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1.
Front Immunol ; 12: 624013, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33828548

RESUMO

Studies in animal models have shown that skin tissue-resident memory T (TRM) cells provide enhanced and immediate effector function at the site of infection. However, analyses of skin TRM cells in humans have been hindered by the lack of an optimized isolation protocol. Here, we present a combinatorial strategy-the 6-h collagenase IV digestion and gentle tissue dissociation - for rapid and efficient isolation of skin TRM cells with skin tissue-specific immune features. In comparison with paired blood circulating memory T cells, these ex vivo isolated skin T cells express typical TRM cell markers and display higher polyfunctional properties. Moreover, these isolated cells can also be assessed for longer periods of time in ex vivo cultures. Thus, the optimized isolation protocol provides a valuable tool for further understanding of human skin TRM cells, especially for direct comparison with peripheral blood T cells at the same sample collection time.

2.
Elife ; 102021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33749591

RESUMO

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress-responses, or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the Förster resonance energy transfer (FRET)-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial immune responses and detrimental autoimmunity.

3.
Nat Commun ; 12(1): 1737, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741932

RESUMO

Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes. Although ILCs are mainly known as tissue-resident cells, their precise localization and interactions with the microenvironment are still unclear. Here we combine a multiplexed immunofluorescence technique and a customized computational, open-source analysis pipeline to unambiguously identify CD127+ ILCs in situ and characterize these cells and their microenvironments. Moreover, we reveal the transcription factor IRF4 as a marker for tonsillar ILC3, and identify conserved stromal landmarks characteristic for ILC localization. We also show that CD127+ ILCs share tissue niches with plasma cells in the tonsil. Our works thus provide a platform for multiparametric histological analysis of ILCs to improve our understanding of ILC biology.


Assuntos
Linfócitos/imunologia , Linfócitos/patologia , Fenótipo , Análise Espacial , Algoritmos , Análise por Conglomerados , Tecido Conjuntivo/diagnóstico por imagem , Tecido Conjuntivo/patologia , Humanos , Processamento de Imagem Assistida por Computador , Imunidade Inata , Fatores Reguladores de Interferon/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Aprendizado de Máquina , Tonsila Palatina/diagnóstico por imagem , Tonsila Palatina/patologia
4.
Artigo em Inglês | MEDLINE | ID: mdl-33653962

RESUMO

OBJECTIVE: Therapies targeting B cells have been used in the clinic for multiple sclerosis (MS). In patients with relapsing MS, anti-CD20 therapy often suppresses relapse activity; yet, their effect on disease progression has been disappointing. Most anti-CD20 therapeutic antibodies are type I, but within the unique microenvironment of the brain, type II antibodies may be more beneficial, as type II antibodies exhibit reduced complement-dependent cytotoxicity and they have an increased capacity to induce direct cell death that is independent of the host immune response. METHODS: We compared the effect of type I with type II anti-CD20 therapy in a new rodent model of secondary progressive MS (SPMS), which recapitulates the principal histopathologic features of MS including meningeal B-cell aggregates. Focal MS-like lesions were induced by injecting heat-killed Mycobacterium tuberculosis into the piriform cortex of MOG-immunized mice. Groups of mice were treated with anti-CD20 antibodies (type I [rituxumab, 10 mg/kg] or type II [GA101, 10 mg/kg]) 4 weeks after lesion initiation, and outcomes were evaluated by immunohistochemistry. RESULTS: Anti-CD20 therapy decreased the extent of glial activation, significantly decreased the number of B and T lymphocytes in the lesion, and resulted in disruption of the meningeal aggregates. Moreover, at the given dose, the type II anti-CD20 therapy was more efficacious than the type I and also protected against neuronal death. CONCLUSIONS: These results indicate that anti-CD20 may be an effective therapy for SPMS with B-cell aggregates and that the elimination of CD20+ B cells alone is sufficient to cause disruption of aggregates in the brain.

