Bacterial invasion of the submicron osteocyte lacuna-canaliculi network (OLCN): a part of osteomyelitis disease biology.

Two chronic osteomyelitis patients, a diabetic foot osteomyelitis patient and a fracture-related infection patient, all with staphylococci-positive microbiology, were examined to confirm the clinical relevance of bacterial invasion of the submicron osteocyte lacuna-canaliculi network (OLCN) in bone tissue. Based on immunohistochemistry and light microscopy both Staphylococcus aureus and Staphylococcus epidermidis were identified within the OLCN of all four patients. The findings consolidate that bacterial OLCN invasion is a clinically relevant part of osteomyelitis disease biology, which from experimental porcine infections, seems to be time depending. The microscopy pictures of the four patients significantly add to visualize the phenomenon of bacterial OLCN invasion.

Gram-Negative Bacteria Are Internalized Into Osteocyte-Like Cells.

While Gram-positive organisms are the most common causative agent of initial bone infections, the percentage of Gram-negative species increases in reoccurring bone infections. As bacterial internalization has been suggested as one cause of reoccurring bone infection, we tested the hypothesis that Gram-negative species of bacteria can be internalized into bone cells. Using the MLO-A5 and the MLO-Y4 cell lines as our cell models, we demonstrated that the Gram-negative species, Proteus mirabilis and Serratia marcescens, can be internalized in these cells using an internalization assay. This rate at which these two species were internalized was both time- and initial concentration-dependent. Confocal analysis demonstrated the presence of internalized bacteria within both cell types. Inhibition of the cellular uptake with methyl-ß-cyclodextrin and chloroquine both reduced internalized bacteria, indicating that this process is, at least in part, cell mediated. Finally, we demonstrated that the presence of internalized P. mirabilis did not impact cell viability, measured either by lactate dehydrogenase (LDH) release or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity, while the presence of S. marcescens, on the other hand, both increased LDH release and reduced MTT activity, indicating a loss of cell viability in response to the organism. These results indicated that both species of Gram-negative bacteria can be internalized by bone cells and that these internalized bacteria could potentially result in reoccurring bone infections. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:861-870, 2020.

Mechanisms of Immune Evasion and Bone Tissue Colonization That Make Staphylococcus aureus the Primary Pathogen in Osteomyelitis.

PURPOSE OF REVIEW: Staphylococcus aureus is the primary pathogen responsible for osteomyelitis, which remains a major healthcare burden. To understand its dominance, here we review the unique pathogenic mechanisms utilized by S. aureus that enable it to cause incurable osteomyelitis. RECENT FINDINGS: Using an arsenal of toxins and virulence proteins, S. aureus kills and usurps immune cells during infection, to produce non-neutralizing pathogenic antibodies that thwart adaptive immunity. S. aureus also has specific mechanisms for distinct biofilm formation on implants, necrotic bone tissue, bone marrow, and within the osteocyte lacuno-canicular networks (OLCN) of live bone. In vitro studies have also demonstrated potential for intracellular colonization of osteocytes, osteoblasts, and osteoclasts. S. aureus has evolved a multitude of virulence mechanisms to achieve life-long infection of the bone, most notably colonization of OLCN. Targeting S. aureus proteins involved in these pathways could provide new targets for antibiotics and immunotherapies.

An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis.

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 µm non-motile cocci, S. aureus deforms smaller than 0.5 µm in the sub-micron channels of the OLCN. Here we present the µSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The µSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the µSiM-CA. However the µSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.

