptores de Lisoesfingolípidos/metabolismo , Flujo Sanguíneo Regional , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transducción de Señal , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Bazo/citologíaRESUMEN
Although oligoclonal bands in the cerebrospinal fluid have been a hallmark of multiple sclerosis diagnosis for over three decades, the role of antibody-secreting cells in multiple sclerosis remains unclear. T and B cells are critical for multiple sclerosis pathogenesis, but increasing evidence suggests that plasma cells also contribute, through secretion of autoantibodies. Long-lived plasma cells are known to drive various chronic inflammatory conditions as e.g. systemic lupus erythematosus, however, to what extent they are present in autoimmune central nervous system inflammation has not yet been investigated. In brain biopsies from multiple sclerosis patients and other neurological diseases, we could detect non-proliferating plasma cells (CD138+Ki67-) in the parenchyma. Based on this finding, we hypothesized that long-lived plasma cells can persist in the central nervous system (CNS). In order to test this hypothesis, we adapted the multiple sclerosis mouse model experimental autoimmune encephalomyelitis to generate a B cell memory response. Plasma cells were found in the meninges and the parenchyma of the inflamed spinal cord, surrounded by tissue areas resembling survival niches for these cells, characterized by an up-regulation of chemokines (CXCL12), adhesion molecules (VCAM-1) and survival factors (APRIL and BAFF). In order to determine the lifetime of plasma cells in the chronically inflamed CNS, we labeled the DNA of proliferating cells with 5-ethynyl-2'-deoxyuridine (EdU). Up to five weeks later, we could detect EdU+ long-lived plasma cells in the murine CNS. To our knowledge, this is the first study describing non-proliferating plasma cells directly in the target tissue of a chronic inflammation in humans, as well as the first evidence demonstrating the ability of plasma cells to persist in the CNS, and the ability of the chronically inflamed CNS tissue to promote this persistence. Hence, our results suggest that the CNS provides survival niches for long-lived plasma cells, similar to the niches found in other organs. Targeting these cells in the CNS offers new perspectives for treatment of chronic autoimmune neuroinflammatory diseases, especially in patients who do not respond to conventional therapies.
Asunto(s)
Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/patología , Tejido Parenquimatoso/patología , Células Plasmáticas/patología , Adulto , Anciano , Animales , Antígenos CD/metabolismo , Proteínas de Unión al Calcio , Quimiocina CXCL12/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Masculino , Ratones , Proteínas de Microfilamentos , Persona de Mediana Edad , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adulto JovenRESUMEN
Plasma cells are terminally differentiated B cells that secrete antibodies, important for immune protection, but also contribute to any allergic and autoimmune disease. There is increasing evidence that plasma cell populations exhibit a considerable degree of heterogeneity with respect to their immunophenotype, migration behavior, lifetime, and susceptibility to immunosuppressive drugs. Pathogenic long-lived plasma cells are refractory to existing therapies. In contrast, short-lived plasma cells can be depleted by steroids and cytostatic drugs. Therefore, long-lived plasma cells are responsible for therapy-resistant autoantibodies and resemble a challenge for the therapy of antibody-mediated autoimmune diseases. Both lifetime and therapy resistance of plasma cells are supported by factors produced within their microenviromental niches. Current results suggest that plasma cell differentiation and survival factors such as IL-6 also signal via mammalian miRNAs within the plasma cell to modulate downstream transcription factors. Recent evidence also suggests that plasma cells and/or their immediate precursors (plasmablasts) can produce important cytokines and act as antigen-presenting cells, exhibiting so far underestimated roles in immune regulation and bone homeostasis. Here, we provide an overview on plasma cell biology and discuss exciting, experimental, and potential therapeutic approaches to eliminate pathogenic plasma cells.