Impaired fracture healing upon neutrophil-specific adrenoreceptor beta 2 knockout in non-osteoporotic and osteoporotic mice

Impaired fracture healing upon neutrophil-specific adrenoreceptor beta 2 knockout in non-osteoporotic and osteoporotic mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Impaired fracture healing upon neutrophil-specific adrenoreceptor beta 2 knockout in non-osteoporotic and osteoporotic mice Sandra Dieterich, Nico Gläser, Christoph Kölbl, Dorothea Gebauer, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8593165/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 16 You are reading this latest preprint version Abstract After bone fracture, osteoporotic mice show delayed healing associated with elevated systemic inflammation and increased neutrophil numbers in the early fracture hematoma. Because short-term propranolol treatment reduced neutrophil recruitment, we hypothesized that deletion of β 2 -adrenoreceptor (Adrb2) signaling in neutrophils would normalize neutrophil recruitment and accelerate fracture healing in osteoporotic mice. A conditional Adrb2 knockout in Ly6G⁺ neutrophils was generated using Ly6G-Cre Adrb2-flox mice. Bone and immune phenotypes were analyzed via µCT and histology under non-fracture conditions and during fracture healing in ovariectomized mice with postmenopausal osteoporosis. Both non-osteoporotic and osteoporotic female Ly6G-Adrb2-KO mice showed impaired fracture healing vs. controls, while only minor bone alterations were observed under non-fracture conditions. Pathway analysis of isolated neutrophils, characterized by RNA-sequencing, suggested reduced neutrophil activation and disturbed neutrophil/mast cell interactions upon Ly6G-Adrb2-KO. In summary, our data demonstrate that Adrb2 signaling is important for neutrophil recruitment and seems to be critical for proper fracture healing. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Biological sciences/Physiology neutrophil granulocytes inflammation adrenergic signaling beta 2 adrenoreceptor osteoporosis fracture healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction It is well established that the skeletal and the immune system are closely interconnected in many aspects, a concept that has given rise to the interdisciplinary field of osteoimmunology. Immune cells such as T cells, macrophages, and neutrophil granulocytes critically influence bone metabolism through the secretion of mediators such as Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL), tumor necrosis factor-alpha (TNF-α), or Interleukin-6 (IL-6), thereby modulating both osteoclastic bone resorption and osteoblastic bone formation [ 1 , 2 ]. One clinical example of a disrupted osteoimmunological balance is postmenopausal osteoporosis, which is characterized by an imbalance in bone metabolism, with increased activity of bone-resorbing osteoclasts relative to bone-forming osteoblasts. This imbalance is driven not only by the direct positive effects of estrogen on bone formation and its inhibitory effects on bone resorption, but also by the fact that estrogen deficiency after menopause induces a chronic low-grade inflammatory state [ 3 , 4 ], which further contributes to impaired bone homeostasis. As a result, osteoporotic bone exhibits an altered microarchitecture that increases fracture risk [ 5 ]. Each year, more than 3.5 million new osteoporotic fractures occur in Europe, and 10–15% of patients die within the first year after suffering from an osteoporotic hip fracture [ 6 , 7 ]. In addition to the increased fracture risk, osteoporotic bone displays a reduced capacity for fracture healing [ 8 ], as demonstrated in numerous animal studies using ovariectomized, estrogen-deficient rodents. These animals exhibit a mechanically weaker fracture callus with increased osteoclast numbers [ 9 – 12 ], reduced expression of cartilage markers [ 13 ], and impaired expression of angiogenic factors [ 14 ]. Furthermore, we previously demonstrated that ovariectomized mice exhibit elevated systemic levels of proinflammatory cytokines and increased numbers of neutrophil granulocytes in the fracture hematoma three days after surgery, which was associated with delayed fracture healing [ 15 ]. However, the mechanisms underlying the enhanced recruitment and/or activity of neutrophils in osteoporosis and osteoporotic fracture healing remain largely unclear. Emerging evidence suggests a role for adrenergic signaling in this process, as neutrophil granulocytes are strongly influenced by catecholamines and adrenergic stimulation [ 16 , 17 ]. Catecholamines play a pivotal physiological role, as they are released in response to external and internal stressors via activation of the hypothalamic-pituitary-adrenal (HPA) axis. By binding to distinct adrenergic receptor subtypes expressed across multiple organs and tissues, catecholamines regulate essential physiological functions, including cardiovascular activity, pupillary diameter, and autonomic control of gastrointestinal processes [ 18 , 19 ]. Bone is also subject to sympathetic regulation, which influences both bone formation and turnover. For example, treatment of stressed male mice with the non-selective β-adrenergic receptor antagonist propranolol immediately before fracture significantly reduced the number of neutrophil granulocytes in the fracture hematoma and improved fracture healing [ 20 ]. In this context, it is important to note that neutrophil granulocytes, similar to osteoblasts and osteoclasts [ 21 ], express the β 2 -adrenoreceptor (Adrb2) [ 22 , 23 ]. This receptor is thought to play a key role in adrenergic regulation of bone metabolism, as previous studies demonstrated that both global [ 24 , 25 ] and osteoblast-specific [ 26 ] Adrb2 knockout (KO) led to increased bone volume in mice. However, the role of Adrb2 signaling in immune cells in the context of bone metabolism and fracture healing remains unknown. Therefore, the aim of this study was to elucidate the role of Adrb2 in Ly6G⁺ neutrophil granulocytes during fracture healing in both non-osteoporotic and osteoporotic mice. We hypothesized that catecholamine release following fracture activates Adrb2 signaling in neutrophils, thereby promoting their enhanced recruitment to the fracture hematoma and contributing to impaired bone regeneration in osteoporotic bone. To test this hypothesis, mice with a neutrophil-specific deletion of Adrb2 were generated. Bone metabolism upon KO was analyzed in both sexes while fracture healing was investigated in female mice after ovariectomy, a model of postmenopausal osteoporosis. Methods Animal housing and transgenic mouse model All animal experiments were conducted in compliance with the European Guidelines for Animal Research on the Protection of Animals and the ARRIVE guidelines and were approved by the local animal welfare authority (Regierungspräsidium Tübingen, No. 1612). Mice were housed in groups of up to five per cage under a 12-hour light/ 12-hour dark cycle with ad libitum access to food and water. The study utilized transgenic mice with a C57BL/6J background carrying a neutrophil-specific KO of the Adrb2 under control of the Cre/loxP system (Ly6G-Cre⁺ Adrb2 flox/flox ). This mouse line was established by breeding Ly6G-Cre mice [ 27 ] with Adrb2flox mice kindly donated by Gerard Karsenty [ 28 ]. Cre-negative littermates (Ly6G-Cre − Adrb2 flox/flox ) were included as controls. Experimental design and surgical procedures In the first part of the study, male Cre + and Cre − mice were euthanized by isoflurane overdose and terminal intracardiac blood withdrawal at the age of 12 weeks to analyze their bone and immune phenotype via µCT analysis, histology and flow cytometry (Fig. 1 ). Male littermate mice were used in accordance with the 3R principle of reduction, as the females were required for the fracture surgeries and the males could be included in the phenotyping study without generating additional breeding pairs. To assess the bone phenotype in female mice, the unfractured contralateral femur was analyzed via µCT. The second part of the study focused on fracture healing in female non-osteoporotic and osteoporotic mice. Twelve-week-old female mice were subjected to either a sham operation or bilateral ovariectomy (OVX) to induce postmenopausal osteoporosis as previously described [ 4 , 15 , 29 ]. Anesthesia was performed starting with 5–6% isoflurane (Forene, Abbott) and maintained with 2% isoflurane (Forene, Abbott) with an oxygen flow rate of 0.7 mL/min. To ensure adequate analgesia, all mice received tramadol (25 mg/L, Tramal, Gruenenthal GmbH) in their drinking water from one day pre- until three days postoperatively. After surgery, normal housing diet (ssniff R/M-H, V1535-300, Ssniff GmbH) was changed to phytoestrogen-low diet (ssniff R/M-H, V1554-300, Ssniff GmbH). Four weeks post-surgery, a standardized, unilateral transverse osteotomy of the right femur was performed, which was stabilized using an external fixator as previously described [ 30 ]. Mice were euthanized by isoflurane overdose on day 3 and 21 post-fracture for subsequent analyses, including µCT analysis, histology, and IHC staining (Fig. 1 ). Biomechanical testing To assess the mechanical properties of the bones, biomechanical testing was conducted on intact femurs from 12-week-old male mice. A destructive three-point bending test was performed following a previously established protocol [ 30 ]. Each bone was subjected to a load of up to 10 N using a materials testing machine (Zwick Roell, Ulm, Germany), while load and deflection were continuously recorded. For each bone the maximal load until failure (F max ) was calculated and the flexural rigidity was determined from the slope of the linear region of the load-deflection curve. µCT analysis Fractured and unfractured femurs were fixed in 4% paraformaldehyde for 48 h prior to micro-computed tomography (µCT) scanning. Imaging was performed using a Skyscan 1172 device (Skyscan, Aartselaar, Belgium) at a peak voltage of 50 kV and a current of 200 µA to assess bone content and mineralization. The isotropic voxel resolution was set to 8 µm. Three-dimensional analysis was conducted using computed tomography analysis (CTAn) and CT volume (CTVol) software (Bruker) in accordance with ASBMR guidelines [ 31 ]. Analyzing the fractured bones, the volume of interest (VOI) was defined as the entire periosteal callus region located between the two inner pinholes of the fixator. Unfractured bones were analyzed in two VOIs: VOI 1 encompassed a region extending 360 µm from the proximal end of the growth plate to 280 µm proximal to the distal end of the bone. VOI 2 covered the area from the proximal end of the trochanter tertius to 80 µm proximal to the distal end. Tissue mineral density was quantified using calibration phantoms containing defined hydroxyapatite (HA) concentrations of 250 mg HA/cm³ and 750 mg HA/cm³. The threshold for mineralized tissue was set at 394 mg HA/cm 3 for trabecular bone and 642 mg HA/cm³ for cortical bone and fracture callus analysis. Histomorphometry Decalcified histological analysis of fractured and unfractured femurs was performed as previously described [ 32 ]. Sections of 4 µm thickness were stained with Safranin O/Fast Green to assess tissue composition and with tartrate-resistant alkaline phosphatase (TRAP) to identify osteoclasts. In the fractured bones, the proportions of bone, cartilage, and fibrous tissue within the fracture callus at day 21 post-fracture were quantified in Safranin O-stained sections using image analysis software (Leica MMAF 1.4.0 Imaging System; Leica, Wetzlar, Germany). The region of interest (ROI) of the fractured samples was defined as the entire fracture callus between the two inner pinholes of the fixator. In addition, osteoblasts and osteoclasts were quantified in sections of fractured and unfractured femora at 20× magnification. In unfractured samples, the ROI (480 x 350 µm) for quantifying osteoblasts and osteoclasts was defined as an area located 960 µm proximal of the distal growth plate and positioned between both cortices in a trabecular-rich area, excluding cortical bone, as previously described [ 18 ]. Osteocytes were quantified at the same longitudinal level but within the cortical bone. All cells fully embedded within the cortical matrix were classified as osteocytes. In fractured samples, the same area was defined in the periosteal callus near the fracture gap. Osteoblasts were identified and counted in Safranin O-stained sections based on their characteristic morphology as cubic-shaped cells lining the bone surface. Osteoclasts were analyzed in TRAP-stained sections and identified by their positive TRAP staining, distinctive multinucleated morphology, size, and localization on the bone surface. Immunohistochemistry Longitudinal sections of 4 µm thickness were prepared for immunohistochemical staining. Detection of Mcpt5 was performed using the primary antibody rabbit anti-mouse MC Protease 5 (1:100; orb11030, Biorbyt, Cambridge, UK), which was incubated overnight at 4°C. The secondary antibody goat anti-rabbit IgG-biotin (1:200; B2770, Life Technologies, Carlsbad, CA, USA) was applied at room temperature (RT) for 1 h. Detection of Ly6G was performed using the primary antibody rat anti-mouse Ly6G (1:300; 127632, BioLegend, San Diego, USA) which was incubated overnight at 4°C. The secondary antibody goat anti-rat IgG-biotin (1:200; #31830, Invitrogen, Carlsbad, CA, USA) was applied at RT for 1 h. For both single staining’s, horseradish peroxidase (HRP)-conjugated streptavidin (PK-6100, VECTASTAIN Elite ABC-HRP Kit, Peroxidase, Vector Laboratories, Burlingame, UK) was used for signal detection according to the manufacturer’s