Biology subjects

Solomon, L. B.

Publications and source records attributed to Solomon, L. B..

11 recordsLinked to original sources

bioRxiv2026

Human Osteocytes Express MHC ClassII and Act as Non-classical Antigen-Presenting Cells During Bacterial Infection

Osteocytes are the most abundant cells in bone and are increasingly recognised not only for their role in skeletal remodelling and inflammatory signalling but also for their potential involvement in immune responses. In this study, we searched available gene expression datasets of human primary osteocyte-like cells exposed acutely to Staphylococcus aureus and identified significantly induced expression of key genes related to antigen processing and presentation. We then confirmed that human bone explant-derived osteoblastic cells, representative of a mature osteoblast-pre-osteocyte stage, expressed, as expected, high cell surface levels of major histocompatibility complex (MHC) Class I but also, low basal levels of the MHC Class II family member, HLA-DR. However, confocal imaging revealed high expression of MHC Class II molecules and the peptide-loading chaperone HLA-DM within the lysosomal compartments, consistent with canonical antigen-processing machinery. Differentiation towards a mature osteocyte phenotype increased MHC Class II protein levels and maintained expression of intracellular HLA-DM. Exposure of mature osteocyte-like cells to S. aureus further up-regulated both intracellular and cell surface MHC Class II expression. Demonstrative of antigen presenting cell functionality, S. aureus-exposed osteocytes induced autologous CD4+ T cell proliferation. Furthermore, MHC Class II expression in osteocytes was detected in bone sampled from patients with periprosthetic joint infections, providing evidence that these mechanisms operate in vivo. Together, our findings reveal that human osteocytes are capable of inducible MHC Class II-associated antigen presentation in response to bacterial challenge, pointing to a novel role for osteocytes in adaptive immune surveillance within bone.

immunology↗

bioRxiv2026

Quantification of bone loss, periosteal bone formation and novel histopathological changes in a mouse implant-related Staphylococcus aureus infection model

Implant-associated bone infection involves a complex interplay between pathogenic stimuli and host cell responses, yet analysis in preclinical models has typically relied on qualitative or semi-quantitative measures. We aimed to establish a quantified evaluation framework to define host-pathogen relationships in a preclinical implant infection model. Staphylococcus aureus-coated stainless-steel implants were inserted trans-cortically in mouse tibiae and bone changes recorded longitudinally by in vivo micro-CT. An automated segmentation task list was developed to independently isolate and quantify cortical, periosteal-reactive, and trabecular bone compartments. RGB trichrome histomorphometry was used to quantify bone matrix integrity, osteocyte lacunar geometry, and osteoclastic activity. Droplet digital PCR was used to determine absolute bacterial and host genome copy number. Infected implants produced marked reductions in trabecular bone volume fraction, number, and bone mineral density (BMD), together with decreased cortical bone volume fraction and increased cortical porosity, accompanied by significant elevations in periosteal bone volume fraction. Histologically, infected bone exhibited increased eroded surface indicative of osteoclastic resorption, extensive degraded bone matrix and pathological remodelling of osteocyte lacunae towards circularity, consistent with an osteocytic osteolysis response. Infection-induced changes to cortical bone structure correlated mostly with host cell rather than bacterial load; however, cortical BMD negatively correlated with the bacterial:host genome ratio. This multifaceted, quantified framework reveals distinct pathobiological effects of implant-associated infection on trabecular, cortical, and periosteal bone compartments, bone matrix and osteocyte and osteoclast populations, consistent with reports in human patients, suggesting that major pathological changes are driven by the host bone cell response to infection.

pathology↗

bioRxiv2025

TWEAK and Fn14 expression in osteoarthritis: evidence for TWEAK-induced RANKL release from chondrocytes

