ABSTRACT
Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the infested human or animal in the long term and, thus, impact agricultural and socio-economic outcomes. However, parasite-driven immune-regulatory effects have been described, with potential therapeutic relevance for autoimmune diseases. While the metabolism in both the host and parasites contributes to their defense and is the basis for nematode survival in the intestine, it has remained largely understudied due to a lack of adequate technologies. We have developed and applied NAD(P)H fluorescence lifetime imaging to explanted murine intestinal tissue during infection with the natural nematode Heligmosomoides polygyrus to study the metabolic processes in both the host and parasites in a spatially resolved manner. The exploitation of the fluorescence lifetime of the co-enzymes nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, which are preserved across species, depends on their binding status and the binding site on the enzymes catalyzing metabolic processes. Focusing on the most abundantly expressed NAD(P)H-dependent enzymes, the metabolic pathways associated with anaerobic glycolysis, oxidative phosphorylation/aerobic glycolysis, and NOX-based oxidative burst, as a major defense mechanism, were distinguished, and the metabolic crosstalk between the host and parasite during infection was characterized.
Subject(s)
Nematode Infections , Parasites , Humans , Animals , Mice , NAD , Oxidative Phosphorylation , Intestines/diagnostic imagingABSTRACT
Affinity maturation of B cell clones within germinal centers constitutes an important mechanism for immune memory. During this process, B cell receptor signaling capacity is tested in multiple rounds of positive selection. Antigen stimulation and co-stimulatory signals mobilize calcium to switch on gene expression leading to proliferation and survival and to differentiation into memory B cells and plasma cells. Additionally, all these processes require adaption of B cell metabolism, and calcium signaling and metabolic pathways are closely interlinked. Mitochondrial adaption, ROS production, and NADPH oxidase activation are involved in cell fate decisions, but it remains elusive to what extent, especially because the analysis of these dynamic processes in germinal centers has to take place in vivo. Here, we introduce a quantitative intravital imaging method for combined measurement of cytoplasmic calcium concentration and enzymatic fingerprinting in germinal center B cells as a possible tool in order to further examine the relationship of calcium signaling and immunometabolism.
Subject(s)
Calcium , NAD , NAD/metabolism , Calcium/metabolism , Fluorescence Resonance Energy Transfer , Germinal Center , Receptors, Antigen, B-Cell/metabolismABSTRACT
Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.
Subject(s)
COVID-19 , Chemokine CCL21 , Chemokines, CC , Humans , COVID-19/immunology , Fibrosis , Lung , T-Lymphocytes/immunologyABSTRACT
Infections with intestinal nematodes have an equivocal impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. One basic mechanism governing parasite and host cellular and tissue functions, metabolism, has largely been neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic activity and possible metabolic pathways in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via metabolic pathways resembling oxidative phosphorylation/aerobic glycolysis features. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis-like and effective oxidative phosphorylation-like metabolic pathways, towards mainly anaerobic glycolysis-like pathways, back to oxidative phosphorylation/aerobic glycolysis-like pathways during their different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of NAD(P)H-FLIM in live tissues constitutes a unique tool to study possible shifts between metabolic pathways in host-parasite crosstalk, in various parasitic intestinal infections.
Subject(s)
Nematospiroides dubius , Parasites , Animals , Mice , NAD/metabolism , NADP/metabolism , Optical Imaging , Parasites/metabolismABSTRACT
Rheumatoid arthritis and osteoarthritis are two related chronic diseases of the musculoskeletal system which are particularly pronounced in the region of joints and bones. Their pathogeneses are associated with chronic inflammation, which can disrupt homeostasis in bones and articular cartilage. Degradation products deriving from articular cartilage can contribute to the exacerbation of inflammation in the joint region. Mechanical stimuli and blood vessels also play a central role in both the regulation of bone growth as well as in the regeneration of bone tissue. Not only chronic inflammatory processes but also hormonal changes after menopause or undesired effects of glucocorticoid therapy have an influence on the balance between bone resorption and deposition, by promoting the former and reducing the latter. This results in decreased bone quality and, in some cases, considerable loss of bone or osteoporosis. An in-depth understanding of these processes at the molecular, cellular, and tissue level, as well as of the changes present in chronic inflammatory diseases, has been the focus of research at the German Rheumatism Research Center (Deutsches Rheuma-Forschungszentrum, DRFZ) since its foundation. Based on an improved understanding of these mechanisms, the DRFZ aims to develop improved prevention and treatment strategies with effects even in early disease stages.
Subject(s)
Cartilage, Articular , Osteoarthritis , Female , Glucocorticoids , Humans , Inflammation , Stromal CellsABSTRACT
Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.
Subject(s)
Cell Communication/genetics , Cellular Microenvironment/genetics , Microscopy, Fluorescence/methods , Molecular Imaging/methods , Algorithms , Cell Line , Fluorescent Dyes/chemistry , PhotonsABSTRACT
Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.
Subject(s)
Fluorescent Antibody Technique/methods , Intravital Microscopy/methods , Microscopy, Fluorescence, Multiphoton/methods , Algorithms , Animals , Cell Line , Data Analysis , Humans , Image Processing, Computer-Assisted , Intravital Microscopy/instrumentation , Mice , Microscopy, Fluorescence, Multiphoton/instrumentationABSTRACT
We aimed to evaluate SIGLEC1 (CD169) as a biomarker in multiple sclerosis (MS) and Neuromyelitis optica spectrum disorder (NMOSD) and to evaluate the presence of SIGLEC1+ myeloid cells in demyelinating diseases. We performed flow cytometry-based measurements of SIGLEC1 expression on monocytes in 86 MS patients, 41 NMOSD patients and 31 healthy controls. Additionally, we histologically evaluated the presence of SIGLEC1+ myeloid cells in acute and chronic MS brain lesions as well as other neurological diseases. We found elevated SIGLEC1 expression in 16/86 (18.6%) MS patients and 4/41 (9.8%) NMOSD patients. Almost all MS patients with high SIGLEC1 levels received exogenous interferon beta as an immunomodulatory treatment and only a small fraction of MS patients without interferon treatment had increased SIGLEC1 expression. In our cohort, SIGLEC1 expression on monocytes was-apart from those patients receiving interferon treatment-not significantly increased in patients with MS and NMOSD, nor were levels associated with more severe disease. SIGLEC1+ myeloid cells were abundantly present in active MS lesions as well as in a range of acute infectious and malignant diseases of the central nervous system, but not chronic MS lesions. The presence of SIGLEC1+ myeloid cells in brain lesions could be used to investigate the activity in an inflammatory CNS lesion.
