[The DRFZ-a pioneer in research on the interaction between immune and stromal cells during de- and regeneration of the musculoskeletal system]. / Das DRFZ ­ ein Vorreiter bei der Erforschung des Zusammenspiels von Immun- und Stromazellen bei der De- und Regeneration des Bewegungsapparats.

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.

Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix.

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.

Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging.

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.

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

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.

Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging.

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.

Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections.

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.

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis.

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions.

Quantitative Imaging of Ca2+ by 3D-FLIM in Live Tissues.

The calcium concentration within living cells is highly dynamic and, for many cell types, a reliable indicator of the functional state of the cells-both of isolated cells, but even, more important, of cells in tissue. In order to dynamically quantify intracellular calcium levels, various genetically encoded calcium sensors have been developed-the best of which are those based on Förster resonant energy transfer (FRET). Here we present a fluorescence lifetime imaging (FLIM) method to measure FRET in such a calcium sensor (TN L15) in neurons of hippocampal slices and of the brain stem of anesthetized mice. The method gives the unique opportunity to determine absolute neuronal calcium concentrations in the living organism.

Static and dynamic components synergize to form a stable survival niche for bone marrow plasma cells.

In the bone marrow (BM), memory plasma cells (PCs) survive for long time periods in dedicated microenvironmental survival niches, resting in terms of proliferation. Several cell types, such as eosinophils and reticular stromal cells, have been reported to contribute to the survival niche of memory PCs. However, until now it has not been demonstrated whether the niche is formed by a fixed cellular microenvironment. By intravital microscopy, we provide for the first time evidence that the direct contacts formed between PCs and reticular stromal cells are stable in vivo, and thus the PCs are sessile in their niches. The majority (∼ 80%) of PCs directly contact reticular stromal cells in a non-random fashion. The mesenchymal reticular stromal cells in contact with memory PCs are not proliferating. On the other hand, we show here that eosinophils in the vicinity of long-lived PCs are vigorously proliferating cells and represent a dynamic component of the survival niche. In contrast, if eosinophils are depleted by irradiation, newly generated eosinophils localize in the vicinity of radiation-resistant PCs and the stromal cells. These results suggest that memory PC niches may provide attraction for eosinophils to maintain stability with fluctuating yet essential accessory cells.

The role of CD8+ T cells and their local interaction with CD4+ T cells in myelin oligodendrocyte glycoprotein35-55-induced experimental autoimmune encephalomyelitis.

T cells have an essential role in the induction of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Although for CD4(+) T cells it is well established that they contribute to the disease, less is known about the role of CD8(+) T cells. Our aim was to determine the individual contribution of CD4(+) and CD8(+) T cells in myelin oligodendrocyte glycoprotein (MOG)35-55-induced EAE. We investigated MOG35-55-activated CD8(+) T cells to clarify their potential to induce or attenuate EAE. We monitored the behavior of CD8(+) T cells and their interaction with CD4(+) T cells directly at the site of inflammation in the CNS using intravital imaging of the brainstem of EAE-affected living anesthetized mice. We found that mice without CD4(+) T cells did not develop relevant clinical signs of disease, although CD8(+) T cells were present in the CNS of these mice. These CD8(+) T cells displayed reduced motility compared with those in the presence of CD4(+) T cells. In mice that harbored CD4(+) and CD8(+) T cells, we saw a similar extent of clinical signs of EAE as in mice with only CD4(+) T cells. Furthermore, the dynamic motility and viability of CD4(+) T cells were not disturbed by CD8(+) T cells in the lesions of these mice. Therefore, we conclude that in MOG35-55-induced EAE, CD8(+) T cell accumulation in the CNS represents instead an epiphenomenon with no impact on clinical disease or on the effects of CD4(+) T cells, the latter being the true inducers of the disease.

Combined FRET-FLIM and NAD(P)H FLIM to Analyze B Cell Receptor Signaling Induced Metabolic Activity of Germinal Center B Cells In Vivo.

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.

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection.

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.

Distinct tissue niches direct lung immunopathology via CCL18 and CCL21 in severe COVID-19.

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.

Intravital two-photon microscopy: focus on speed and time resolved imaging modalities.

Initially used mainly in the neurosciences, two-photon microscopy has become a powerful tool for the analysis of immunological processes. Here, we describe currently available two-photon microscopy techniques with a focus on novel approaches that allow very high image acquisition rates compared with state-of-the-art systems. This improvement is achieved through a parallelization of the excitation process: multiple beams scan the sample simultaneously, and the fluorescence is collected with sensitive charge-coupled device (CCD)-based line or field detectors. The new technique's performance is compared with conventional single beam laser-scanning systems that detect signals by means of photomultipliers. We also discuss the use of time- and polarization-resolved fluorescence detection, especially fluorescence lifetime imaging (FLIM), which goes beyond simple detection of cells and tissue structures and allows insight into cellular physiology. We focus on the analysis of endogenous fluorophores such as NAD(P)H as a way to analyze the redox status in cells with subcellular resolution. Here, high-speed imaging setups in combination with novel ways of data analysis allow the generation of FLIM data sets almost in real time. The implications of this technology for the analysis of immune reactions and other cellular processes are discussed.

NAD(P)H fluorescence lifetime imaging of live intestinal nematodes reveals metabolic crosstalk between parasite and host.

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.

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity.

BACKGROUND: Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated. METHODS: We applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures. RESULTS: By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks. CONCLUSIONS: The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.

Recent advances in dynamic intravital multi-photon microscopy.

Standard multiphoton laser scanning microscopy (MPLSM) has revolutionized our view of physiologic and pathologic processes in living organisms by enlightening different aspects of cellular choreography in immune responses, that is, cellular motility and co-localization. To understand cellular communication on a molecular level, novel transgenic reporter mice have been generated. In parallel, MPLSM systems have been developed, which make it possible for this technique to be more widely used to address crucial immunological questions. Here, we review the latest progress concerning transgenic mouse technology and multiphoton imaging capacities and discuss further developments which will enable us to visualize all around monitoring and quantification of cellular function at a molecular level directly in vivo.

Imaging of Bone Marrow Plasma Cells and of Their Niches.

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.

Method for Multiplexed Dynamic Intravital Multiphoton Imaging.

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.