ObjectiveA role for TWEAK expression and that of its receptor Fn14 by osteoblasts and synovial tissue has been proposed in the pathogenesis of osteoarthritis (OA). Here, we examined whether the cartilage in OA was also a source and target of TWEAK. DesignArticular cartilage samples from 24 patients undergoing hip or knee replacement surgery for OA were investigated for TWEAK and Fn14 expression by both immunostaining and real-time RT-PCR. Samples were graded according to the Pritzker scale from grade 0 (normal) through increasing levels of cartilage damage (grades 1-3). Human primary chondrocytes isolated from OA cartilage were treated with combinations of recombinant TWEAK and TNF and examined for acute effects on TWEAK, FN14, RANKL and ADAM17 mRNA levels. Released soluble RANKL levels were measured by ELISA. ResultsImmunostaining indicated that the majority (21/24) of OA cartilage samples expressed low levels of TWEAK protein and high levels of Fn14, however, expression did not appear to vary with respect to OA grade. TWEAK mRNA levels were elevated in grade 2 OA cartilage compared to grade 0 (p < 0.05), while Fn14 mRNA levels appeared elevated across all damage grades, significantly in grade 2 samples. Primary chondrocytes treated with TNF, alone or in combination with TWEAK, exhibited early upregulated Fn14 mRNA expression. TWEAK induced RANKL mRNA expression. while both TWEAK and TNF induced the mRNA expression of the RANKL sheddase, ADAM17. Consistent with these findings, TWEAK and TNF both induced soluble RANKL release by chondrocytes. ConclusionsThe expression and regulation of TWEAK and Fn14 is a feature of human OA articular cartilage, suggesting a pathogenetic role. TNF may prime chondrocytes for TWEAK reactivity by upregulating Fn14 expression. The TWEAK and TNF induction of ADAM17 expression and sRANKL release by chondrocytes points to a further disease pathway in human OA.

pathology↗

bioRxiv2025

Chronic Staphylococcus aureus infection of osteocytes promotes an intracellular viable-but-non-culturable phenotype

Osteomyelitis associated with periprosthetic joint infection (PJI) is a serious and growing complication of orthopaedic joint replacement surgery, with a failure to cure rate within 2-years of between 15-46%. PJI is a severe chronic infectious disease with a 20% recurrence rate and culture-negativity in 7-39% of cases, of which 14-27% are PCR-positive, findings that remain poorly explained. Osteocytes, the most numerous bone cell type, have been identified as potential long-term intracellular reservoirs for Staphylococcus aureus, shielding it from antimicrobial treatment. Here we demonstrate that in chronic intra-osteocytic infections, S. aureus consistently adopts a viable-but-non-culturable (VBNC) state, capable of spontaneous host cell escape and reversion to active growth. Whole-genome sequencing identified single nucleotide polymorphisms (SNPs) in gene pairs between clones. Two revertant clones exhibited SNPs in srrB and murA2, and five exhibited SNPs in garR and upstream of tatB. While the functional importance of these changes is uncertain, they suggest that diverse genetic mechanisms underly entrance into and exit from the intracellular VBNC state. These findings provide a compelling explanation for culture-negative, PCR-positive osteomyelitis and have direct implications for the diagnosis and treatment of PJI.

microbiology↗

bioRxiv2023

Antibiotics at Clinical Concentrations Show Limited Effectivity Against Acute and Chronic Intracellular S. aureus Infections in Osteocytes

ObjectivesCase numbers of osteomyelitis are rising and chronic infections remain difficult to cure. While it is known that the major pathogen Staphylococcus aureus can persist intracellularly in osteocytes, the effectivity of antibiotics against this condition remains largely unknown. We sought to determine if current clinically utilised antibiotics were capable of clearing an intracellular osteocyte S. aureus infection. MethodsRifampicin, vancomycin, levofloxacin, ofloxacin, amoxicillin, oxacillin, doxycycline, linezolid, gentamicin and tigecycline were assessed for their MIC and minimum bactericidal concentrations (MBC) against 11 S. aureus clinical isolates and the reference strain ATCC 25923, at pH 5.0 and 7.2 to mimic lysosomal and cytoplasmic environments, respectively. Those antibiotics whose bone achievable concentration was commonly above their respective MICs for the strains tested were further assayed in a human osteocyte infection model under either acute or chronic conditions. Osteocyte-like cells were treated at 1, 4 and 10x the MIC for 1 and 7 days following infection (acute model), or after 14 days of infection (chronic model). The intracellular effectivity of each antibiotic was measured in terms of colony forming unit (CFU) reduction, small colony variant (SCV) formation and bacterial mRNA expression change. ResultsOnly rifampicin, levofloxacin and linezolid reduced intracellular CFU numbers significantly in the acute model. The effect was larger after 7 days compared to 1 day of treatment. However, no treatment reduced the quantity of bacterial mRNA, nor prevented non-culturable bacteria from returning to a culturable state. DiscussionThese findings indicate that S. aureus adapts phenotypically during intracellular infection of osteocytes, adopting a reversible quiescent state which is protected against antibiotics, even at 10x their MIC. Thus, new therapeutic approaches are necessary to cure S. aureus intracellular infections in osteocytes.