instructions. NovaRED (SK-4800, Vector NovaRED Substrate Kit, Peroxidase (HRP), Vector Laboratories) served as the chromogen, and sections were counterstained with hematoxylin (1:2000; 2C-306, Waldeck, Münster, Germany). Species-specific, non-targeting immunoglobulins were used as isotype controls. A total of 4–8 mice per group were analyzed. Neutrophils and mast cells were quantified within a defined volume of interest (480 × 350 µm) in an area of the fracture callus with maximum amount of cells at 20× magnification. Identification was based on characteristic staining, morphology, and cell size. Immunofluorescent staining Ly6G-Avidin A double staining of neutrophils and mast cells in the fracture callus 3 days after osteotomy was performed following the subsequent protocol. Detection of Ly6G was performed using the primary antibody rat anti-mouse Ly6G (1:300; #127632, BioLegend, San Diego, USA) which was incubated overnight at 4°C. The secondary antibody rabbit anti-rat IgG (H + L) Alexa Flour 488 (A-21210; Thermo Fisher, Waltham, Massachusetts, USA) and Avidin Texas Red (A820, Invitrogen, Carlsbad, CA, USA), which stains mast cell granules, were applied at RT for 1 h. Nuclei were stained with Hoechst (33258; Sigma-Aldrich, St. Louis, Missouri, USA) for 10 min at RT. Species-specific, non-targeting immunoglobulins were used as isotype controls. Stained slides were screened for possible mast cell intracellular trap (MIT) formation. Flow cytometry Flow cytometry was performed to characterize immune cell populations in the bone marrow and spleen of Cre - and Cre + 12-week-old male mice. The bone marrow was flushed out of the left femur using 10 ml phosphate-buffered saline (PBS). The spleen was harvested and passed through a 70-µm cell strainer (Corning Inc., Durham, NC, USA). Cells from both the spleen and bone marrow underwent erythrolysis to remove red blood cells. For immunophenotyping, macrophages (F4/80 + ), neutrophils (Ly-6G + ), inflammatory monocytes (CD11b + ), B-lymphocytes (CD19 + ), T-lymphocytes (CD3 + ), cytotoxic T-lymphocytes (CD3 + , CD8 + ), and T-helper lymphocytes (CD3 + , CD4 + ) were identified using the antibodies listed in Table 1 . Isotype-matched immunoglobulin antibodies (Table 1 ) served as negative controls. The isolated cells were incubated with the respective antibodies for 30 min on ice. Dead-cell discrimination was performed using 7-aminoactinomycin D (7-AAD, Sigma, Steinheim, Germany). Flow cytometric analysis was conducted on a FACSLyric flow cytometer (BD Bioscience), and data were analyzed using FlowJo software v10 (FlowJo LLC, Ashland, OR). Table 1 Flow cytometry antibodies Antibody Label Product Company Dilution CD11b (rat anti-mouse) Alexa Fluor® 700 56-0112-82 Thermo Fisher Scientific, Inc. 1:200 CD19 (rat anti-mouse) PE 12-0193-81 Thermo Fisher Scientific, Inc. 1:200 CD3e (rat anti-mouse) PE-Cyanine7 25-0031-82 Thermo Fisher Scientific, Inc. 1:200 CD4 (rat anti-mouse) APC-e-Fluor® 780 47-0042-82 Thermo Fisher Scientific, Inc. 1:200 CD8a (rat anti-mouse) APC 17-0081-81 Thermo Fisher Scientific, Inc. 1:200 F4/80 (rat anti-mouse) FITC 11-4701-82 Thermo Fisher Scientific, Inc. 1:50 Ly6G (rat anti-mouse) V450 11-4801-82 BD Bioscience 1:200 IgG Isotype (Armenian hamster) PE-Cyanine7 25-4888-81 Thermo Fisher Scientific, Inc. 1:200 IgG2a K Isotype (rat) Alexa Fluor® 700 IC006N R&D Systems, Inc. 1:50 IgG2a K Isotype (rat) APC 17-4321-81 Thermo Fisher Scientific, Inc. 1:200 IgG2a K Isotype (rat) PE 12-4321-81 Thermo Fisher Scientific, Inc. 1:200 IgG2a K Isotype (rat) FITC 11-4321-42 Thermo Fisher Scientific, Inc. 1:100 IgG2b K Isotype (rat) APC-eFluor® 780 47-4031-82 Thermo Fisher Scientific, Inc. 1:200 IgG2a K Isotype (rat) V450 560377 BD Bioscience 1:200 Magnetic-activated cell sorting (MACS) of bone marrow neutrophils Following bone marrow flushing from long bones of intact mice, red blood cells were lysed using 5 mL erythrocyte lysis buffer (5 min, 37°C). The suspension was subsequently centrifuged at 1500 rpm for 5 min at 4°C. After removing the supernatant, the cell pellet was resuspended in 200 µL MACS buffer. The suspension was filtered through a 30-µm cell strainer with additionally 400 µL MACS buffer, and the total cell number was determined. 5 × 10⁷ cells were centrifuged at 300 × g for 10 min at 4°C, and the supernatant was discarded. For negative selection of neutrophils, 50 µL of a neutrophil-specific biotin antibody cocktail was added to the cell pellet, mixed, and incubated for 10 minutes at 2–8°C. This antibody cocktail binds to all non-neutrophilic cells in the suspension, leaving neutrophils untouched. After washing with 5–10 mL MACS buffer, cells were centrifuged (300 × g, 10 min), the supernatant was aspirated, and the pellet was resuspended in 400 µL MACS buffer. Next, 100 µL of anti-biotin MicroBeads were added, mixed, and incubated for 15 min at 2–8°C, allowing them to bind to the biotinylated antibodies. After an additional wash with 5–10 mL MACS buffer and a final centrifugation (300 × g, 10 min), the supernatant was discarded, and the pellet was resuspended in 500 µL MACS buffer. For magnetic cell separation, the cell suspension was applied to an LS-MACS column pre-rinsed with 3 mL MACS buffer and placed in a QuadroMACS separator. This step allowed the separation of neutrophils from other labeled cells, which were retained by the column, while neutrophils passed through and were collected for further analysis. RNAseq analysis of isolated neutrophils Neutrophils were isolated from the long bones of three Cre - and three Cre + mice by MACS as described above. For each mouse, the isolated neutrophils were split into two wells. One well was stimulated for 3 h with 10 − 5 M norepinephrine at 37°C, while the other well served as an unstimulated control. For RNA isolation using the RNeasy Mini Kit (Qiagen), neutrophils were resuspended in 400 µL RPMI medium supplemented with 10% (v/v) FCS after two rounds of centrifugation (300 × g, 10 min) and washing in PBS. Cell numbers were determined prior to stimulation. After 3 h stimulation period, cells were centrifuged (300 × g, 10 min), washed with 200 µL PBS, and centrifuged again (300 × g, 10 min). The supernatant was removed, and the final cell pellet of each subgroup was resuspended in 350 µL RLT buffer for RNA isolation. Lysates were stored at − 80°C until RNA sequencing. RNAseq was performed by the Novogene Corporation (Munich, Germany). The workflow began with sample quality control (Sample QC) to ensure that the samples met the RNAseq criteria, including RNA quantity and RNA integrity number (RIN). Following this, the appropriate RNA library was prepared and tested for quality (Library QC). The RNA library was constructed through polyadenylated (polyA) capture or ribosomal RNA (rRNA) removal, followed by reverse transcription to cDNA. Sequencing was conducted using Illumina PE150 technology (Illumina, San Diego, CA, USA) with a paired-end 150-bp sequencing strategy. The resulting data were subjected to quality control (Data QC). Gene expression differences with a p-value of less than 0.05 between the groups were considered differentially regulated. Bioinformatic analyses were carried out on the differentially regulated genes, including Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. ClusterProfiler software was used for enrichment analysis. GO ( http://www.geneontology.org/ ) is a widely used bioinformatics classification system that categorizes gene properties across species into three main branches: cellular component, molecular function, and biological process. GO terms with an adjusted probability (padj) < 0.05 were considered significantly enriched. KEGG is a curated database containing genomic, biological pathway, and disease information. Pathway enrichment analysis identifies significantly enriched metabolic or signaling pathways associated with differentially expressed genes, comparing them to the entire genomic background. KEGG pathways with padj < 0.05 were considered significantly enriched. Statistics Statistical analyses were conducted using unpaired t-test and Two-way analysis of variance (ANOVA) followed by Šidák’s post hoc test. A significance level of p < 0.05 was applied. Data in figures and tables are depicted as bars with mean ± standard deviation. Each experimental group consisted of 3–8 animals, with exact group sizes provided in the figure legends. Results First, the immune and bone phenotype of 12-week-old male Ly6G-Cre/Adrb2-flox mice was assessed to analyze differences under non-fracture conditions upon KO (Fig. 1 ). Immune cell analysis showed no differences in CD19 + , CD11b + /Ly6G + , CD11b + /F4/80 + , CD3 + /CD4 + , and CD3 + /CD8 + cells in spleen and bone marrow in Adrb2 KO mice compared to control mice (Table 2 ). Bone phenotyping revealed that the bone parameters femur length (Fig. 2 , a), maximal load to failure (Fig. 2 , b), bending stiffness (Fig. 2 , c), trabecular bone volume fraction (BV/TV)(Fig. 2 , g), trabecular number (Tb.N)(Fig. 2 , h), trabecular thickness (Tb.Th)(Fig. 2 , i), trabecular separation (Tb.Sp)(Fig. 2 , j), osteoblast activity (Supp. Figure 1 , a), and osteoclast activity (Supp. Figure 1 , b) were unaffected. In addition, assessment of the lumbar spine showed no differences in BV/TV, Tb.N, Tb.Th and Tb.Sp (Supp. Figure 2 ). Cre + male mice showed a slightly increased trabecular tissue mineral density (TMD) in the femur (Fig. 2 , e) and increased cortical thickness (C.Th) (Fig. 2 , d) with increased numbers of osteocytes in the cortex (Supp. Figure 1 , c) compared to Cre − littermates. Table 2 Immune cell phenotyping using flow cytometry. Percentage of CD19 + , CD11b + /Ly6G + , CD11b + /F4/80 + , CD3 + /CD4 + , and CD3 + /CD8 + cells. Statistical significance was determined by unpaired t-test (comparison Cre − vs. Cre + ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ( N = 6; males) Spleen Bone marrow Cell type/ Genotype CD19 + CD11b + / Ly6G + CD11b + / F4/80 + CD3 + / CD4 + CD3 + / CD8 + CD19 + CD11b + / Ly6G + CD11b + / F4/80 + CD3 + / CD4 + CD3 + / CD8 + Cre − 50.9 ± 4.6 2.6 ± 0.8 6.5 ± 1.4 16.8 ± 3.0 20.7 ± 5.3 17.6 ± 2.7 35.4 ± 8.1 12.1 ± 3.2 0.9 ± 0.2 1.1 ± 0.2 Cre + 50.1 ± 3.5 3.4 ± 1.4 4.8 ± 1.4 15.4 ± 2.5 18.5 ± 3.9 14.2 ± 3.7 32.1 ± 6.5 11.4 ± 3.0 1.0 ± 0.4 1.1 ± 0.4 To investigate the role of Adrb2-signaling on neutrophils during fracture healing in non-osteoporotic and osteoporotic bone, 12-week-old female mice underwent OVX to induce an osteoporotic bone phenotype driven by estrogen depletion. The mice were randomly distributed to the different groups and did not display differences in the body weight 21 days after fracture surgery (Fig. 3 , a). Successful OVX was confirmed by a reduced uterus weight in OVX mice (Fig. 3 , b). Furthermore, µCT analysis of the contralateral unfractured femur revealed a significant reduction of BV/TV (Fig. 3 , c), and Tb.N (Fig. 3 , e) while Tb.Sp (Fig. 3 , g) was increased in both Cre − and Cre + OVX mice compared to the respective sham group (Fig. 3 , d). This indicates that OVX induced an osteoporotic bone phenotype irrespective of the genotype. In addition, we observed that Cre + female mice display no bone phenotype as demonstrated by absence of differences in BV/TV, Tb.N, Tb.Th and Tb.Sp of the unfractured femur compared to Cre − mice (Fig. 3 , c, e-g). Fracture healing outcome 21 days after surgery was investigated using µCT analysis and histomorphometry. 3D µCT analysis depicted significantly reduced BV/TV and bone volume (BV) in OVX mice compared to sham mice while tissue volume (TV) was unaffected (Fig. 4 , a - c), irrespective of genotype, indicating delayed healing due to OVX in both groups. More strikingly, BV/TV and BV were significantly decreased in both Cre + groups compared to their respective Cre − group (Fig. 4 , a, b), indicating delayed healing due to the Adrb2-KO. Representative 3D reconstruction images of the fracture calli are depicted in Fig. 4 , d. In addition, two-dimensional histomorphometry revealed a significant decreased bone and increased connective tissue content in the fracture gap of sham Cre + mice compared to sham Cre − mice supporting the µCT data (Fig. 4 , e - g). To further elucidate the role of Adrb2 deletion in neutrophils and investigate underlying molecular mechanisms of the delayed healing in Cre + mice, RNA-Sequencing of isolated neutrophils from the bone marrow of Cre − and Cre + male mice was conducted. Half of the cells were stimulated with 10 –5 M norepinephrine (Fig. 5 , a) to induce the Adrb2 pathway. Unstimulated neutrophils revealed 48 upregulated and 86 downregulated genes upon Adrb2 KO while an additional noradrenaline stimulation resulted in 148 upregulated and 135 downregulated genes (Suppl. Figure 3). A list of all significantly regulated genes for unstimulated and stimulated neutrophils is provided in the supplements. Within those, the most interesting genes were Janus kinase 3 (Jak3) , Elastase (Elane) , the Adrenergic receptor beta 2 (Adrb2) , Chemokine (C-C motif) receptor 10 (Ccr10) , Gata2 (Gata2) , Fc receptor (Fcer1a) (Fig. 5 , b). Jak3 , Elane , Adrb2 and Ccr10 were significantly downregulated in unstimulated Cre + neutrophils (Fig. 5 , b) while Gata2 , Fcer1a and Adrb2 showed significantly downregulated gene expressions in stimulated Cre + neutrophils (Fig. 5 , b). GO enrichment analysis of unstimulated neutrophils indicated alteration in neutrophil cellular functions upon KO (Fig. 5 , c), while GO enrichment analysis of stimulated neutrophils display several mast cell related terms (Fig. 5 , d). The significantly downregulated pathways “(positive) regulation of mast cell activation” in stimulated Cre + neutrophils could imply a potentially affected neutrophil/mast