Inmunopatología de la brucelosis osteoarticular / Immunopathology of osteoarticular brucelosis

La brucelosis es una de las enfermedades zoonóticas más importantes a nivel mundial capaz de producir enfermedad crónica en los seres humanos. La localización osteoarticular es la presentación más común de la enfermedad activa en el hombre. Sin embargo, algunos de los mecanismos moleculares implicados en la enfermedad osteoarticular han comenzado a dilucidarse recientemente. Brucella abortus induce daño óseo a través de diversos mecanismos en los cuales están implicados TNF-α y RANKL. En estos procesos participan células inflamatorias que incluyen monocitos/macrófagos, neutrófilos, linfocitos T del tipo Th17 y linfocitos B. Además, B. abortus puede afectar directamente las células osteoarticulares. La bacteria inhibe la deposición de la matriz ósea por los osteoblastos y modifica el fenotipo de estas células para producir metaloproteinasas de matriz (MMPs) y la secreción de citoquinas que contribuyen a la degradación del hueso. Por otro lado, la infección por B. abortus induce un aumento en la osteoclastogénesis, lo que aumenta la resorción de la matriz ósea orgánica y mineral y contribuye al daño óseo. Dado que la patología inducida por Brucella afecta el tejido articular, se estudió el efecto de la infección sobre los sinoviocitos. Estos estudios revelaron que, además de inducir la activación de estas células para secretar quemoquinas, citoquinas proinflamatorias y MMPs, la infección inhibe la muerte por apoptosis de los sinoviocitos. Brucella es una bacteria intracelular que se replica en el retículo endoplásmico de los macrófagos. El análisis de los sinoviocitos infectados con B. abortus indicó que las bacterias también se multiplican en el retículo endoplasmático, lo que sugiere que la bacteria podría usar este tipo celular para la multiplicación intracelular durante la localización osteoarticular de la enfermedad. Los hallazgos presentados en esta revisión intentan responder a preguntas sobre los mediadores inflamatorios implicados en el daño osteoarticular causado por Brucella. (AU)

Brucellosis is one of the most important zoonotic diseases that can produce chronic disease in humans worldwide. Osteoarticular involvement is the most common presentation of human active disease. The molecular mechanisms implicated in bone damage have started to be elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and RANKL are implicated. These processes are driven by inflammatory cells, including monocytes/macrophages, neutrophils, Th17 lymphocytes and B cells. Also, Brucella abortus (B. abortus) can directly affect osteoarticular cells. The bacterium inhibits bone matrix deposition by osteoblast and modifies the phenotype of these cells to produce matrix methalloproteinases (MMPs) and cytokine secretion that contribute to bone matrix degradation. B. abortus also affects osteoclast increasing mineral and organic bone matrix resorption and contributing to bone damage. Since the pathology induced by Brucella species involves joint tissue, experiments conducted in sinoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits sinoviocyte apoptosis. Brucella is an intracellular bacterium that replicate in the endoplasmic reticulum of macrophages. The analysis of B. abortus infected sinoviocytes indicated that bacteria also replicate in their reticulum suggesting that the bacterium could use this cell type for intracellular replication during the osteoarticular localization of the disease. The findings presented in this review try to answer key questions about the inflammatory mediators involved in osteoarticular damage caused by Brucella. (AU)

Endocrine modulation of Brucella abortus-infected osteocytes function and osteoclastogenesis via modulation of RANKL/OPG.

Osteoarticular brucellosis is the most frequent complication of active disease. A large amount of cells in bone are osteocytes. Since bone remodeling process is regulated by hormones we sought to study the effect of cortisol and DHEA in Brucella abortus-infected osteocytes. Cortisol treatment inhibited the expression of IL-6, TNF-α, MMP-2 and RANKL in B. abortus-infected osteocytes. DHEA could reverse the inhibitory effect of cortisol on MMP-2 production. B. abortus infection inhibited connexin 43 (Cx43) expression in osteocytes. This expression was increased when cortisol was incorporated during the infection and DHEA treatment partially reversed the effect of cortisol. Osteocytes-infected with B. abortus induced osteoclast's differentiation. Yet, the presence of cortisol, but not DHEA, during osteocyte infection inhibited osteoclastogenesis. Glucocorticoid receptor (GR) is implicated in the signaling of cortisol. Infection with B. abortus was able to increase GRα/ß ratio. Levels of intracellular cortisol are not only dependent on GR expression but also a result of the activity of the isoenzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 (cortisone to cortisol conversion), 11ß-HSD2 (cortisol to cortisone conversion). B. abortus infection increased 11ß-HSD 1/2 ratio and cortisone mimicked the effect of cortisol. Our results indicated that cortisol and DHEA could modulate osteocyte responses during B. abortus infection.