microbiology↗

bioRxiv2023

Beyond the Colony-Forming-Unit: Rapid Bacterial Evaluation in Osteomyelitis

Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony-forming-unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.

microbiology↗

bioRxiv2023

Failure to clear intra-osteocytic Staphylococcus aureus is not the result of antibiotic-mediated autophagy dysregulation

BackgroundStaphylococcus aureus is a major causative pathogen of osteomyelitis. The intracellular infection of osteocytes and related bone cells can persist despite application of gold-standard clinical interventions. The mechanisms by which intracellular S. aureus persists during antibiotic therapy are unknown. In this study, we apply S. aureus to an in vitro model of differentiated osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. MethodsHuman osteocyte-like cells were exposed to combinations of rifampicin, vancomycin and modulators of autophagy, in the presence or absence of S. aureus. Intracellular bacterial growth characteristics were assessed through CFU analysis, viable bacterial DNA abundance and the rate of escape into antibiotic-free medium, in parallel with measures of host cell autophagic flux. ResultsRifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of the intracellular bacterial community, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, while the modulation of autophagic flux affected bacterial culturability, this modulation did not affect viable bacterial DNA levels. ConclusionsAutophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. Whilst rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability while failing to clear bacterial DNA and hence intracellular bacterial load. Thus, whilst rifampicin and vancomycin exhibited off-target effects that modulated autophagy in osteocyte-like cells, this could not explain the persistent infection observed for S. aureus.

microbiology↗

bioRxiv2023

Culture of Saos-2 cells under hypoxic conditions stimulates rapid differentiation to an osteocyte-like stage

1.Few human osteocyte in vitro models exist and the differentiation of immature osteoblasts to an osteocyte stage typically takes at least 4-weeks of culture, making the study of this process challenging and time consuming. The osteosarcoma cell line Saos-2 has proved to be a useful model of human osteoblast differentiation through to a mature osteocyte-like stage. Culture under osteogenic conditions in a standard 5% CO2 and normoxic (21% O2) atmosphere results in reproducible mineralisation and acquisition of mature osteocyte markers over the expected 28-35 day culture period. In order to expedite experimental assays, we tested whether reducing available oxygen to mimic concentrations experienced by osteocytes in vivo would increase the rate of differentiation of Saos-2 cells. Cells cultured in a 5% CO2, 1% O2 atmosphere exhibited accelerated deposition of mineral, reaching near saturation by 14 days as demonstrated with the Alizarin Red and Von Kossa staining. The gene expression of the major hypoxia-induced transcription factor HIF1 and the key osteogenic transcription factor RUNX2 were both elevated under 1% O2. Early (COLA1, MEPE) and mature (PHEX, DMP1 and SOST) osteocyte markers were also upregulated earlier under hypoxic compared to normoxic growth conditions. Thus, culture under low oxygen accelerates key markers of osteocyte differentiation, resulting in a useful human osteocyte-like in vitro cell model within 14 days.

cell biology↗

bioRxiv2023

Microstructural and cellular characterisation of the subchondral trabecular bone in human knee and hip osteoarthritis using synchrotron tomography

ObjectiveIt is unclear if different factors influence osteoarthritis (OA) progression and the changes characterising OA disease in hip and knee. We investigated the difference between hip OA and knee OA at the subchondral bone tissue and cellular level, relative to the degree of cartilage degeneration. DesignBone samples were collected from 11 patients (aged 70{+/-}8 years) undergoing knee arthroplasty and 8 patients (aged 64{+/-}12 years) undergoing hip arthroplasty surgery. Bone microstructure, osteocyte-lacunar network and bone matrix vascularity were evaluated using synchrotron micro-CT imaging. Samples were additionally examined histologically to determine osteocyte density, viability, and connectivity. ResultsAfter adjustment for donor gender and age, associations between the extent of cartilage degeneration, bone volume fraction [8.7, 95% CI (3.4, 14.1)], trabecular number [1.5, 95% CI (0.8, 2.3)], osteocyte lacunar density [4714.9; 95% CI (2079.1, 7350.6)] and trabecular separation [-0.06, 95% CI (0.01, 0.1)] were found in both knee and hip OA. When compared to knee OA, hip OA was characterised by higher trabecular thickness [0.006, 95% CI (-4, 0.01)], larger but less spheric osteocyte lacunae [47.3; 95% CI (11.2, 83.4), -0.04; 95% CI (-0.6, -0.01), respectively], lower vascular canal density [-22.8; 95% CI (-35.4, -10.3)] lower osteocyte density [-84.9; 95% CI (-102.4, -67.4)], and less senescent but more apoptotic osteocytes [-2.4; 95% CI (-3.6, -1.2), 24.9; 95% CI (17.7, 32.1)], respectively. ConclusionSubchondral bone from hip OA and knee OA exhibits different characteristics at the tissue and cellular levels, suggesting different mechanisms of OA progression between the hip and knee joints.