cell interaction upon neutrophil specific Adrb2 KO (Fig. 5 , d). Because of the finding that Adrb2 deletion on neutrophils might affect neutrophil and mast cell recruitment/activation, we analyzed mast cell numbers in the fracture callus of sham and OVX female mice 21-days post-fracture (Fig. 6 , a). The analysis revealed no significant differences between sham and OVX mice of both genotypes (Fig. 6 , a). However, female Cre + OVX mice showed a significantly reduced percentage of mast cells compared to Cre − OVX mice (Fig. 6 , a). This is in line with significantly reduced mast cell numbers in the intact femur of Cre + male mice (Fig. 6 , b). To further investigate the effect of Adrb2 deletion on neutrophils, we performed a neutrophil (Ly6G) staining in the fracture callus 3 days post-fracture where we observed a strong reduction of neutrophil recruitment in Cre + mice compared to Cre − littermates (Fig. 6 , c, d). In addition, we performed a double staining of neutrophils (Ly6G) and mast cells (Avidin) to investigate the recently described mechanism of MIT formation, where mast cells engulf neutrophils, during fracture healing. And indeed, we could show neutrophils trapped intracellularly in mast cells in the early fracture hematoma (Fig. 6 , e). Taken together, these data indicate that neutrophil-specific deletion of the Adrb2 may impair neutrophil recruitment and reduce mast cell numbers by a so far unknown mechanism. Discussion The aim of this study was to investigate the effect of a neutrophil specific Adrb2 deletion on bone homeostasis and fracture healing in non-osteoporotic and osteoporotic bone. Our hypothesis was that the deletion of the Adrb2 accelerates fracture healing specifically under osteoporotic conditions due to reduced neutrophil recruitment. In contrast to this initial hypothesis, we could show that Adrb2-KO on neutrophils had a minor positive effect on intact bone, while fracture healing was significantly delayed in both non-osteoporotic and osteoporotic conditions. A conditional KO of the Adrb2 on Ly6G expressing cells was generated using the Cre/loxP system which results in reduced Adrb2 expression on neutrophils confirmed by our RNASeq data. First, the immune and bone phenotype of Cre − and Cre + male littermates was assessed. Male mice were chosen to address 3R principles, as we used their female littermates for the fracture healing study. Nevertheless, the bone phenotype of female Cre − and Cre + mice was also analyzed in this study using the contralateral intact left femur. Flow cytometric analysis of male mice revealed no significant differences in immune cell populations following Adrb2 knockout (KO). In contrast, a minor bone phenotype was observed, characterized by increased cortical thickness, elevated osteocyte numbers, and higher trabecular TMD. Although these alterations were modest, they suggest that neutrophil-specific deletion of Adrb2 may exert subtle positive effects on bone homeostasis under non-fracture conditions in male mice. One possible explanation is that altered neutrophil signaling indirectly affects osteocyte function or bone remodeling dynamics through changes in local paracrine communication, inflammatory mediator release, or coupling signals between bone-resorbing and bone-forming cells. However, as the underlying mechanisms were not directly investigated in our study, further targeted analyses will be required to fully elucidate the pathways responsible for these bone-specific alterations in Cre + male mice. In contrast, female Cre + mice with a femur facture did not display a bone phenotype upon neutrophil-specific Adrb2 KO in their contralateral non-fractured femur. It has been shown previously that a fracture does also affect the rest of the skeleton, which might explain the differences between male and female bone phenotype in our study [ 33 ]. Next, we investigated fracture healing, beginning with the induction of postmenopausal osteoporosis at 12 weeks of age by ovariectomy, followed by a standardized femoral osteotomy stabilized with an external fixator at 16 weeks of age. Analysis of uterine weight confirmed the expected uterine atrophy in both Cre⁻ and Cre⁺ ovariectomized (OVX) mice. Consistently, µCT analysis of the unfractured femur demonstrated the characteristic decrease in bone volume fraction (BV/TV) in OVX mice, independent of genotype. These findings indicate that neutrophil-specific deletion of Adrb2 does not protect mice from the development of osteoporosis. Although osteoporosis has been associated with an increased sympathetic tone [ 34 ], our data suggest that adrenergic signaling in neutrophils does not contribute to osteoporosis development. In contrast, in osteoblasts, β2-adrenergic signaling is known to play a critical role in the pathogenesis of osteoporosis [ 26 ]. µCT analysis of the fracture calli at 21 days post-fracture revealed significantly reduced bone volume fraction (BV/TV) and bone volume (BV) in OVX mice compared with sham-operated mice of both genotypes, indicating impaired fracture healing in Cre⁻ and Cre⁺ osteoporotic mice. These findings suggest that neutrophil-specific Adrb2 knockout does not rescue delayed fracture healing in osteoporotic mice. In contrast, Cre⁺ sham and OVX mice exhibited significantly poorer fracture healing than their respective Cre⁻ controls, indicating that neutrophil-specific deletion of Adrb2 impairs fracture healing under both physiological and osteoporotic conditions. These findings are contradictory to our initial hypothesis and indicate that Adrb2 signaling specifically in neutrophils is of great importance for successful fracture healing. In line with our findings, a recent study by Jahn et al. has demonstrated that treatment with propranolol, an unspecific beta adrenergic blocker, impaired fracture healing in mice [ 35 ], although previous studies observed opposite results [ 20 , 34 , 36 ]. The different results of these studies may depend on different study designs and may indicate a time and/or concentration dependent influence of drug treatment. To further elucidate our findings, we performed a more detailed characterization of neutrophils using an in vitro culture approach followed by RNA sequencing. Neutrophils were isolated from the bone marrow of Cre⁻ and Cre⁺ mice and either stimulated with noradrenaline or cultured in medium alone as a control. RNA sequencing identified several differentially expressed genes in both unstimulated and noradrenaline-stimulated Cre⁺ neutrophils compared with Cre⁻ neutrophils, including Jak3 , Elane , Adrb2 , Ccr10 , Gata2 , and Fcer1a . Jak3 encodes a non-receptor tyrosine kinase that plays a critical role in neutrophil activation in response to interleukin-8 (IL-8) during inflammatory reactions [ 37 ]. Moreover, Jak3 has been shown to be essential for neutrophil chemotaxis [ 37 , 38 ]. This indicates that neutrophil chemotaxis might be affected upon Adrb 2 KO. Ccr10 is known as an important receptor for the immune response and plays a role in the recruitment of immune cells but less is known about its function in neutrophils [ 39 ]. In addition, Elane encodes for neutrophil expressed elastase which is known as an important factor of promoting inflammation e.g. in various lung diseases [ 40 ]. Other downregulated candidate genes were Fcer1a and Gata2 . Fcer1a encodes for a high affinity IgE receptor playing a central role in allergic diseases and is mainly expressed on mast cells but in a smaller manner also on neutrophils [ 40 , 41 ]. Gata2 is an important transcription factor regulating Fcer1a expression which means that the downregulation of Gata2 results in a downregulation of Fcer1a expression [ 42 ]. GO enrichment analysis of noradrenaline-stimulated neutrophils further indicates the possibility of a disturbed neutrophil/mast cell interaction since the GO term “(positive) regulation of mast cell activation” was significantly downregulated upon Ly6G-Adrb2-KO. Elastase could be involved in this regulation, as it is known to recruit mast cells. In general, mast cells derive from hematopoietic stem cells of the bone marrow and are mainly known for their function in IgE mediated allergies and tissue repair [ 43 , 44 ]. But they are also associated with age-related and postmenopausal osteoporosis as indicated by increased mast cell numbers and the ability to regulate osteoclastic activity [ 45 – 47 ]. Using mast cell deficient mice, we demonstrated previously that mast cells activate osteoclastic bone resorption in postmenopausal osteoporosis and are critically involved in osteoporotic fracture healing [ 48 – 50 ]. Furthermore, severely injured mice showed increased mast cell numbers in the fracture hematoma while mast cell–depleted mice were protected against severe injury–induced impairment of fracture healing [ 51 ]. In summary, these data demonstrate that increased mast cell numbers under conditions of hyperinflammation can be detrimental to bone regeneration. Based on these observations, we decided to further investigate mast cell populations in our mouse model. Immunohistochemical staining was performed to quantify mast cells in the intact femur of male mice and in the fracture callus of female mice. Our data revealed that, Cre⁺ male mice exhibited significantly reduced mast cell numbers in the intact bone. In the fracture callus, this reduction of mast cells was also observed in Cre⁺ osteoporotic mice which displayed significantly fewer mast cells in the fracture callus compared to Cre⁻ osteoporotic controls. Despite the reduction of mast cell numbers, Cre⁺ mice still exhibited delayed fracture healing, which is challenging to reconcile since in previous fracture healing studies in models of hyperinflammation (e.g. osteoporosis) mast cell deficiency resulted in improved fracture healing. Currently, we have no proven explanation for our findings. One possible explanation could be that the neutrophil–mast cell crosstalk is disrupted in this model and that a balanced neutrophil–mast cell crosstalk is important for fracture healing. Previous studies have shown that neutrophils recruit mast cells to sites of inflammation through the secretion of chemokines such as CXCL1 and CXCL12 [ 52 ]. An altered chemokine expression or signaling in Adrb2-deficient neutrophils could therefore impair mast cell recruitment and function, leading to a dysregulated inflammatory response and ultimately compromises fracture healing. To further understand this neutrophil/mast cell interaction we stained neutrophils in the fracture callus 3 days post-fracture where we observed a drastically reduced neutrophil recruitment in Cre + mice. This implies that Ly6G-Adrb2 KO mice exhibit disturbed neutrophil activation and an impaired neutrophil/mast cell crosstalk, resulting in reduced numbers of both cell types in the fracture callus. As previously described by Kovtun et al. , neutrophils play a crucial role in bone fracture healing [ 53 ]. Treatment with a Ly6G antibody led to impaired bone regeneration highlighting the importance of undisturbed neutrophil recruitment and function in the early inflammatory phase of fracture healing [ 53 ]. This imbalance of neutrophils and mast cells in our mouse model may explain the poor fracture healing outcome observed in Cre + mice, as a finely tuned activation and interaction has been shown to be essential for successful fracture repair. Despite their central role in innate immunity - mediating antimicrobial defense through phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs) - neutrophils also appear to exert important immunomodulatory functions during tissue repair [ 54 ]. In this context, Mihlan et al. recently described a process termed mast cell–induced neutrophil trapping (MIT), in which degranulating mast cells secrete leukotriene B4 to attract neutrophils and retain them within mast cells. The trapped neutrophils subsequently undergo cell death, while their components are retained by mast cells, thereby enhancing mast cell metabolic fitness and functional capacity [ 55 ]. Notably, we were able to detect MIT formation in the fracture callus at 3 days post-fracture using immunofluorescent double staining for neutrophils and mast cells. Nevertheless, this observation alone does not explain the impaired fracture healing observed in Ly6G-Adrb2 KO mice. One plausible explanation could be that reduced neutrophil activation and recruitment in these mice limits the initiation and regulation of early inflammatory signaling cascades required for effective mast cell activation, angiogenesis, and subsequent tissue remodeling. Consequently, insufficient early inflammatory signaling may lead to a failure to properly transition from the inflammatory to the reparative phase of fracture healing. In conclusion, our findings highlight the importance of a tightly regulated neutrophil–mast cell interaction during bone regeneration and suggest that both insufficient and excessive immune cell activity can be detrimental to fracture healing. Adrb2 signaling on neutrophils seems to be important in that context. Further studies are clearly required to dissect the molecular mechanisms underlying this bidirectional crosstalk and to determine how its dysregulation contributes to impaired bone repair in osteoporotic conditions. Limitations of our study are that our Ly6G-Cre model affects not only neutrophils but also other myeloid derived cells expressing Ly6G, e.g. some types of monocytes or myeloid-derived suppressor cells. Furthermore, investigating earlier time points after fracture, neutrophil functions and other involved immune cells, e.g. T- and B-cells, to further characterize the neutrophil/mast cell crosstalk during bone healing would be needed for future studies. Conclusion In summary, our data show that Adrb2-signaling on neutrophils is not relevant for the development of postmenopausal osteoporosis, while it seems to be critical for fracture healing in both non-osteoporotic and osteoporotic mice. This could be due to an impaired activation of neutrophils and a disturbed interaction of neutrophils and mast cells upon Ly6G-Adrb2-KO which needs to be further investigated. Declarations Author contributions S.D. and N.G. contributed equally. S.D., N.G and M.H.