Novel Insights into Staphylococcus aureus Deep Bone Infections: the Involvement of Osteocytes.

Periprosthetic joint infection (PJI) is a potentially devastating complication of orthopedic joint replacement surgery. PJI with associated osteomyelitis is particularly problematic and difficult to cure. Whether viable osteocytes, the predominant cell type in mineralized bone tissue, have a role in these infections is not clear, although their involvement might contribute to the difficulty in detecting and clearing PJI. Here, using Staphylococcus aureus, the most common pathogen in PJI, we demonstrate intracellular infection of human-osteocyte-like cells in vitro and S. aureus adaptation by forming quasi-dormant small-colony variants (SCVs). Consistent patterns of host gene expression were observed between in vitro-infected osteocyte-like cultures, an ex vivo human bone infection model, and bone samples obtained from PJI patients. Finally, we confirm S. aureus infection of osteocytes in clinical cases of PJI. Our findings are consistent with osteocyte infection being a feature of human PJI and suggest that this cell type may provide a reservoir for silent or persistent infection. We suggest that elucidating the molecular/cellular mechanism(s) of osteocyte-bacterium interactions will contribute to better understanding of PJI and osteomyelitis, improved pathogen detection, and treatment.IMPORTANCE Periprosthetic joint infections (PJIs) are increasing and are recognized as one of the most common modes of failure of joint replacements. Osteomyelitis arising from PJI is challenging to treat and difficult to cure and increases patient mortality 5-fold. Staphylococcus aureus is the most common pathogen causing PJI. PJI can have subtle symptoms and lie dormant or go undiagnosed for many years, suggesting persistent bacterial infection. Osteocytes, the major bone cell type, reside in bony caves and tunnels, the lacuno-canalicular system. We report here that S. aureus can infect and reside in human osteocytes without causing cell death both experimentally and in bone samples from patients with PJI. We demonstrate that osteocytes respond to infection by the differential regulation of a large number of genes. S. aureus adapts during intracellular infection of osteocytes by adopting the quasi-dormant small-colony variant (SCV) lifestyle, which might contribute to persistent or silent infection. Our findings shed new light on the etiology of PJI and osteomyelitis in general.

Chronic Osteomyelitis with Staphylococcus aureus Deformation in Submicron Canaliculi of Osteocytes: A Case Report.

CASE: A patient presenting with an infected diabetic foot ulcer and Staphylococcus aureus chronic osteomyelitis was studied to validate the clinical importance of bacterial colonization of osteocytic-canalicular networks, as we recently reported in a mouse model. We utilized transmission electron microscopy to describe the deformation of S. aureus, from round cocci to rod-shaped bacteria, in the submicron osteocytic-canalicular networks of amputated bone tissue. CONCLUSION: To our knowledge, this is the first evidence of S. aureus deformation and invasion of the osteocytic-canalicular system in human bone, which supports a new mechanism of persistence in the pathogenesis of chronic osteomyelitis.

Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion.

Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-ß, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage.

Autogenous bone grafts contamination after exposure to the oral cavity.

The purpose of this paper was to analyze specimens of autogenous bone block grafts exposed to the oral cavity after ridge reconstructions. Specimens of chronic suppurative osteomyelitis (CSO) of the jaws were used as comparison for bacterial colonization pattern. For this, 5 specimens of infected autogenous bone grafts were used and 10 specimens of CSO embedded in paraffin were stained with Brown and Brenn technique and analyzed under light microscopy. The results showed a similar colonization pattern in both situations, with the establishment of bacterial biofilm and the predominance of Gram-positive bacteria. The conclusion was that the similarity in bacterial distribution and colonization between autogenous bone grafts and CSO stresses the necessity of more invasive procedures for the treatment of the autogenous bone grafts early exposed to the oral cavity.

Molecular characterization of Treponema denticola infection-induced bone and soft tissue transcriptional profiles.

