Resting collecting lymphatic vessels (cLVs) sense edema in distal joints and initiate contractions via unknown mechanisms. Rheumatoid arthritis (RA) patients have lymphatic drainage deficiencies from affected joints, and defects in the synovial lymphatic system exacerbate inflammatory-erosive arthritis in animal models. To understand this, we generated Efhd1-CreERT2 and Myoc-CreERT2 mice for cell-specific genetic gain and loss of function studies. These mice were crossed with tdTomato reporter (Ai9) mice, and studies showed selective tamoxifen-induced transgene expression in CD31-/CD34+ telocyte-like cells in knee and ankle synovium, and in networks physically associated with mast cells proximal to popliteal lymphatic vessels (PLVs). Consistent with the known loss of CD31-/CD34+ telocyte in RA synovium, these cells were also decreased in TNF-tg knees and partially recovered by anti-TNF treatment. Ultrastructural and gene expression studies confirmed a distinct telocyte phenotype versus closely related fibroblasts. In vivo depletion studies in tamoxifen-treated Efhd1-CreERT2 and Myoc-CreERT2 mice crossed to diphtheria toxin alpha-floxed (DTAflox) mice demonstrated telocyte requirements for physiologic lymphatic drainage and resolution of joint inflammation and focal erosions from zymosan-induced arthritis in the knee. In vitro studies demonstrated increased sensitivity to osmotic shock and decreased motility versus fibroblasts, and telocyte potential to differentiate into myofibroblasts on stiff matrix. Collectively, these findings support a model of joint homeostasis in which osmotic pressure-sensing telocyte networks extend from the synovium into mast cells proximal to joint-draining cLVs, and telocyte loss is associated with defects in the synovial lymphatic system and increased susceptibility to joint inflammation and structural damage from arthritis.

Abstract Introduction Staphylococcus aureus is the leading cause of implant-associated osteomyelitis. It has a high recurrence rate, a low post-operative cure rate, and can lead to sepsis, multiorgan failure, and death. This indicates a need for future innovation in therapeutics and vaccines. With our humanized mouse model, we aimed to investigate the human T cell response during infection, as T cells play a crucial role in controlling bacterial growth during chronic infections. Using immunohistochemistry, we have found preliminary evidence of T cell dysfunction in the bone marrow niche. This has led to our hypothesis that CD4 T cells are becoming exhausted in the bone marrow due to chronic infection. Methods Female humanized NSG-SGM3 BLT mice (20-24 weeks old) underwent transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux) or sham surgery. At fourteen days post-infection, bone marrow cells were isolated and subjected to flow cytometry and single-cell RNA sequencing. Results In the bone, we observed an influx of Th1/Th17 cells through single-cell RNA sequencing. Using spectral flow cytometry, we observed increases in immune checkpoint proteins LAG-3, PD-1, and TIM-3 on CD4 T cells. Interestingly, we also discovered that TIM-3-positive CD4 T cells exhibited reduced Ki67 staining, suggesting potentially impaired functional capacity. To explore this further, we examined the effector profile (IFN-γ, IL-17A, and TNF-α) of these cells and observed diminished cytokine production in TIM-3-positive cells. Finally, we analyzed human serum from arthroplasty patients and found TIM-3 levels to be highly predictive of adverse outcomes. Conclusion These results suggest CD4 T cells may be dysfunctional and contribute to the chronicity of infection in S. aureus osteomyelitis. Ultimately, this work will provide novel mechanistic insights into bacteria-T cell interactions during S. aureus bone infections, hoping to inform better diagnostics and therapeutics. Funding Source NIAMS Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Quantitative description of complex anatomical structures remains challenging due to the expertise necessary for manual segmentation, labor, and interobserver variability. To overcome this, automated detection of specific landmarks can be accomplished by digital image analysis techniques, including deep learning (DL) models. To this end, we undertook supervised automated analysis of micro-computed tomography (micro-CT) datasets of murine hindpaws and forepaws. Advancing beyond previously published semi-automated (SA) marker-based watershed algorithms, we added structure enhancement, tensor voting, and output dilation to identify joint spaces. Segmentation was enhanced by utilizing a DL joint space prediction model (3D U-Net architecture, ResNet-18 backbone) using wild-type (WT) hindpaw labels as ground truth. Prediction was extended to hindpaws and forepaws from WT and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis of both sexes across age. Segmentation accuracy improved dramatically using the DL methodology. Accuracy declined with increased disease severity and age in TNF-Tg mice. Subsequent testing in forepaws also displayed progressive reduction in accuracy with increasing arthritic severity. Overall, this supervised automated model outperforms recent SA approaches in healthy joints to enhance the investigation of complex bone anatomy. Although flexible application to novel and disease-modified datasets demonstrates deprecated performance, utilization may nonetheless catalyze structure-specific segmentation model development.