-L. wrote the manuscript text and prepared the figures. Data collection and analysis was performed by S.D. and N.G. Data interpretation was done by S.D., N.G., C.K., D.G., J.B., O.K., V.F., A.I. and M.H.-L. Surgery assistance: C.K., D.G., J.B. and O.K. Project supervision and funding was done by M.H.-L. All authors reviewed the manuscript. Conflict of interest The authors declare no conflict of interest. Funding This study was funded by the German Research Foundation (DFG, grant number HA 8470/2-1). Acknowledgements We would like to thank Tina Vogel, Dr. Justyna Pawlak-Wurster, Iris Baum, Andrea Böhmler and Sandra Richter for their excellent work as technicians. In addition, we would like to thank the animal keepers Ricco Richter, Jasmin Jucha and Herrmann Klassen at the TFZ in Ulm for taking care of the animals. References Takayanagi, H., Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems . Nat Rev Immunol, 2007. 7(4): p. 292–304. 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Supplementary Files Supplemental.docx KOCTLvsWTCTLdegall.xls KONAvsWTNAdegall.xls Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Feb, 2026 Reviews received at journal 18 Feb, 2026 Reviews received at journal 17 Feb, 2026 Reviews received at journal 16 Feb, 2026 Reviews received at journal 16 Feb, 2026 Reviewers agreed at journal 09 Feb, 2026 Reviewers agreed at journal 09 Feb, 2026 Reviewers agreed at journal 09 Feb, 2026 Reviewers agreed at journal 06 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviewers agreed at journal 04 Feb, 2026 Reviewers agreed at journal 04 Feb, 2026 Reviewers invited by journal 21 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 13 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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01:29:00","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135332,"visible":true,"origin":"","legend":"","description":"","filename":"44d54ad9a05741b9889dee618e9aeb421structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/5be08675c4390d5d1adda79d.xml"},{"id":101021148,"identity":"b19295ed-5447-43b2-83ed-d2ebd1565253","added_by":"auto","created_at":"2026-01-24 01:29:00","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147306,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/efa9a770855d90b8d3b66c70.html"},{"id":101296566,"identity":"90f5e577-e8e6-4d99-b3ac-9ae5f02692b4","added_by":"auto","created_at":"2026-01-28 09:15:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":415992,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental setup of the phentoyping procedure using male mice (♂︎) and the fracture healing study using female mice (♀). Created in BioRender. Ohmayer, W. (2026) https://BioRender.com/p4jqfzh\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/52092e0e4c2624bf0f2211d1.png"},{"id":101021119,"identity":"17b4e89b-da0a-4f83-90d7-60158632c70e","added_by":"auto","created_at":"2026-01-24 01:28:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":313978,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBone phenotyping of 12 week old male Ly6G-Cre/Adrb2-flox KO mice.\u003c/strong\u003e Following parameters were evaulated: \u003cstrong\u003ea)\u003c/strong\u003e Femur length in mm. \u003cstrong\u003eb)\u003c/strong\u003e \u0026nbsp;Fmax in N. \u003cstrong\u003ec)\u003c/strong\u003e EI. \u003cstrong\u003ed)\u003c/strong\u003e cortical TMD in mgHA/ccm. \u003cstrong\u003ee) \u003c/strong\u003ecortical thickness (C.Th). \u003cstrong\u003ef)\u003c/strong\u003e trabecular TMD in mgHA/ccm. \u003cstrong\u003eg)\u003c/strong\u003e femoral trabecular BV/TV in %. \u003cstrong\u003eh)\u003c/strong\u003e femoral trabecular number in 1/mm. \u003cstrong\u003ei)\u003c/strong\u003e femoral trabecular thickness in mm. \u003cstrong\u003ej)\u003c/strong\u003e\u0026nbsp;femoral trabecular separation in mm. Statistical significance was determined by unpaired t-test (comparison Cre\u003csup\u003e-\u003c/sup\u003e vs. Cre\u003csup\u003e+\u003c/sup\u003e). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. (\u003cem\u003eN\u003c/em\u003e=6-8; males)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/7532c0b17716711e09224343.png"},{"id":101204631,"identity":"bd714def-cd91-4f8e-8839-ebee4c323f18","added_by":"auto","created_at":"2026-01-27 09:43:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of body weight, uterus weight and trabecular bone parameters of the contralateral unfractured femur 21 days after fracture surgery.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e Mouse body weight in g. \u003cstrong\u003eb)\u003c/strong\u003e Uterus weight in g; \u0026nbsp;\u003cstrong\u003ec)\u003c/strong\u003e BV/TV in %. \u003cstrong\u003ed)\u003c/strong\u003e 3D-reconstruction images of the trabecular bone. \u003cstrong\u003ee)\u003c/strong\u003eTb.N in 1/mm. \u003cstrong\u003ef)\u003c/strong\u003e Tb.Th in mm. \u003cstrong\u003eg)\u003c/strong\u003e Tb.Sp in mm. Statistical significance was determined by Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. (\u003cem\u003eN\u003c/em\u003e=5-8; females)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/f5e565253e18ed6b4d3b3b06.png"},{"id":101203956,"identity":"63f4bb1e-0f03-49a7-a273-c0859a3dd8c5","added_by":"auto","created_at":"2026-01-27 09:41:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":310209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eµCT analysis, and histomorphometry at day 21 after fracture surgerym\u003c/strong\u003e. \u003cstrong\u003ea) \u003c/strong\u003eCallus bone volume per tissue volume (BV/TV) in %. \u003cstrong\u003eb)\u003c/strong\u003e Callus bone volume (BV). \u003cstrong\u003ec)\u003c/strong\u003eCallus tissue volume (TV). \u003cstrong\u003ed)\u003c/strong\u003e 3D-reconstruction images of the fracture callus from representative mice of each group. yellow = mature bone; red = newly formed bone. \u003cstrong\u003ee, f and g) \u003c/strong\u003eHistomorphometry; Percentage of bone (e), cartilage (f) and connective tissue (g) in the newly formed callus gap. Statistical significance was determined by Two-way ANOVA. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. (\u003cem\u003eN\u003c/em\u003e=6-8; females)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/09c5d500ad21e5ae300f22b9.png"},{"id":101204241,"identity":"3517c45f-4762-418b-8381-853eec4d1626","added_by":"auto","created_at":"2026-01-27 09:42:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":743234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophil isolation via Magnetic Activated Cell Sorting (MACS), stimulation with norepinephrine and RNA-Sequencing analysis.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e Experimental design. \u003cstrong\u003eb)\u003c/strong\u003e Differentially expressed genes in the comparison of unstimulated and stimulated Cre- and Cre+ neutrophils. \u003cstrong\u003ec) \u003c/strong\u003eGO enrichment analysis from significantly downregulated genes in unstimulated Cre\u003csup\u003e-\u003c/sup\u003e vs. Cre\u003csup\u003e+\u003c/sup\u003e neutrophils. \u003cstrong\u003ed)\u003c/strong\u003e GO enrichment analysis from significantly downregulated genes in stimulated Cre\u003csup\u003e-\u003c/sup\u003e vs. Cre\u003csup\u003e+\u003c/sup\u003e neutrophils. \u003cstrong\u003ec)\u003c/strong\u003e and \u003cstrong\u003ed):\u003c/strong\u003e The abscissa is the ratio of the number of differential genes linked with the GO pathway to the total number of differential genes. The ordinate is GO Pathway. The size of a point represents the number of genes annotated to a specific GO pathway. The color scale represents the significant level of the enrichment. (\u003cem\u003eN\u003c/em\u003e=3; males)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/a2527db00b4a282004b78a92.png"},{"id":101204582,"identity":"760bee6f-4487-4a3b-a495-816afaa090a6","added_by":"auto","created_at":"2026-01-27 09:43:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":276373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry staining’s of neutrophils and mast cells.\u003c/strong\u003e \u003cstrong\u003ea) \u003c/strong\u003eMast cell ratio (mast cell numbers/bone area) in the fracture callus of female mice 21 days post-fracture. \u003cstrong\u003eb)\u003c/strong\u003e Mast cell numbers in the intact femur of male mice; grey = Cre\u003csup\u003e-\u003c/sup\u003e; blue = Cre\u003csup\u003e+\u003c/sup\u003e. \u003cstrong\u003ec)\u003c/strong\u003e Neutrophils per callus area in the fracture callus of female mice 3 days post-fracture\u003cstrong\u003e. d)\u003c/strong\u003e Representative images of the neutrophil (Ly6G) staining in the fracture callus of female mice 3 days post-fracture. \u003cstrong\u003ee)\u003c/strong\u003e + = Doublestaining of neutrophils (Ly6G; green) and mast cells (Avidin; red) showing MIT formation (highlighted with an arrow) in the fracture callus of female mice 3 days post-fracture. Nuclei are stained in blue with Hoechst. - = negative control with species-specific, non-targeting immunoglobulins. Statistical significance was determined by unpaired t-test (comparison Cre\u003csup\u003e-\u003c/sup\u003e vs. Cre\u003csup\u003e+\u003c/sup\u003e)(C) and Two-way ANOVA (A + D). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P\u0026nbsp;\u0026lt;\u0026nbsp;0.001, ****P \u0026lt; 0.0001. (\u003cem\u003eN\u003c/em\u003e=4-8)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/e671ddac873422928994a4c6.png"},{"id":101398831,"identity":"5a744e28-08e1-4eea-b188-5f2cb7558a9e","added_by":"auto","created_at":"2026-01-29 09:48:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3500812,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/d9ced1bf-39dc-4217-9ac8-bf83406743ee.pdf"},{"id":101397662,"identity":"fc4cedc2-8567-4bfd-9df9-36cc4a9c1cfb","added_by":"auto","created_at":"2026-01-29 09:34:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":326624,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemental.docx","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/d7ea2bedbdc287d23df36588.docx"},{"id":101204136,"identity":"008c3515-b00e-46a5-809a-047ad1df03b3","added_by":"auto","created_at":"2026-01-27 09:41:43","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":58079,"visible":true,"origin":"","legend":"","description":"","filename":"KOCTLvsWTCTLdegall.xls","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/3c3d4c2834e085eaf31cef45.xls"},{"id":101204487,"identity":"4aba6f53-9dea-460a-9d4d-1e27358a0af5","added_by":"auto","created_at":"2026-01-27 09:43:20","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":126516,"visible":true,"origin":"","legend":"","description":"","filename":"KONAvsWTNAdegall.xls","url":"https://assets-eu.researchsquare.com/files/rs-8593165/v1/85212d11a0b084bc998f92e0.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impaired fracture healing upon neutrophil-specific adrenoreceptor beta 2 knockout in non-osteoporotic and osteoporotic mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIt is well established that the skeletal and the immune system are closely interconnected in many aspects, a concept that has given rise to the interdisciplinary field of osteoimmunology. Immune cells such as T cells, macrophages, and neutrophil granulocytes critically influence bone metabolism through the secretion of mediators such as Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL), tumor necrosis factor-alpha (TNF-α), or Interleukin-6 (IL-6), thereby modulating both osteoclastic bone resorption and osteoblastic bone formation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. One clinical example of a disrupted osteoimmunological balance is postmenopausal osteoporosis, which is characterized by an imbalance in bone metabolism, with increased activity of bone-resorbing osteoclasts relative to bone-forming osteoblasts. This imbalance is driven not only by the direct positive effects of estrogen on bone formation and its inhibitory effects on bone resorption, but also by the fact that estrogen deficiency after menopause induces a chronic low-grade inflammatory state [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which further contributes to impaired bone homeostasis. As a result, osteoporotic bone exhibits an altered microarchitecture that increases fracture risk [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEach year, more than 3.5\u0026nbsp;million new osteoporotic fractures occur in Europe, and 10\u0026ndash;15% of patients die within the first year after suffering from an osteoporotic hip fracture [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition to the increased fracture risk, osteoporotic bone displays a reduced capacity for fracture healing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], as demonstrated in numerous animal studies using ovariectomized, estrogen-deficient rodents. These animals exhibit a mechanically weaker fracture callus with increased osteoclast numbers [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], reduced expression of cartilage markers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and impaired expression of angiogenic factors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, we previously demonstrated that ovariectomized mice exhibit elevated systemic levels of proinflammatory cytokines and increased numbers of neutrophil granulocytes in the fracture hematoma three days after surgery, which was associated with delayed fracture healing [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the mechanisms underlying the enhanced recruitment and/or activity of neutrophils in osteoporosis and osteoporotic fracture healing remain largely unclear.