5.
Acta Biomater ; 2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33636402

RESUMO

Immunotherapy stands out as a powerful and promising therapeutic strategy in the treatment of cancer, infections, and autoimmune diseases. Adoptive immune therapies are usually centered on modified T cells and their specific expansion towards antigen-specific T cells against cancer and other diseases. However, despite their unmatched features, the potential of B cells in immunotherapy is just beginning to be explored. The main role of B cells in the immune response is to secrete antigen-specific antibodies and provide long-term protection against foreign pathogens. They further function as antigen-presenting cells (APCs) and secrete pro- and anti-inflammatory cytokines and thus exert positive and negative regulatory stimuli on other cells involved in the immune response such as T cells. Therefore, while hyperactivation of B cells can cause autoimmunity, their dysfunctions lead to severe immunodeficiencies. Only suitably activated B cells can play an active role in the treatment of cancers, infections, and autoimmune diseases. As a result, studies have focused on B cell-targeted immunotherapies in recent years. For this, the development, functions, interactions with the microenvironment, and clinical importance of B cells should be well understood. In this review, we summarize the main events during B cell activation. From the viewpoint of mechanobiology we discuss the translation of external cues such as surface topology, substrate stiffness, and biochemical signaling into B cell functions. We further dive into current B cell-targeted therapy strategies and their clinical applications. STATEMENT OF SIGNIFICANCE: B cells are proving as a promising tool in the field of immunotherapy. B cells exhibit various functions such as antibody production, antigen presentation or secretion of immune-regulatory factors which can be utilized in the fight against oncological or immunological disorders. In this review we discuss the importance of external mechanobiological cues such as surface topology, substrate stiffness, and biochemical signaling on B cell function. We further summarize B cell-targeted therapy strategies and their clinical applications, as in the context of anti-tumor responses and autoimmune diseases.

6.
Cell Rep ; 32(6): 108030, 2020 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-32783949

RESUMO

Plasma cells secreting affinity-matured antibodies develop in germinal centers (GCs), where B cells migrate persistently and directionally over defined periods of time. How modes of GC B cell migration influence plasma cell development remained unclear. Through genetic deletion of the F-actin bundling protein Swiprosin-1/EF-hand domain family member 2 (EFhd2) and by two-photon microscopy, we show that EFhd2 restrains B cell speed in GCs and hapten-specific plasma cell output. Modeling the GC reaction reveals that increasing GC B cell speed promotes plasma cell generation. Lack of EFhd2 also reduces contacts of GC B cells with follicular dendritic cells in vivo. Computational modeling uncovers that both GC output and antibody affinity depend quantitatively on contacts of GC B cells with follicular dendritic cells when B cells migrate more persistently. Collectively, our data explain how GC B cells integrate speed and persistence of cell migration with B cell receptor affinity.

7.
Ther Adv Chronic Dis ; 11: 2040622320944773, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32850106

RESUMO

Mitochondrial dysfunction is a common pathological hallmark in various inflammatory and degenerative diseases of the central nervous system, including multiple sclerosis (MS). We previously showed that oxidative stress alters axonal mitochondria, limiting their transport and inducing conformational changes that lead to axonal damage. Teriflunomide (TFN), an oral immunomodulatory drug approved for the treatment of relapsing forms of MS, reversibly inhibits dihydroorotate dehydrogenase (DHODH). DHODH is crucial for de novo pyrimidine biosynthesis and is the only mitochondrial enzyme in this pathway, thus conferring a link between inflammation, mitochondrial activity and axonal integrity. Here, we investigated how DHODH inhibition may affect mitochondrial behavior in the context of oxidative stress. We employed a model of transected murine spinal roots, previously developed in our laboratory. Using confocal live imaging of axonal mitochondria, we showed that in unmanipulated axons, TFN increased significantly the mitochondria length without altering their transport features. In mitochondria challenged with 50 µM hydrogen peroxide (H2O2) to induce oxidative stress, the presence of TFN at 1 µM concentration was able to restore mitochondrial shape, motility, as well as mitochondrial oxidation potential to control levels. No effects were observed at 5 µM TFN, while some shape and motility parameters were restored to control levels at 50 µM TFN. Thus, our data demonstrate an undescribed link between DHODH and mitochondrial dynamics and point to a potential neuroprotective effect of DHODH inhibition in the context of oxidative stress-induced damage of axonal mitochondria.