pathology↗

bioRxiv2022

The Effects of Vitamin E Analogues α-Tocopherol and γ-Tocotrienol on the Human Osteocyte Response to Ultra-high Molecular Weight Polyethylene Wear Particles

Polyethylene (PE) liners are a common bearing surface of orthopaedic prostheses. Wear particles of ultra-high molecular weight PE (UHMWPE) contribute to periprosthetic osteolysis, a major cause of aseptic loosening. Vitamin E is added to some PE liners to prevent oxidative degradation. Osteocytes, an important cell type for controlling both bone mineralisation and bone resorption, have been shown to respond UHMWPE particles by upregulating pro-osteoclastogenic and osteocytic osteolysis. Here, we examined the effects of the vitamin E analogues -tocopherol and {gamma}-tocotrienol alone or in the context of UHMWPE particles on human osteocyte gene expression and mineralisation behaviour. Human osteoblasts differentiated to an osteocyte-like stage were exposed to UHMWPE wear particles in the presence or absence of either -Tocopherol or {gamma}-Tocotrienol. Both -Tocopherol and {gamma}-Tocotrienol induced antioxidant-related gene expression. UHMWPE particles independently upregulated antioxidant gene expression, suggesting an effect of wear particles on oxidative stress. Both vitamin E analogues strongly induced OPG mRNA expression and {gamma}-Tocotrienol also inhibited RANKL mRNA expression, resulting in a significantly reduced RANKL:OPG mRNA ratio (p < 0.01) overall. UHMWPE particles reversed the suppressive effect of -Tocopherol but not of {gamma}-Tocotrienol on this pro-osteoclastogenic index. UHMWPE particles also upregulated osteocytic-osteolysis related gene expression. Vitamin E analogues alone or in combination with UHMWPE particles also resulted in upregulation of these genes. Consistent with this, both vitamin E analogues promoted calcium release from mineralised cultures of osteocyte-like cells. Our findings suggest that while vitamin E may suppress osteocyte support of osteoclastogenesis in the presence of UHMWPE particles, the antioxidant effect may induce osteocytic osteolysis, which could promote periprosthetic osteolysis. It will be important to conduct further studies of vitamin E to determine the long-term effects of its inclusion in prosthetic materials.

cell biology↗

bioRxiv2020

Relationships between the Bone Expression of Alzheimer Disease-Related Genes, Bone Remodelling Genes and Cortical Bone Structure in Neck of Femur Fracture

Neck of femur (NOF) fracture is a prevalent fracture type amongst the ageing and osteoporotic populations, commonly requiring total hip replacement (THR) surgery. Increased fracture risk has also been associated with Alzheimer disease (AD) in the aged. Here, we sought to identify possible relationships between the pathologies of osteoporosis and dementia by analysing bone expression of neurotropic or dementia-related genes in patients undergoing THR surgery for NOF fracture. Femoral bone samples from 66 NOF patients were examined for expression of the neurotropic genes amyloid precursor protein (APP), APP-like protein-2 (APLP2), Beta Secretase Cleaving Enzyme-1 (BACE1) and nerve growth factor (NGF). Relationships were examined between the expression of these and of bone regulatory genes, systemic factors and bone structural parameters ascertained from plain radiographs. We found strong relative levels of expression and positive correlations between APP, APLP2, BACE1 and NGF levels in NOF bone. Significant correlations were found between APP, APLP2, BACE1 mRNA levels and bone remodelling genes TRAP, RANKL, and the RANKL:OPG mRNA ratio, indicative of potential functional relationships at the time of fracture. Analysis of the whole cohort, as well as non-dementia and dementia sub-groups, revealed structural relationships between APP and APLP2 mRNA expression and lateral femoral cortical thickness. These findings suggest that osteoporosis and AD may share common molecular pathways of disease progression, perhaps explaining the common risk factors associated with these diseases. The observation of a potential pathologic role for AD-related genes in bone may also provide alternative treatment strategies for osteoporosis and fracture prevention.

physiology↗