\u003c/p\u003e \u003cp\u003eEmerging evidence suggests a role for adrenergic signaling in this process, as neutrophil granulocytes are strongly influenced by catecholamines and adrenergic stimulation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Catecholamines play a pivotal physiological role, as they are released in response to external and internal stressors via activation of the hypothalamic-pituitary-adrenal (HPA) axis. By binding to distinct adrenergic receptor subtypes expressed across multiple organs and tissues, catecholamines regulate essential physiological functions, including cardiovascular activity, pupillary diameter, and autonomic control of gastrointestinal processes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Bone is also subject to sympathetic regulation, which influences both bone formation and turnover. For example, treatment of stressed male mice with the non-selective β-adrenergic receptor antagonist propranolol immediately before fracture significantly reduced the number of neutrophil granulocytes in the fracture hematoma and improved fracture healing [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this context, it is important to note that neutrophil granulocytes, similar to osteoblasts and osteoclasts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], express the β\u003csub\u003e2\u003c/sub\u003e-adrenoreceptor (Adrb2) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This receptor is thought to play a key role in adrenergic regulation of bone metabolism, as previous studies demonstrated that both global [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and osteoblast-specific [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] Adrb2 knockout (KO) led to increased bone volume in mice. However, the role of Adrb2 signaling in immune cells in the context of bone metabolism and fracture healing remains unknown.\u003c/p\u003e \u003cp\u003eTherefore, the aim of this study was to elucidate the role of Adrb2 in Ly6G⁺ neutrophil granulocytes during fracture healing in both non-osteoporotic and osteoporotic mice. We hypothesized that catecholamine release following fracture activates Adrb2 signaling in neutrophils, thereby promoting their enhanced recruitment to the fracture hematoma and contributing to impaired bone regeneration in osteoporotic bone. To test this hypothesis, mice with a neutrophil-specific deletion of Adrb2 were generated. Bone metabolism upon KO was analyzed in both sexes while fracture healing was investigated in female mice after ovariectomy, a model of postmenopausal osteoporosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal housing and transgenic mouse model\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in compliance with the European Guidelines for Animal Research on the Protection of Animals and the ARRIVE guidelines and were approved by the local animal welfare authority (Regierungspr\u0026auml;sidium T\u0026uuml;bingen, No. 1612). Mice were housed in groups of up to five per cage under a 12-hour light/ 12-hour dark cycle with ad libitum access to food and water. The study utilized transgenic mice with a C57BL/6J background carrying a neutrophil-specific KO of the Adrb2 under control of the Cre/loxP system (Ly6G-Cre⁺ Adrb2\u003csup\u003eflox/flox\u003c/sup\u003e). This mouse line was established by breeding Ly6G-Cre mice [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] with Adrb2flox mice kindly donated by Gerard Karsenty [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Cre-negative littermates (Ly6G-Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e Adrb2\u003csup\u003eflox/flox\u003c/sup\u003e) were included as controls.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design and surgical procedures\u003c/h3\u003e\n\u003cp\u003eIn the first part of the study, male Cre\u003csup\u003e+\u003c/sup\u003e and Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e mice were euthanized by isoflurane overdose and terminal intracardiac blood withdrawal at the age of 12 weeks to analyze their bone and immune phenotype via \u0026micro;CT analysis, histology and flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Male littermate mice were used in accordance with the 3R principle of reduction, as the females were required for the fracture surgeries and the males could be included in the phenotyping study without generating additional breeding pairs. To assess the bone phenotype in female mice, the unfractured contralateral femur was analyzed via \u0026micro;CT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe second part of the study focused on fracture healing in female non-osteoporotic and osteoporotic mice. Twelve-week-old female mice were subjected to either a sham operation or bilateral ovariectomy (OVX) to induce postmenopausal osteoporosis as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Anesthesia was performed starting with 5\u0026ndash;6% isoflurane (Forene, Abbott) and maintained with 2% isoflurane (Forene, Abbott) with an oxygen flow rate of 0.7 mL/min. To ensure adequate analgesia, all mice received tramadol (25 mg/L, Tramal, Gruenenthal GmbH) in their drinking water from one day pre- until three days postoperatively. After surgery, normal housing diet (ssniff R/M-H, V1535-300, Ssniff GmbH) was changed to phytoestrogen-low diet (ssniff R/M-H, V1554-300, Ssniff GmbH). Four weeks post-surgery, a standardized, unilateral transverse osteotomy of the right femur was performed, which was stabilized using an external fixator as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Mice were euthanized by isoflurane overdose on day 3 and 21 post-fracture for subsequent analyses, including \u0026micro;CT analysis, histology, and IHC staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBiomechanical testing\u003c/h3\u003e\n\u003cp\u003eTo assess the mechanical properties of the bones, biomechanical testing was conducted on intact femurs from 12-week-old male mice. A destructive three-point bending test was performed following a previously established protocol [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Each bone was subjected to a load of up to 10 N using a materials testing machine (Zwick Roell, Ulm, Germany), while load and deflection were continuously recorded. For each bone the maximal load until failure (F\u003csub\u003emax\u003c/sub\u003e) was calculated and the flexural rigidity was determined from the slope of the linear region of the load-deflection curve.\u003c/p\u003e\n\u003ch3\u003eµCT analysis\u003c/h3\u003e\n\u003cp\u003eFractured and unfractured femurs were fixed in 4% paraformaldehyde for 48 h prior to micro-computed tomography (\u0026micro;CT) scanning. Imaging was performed using a Skyscan 1172 device (Skyscan, Aartselaar, Belgium) at a peak voltage of 50 kV and a current of 200 \u0026micro;A to assess bone content and mineralization. The isotropic voxel resolution was set to 8 \u0026micro;m. Three-dimensional analysis was conducted using computed tomography analysis (CTAn) and CT volume (CTVol) software (Bruker) in accordance with ASBMR guidelines [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Analyzing the fractured bones, the volume of interest (VOI) was defined as the entire periosteal callus region located between the two inner pinholes of the fixator. Unfractured bones were analyzed in two VOIs: VOI 1 encompassed a region extending 360 \u0026micro;m from the proximal end of the growth plate to 280 \u0026micro;m proximal to the distal end of the bone. VOI 2 covered the area from the proximal end of the trochanter tertius to 80 \u0026micro;m proximal to the distal end. Tissue mineral density was quantified using calibration phantoms containing defined hydroxyapatite (HA) concentrations of 250 mg HA/cm\u0026sup3; and 750 mg HA/cm\u0026sup3;. The threshold for mineralized tissue was set at 394 mg HA/cm\u003csup\u003e3\u003c/sup\u003e for trabecular bone and 642 mg HA/cm\u0026sup3; for cortical bone and fracture callus analysis.\u003c/p\u003e\n\u003ch3\u003eHistomorphometry\u003c/h3\u003e\n\u003cp\u003eDecalcified histological analysis of fractured and unfractured femurs was performed as previously described [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Sections of 4 \u0026micro;m thickness were stained with Safranin O/Fast Green to assess tissue composition and with tartrate-resistant alkaline phosphatase (TRAP) to identify osteoclasts. In the fractured bones, the proportions of bone, cartilage, and fibrous tissue within the fracture callus at day 21 post-fracture were quantified in Safranin O-stained sections using image analysis software (Leica MMAF 1.4.0 Imaging System; Leica, Wetzlar, Germany). The region of interest (ROI) of the fractured samples was defined as the entire fracture callus between the two inner pinholes of the fixator.\u003c/p\u003e \u003cp\u003eIn addition, osteoblasts and osteoclasts were quantified in sections of fractured and unfractured femora at 20\u0026times; magnification. In unfractured samples, the ROI (480 x 350 \u0026micro;m) for quantifying osteoblasts and osteoclasts was defined as an area located 960 \u0026micro;m proximal of the distal growth plate and positioned between both cortices in a trabecular-rich area, excluding cortical bone, as previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Osteocytes were quantified at the same longitudinal level but within the cortical bone. All cells fully embedded within the cortical matrix were classified as osteocytes. In fractured samples, the same area was defined in the periosteal callus near the fracture gap. Osteoblasts were identified and counted in Safranin O-stained sections based on their characteristic morphology as cubic-shaped cells lining the bone surface. Osteoclasts were analyzed in TRAP-stained sections and identified by their positive TRAP staining, distinctive multinucleated morphology, size, and localization on the bone surface.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eLongitudinal sections of 4 \u0026micro;m thickness were prepared for immunohistochemical staining. Detection of Mcpt5 was performed using the primary antibody rabbit anti-mouse MC Protease 5 (1:100; orb11030, Biorbyt, Cambridge, UK), which was incubated overnight at 4\u0026deg;C. The secondary antibody goat anti-rabbit IgG-biotin (1:200; B2770, Life Technologies, Carlsbad, CA, USA) was applied at room temperature (RT) for 1 h. Detection of Ly6G was performed using the primary antibody rat anti-mouse Ly6G (1:300; 127632, BioLegend, San Diego, USA) which was incubated overnight at 4\u0026deg;C. The secondary antibody goat anti-rat IgG-biotin (1:200; #31830, Invitrogen, Carlsbad, CA, USA) was applied at RT for 1 h. For both single staining\u0026rsquo;s, horseradish peroxidase (HRP)-conjugated streptavidin (PK-6100, VECTASTAIN Elite ABC-HRP Kit, Peroxidase, Vector Laboratories, Burlingame, UK) was used for signal detection according to the manufacturer\u0026rsquo;s instructions. NovaRED (SK-4800, Vector NovaRED Substrate Kit, Peroxidase (HRP), Vector Laboratories) served as the chromogen, and sections were counterstained with hematoxylin (1:2000; 2C-306, Waldeck, M\u0026uuml;nster, Germany). Species-specific, non-targeting immunoglobulins were used as isotype controls. A total of 4\u0026ndash;8 mice per group were analyzed. Neutrophils and mast cells were quantified within a defined volume of interest (480 \u0026times; 350 \u0026micro;m) in an area of the fracture callus with maximum amount of cells at 20\u0026times; magnification. Identification was based on characteristic staining, morphology, and cell size.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescent staining Ly6G-Avidin\u003c/h3\u003e\n\u003cp\u003eA double staining of neutrophils and mast cells in the fracture callus 3 days after osteotomy was performed following the subsequent protocol. Detection of Ly6G was performed using the primary antibody rat anti-mouse Ly6G (1:300; #127632, BioLegend, San Diego, USA) which was incubated overnight at 4\u0026deg;C. The secondary antibody rabbit anti-rat IgG (H\u0026thinsp;+\u0026thinsp;L) Alexa Flour 488 (A-21210; Thermo Fisher, Waltham, Massachusetts, USA) and Avidin Texas Red (A820, Invitrogen, Carlsbad, CA, USA), which stains mast cell granules, were applied at RT for 1 h. Nuclei were stained with Hoechst (33258; Sigma-Aldrich, St. Louis, Missouri, USA) for 10 min at RT. Species-specific, non-targeting immunoglobulins were used as isotype controls. Stained slides were screened for possible mast cell intracellular trap (MIT) formation.