Chemokines are essential mediators of immune responses, and the CCL20/CCR6 chemokine signaling axis is known to be involved in inflammation, infectious diseases, and cancer progression. However, the role of the CCL20/CCR6 axis in host defense against Staphylococcus aureus osteomyelitis remains unknown. We hypothesized that the CCL20/CCR6 axis is critical for the recruitment and activation of immune cells against S. aureus, and the lack of CCL20 or its monogamous receptor CCR6 leads to exacerbation of S. aureus osteomyelitis. In vitro studies confirmed that osteoblasts and macrophages (M0 and M2 subtypes) secrete CCL20 following S. aureus exposure. Implant-associated osteomyelitis in C57BL/6, CCL20-/-, and CCR6-/- mice revealed an early increase in planktonic bacterial growth on day 1 and increased bacterial loads in soft tissue and bone on day 14 post-infection in both CCL20-/- and CCR6-/- mice. Immunohistochemistry and flow cytometry revealed that CCL20-/- and CCR6-/- mice have impaired recruitment of T cells, especially CCR6+ T cells, to the site of infection. Interestingly, CCR6-/- mice exhibited increases in osteoclast numbers, reactive bone formation, and reduced bone mineral density. In a clinical pilot study, we observed a fivefold increase in serum CCL20 levels (P < 0.05) in S. aureus osteomyelitis patients (n = 23) vs uninfected controls (n = 10). Remarkably, serum CCL20 levels immediately following septic death were 100-fold higher vs uninfected patients (P < 0.05). Collectively, these results highlight the critical role of CCL20/CCR6-mediated host immunity during the establishment of S. aureus osteomyelitis and the potential of CCL20 as a biomarker of osteomyelitis-induced sepsis.IMPORTANCEStaphylococcus aureus is the most common pathogen in orthopedic infections, and hard-to-treat strains (methicillin-resistant S. aureus) cause >50% of these infections. Thus, there is an urgent need to develop immunotherapies to treat these life-threatening infections. The role of the CCL20/CCR6 chemokine signaling axis on S. aureus osteomyelitis is unknown. In our efforts to uncover its role, we reveal that osteoblasts and macrophages secrete CCL20 in response to infection, and mice lacking CCL20 or its monogamous receptor CCR6 are more susceptible to S. aureus osteomyelitis. Mechanistically, we observed that increased infection severity in the knockout mice is associated with decreased T cell recruitment and increased osteoclastogenesis at the bone infection site. Importantly, in a clinical pilot study, we observed that CCL20 can be a useful biomarker of osteomyelitis-induced septic death. Overall, our study highlights the crucial immunomodulatory role that the CCL20/CCR6 axis plays during osteomyelitis.

