Biology subjects

Duda, G.

Publications and source records attributed to Duda, G..

4 recordsLinked to original sources

bioRxiv2025

Using artificial intelligence-based software for an unbiased discrimination of immune cell subtypes in the fracture hematoma and bone marrow of non-osteoporotic and osteoporotic mice

It is well established that the early inflammatory response following fracture is essential for initiating subsequent bone regeneration. An imbalance in inflammation, whether within the innate or adaptive immune response, can result in impaired fracture healing. In our previous studies, we demonstrated that, for example, mice with ovariectomy-induced osteoporosis exhibit altered immune cell populations in the early fracture hematoma and bone marrow, leading to delayed healing. These analyses were conducted using conventional FACS/flow cytometry software, where surface marker expression was assessed using a single threshold based on isotype controls--a binary "yes or no" decision. Recent advances have highlighted that immune cell populations are often more heterogeneous, with distinct phenotypic subgroups depending on their polarization status. This has been particularly well documented for macrophage subpopulations (M1, M2, and intermediate polarization states). In light of this, we employed a commercially available artificial intelligence-based clustering software (Cytolution) to more accurately and objectively identify immune cell subpopulations. We re-analyzed flow cytometry raw data from fracture hematoma and bone marrow of non-osteoporotic and osteoporotic mice at day 1 after fracture. Our findings revealed distinct subclusters for granulocytes (27 subclusters), macrophages (7 subclusters), B cells (4 subclusters), and T cells (6 subclusters) within the fracture hematoma and bone marrow. Comparing osteoporotic and non-osteoporotic mice, we observed an increased abundance of a specific B cell subpopulation in osteoporotic mice, alongside a significant reduction of a particular granulocyte subpopulation in the early fracture hematoma. Several subclusters of granulocytes, T cells, and macrophages were also altered in the bone marrow. The specific role of these immune cell subclusters remains to be investigated in the future. These results suggest that AI-based clustering may provide a powerful tool for identifying immune cell phenotypes during bone regeneration, offering a more nuanced understanding of flow cytometry data.

immunology↗

bioRxiv2023

In vivo optimized three-photon imaging of intact mouse tibia links plasma cell motility to functional states in the bone marrow.

Intravital multi-photon imaging of the bone marrow is crucial to the study of cellular dynamics, communication with the microenvironment and functions, however, imaging of deep tissue areas is challenging and minimally invasive methods for deep-marrow imaging in intact long bones are needed. We developed a high pulse energy 1650 nm laser prototype, which permits to surpass >100 {micro}m thick cortical bone and to perform three-photon microscopy (3PM) in more than 400 {micro}m depth in the marrow cavity of intact mouse tibia in vivo. Its unique 3 and 4 MHz laser repetition rates allowed us to analyze motility patterns of rare cells over large fields of view deep within the unperturbed marrow. In this way, we found a bi-modal migratory behavior of marrow plasma cells. Besides, the analysis of third harmonics generation (THG) in the tibia identified this signal to be a label-free indicator of the abundance of cellular organelles, in particular the endoplasmic reticulum, reflecting protein biosynthesis capacity. We found that only one third of the plasma cells in the tibia marrow of adult mice have a strong THG signal and, thus, a high protein synthesis capacity, while the other two thirds of plasma cells display a low THG signal. Finally, we identified an inverse link between migratory behavior and THG signal strength in marrow plasma cells. As in these cells, the protein biosynthesis capacity indicated by a strong THG signal is mainly associated with antibody secretion, we could relate motility to functional states of plasma cells in vivo. Our 3PM method retains the ability to connect cellular dynamics to protein biosynthesis capacity in various marrow cell types beyond plasma cells, as THG is a ubiquitous signal, opening new perspectives on understanding how tissue microenvironment impacts on cellular functions in the bone marrow.

biophysics↗

bioRxiv2023

Increased beta2-adrenergic signaling is a targetable stimulus essential for bone healing by promoting callus neovascularization

Traumatic brain injury (TBI) is associated with a hyperadrenergic state and paradoxically causes systemic bone loss while accelerating fracture healing. Here, we identify the beta2-adrenergic receptor (Adrb2) as a central mediator of these skeletal manifestations. While the negative effects of TBI on the unfractured skeleton can be explained by the established impact of Adrb2 signaling on bone formation, Adrb2 promotes neovascularization of the fracture callus under conditions of high sympathetic tone, including TBI and advanced age. Mechanistically, norepinephrine stimulates the expression of Vegfa and Cgrp primarily in periosteal cells via Adrb2, both of which synergistically promote the formation of osteogenic type-H vessels in the fracture callus. Accordingly, the beneficial effect of TBI on bone repair is abolished in mice lacking Adrb2 or Cgrp, and aged Adrb2-deficient mice without TBI develop fracture nonunions despite high bone formation in uninjured bone. Pharmacologically, the Adrb2 antagonist propranolol impairs, and the agonist formoterol promotes fracture healing in aged mice by regulating callus neovascularization. Clinically, intravenous beta-adrenergic sympathomimetics are associated with improved callus formation in trauma patients with long bone fractures. Thus, Adrb2 is a novel target for promoting bone healing, and widely used beta-blockers may cause fracture nonunion under conditions of increased sympathetic tone. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/548550v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@cea57corg.highwire.dtl.DTLVardef@d29642org.highwire.dtl.DTLVardef@186ea5aorg.highwire.dtl.DTLVardef@83eaae_HPS_FORMAT_FIGEXP M_FIG Artwork was created in BioRender. C_FIG

cell biology↗

bioRxiv2020

Functionalized bead assay to measure 3-dimensional traction forces during T-cell activation

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination process. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Common approaches such as traction force microscopy (TFM) employ a global readout of the force fields, e.g. by measuring the displacements of hydrogel-embedded marker beads. Recent data, however, indicated that T-cells exert tensile forces locally via TCR-enriched microvilli while scanning the surface of antigen-presenting cells. Here, we developed a traction force microscopy platform, which allows for quantifying the pulls exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. For this, we immobilized specific T-cell activating antibodies directly on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas, CD45 is excluded from bead-microvilli contacts. Significance statementDuring the antigen recognition process, T-cells explore and probe their environment via microvilli, which exert local pushes and pulls at the surface of the antigen presenting cell. It is currently believed that these forces influence or even enable the antigen recognition process. Here, we describe the development of a platform, which allows us to quantify the magnitude and direction of traction forces exerted locally by T cell microvilli. Simultaneous Ca2+ imaging was used to link the measured forces to the overall T cell activation status. Superresolution microscopy resolved the contact sites of bead-microvilli contact at the nanoscale: cells contacted beads via actin vortex-like structures, which excluded the phosphatase CD45 from the contacts.

biophysics↗