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eFlow cytometry was performed to characterize immune cell populations in the bone marrow and spleen of Cre\u003csup\u003e-\u003c/sup\u003e and Cre\u003csup\u003e+\u003c/sup\u003e 12-week-old male mice. The bone marrow was flushed out of the left femur using 10 ml phosphate-buffered saline (PBS). The spleen was harvested and passed through a 70-\u0026micro;m cell strainer (Corning Inc., Durham, NC, USA). Cells from both the spleen and bone marrow underwent erythrolysis to remove red blood cells. For immunophenotyping, macrophages (F4/80\u003csup\u003e+\u003c/sup\u003e), neutrophils (Ly-6G\u003csup\u003e+\u003c/sup\u003e), inflammatory monocytes (CD11b\u003csup\u003e+\u003c/sup\u003e), B-lymphocytes (CD19\u003csup\u003e+\u003c/sup\u003e), T-lymphocytes (CD3\u003csup\u003e+\u003c/sup\u003e), cytotoxic T-lymphocytes (CD3\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e+\u003c/sup\u003e), and T-helper lymphocytes (CD3\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e) were identified using the antibodies listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Isotype-matched immunoglobulin antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) served as negative controls. The isolated cells were incubated with the respective antibodies for 30 min on ice. Dead-cell discrimination was performed using 7-aminoactinomycin D (7-AAD, Sigma, Steinheim, Germany). Flow cytometric analysis was conducted on a FACSLyric flow cytometer (BD Bioscience), and data were analyzed using FlowJo software v10 (FlowJo LLC, Ashland, OR).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFlow cytometry antibodies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLabel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCompany\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD11b (rat anti-mouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlexa Fluor\u0026reg; 700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56-0112-82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD19 (rat anti-mouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12-0193-81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD3e (rat anti-mouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePE-Cyanine7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25-0031-82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD4 (rat anti-mouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC-e-Fluor\u0026reg; 780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47-0042-82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD8a (rat anti-mouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17-0081-81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF4/80 (rat anti-mouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11-4701-82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLy6G (rat anti-mouse)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eV450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11-4801-82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBD Bioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG Isotype (Armenian hamster)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePE-Cyanine7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25-4888-81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG2a K Isotype (rat)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlexa Fluor\u0026reg; 700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIC006N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u0026amp;D Systems, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG2a K Isotype (rat)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17-4321-81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG2a K Isotype (rat)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12-4321-81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG2a K Isotype (rat)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFITC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11-4321-42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG2b K Isotype (rat)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAPC-eFluor\u0026reg; 780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47-4031-82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThermo Fisher Scientific, Inc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG2a K Isotype (rat)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eV450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e560377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBD Bioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMagnetic-activated cell sorting (MACS) of bone marrow neutrophils\u003c/h2\u003e \u003cp\u003eFollowing bone marrow flushing from long bones of intact mice, red blood cells were lysed using 5 mL erythrocyte lysis buffer (5 min, 37\u0026deg;C). The suspension was subsequently centrifuged at 1500 rpm for 5 min at 4\u0026deg;C. After removing the supernatant, the cell pellet was resuspended in 200 \u0026micro;L MACS buffer. The suspension was filtered through a 30-\u0026micro;m cell strainer with additionally 400 \u0026micro;L MACS buffer, and the total cell number was determined. 5 \u0026times; 10⁷ cells were centrifuged at 300 \u0026times; g for 10 min at 4\u0026deg;C, and the supernatant was discarded. For negative selection of neutrophils, 50 \u0026micro;L of a neutrophil-specific biotin antibody cocktail was added to the cell pellet, mixed, and incubated for 10 minutes at 2\u0026ndash;8\u0026deg;C. This antibody cocktail binds to all non-neutrophilic cells in the suspension, leaving neutrophils untouched. After washing with 5\u0026ndash;10 mL MACS buffer, cells were centrifuged (300 \u0026times; g, 10 min), the supernatant was aspirated, and the pellet was resuspended in 400 \u0026micro;L MACS buffer. Next, 100 \u0026micro;L of anti-biotin MicroBeads were added, mixed, and incubated for 15 min at 2\u0026ndash;8\u0026deg;C, allowing them to bind to the biotinylated antibodies. After an additional wash with 5\u0026ndash;10 mL MACS buffer and a final centrifugation (300 \u0026times; g, 10 min), the supernatant was discarded, and the pellet was resuspended in 500 \u0026micro;L MACS buffer. For magnetic cell separation, the cell suspension was applied to an LS-MACS column pre-rinsed with 3 mL MACS buffer and placed in a QuadroMACS separator. This step allowed the separation of neutrophils from other labeled cells, which were retained by the column, while neutrophils passed through and were collected for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNAseq analysis of isolated neutrophils\u003c/h2\u003e \u003cp\u003eNeutrophils were isolated from the long bones of three Cre\u003csup\u003e-\u003c/sup\u003e and three Cre\u003csup\u003e+\u003c/sup\u003e mice by MACS as described above. For each mouse, the isolated neutrophils were split into two wells. One well was stimulated for 3 h with 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M norepinephrine at 37\u0026deg;C, while the other well served as an unstimulated control. For RNA isolation using the RNeasy Mini Kit (Qiagen), neutrophils were resuspended in 400 \u0026micro;L RPMI medium supplemented with 10% (v/v) FCS after two rounds of centrifugation (300 \u0026times; g, 10 min) and washing in PBS. Cell numbers were determined prior to stimulation. After 3 h stimulation period, cells were centrifuged (300 \u0026times; g, 10 min), washed with 200 \u0026micro;L PBS, and centrifuged again (300 \u0026times; g, 10 min). The supernatant was removed, and the final cell pellet of each subgroup was resuspended in 350 \u0026micro;L RLT buffer for RNA isolation. Lysates were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until RNA sequencing. RNAseq was performed by the Novogene Corporation (Munich, Germany). The workflow began with sample quality control (Sample QC) to ensure that the samples met the RNAseq criteria, including RNA quantity and RNA integrity number (RIN). Following this, the appropriate RNA library was prepared and tested for quality (Library QC). The RNA library was constructed through polyadenylated (polyA) capture or ribosomal RNA (rRNA) removal, followed by reverse transcription to cDNA. Sequencing was conducted using Illumina PE150 technology (Illumina, San Diego, CA, USA) with a paired-end 150-bp sequencing strategy. The resulting data were subjected to quality control (Data QC). Gene expression differences with a p-value of less than 0.05 between the groups were considered differentially regulated. Bioinformatic analyses were carried out on the differentially regulated genes, including Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. ClusterProfiler software was used for enrichment analysis. GO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org/\u003c/span\u003e\u003cspan address=\"http://www.geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) is a widely used bioinformatics classification system that categorizes gene properties across species into three main branches: cellular component, molecular function, and biological process. GO terms with an adjusted probability (padj)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significantly enriched. KEGG is a curated database containing genomic, biological pathway, and disease information. Pathway enrichment analysis identifies significantly enriched metabolic or signaling pathways associated with differentially expressed genes, comparing them to the entire genomic background. KEGG pathways with padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significantly enriched.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using unpaired t-test and Two-way analysis of variance (ANOVA) followed by Šid\u0026aacute;k\u0026rsquo;s post hoc test. A significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied. Data in figures and tables are depicted as bars with mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Each experimental group consisted of 3\u0026ndash;8 animals, with exact group sizes provided in the figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eFirst, the immune and bone phenotype of 12-week-old male Ly6G-Cre/Adrb2-flox mice was assessed to analyze differences under non-fracture conditions upon KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Immune cell analysis showed no differences in CD19\u003csup\u003e+\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e/Ly6G\u003csup\u003e+\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e/F4/80\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003e, and CD3\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e cells in spleen and bone marrow in \u003cem\u003eAdrb2\u003c/em\u003e KO mice compared to control mice (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Bone phenotyping revealed that the bone parameters femur length (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, a), maximal load to failure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, b), bending stiffness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, c), trabecular bone volume fraction (BV/TV)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, g), trabecular number (Tb.N)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, h), trabecular thickness (Tb.Th)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, i), trabecular separation (Tb.Sp)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, j), osteoblast activity (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, a), and osteoclast activity (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, b) were unaffected. In addition, assessment of the lumbar spine showed no differences in BV/TV, Tb.N, Tb.Th and Tb.Sp (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cre\u003csup\u003e+\u003c/sup\u003e male mice showed a slightly increased trabecular tissue mineral density (TMD) in the femur (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, e) and increased cortical thickness (C.Th) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, d) with increased numbers of osteocytes in the cortex (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, c) compared to Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e littermates.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmune cell phenotyping using flow cytometry. Percentage of CD19\u003csup\u003e+\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e/Ly6G\u003csup\u003e+\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e/F4/80\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003e, and CD3\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e cells. Statistical significance was determined by unpaired t-test (comparison Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e vs. Cre\u003csup\u003e+\u003c/sup\u003e). *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6; males)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eSpleen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c11\" namest=\"c7\"\u003e \u003cp\u003eBone marrow\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell type/\u003c/p\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD19\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCD11b\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eLy6G\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCD11b\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eF4/80\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCD3\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCD3\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCD19\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCD11b\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eLy6G\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCD11b\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eF4/80\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCD3\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCD3\u003csup\u003e+\u003c/sup\u003e/\u003c/p\u003e \u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCre\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003c/p\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003c/p\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e35.