Background/Objectives: Hydroxybisphosphonate-conjugated sitafloxacin (HBCS) was developed to achieve higher antibiotic concentrations within infected bone. Small animal studies supported further development, but the feasibility of HBCS treatment in a more clinically relevant and larger animal model is unknown. Methods: In this study, we present case reports on four sheep, each receiving four MRSA-contaminated tibial screws treated with different regimens of intravenous antibiotics. The first two sheep received two screws contaminated with 103 CFU and two screws contaminated with 105 CFU. Sheep 1 only received vancomycin, starting on day two. Sheep 2 received vancomycin, starting on day 2, but also received 7 doses of HBCS (2 mg/kg/48 h). The protocol for the final two sheep was revised, and both received four screws contaminated with 103 CFU, and vancomycin was started preoperatively. Sheep 3 and 4 received 7 doses (starting on day 6) and 9 doses (starting on day 2) of HBCS (4 mg/kg/48 h), respectively. Bacteriology was performed on three screws per animal. Longitudinal radiography and histology (n = 1 screw) were assessed for signs of osteolysis and reactive bone formation. Electron microscopy (EM) was performed in the first two sheep to evaluate antibiotic-induced bacterial damage. Results: All sheep tolerated HBCS infusion without clinical signs of discomfort. In addition to a high bacterial load (~104 CFU on all screws), Sheep 1 displayed extensive radiographic and histologic evidence of peri-implant osteolysis and reactive bone formation. Despite having a high bacterial load (~104 CFU on all screws), Sheep 2 displayed only mild radiographic and histologic evidence of peri-implant osteolysis and periosteal reactive bone formation. Bacteriology in Sheep 3 and 4 demonstrated near MRSA eradication (<100 CFU on 2 screws). Both sheep displayed no evidence of osteolysis or new bone formation adjacent to the screw head. EM confirmed the presence of bacteria resorbing bone and replicating in biofilm in Sheep 1, while antibiotic-killed bacteria with ruptured septal planes were seen in Sheep 2. Conclusions: This study demonstrates the feasibility of HBCS therapy in a clinically relevant animal model and provides guidance on future efficacy studies, such as the use of an inoculum of 103 CFU per screw, the initiation of antibiotic treatment commencing at the time of surgery, and the usability of antibiotic-killed bacteria within altered glycocalyx observed by TEM as a potential biomarker for HBCS efficacy.

Although vaccines have been hailed as one of the greatest advances in medicine based on their unparalleled cost-effectiveness in eradicating life-threatening infectious diseases, their role in orthopedic trauma-related infections is unclear. This is largely because vaccines are primarily made against pathogens that cause communicable diseases rather than opportunistic infections secondary to trauma, and most successful vaccines are against viruses rather than biofilm forming bacteria. Nonetheless, the tremendous costs to patients and healthcare systems warrant orthopedic trauma vaccine research, which has been a focal topic in recent international consensus meetings on musculoskeletal infection. This subject was also covered at the 2023 Osteosynthesis and Trauma Care Foundation (OTCF) meeting in Rome, Italy, and the purpose of this supplement article is to (1) highlight the osteoimmunology, animal models, translational research and clinical pilots that were discussed, (2) the proposed future directions that could lead to diagnostics and prognostics that are critically needed for evidence-based decision making, and (3) vaccines and passive-immunization strategies that could potentially be utilized to treat patients with orthopedic infections.

In vitro and in vivo studies are critical for the preclinical efficacy assessment of novel therapies targeting musculoskeletal infections (MSKI). Many preclinical models have been developed and applied as a prelude to evaluating safety and efficacy in human clinical trials. In performing these studies, there is both a requirement for a robust assessment of efficacy, as well as a parallel responsibility to consider the burden on experimental animals used in such studies. Since MSKI is a broad term encompassing infections varying in pathogen, anatomical location, and implants used, there are also a wide range of animal models described modeling these disparate infections. Although some of these variations are required to adequately evaluate specific interventions, there would be enormous value in creating a unified and standardized criteria to animal testing in the treatment of MSKI. The Treatment Workgroup of the 2023 International Consensus Meeting on Musculoskeletal Infection was responsible for questions related to preclinical models for treatment of MSKI. The main objective was to review the literature related to priority questions and estimate consensus opinion after voting. This document presents that process and results for preclinical models related to (1) animal model considerations, (2) outcome measurements, and (3) imaging.

Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real-time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real-time quantification of green fluorescent protein (GFP+) host cells and enhanced cyan fluorescent protein (ECFP+) or red fluorescent protein (RFP+) methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with similar to 10(5) CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90 degrees angle similar to 0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 +/- 0.000675 mm(3) during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h, 56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 h in the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by similar to 2 x 10(4) CFU/h on the implant from 2 to 12 h post-op (p < 0.05; r(2) > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

Implant-associated Staphylococcus aureus (S. aureus) osteomyelitis (IASO) leads to high orthopedic implant failure rates due to the formation of Staphylococcal abscess community within the bone marrow and bacterial colonization in the osteocyte lacuno-canalicular network (OLCN). To address this, antimicrobial peptides (HHC36)-loaded titania nanotubes (NTs) are developed on titanium screws (Ti-NTs-P-A), which integrate pH-responsive polymethacrylic acid to control HHC36 release for eradicating bacteria in IASO. Colony-forming unit assay confirmed that Ti-NTs-P-A screws maintained sustainable antibacterial effectiveness, killing over 65% of S. aureus even after multiple bacterial solution replacements. Notably, Ti-NTs-P-A screws exhibit significant pH-responsive HHC36 release behavior and bactericidal activity, consistent with the phenotype of peptides-killed bacteria from scanning electron microscopy. Transcriptome sequencing results reveal that Ti-NTs-P-A screws interfered with ribosome formation and disrupted the arginine biosynthesis, which is crucial for bacterial survival in acidic environments. In the non-infected implant model, the bone-implant contact ratio of the Ti-NTs-P-A screw is 2.3 times that of the clinically used titanium screw. In an IASO model, Ti-NTs-P-A screws effectively eradicated bacteria within the OLCN, achieving an 80% infection control rate and desirable osteointegration. Collectively, Ti-NTs-P-A screws with pH-responsive antibacterial properties exhibit great potential for eradicating bacteria and achieving osseointegration in IASO.

Rheumatoid arthritis (RA) is a complex immune-mediated inflammatory disorder in which patients suffer from inflammatory-erosive arthritis. Recent advances on histopathology heterogeneity of RA synovial tissue revealed three distinct phenotypes based on cellular composition (pauci-immune, diffuse and lymphoid), suggesting that distinct etiologies warrant specific targeted therapy which motivates a need for cost effective phenotyping tools in preclinical and clinical settings. To this end, we developed an automated multi-scale computational pathotyping (AMSCP) pipeline for both human and mouse synovial tissue with two distinct components that can be leveraged together or independently: (1) segmentation of different tissue types to characterize tissue-level changes, and (2) cell type classification within each tissue compartment that assesses change across disease states. Here, we demonstrate the efficacy, efficiency, and robustness of the AMSCP pipeline as well as the ability to discover novel phenotypes. Taken together, we find AMSCP to be a valuable cost-effective method for both pre-clinical and clinical research.

Rheumatoid arthritis (RA) is characterized by erosive pathology associated with joint inflammation and a sexual dimorphism with increased prevalence in females. Here, we aim to determine whether androgen is protective against inflammatory-erosive disease in TNF-transgenic (TNF-Tg) mice. Wild-type (WT) and TNF-Tg male mice underwent sham (WT, n = 3; TNF-Tg, n = 7) or orchiectomy (WT, n = 3; TNF-Tg, n = 7) surgery at 1 month old to remove androgen production confirmed by serum testosterone concentration. Cohorts of orchiectomized TNF-Tg males were treated with either 5 alpha-dihydrotestosterone (.025 mg/day) (n = 3) or placebo (n = 3) via subcutaneous pellet insertion. Weekly clinical measures, along with mid-hindpaw bone volumes and ankle histology at 3 months old were evaluated for all groups. Orchiectomies in TNF-Tg males significantly decreased serum testosterone (P < .05), weight gain (P < .001), and mid-hindpaw bone volumes (P < .05) in comparison to sham TNF-Tg mice. The cuboid bone also had increased synovitis by histology with the loss of androgen (P < .05). Treatment of orchiectomized TNF-Tg males with 5 alpha-dihydrotestosterone protected against the changes in weight gain (P < .01) and bone erosion (P < .05) associated with decreased osteoclast number in the cuboid (P < .01). In the TNF-Tg model of chronic inflammatory arthritis, androgen is protective in erosive disease. The loss of endogenous androgen significantly accelerated the progression of inflammatory-erosive arthritis in male TNF-Tg mice to a similar severity as age-matched female mice. In addition, treatment with exogenous androgen prevented this observed bone loss in orchiectomized TNF-Tg males. Overall, androgen delays and limits bone erosion even in the presence of active inflammation and future studies are warranted to elucidate the associated mechanisms.