Subject(s)
Brain/metabolism , Multiple Sclerosis/metabolism , Sialic Acid Binding Ig-like Lectin 1/metabolism , Biomarkers/metabolism , Case-Control Studies , Flow Cytometry , Humans , Interferon-beta/therapeutic use , Multiple Sclerosis/blood , Multiple Sclerosis/drug therapyABSTRACT
Decade-long survival of plasma cells in the bone marrow has long been a puzzling matter. To understand how plasma cells are maintained and supported by survival-niches to account for lifelong antibody production demands new intravital imaging techniques that are able to follow up a single cell and their interaction with other cell types in situ. We achieved to successfully establish longitudinal imaging of the bone marrow (LIMB) that is based on an implantable endoscopic device. In this chapter, basic approaches on how to investigate plasma cell-stroma interaction and surgical implantation procedures are introduced.
Subject(s)
Bone Marrow Cells/physiology , Bone Marrow/physiology , Cellular Microenvironment , Image Processing, Computer-Assisted , Intravital Microscopy , Microscopy, Fluorescence, Multiphoton , Plasma Cells/physiology , Adoptive Transfer , Animals , Bone Marrow/metabolism , Bone Marrow Cells/metabolism , Bone Marrow Transplantation , Cell Separation , Genes, Reporter , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice, Transgenic , Plasma Cells/metabolism , Positive Regulatory Domain I-Binding Factor 1/genetics , Positive Regulatory Domain I-Binding Factor 1/metabolismABSTRACT
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.
Subject(s)
B-Lymphocytes/metabolism , Fluorescence Resonance Energy Transfer/methods , Lymphocyte Activation , Animals , Female , Male , MiceABSTRACT
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.
Subject(s)
B-Lymphocytes/immunology , Calcium-Binding Proteins/immunology , Dendritic Cells, Follicular/immunology , Germinal Center/immunology , Plasma Cells/immunology , Animals , Calcium-Binding Proteins/deficiency , Cell Differentiation , Cell Movement/immunology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Peptide Elongation Factor 2ABSTRACT
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.
Subject(s)
Microscopy , Tomography, Optical Coherence , Animals , Cell Movement , Collagen , Mice , PhotonsABSTRACT
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.
Subject(s)
Lens, Crystalline , Lenses , Animals , Bone Marrow , Mice , Mice, Transgenic , OsteotomyABSTRACT
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.
Subject(s)
Biomarkers , Granulocytes/metabolism , Multiple Sclerosis/diagnosis , Multiple Sclerosis/metabolism , Neuromyelitis Optica/diagnosis , Neuromyelitis Optica/metabolism , Adult , Autoimmunity , Case-Control Studies , Disease Susceptibility , Female , Humans , Immunophenotyping , Leukocyte Count , Leukocytes, Mononuclear/metabolism , Lupus Erythematosus, Systemic , Male , Middle Aged , Multiple Sclerosis/etiology , Neuromyelitis Optica/etiologyABSTRACT
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.
Subject(s)
Enzymes/metabolism , NADP/metabolism , NAD/metabolism , Optical Imaging , Protein Interaction Mapping , 3T3-L1 Cells , Animals , MiceABSTRACT
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.
Subject(s)
Immunity, Humoral , Plasma Cells/immunology , Plasma Cells/metabolism , Antibody Formation , Bone Marrow/immunology , Bone Marrow/metabolism , Cell Movement/immunology , Cell Survival , Cellular Microenvironment/immunology , Disease Susceptibility , Energy Metabolism , Humans , Intestines/immunologyABSTRACT
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.
Subject(s)
Bony Callus/blood supply , Bony Callus/metabolism , Cell Communication , Macrophages/metabolism , Neovascularization, Physiologic , Animals , Biomarkers , Bone Regeneration , Endothelial Cells/metabolism , Endothelium , Macrophages/immunology , Mice , Models, Animal , Osteoblasts , OsteogenesisABSTRACT
The bone marrow (BM) consists of multiple, structured micro-environmental entities-the so called niches, which contain hematopoietic cells as well as stromal cells. These niches fulfill a variety of functions, such as control of the hematopoietic stem cell pool, differentiation of hematopoietic cells, and maintenance of immunological memory. However, due to the molecular and cellular complexity and a lack of suitable histological multiplexing methods, the composition of the various BM niches is still elusive. In this study, we apply multiepitope-ligand-cartography (MELC) on bone sections from mice. We combine multiplexed immunofluorescence histology data with various object-based segmentation approaches in order to define irregularly shaped, net-like structures of stromal cells. We confirm MELC as a robust histological method and validate our automated segmentation algorithms using flow cytometry and manual evaluation. By means of MELC multiplexing, we reveal heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network. Moreover, we demonstrate by quantification a preferential contact of B cell subsets as well as of plasma cells to processes of CXCL12-expressing stromal cells, compared with stromal somata. In summary, our approach is suitable for spatial analysis of complex tissue structures.