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003c/p\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCre\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003c/p\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003c/p\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003c/p\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of Adrb2-signaling on neutrophils during fracture healing in non-osteoporotic and osteoporotic bone, 12-week-old female mice underwent OVX to induce an osteoporotic bone phenotype driven by estrogen depletion. The mice were randomly distributed to the different groups and did not display differences in the body weight 21 days after fracture surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, a). Successful OVX was confirmed by a reduced uterus weight in OVX mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, b). Furthermore, \u0026micro;CT analysis of the contralateral unfractured femur revealed a significant reduction of BV/TV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, c), and Tb.N (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, e) while Tb.Sp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, g) was increased in both Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e and Cre\u003csup\u003e+\u003c/sup\u003e OVX mice compared to the respective sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, d). This indicates that OVX induced an osteoporotic bone phenotype irrespective of the genotype. In addition, we observed that Cre\u003csup\u003e+\u003c/sup\u003e female mice display no bone phenotype as demonstrated by absence of differences in BV/TV, Tb.N, Tb.Th and Tb.Sp of the unfractured femur compared to Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, c, e-g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFracture healing outcome 21 days after surgery was investigated using \u0026micro;CT analysis and histomorphometry. 3D \u0026micro;CT analysis depicted significantly reduced BV/TV and bone volume (BV) in OVX mice compared to sham mice while tissue volume (TV) was unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, a - c), irrespective of genotype, indicating delayed healing due to OVX in both groups. More strikingly, BV/TV and BV were significantly decreased in both Cre\u003csup\u003e+\u003c/sup\u003e groups compared to their respective Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, a, b), indicating delayed healing due to the Adrb2-KO. Representative 3D reconstruction images of the fracture calli are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, d. In addition, two-dimensional histomorphometry revealed a significant decreased bone and increased connective tissue content in the fracture gap of sham Cre\u003csup\u003e+\u003c/sup\u003e mice compared to sham Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e mice supporting the \u0026micro;CT data (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, e - g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the role of \u003cem\u003eAdrb2\u003c/em\u003e deletion in neutrophils and investigate underlying molecular mechanisms of the delayed healing in Cre\u003csup\u003e+\u003c/sup\u003e mice, RNA-Sequencing of isolated neutrophils from the bone marrow of Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e and Cre\u003csup\u003e+\u003c/sup\u003e male mice was conducted. Half of the cells were stimulated with 10\u003csup\u003e\u0026ndash;5\u003c/sup\u003e M norepinephrine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, a) to induce the Adrb2 pathway. Unstimulated neutrophils revealed 48 upregulated and 86 downregulated genes upon Adrb2 KO while an additional noradrenaline stimulation resulted in 148 upregulated and 135 downregulated genes (Suppl. Figure\u0026nbsp;3). A list of all significantly regulated genes for unstimulated and stimulated neutrophils is provided in the supplements. Within those, the most interesting genes were Janus kinase 3 \u003cem\u003e(Jak3)\u003c/em\u003e, Elastase \u003cem\u003e(Elane)\u003c/em\u003e, the Adrenergic receptor beta 2 \u003cem\u003e(Adrb2)\u003c/em\u003e, Chemokine (C-C motif) receptor 10 \u003cem\u003e(Ccr10)\u003c/em\u003e, Gata2 \u003cem\u003e(Gata2)\u003c/em\u003e, Fc receptor \u003cem\u003e(Fcer1a)\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, b). \u003cem\u003eJak3\u003c/em\u003e, \u003cem\u003eElane\u003c/em\u003e, \u003cem\u003eAdrb2\u003c/em\u003e and \u003cem\u003eCcr10\u003c/em\u003e were significantly downregulated in unstimulated Cre\u003csup\u003e+\u003c/sup\u003e neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, b) while \u003cem\u003eGata2\u003c/em\u003e, \u003cem\u003eFcer1a and Adrb2\u003c/em\u003e showed significantly downregulated gene expressions in stimulated Cre\u003csup\u003e+\u003c/sup\u003e neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, b). GO enrichment analysis of unstimulated neutrophils indicated alteration in neutrophil cellular functions upon KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, c), while GO enrichment analysis of stimulated neutrophils display several mast cell related terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, d). The significantly downregulated pathways \u0026ldquo;(positive) regulation of mast cell activation\u0026rdquo; in stimulated Cre\u0026thinsp;+\u0026thinsp;neutrophils could imply a potentially affected neutrophil/mast cell interaction upon neutrophil specific Adrb2 KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBecause of the finding that \u003cem\u003eAdrb2\u003c/em\u003e deletion on neutrophils might affect neutrophil and mast cell recruitment/activation, we analyzed mast cell numbers in the fracture callus of sham and OVX female mice 21-days post-fracture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a). The analysis revealed no significant differences between sham and OVX mice of both genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a). However, female Cre\u003csup\u003e+\u003c/sup\u003e OVX mice showed a significantly reduced percentage of mast cells compared to Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e OVX mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a). This is in line with significantly reduced mast cell numbers in the intact femur of Cre\u003csup\u003e+\u003c/sup\u003e male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, b). To further investigate the effect of Adrb2 deletion on neutrophils, we performed a neutrophil (Ly6G) staining in the fracture callus 3 days post-fracture where we observed a strong reduction of neutrophil recruitment in Cre\u003csup\u003e+\u003c/sup\u003e mice compared to Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, c, d). In addition, we performed a double staining of neutrophils (Ly6G) and mast cells (Avidin) to investigate the recently described mechanism of MIT formation, where mast cells engulf neutrophils, during fracture healing. And indeed, we could show neutrophils trapped intracellularly in mast cells in the early fracture hematoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, e). Taken together, these data indicate that neutrophil-specific deletion of the Adrb2 may impair neutrophil recruitment and reduce mast cell numbers by a so far unknown mechanism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of this study was to investigate the effect of a neutrophil specific \u003cem\u003eAdrb2\u003c/em\u003e deletion on bone homeostasis and fracture healing in non-osteoporotic and osteoporotic bone. Our hypothesis was that the deletion of the Adrb2 accelerates fracture healing specifically under osteoporotic conditions due to reduced neutrophil recruitment. In contrast to this initial hypothesis, we could show that Adrb2-KO on neutrophils had a minor positive effect on intact bone, while fracture healing was significantly delayed in both non-osteoporotic and osteoporotic conditions.\u003c/p\u003e \u003cp\u003eA conditional KO of the \u003cem\u003eAdrb2\u003c/em\u003e on Ly6G expressing cells was generated using the Cre/loxP system which results in reduced Adrb2 expression on neutrophils confirmed by our RNASeq data. First, the immune and bone phenotype of Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e and Cre\u003csup\u003e+\u003c/sup\u003e male littermates was assessed. Male mice were chosen to address 3R principles, as we used their female littermates for the fracture healing study. Nevertheless, the bone phenotype of female Cre\u003csup\u003e\u0026minus;\u003c/sup\u003e and Cre\u003csup\u003e+\u003c/sup\u003e mice was also analyzed in this study using the contralateral intact left femur. Flow cytometric analysis of male mice revealed no significant differences in immune cell populations following Adrb2 knockout (KO). In contrast, a minor bone phenotype was observed, characterized by increased cortical thickness, elevated osteocyte numbers, and higher trabecular TMD. Although these alterations were modest, they suggest that neutrophil-specific deletion of Adrb2 may exert subtle positive effects on bone homeostasis under non-fracture conditions in male mice. One possible explanation is that altered neutrophil signaling indirectly affects osteocyte function or bone remodeling dynamics through changes in local paracrine communication, inflammatory mediator release, or coupling signals between bone-resorbing and bone-forming cells. However, as the underlying mechanisms were not directly investigated in our study, further targeted analyses will be required to fully elucidate the pathways responsible for these bone-specific alterations in Cre\u003csup\u003e+\u003c/sup\u003e male mice. In contrast, female Cre\u003csup\u003e+\u003c/sup\u003e mice with a femur facture did not display a bone phenotype upon neutrophil-specific Adrb2 KO in their contralateral non-fractured femur. It has been shown previously that a fracture does also affect the rest of the skeleton, which might explain the differences between male and female bone phenotype in our study [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNext, we investigated fracture healing, beginning with the induction of postmenopausal osteoporosis at 12 weeks of age by ovariectomy, followed by a standardized femoral osteotomy stabilized with an external fixator at 16 weeks of age. Analysis of uterine weight confirmed the expected uterine atrophy in both Cre⁻ and Cre⁺ ovariectomized (OVX) mice. Consistently, \u0026micro;CT analysis of the unfractured femur demonstrated the characteristic decrease in bone volume fraction (BV/TV) in OVX mice, independent of genotype. These findings indicate that neutrophil-specific deletion of Adrb2 does not protect mice from the development of osteoporosis. Although osteoporosis has been associated with an increased sympathetic tone [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], our data suggest that adrenergic signaling in neutrophils does not contribute to osteoporosis development. In contrast, in osteoblasts, β2-adrenergic signaling is known to play a critical role in the pathogenesis of osteoporosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e\u0026micro;CT analysis of the fracture calli at 21 days post-fracture revealed significantly reduced bone volume fraction (BV/TV) and bone volume (BV) in OVX mice compared with sham-operated mice of both genotypes, indicating impaired fracture healing in Cre⁻ and Cre⁺ osteoporotic mice. These findings suggest that neutrophil-specific Adrb2 knockout does not rescue delayed fracture healing in osteoporotic mice. In contrast, Cre⁺ sham and OVX mice exhibited significantly poorer fracture healing than their respective Cre⁻ controls, indicating that neutrophil-specific deletion of Adrb2 impairs fracture healing under both physiological and osteoporotic conditions. These findings are contradictory to our initial hypothesis and indicate that Adrb2 signaling specifically in neutrophils is of great importance for successful fracture healing. In line with our findings, a recent study by Jahn et al. has demonstrated that treatment with propranolol, an unspecific beta adrenergic blocker, impaired fracture healing in mice [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], although previous studies observed opposite results [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The different results of these studies may depend on different study designs and may indicate a time and/or concentration dependent influence of drug treatment.