9.
Front Mol Biosci ; 7: 62, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32426367

RESUMO

The multiple sclerosis therapeutic teriflunomide is known to block the de novo synthesis of pyrimidine in mitochondria by inhibiting the enzyme dihydroorotate-dehydrogenase (DHODH). The metabolic processes of oxidative phosphorylation and glycolysis are further possible downstream targets. In healthy adult mice, high levels of dihydroorotate-dehydrogenase (DHODH) activity are measured in the central nervous system (CNS), and DHODH inhibition may cause indirect effects on reactive oxygen species production and NADPH oxidase (NOX) mediated oxidative stress, known to be key aspects of the inflammatory response of the CNS. However, little is known about the effect of teriflunomide on the dynamics of NOX activation in CNS cells and subsequent alterations of neuronal function in vivo. In this study, we employed fluorescence lifetime imaging (FLIM) and phasor analysis of the endogeneous fluorescence of NAD(P)H (nicotinamide adenine dinucleotide phosphate) in the brain stem of mice to visualize the effect of teriflunomide on cellular metabolism. Furthermore, we simultaneously studied neuronal Ca2+ signals in transgenic mice with a FRET-based Troponin C Ca2+ sensor based (CerTN L15) quantified using FRET-FLIM. Hence, we directly correlated neuronal (dys-)function indicated by steadily elevated calcium levels with metabolic activity in neurons and surrounding CNS tissue. Employing our intravital co-registered imaging approach, we could not detect any significant alteration of NOX activation after incubation of the tissue with teriflunomide. Furthermore, we could not detect any changes of the inflammatory induced neuronal dysfunction due to local treatment with teriflunomide. Concerning drug safety, we can confirm that teriflunomide has no metabolic effects on neuronal function in the CNS tissue during neuroinflammation at concentrations expected in orally treated patients. The combined endogenous FLIM and calcium imaging approach developed by us and employed here uniquely meets the need to monitor cellular metabolism as a basic mechanism of tissue functions in vivo.

10.
Nat Commun ; 11(1): 2570, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444631

RESUMO

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.


Assuntos
Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Switching de Imunoglobulina , Memória Imunológica , Baço/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Animais Selvagens/imunologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Células da Medula Óssea/citologia , Ciclo Celular , Proliferação de Células/genética , Regulação da Expressão Gênica/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Baço/citologia , Células Estromais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
11.
Life Sci Alliance ; 3(6)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32341085

RESUMO

The major function of B lymphocytes is to sense antigens and to produce protective antibodies after activation. This function requires the expression of a B-cell antigen receptor (BCR), and evolutionary conserved mechanisms seem to exist that ensure that B cells without a BCR do not develop nor survive in the periphery. Here, we show that the loss of BCR expression on Burkitt lymphoma cells leads to decreased mitochondrial function and impaired metabolic flexibility. Strikingly, this phenotype does not result from the absence of a classical Syk-dependent BCR signal but rather from compromised ER expansion. We show that the reexpression of immunoglobulins (Ig) in the absence of the BCR signaling subunits Igα and Igß rescues the observed metabolic defects. We demonstrate that immunoglobulin expression is needed to maintain ER homeostasis not only in lymphoma cells but also in resting B cells. Our study provides evidence that the expression of BCR components, which is sensed in the ER and shapes mitochondrial function, represents a novel mechanism of metabolic control in B cells.

12.
Cytometry A ; 97(5): 515-527, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32293804

RESUMO

Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 µm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

13.
Cytometry A ; 97(5): 483-495, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32196971

RESUMO

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

14.
Microorganisms ; 8(1)2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31968636

RESUMO

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet's intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring.

15.
Front Immunol ; 10: 2725, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849944

RESUMO

Objective: To investigate whether low-density granulocytes (LDGs) are an immunophenotypic feature of patients with multiple sclerosis (MS) or neuromyelitis optica spectrum disorder (NMOSD). Methods: Blood samples were collected from 20 patients with NMOSD and 17 patients with MS, as well as from 15 patients with Systemic Lupus Erythematosus (SLE) and 23 Healthy Donors (HD). We isolated peripheral blood mononuclear cells (PBMCs) with density gradient separation and stained the cells with antibodies against CD14, CD15, CD16, and CD45, and analyzed the cells by flow cytometry or imaging flow cytometry. We defined LDGs as CD14-CD15high and calculated their share in total PBMC leukocytes (CD45+) as well as the share of CD16hi LDGs. Clinical data on disease course, medication, and antibody status were obtained. Results: LDGs were significantly more common in MS and NMOSD than in HDs, comparable to SLE samples (median values HD 0.2%, MS 0.9%, NMOSD 2.1%, SLE 4.3%). 0/23 of the HDs, but 17/20 NMOSD and 11/17 MS samples as well as 13/15 SLE samples had at least 0.7 % LDGs. NMOSD patients without continuous immunosuppressive treatment had significantly more LDGs compared to their treated counterparts. LDG nuclear morphology ranged from segmented to rounded, suggesting a heterogeneity within the group. Conclusion: LDGs are a feature of the immunophenotype in some patients with MS and NMOSD.