Although the twenty-first century has seen major advances in evidence-based medicine to improve health, athletic performance, and injury prevention, our inability to implement these best practices across underserved American communities has limited the impact of these breakthroughs in sports medicine. Rochester, NY is stereotypical of American communities in which an economically challenged racially diverse urban center with grossly underperforming public schools is surrounded by adequately resourced predominantly Caucasian state-of-the-art education systems. As these great disparities perpetuate and further degrade our society in the absence of interventions, the need for community engagement initiatives is self-evident.

BACKGROUND:Development of reliable disease activity biomarkers is critical for diagnostics, prognostics, and novel drug development. Although computed tomography (CT) is the gold-standard for quantification of bone erosions, there are no consensus approaches or rationales for utilization of specific outcome measures of erosive arthritis in complex joints. In the case of preclinical models, such as sexually dimorphic tumor necrosis factor transgenic (TNF-Tg) mice, disease severity is routinely quantified in the ankle through manual segmentation of the talus or small regions of adjacent bones primarily due to the ease in measurement. Herein, we sought to determine the particular hindpaw bones that represent reliable biomarkers of sex-dependent disease progression to guide future investigation and analysis. METHODS:Hindpaw micro-CT was performed on wild-type (n = 4 male, n = 4 female) and TNF-Tg (n = 4 male, n = 7 female) mice at monthly intervals from 2-5 (females) and 2-8-months (males) of age, since female TNF-Tg mice exhibit early mortality from cardiopulmonary disease at approximately 5-6-months. Further, 8-month-old WT (n = 4) and TNF-Tg males treated with anti-TNF monoclonal antibodies (n = 5) or IgG placebo isotype controls (n = 6) for 6-weeks were imaged with micro-CT every 3-weeks. For image analysis, we utilized our recently developed high-throughput and semi-automated segmentation strategy in Amira software. Synovial and osteoclast histology of ankle joints was quantified using Visiopharm. RESULTS:First, we demonstrated that the accuracy of automated segmentation, determined through analysis of ~9000 individual bones by a single user, was comparable in wild-type and TNF-Tg hindpaws before correction (79.2±8.9% vs 80.1±5.1%, p = 0.52). Compared to other bone compartments, the tarsal region demonstrated a sudden, specific, and significant bone volume reduction in female TNF-Tg mice, but not in males, by 5-months (4-months 4.3± 0.22 vs 5-months 3.4± 0.62 mm3, p<0.05). Specifically, the cuboid showed significantly reduced bone volumes at early timepoints compared to other tarsals (i.e., 4-months: Cuboid -24.1±7.2% vs Talus -9.0±5.9% of 2-month baseline). Additional bones localized to the anterolateral region of the ankle also exhibited dramatic erosions in the tarsal region of females, coinciding with increased synovitis and osteoclasts. In TNF-Tg male mice with severe arthritis, the talus and calcaneus exhibited the most sensitive response to anti-TNF therapy measured by effect size of bone volume change over treatment period. CONCLUSIONS:We demonstrated that sexually dimorphic changes in arthritic hindpaws of TNF-Tg mice are bone-specific, where the cuboid serves as a reliable early biomarker of erosive arthritis in female mice. Adoption of automated segmentation approaches in pre-clinical or clinical models has potential to translate quantitative biomarkers to monitor bone erosions in disease and evaluate therapeutic efficacy.