\u003c/p\u003e \u003cp\u003eTo further elucidate our findings, we performed a more detailed characterization of neutrophils using an in vitro culture approach followed by RNA sequencing. Neutrophils were isolated from the bone marrow of Cre⁻ and Cre⁺ mice and either stimulated with noradrenaline or cultured in medium alone as a control. RNA sequencing identified several differentially expressed genes in both unstimulated and noradrenaline-stimulated Cre⁺ neutrophils compared with Cre⁻ neutrophils, including \u003cem\u003eJak3\u003c/em\u003e, \u003cem\u003eElane\u003c/em\u003e, \u003cem\u003eAdrb2\u003c/em\u003e, \u003cem\u003eCcr10\u003c/em\u003e, \u003cem\u003eGata2\u003c/em\u003e, and \u003cem\u003eFcer1a\u003c/em\u003e. \u003cem\u003eJak3\u003c/em\u003e encodes a non-receptor tyrosine kinase that plays a critical role in neutrophil activation in response to interleukin-8 (IL-8) during inflammatory reactions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, \u003cem\u003eJak3\u003c/em\u003e has been shown to be essential for neutrophil chemotaxis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This indicates that neutrophil chemotaxis might be affected upon \u003cem\u003eAdrb\u003c/em\u003e2 KO. Ccr10 is known as an important receptor for the immune response and plays a role in the recruitment of immune cells but less is known about its function in neutrophils [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In addition, \u003cem\u003eElane\u003c/em\u003e encodes for neutrophil expressed elastase which is known as an important factor of promoting inflammation e.g. in various lung diseases [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Other downregulated candidate genes were \u003cem\u003eFcer1a\u003c/em\u003e and \u003cem\u003eGata2\u003c/em\u003e. \u003cem\u003eFcer1a\u003c/em\u003e encodes for a high affinity IgE receptor playing a central role in allergic diseases and is mainly expressed on mast cells but in a smaller manner also on neutrophils [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eGata2\u003c/em\u003e is an important transcription factor regulating \u003cem\u003eFcer1a\u003c/em\u003e expression which means that the downregulation of \u003cem\u003eGata2\u003c/em\u003e results in a downregulation of \u003cem\u003eFcer1a\u003c/em\u003e expression [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. GO enrichment analysis of noradrenaline-stimulated neutrophils further indicates the possibility of a disturbed neutrophil/mast cell interaction since the GO term \u0026ldquo;(positive) regulation of mast cell activation\u0026rdquo; was significantly downregulated upon Ly6G-Adrb2-KO. Elastase could be involved in this regulation, as it is known to recruit mast cells. In general, mast cells derive from hematopoietic stem cells of the bone marrow and are mainly known for their function in IgE mediated allergies and tissue repair [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. But they are also associated with age-related and postmenopausal osteoporosis as indicated by increased mast cell numbers and the ability to regulate osteoclastic activity [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Using mast cell deficient mice, we demonstrated previously that mast cells activate osteoclastic bone resorption in postmenopausal osteoporosis and are critically involved in osteoporotic fracture healing [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Furthermore, severely injured mice showed increased mast cell numbers in the fracture hematoma while mast cell\u0026ndash;depleted mice were protected against severe injury\u0026ndash;induced impairment of fracture healing [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In summary, these data demonstrate that increased mast cell numbers under conditions of hyperinflammation can be detrimental to bone regeneration. Based on these observations, we decided to further investigate mast cell populations in our mouse model. Immunohistochemical staining was performed to quantify mast cells in the intact femur of male mice and in the fracture callus of female mice. Our data revealed that, Cre⁺ male mice exhibited significantly reduced mast cell numbers in the intact bone. In the fracture callus, this reduction of mast cells was also observed in Cre⁺ osteoporotic mice which displayed significantly fewer mast cells in the fracture callus compared to Cre⁻ osteoporotic controls. Despite the reduction of mast cell numbers, Cre⁺ mice still exhibited delayed fracture healing, which is challenging to reconcile since in previous fracture healing studies in models of hyperinflammation (e.g. osteoporosis) mast cell deficiency resulted in improved fracture healing. Currently, we have no proven explanation for our findings. One possible explanation could be that the neutrophil\u0026ndash;mast cell crosstalk is disrupted in this model and that a balanced neutrophil\u0026ndash;mast cell crosstalk is important for fracture healing. Previous studies have shown that neutrophils recruit mast cells to sites of inflammation through the secretion of chemokines such as CXCL1 and CXCL12 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. An altered chemokine expression or signaling in Adrb2-deficient neutrophils could therefore impair mast cell recruitment and function, leading to a dysregulated inflammatory response and ultimately compromises fracture healing. To further understand this neutrophil/mast cell interaction we stained neutrophils in the fracture callus 3 days post-fracture where we observed a drastically reduced neutrophil recruitment in Cre\u003csup\u003e+\u003c/sup\u003e mice. This implies that Ly6G-Adrb2 KO mice exhibit disturbed neutrophil activation and an impaired neutrophil/mast cell crosstalk, resulting in reduced numbers of both cell types in the fracture callus. As previously described by Kovtun \u003cem\u003eet al.\u003c/em\u003e, neutrophils play a crucial role in bone fracture healing [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Treatment with a Ly6G antibody led to impaired bone regeneration highlighting the importance of undisturbed neutrophil recruitment and function in the early inflammatory phase of fracture healing [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This imbalance of neutrophils and mast cells in our mouse model may explain the poor fracture healing outcome observed in Cre\u003csup\u003e+\u003c/sup\u003e mice, as a finely tuned activation and interaction has been shown to be essential for successful fracture repair.\u003c/p\u003e \u003cp\u003eDespite their central role in innate immunity - mediating antimicrobial defense through phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs) - neutrophils also appear to exert important immunomodulatory functions during tissue repair [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this context, Mihlan et al. recently described a process termed mast cell\u0026ndash;induced neutrophil trapping (MIT), in which degranulating mast cells secrete leukotriene B4 to attract neutrophils and retain them within mast cells. The trapped neutrophils subsequently undergo cell death, while their components are retained by mast cells, thereby enhancing mast cell metabolic fitness and functional capacity [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Notably, we were able to detect MIT formation in the fracture callus at 3 days post-fracture using immunofluorescent double staining for neutrophils and mast cells. Nevertheless, this observation alone does not explain the impaired fracture healing observed in Ly6G-Adrb2 KO mice. One plausible explanation could be that reduced neutrophil activation and recruitment in these mice limits the initiation and regulation of early inflammatory signaling cascades required for effective mast cell activation, angiogenesis, and subsequent tissue remodeling. Consequently, insufficient early inflammatory signaling may lead to a failure to properly transition from the inflammatory to the reparative phase of fracture healing.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings highlight the importance of a tightly regulated neutrophil\u0026ndash;mast cell interaction during bone regeneration and suggest that both insufficient and excessive immune cell activity can be detrimental to fracture healing. Adrb2 signaling on neutrophils seems to be important in that context. Further studies are clearly required to dissect the molecular mechanisms underlying this bidirectional crosstalk and to determine how its dysregulation contributes to impaired bone repair in osteoporotic conditions.\u003c/p\u003e \u003cp\u003eLimitations of our study are that our Ly6G-Cre model affects not only neutrophils but also other myeloid derived cells expressing Ly6G, e.g. some types of monocytes or myeloid-derived suppressor cells. Furthermore, investigating earlier time points after fracture, neutrophil functions and other involved immune cells, e.g. T- and B-cells, to further characterize the neutrophil/mast cell crosstalk during bone healing would be needed for future studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our data show that Adrb2-signaling on neutrophils is not relevant for the development of postmenopausal osteoporosis, while it seems to be critical for fracture healing in both non-osteoporotic and osteoporotic mice. This could be due to an impaired activation of neutrophils and a disturbed interaction of neutrophils and mast cells upon Ly6G-Adrb2-KO which needs to be further investigated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.D. and N.G. contributed equally. S.D., N.G and M.H.-L. wrote the manuscript text and prepared the figures. Data collection and analysis was performed by S.D. and N.G. Data interpretation was done by S.D., N.G., C.K., D.G., J.B., O.K., V.F., A.I. and\u0026nbsp;\u003cbr\u003e\u0026nbsp;M.H.-L. Surgery assistance: C.K., D.G., J.B. and O.K. Project supervision and funding was done by M.H.-L. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the German Research Foundation (DFG, grant number HA 8470/2-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Tina Vogel, Dr. Justyna Pawlak-Wurster, Iris Baum, Andrea B\u0026ouml;hmler and Sandra Richter for their excellent work as technicians. In addition, we would like to thank the animal keepers Ricco Richter, Jasmin Jucha and Herrmann Klassen at the TFZ in Ulm for taking care of the animals.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTakayanagi, H., \u003cem\u003eOsteoimmunology: shared mechanisms and crosstalk between the immune and bone systems\u003c/em\u003e. Nat Rev Immunol, 2007. 7(4): p. 292\u0026ndash;304.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLorenzo, J., M. Horowitz, and Y. Choi, \u003cem\u003eOsteoimmunology: interactions of the bone and immune system\u003c/em\u003e. Endocr Rev, 2008. 29(4): p. 403\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreuil, V., et al., \u003cem\u003eImmune changes in post-menopausal osteoporosis: the Immunos study\u003c/em\u003e. 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Cell, 2024. 187(19): p. 5316\u0026ndash;5335 e28.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"neutrophil granulocytes, inflammation, adrenergic signaling, beta 2 adrenoreceptor, osteoporosis, fracture healing","lastPublishedDoi":"10.21203/rs.3.rs-8593165/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8593165/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAfter bone fracture, osteoporotic mice show delayed healing associated with elevated systemic inflammation and increased neutrophil numbers in the early fracture hematoma. Because short-term propranolol treatment reduced neutrophil recruitment, we hypothesized that deletion of β\u003csub\u003e2\u003c/sub\u003e-adrenoreceptor (Adrb2) signaling in neutrophils would normalize neutrophil recruitment and accelerate fracture healing in osteoporotic mice. A conditional Adrb2 knockout in Ly6G⁺ neutrophils was generated using Ly6G-Cre Adrb2-flox mice. Bone and immune phenotypes were analyzed via \u0026micro;CT and histology under non-fracture conditions and during fracture healing in ovariectomized mice with postmenopausal osteoporosis. Both non-osteoporotic and osteoporotic female Ly6G-Adrb2-KO mice showed impaired fracture healing vs. controls, while only minor bone alterations were observed under non-fracture conditions. Pathway analysis of isolated neutrophils, characterized by RNA-sequencing, suggested reduced neutrophil activation and disturbed neutrophil/mast cell interactions upon Ly6G-Adrb2-KO. In summary, our data demonstrate that Adrb2 signaling is important for neutrophil recruitment and seems to be critical for proper fracture healing.\u003c/p\u003e","manuscriptTitle":"Impaired fracture healing upon neutrophil-specific adrenoreceptor beta 2 knockout in non-osteoporotic and osteoporotic mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-24 01:28:55","doi":"10.21203/rs.3.rs-8593165/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-19T19:23:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T22:11:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T16:04:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T02:05:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-16T15:06:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109966990025300904112487030401373155977","date":"2026-02-09T21:28:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154708662525424824250734711210478147478","date":"2026-02-09T14:49:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56960936748751252206581075296769810139","date":"2026-02-09T09:15:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197291471713108087009485741261102734671","date":"2026-02-06T16:34:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136168087532617373676725320186776335026","date":"2026-02-05T18:13:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1284817253759606121220482701363469893","date":"2026-02-04T14:34:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119808678007919420187473506331395018858","date":"2026-02-04T14:28:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-22T04:04:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T00:24:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T10:05:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Regenerative Medicine","date":"2026-01-13T14:13:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-regenerative-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjregenmed","sideBox":"Learn more about [npj Regenerative Medicine](http://www.nature.com/npjregenmed/)","snPcode":"41536","submissionUrl":"https://mts-npjregenmed.nature.com/cgi-bin/main.plex","title":"npj Regenerative Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0b377b6-8cef-4207-80ea-c956fe21853d","owner":[],"postedDate":"January 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61548489,"name":"Health sciences/Diseases"},{"id":61548490,"name":"Biological sciences/Immunology"},{"id":61548491,"name":"Health sciences/Medical research"},{"id":61548492,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-11T10:23:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-24 01:28:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8593165","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8593165","identity":"rs-8593165","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}