Assuntos
Biomarcadores , Granulócitos/metabolismo , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/metabolismo , Neuromielite Óptica/diagnóstico , Neuromielite Óptica/metabolismo , Adulto , Autoimunidade , Estudos de Casos e Controles , Suscetibilidade a Doenças , Feminino , Humanos , Imunofenotipagem , Contagem de Leucócitos , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/etiologia , Neuromielite Óptica/etiologia
16.
Int J Mol Sci ; 20(22)2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703416

RESUMO

In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.


Assuntos
Enzimas/metabolismo , NADP/metabolismo , NAD/metabolismo , Imagem Óptica , Mapeamento de Interação de Proteínas , Células 3T3-L1 , Animais , Camundongos
17.
Eur J Immunol ; 49(9): 1372-1379, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31149730

RESUMO

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells.


Assuntos
Células da Medula Óssea/citologia , Medula Óssea/fisiologia , Células Estromais/citologia , Transcriptoma/genética , Animais , Fator Ativador de Células B/genética , Células Cultivadas , Citocinas/genética , Células-Tronco Hematopoéticas/citologia , Interleucina-15/genética , Interleucina-7/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA/métodos
18.
Front Immunol ; 10: 788, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068930

RESUMO

Plasma cells (PCs), the B lineage cells responsible for producing and secreting antibodies (Abs), are critical cellular components of the humoral immune system. While most of the antibody-secreting cells in the body have a rather short lifetime of a few days, some of them can become long-lived and persist in the body over the entire life span of an individual. The majority of these long-lived plasma cells secretes protective antibodies against pathogens, and are thereby crucial for the humoral component of immunological memory. The generation of these protective antibody-secreting cells can be triggered by an exposure to pathogens, and also by vaccination. Although the majority of plasma cells are protective, sometimes long-lived plasma cells produce autoreactive antibodies, which contribute to the pathogenesis and perpetuation of chronic autoimmune diseases, including lupus erythematosus, rheumatoid arthritis, or multiple sclerosis. In order to promote the formation of protective antibody-secreting cells and to target pathogenic plasma cells, it is crucial to understand the signals which promote their longevity and allow them to exert their function. In recent years, it has become clear that plasma cells depend on extrinsic factors for their survival, leading to the concept that certain tissue microenvironments promote plasma cell retention and longevity. However, these niches are not static structures, but also have dynamic features with respect to their cellular composition. Here, we review what is known about the molecular and cellular composition of the niches, and discuss the impact of dynamic changes within these microenvironments on plasma cell function. As plasma cell metabolism is tightly linked to their function, we present new tools, which will allow us to analyze metabolic parameters in the plasma cell niches in vivo over time.


Assuntos
Imunidade Humoral , Plasmócitos/imunologia , Plasmócitos/metabolismo , Formação de Anticorpos , Medula Óssea/imunologia , Medula Óssea/metabolismo , Movimento Celular/imunologia , Sobrevivência Celular , Microambiente Celular/imunologia , Suscetibilidade a Doenças , Metabolismo Energético , Humanos , Intestinos/imunologia
19.
Acta Biomater ; 86: 171-184, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30616076

RESUMO

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.


Assuntos
Colágeno Tipo I/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Osteotomia , Tecidos Suporte/química , Animais , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/patologia , Calcificação Fisiológica/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/patologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/ultraestrutura , Modelos Animais de Doenças , Endotélio/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-X
20.
Front Immunol ; 10: 2588, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31956322

RESUMO

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration-the expansion phase (d1-d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14-d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2+, Osx+) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization.


Assuntos
Calo Ósseo/irrigação sanguínea , Calo Ósseo/metabolismo , Comunicação Celular , Macrófagos/metabolismo , Neovascularização Fisiológica , Animais , Biomarcadores , Regeneração Óssea , Células Endoteliais/metabolismo , Endotélio , Macrófagos/imunologia , Camundongos , Modelos Animais , Osteoblastos , Osteogênese
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