Treponema denticola is associated with subgingival biofilms in adult periodontitis and with acute necrotizing ulcerative gingivitis. However, the molecular mechanisms by which T. denticola impacts periodontal inflammation and alveolar bone resorption remain unclear. Here, we examined changes in the host transcriptional profiles during a T. denticola infection using a murine calvarial model of inflammation and bone resorption. T. denticola was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and the calvarial bones were excised. RNA was isolated and analysed for transcript profiling using Murine GeneChip arrays. Following T. denticola infection, 2905 and 1234 genes in the infected calvarial bones and soft tissues, respectively, were differentially expressed (P

Detection of canine distemper virus in bone cells in the metaphyses of distemper-infected dogs.

In the light of recent evidence implicating canine distemper virus (CDV) as a possible etiologic agent in Paget's disease of bone, we thought that it would be of interest to examine distemper-infected bone in the natural host. Samples from the long bones, spleen, and bladder of four distemper-infected and three uninfected dogs were examined for the presence of CDV nucleocapsid and phosphoprotein genes and the measles virus (MV) nucleocapsid gene using the technique of in situ hybridization with radioactively labeled riboprobes. Two of the four distemper-infected dogs showed strongly positive hybridization with both of the CDV probes. The signal was present in marrow cells, in osteoblasts, in osteocytes, and particularly in osteoclasts. No hybridization was seen over the cartilage cells of the growth plate, and there was a clear line of demarcation at the point of invasion of osteoclasts and vascularization. The spleen and bladder samples from infected dogs also showed positive hybridization. There was no hybridization with the MV probe in any of the distemper-infected tissue. Samples from the uninfected dogs showed no evidence of hybridization with either the CDV or MV probes. These results show that CDV can infect bone cells of the natural host and provide further support for the theory that CDV may play a role in human Paget's disease of bone.

Retrovirus-induced osteopetrosis in mice. Ultrastructural evidence of early virus production in osteoblasts and osteocytes.

Newborn female strain NMRI mice were given injections of a mouse retrovirus (OA MuLV) known to induce osteopetrosis, osteoma, and lymphoma. Femur metaphyses and lumbar vertebrae were investigated ultrastructurally 3 d, 7 d and 28 d after infection. Budding, immature and mature virus was observed associated with osteoblasts and osteocytes, but not with osteoclasts or chondrocytes, 28 d after infection with the virus. No production of virus particles was observed in bone-tissue in mock-treated controls. Thus, the primary target cell for OA virus in bone appears to belong to the osteoblastic/osteocytic cell lineage.

Measles virus RNA detected in Paget's disease bone tissue by in situ hybridization.

Morphological and immunocytological studies have demonstrated the presence of paramyxovirus antigens in Paget's bone disease tissue and in particular antigens related to measles virus and respiratory syncytial virus. To examine the relationship between measles virus and Paget's bone disease we used in situ hybridization and a cloned measles virus DNA probe specific for the nucleocapsid protein to detect and locate measles virus RNA sequences in Paget's bone tissue. In five patients with the disease, measles virus RNA sequences were detected not only in 80 to 90% of the multinucleated osteoclasts where there is morphological and immunocytological evidence of measles virus activity but also in 30 to 40% of mononucleated bone cells, mainly osteoblasts, osteocytes, fibroblasts and lympho-monocytes. In contrast, no hybridization was observed in bone tissue from three control patients without signs of Paget's bone disease. These results indicate that the host cell range for measles virus in Paget's disease is more widespread than has been supposed. They also demonstrate the usefulness of the in situ hybridization method to detect viral genetic information in cells where viral antigenic activity is not detectable. These observations further support the hypothesis that measles virus is involved in the pathogenesis of Paget's bone disease.

Pleomorphic C-type virus particles in bone tissue of strain 101 mice and (C3H X 101)F1 hybrids.

The proximal tibial metaphysis of apparently healthy strain 101 mice, 3-4 weeks old, and (C3H X 101)F1 hybrids, 3-48 weeks old, was studied by electron microscopy. Budding, immature, and mature (C-type virus particles were found within trabecular bone tissue of 3 of 8 strain 101 and 4 of 12 (C3H X 101)F1 mice. The particles were most common in lacunae of aging osteocytes and were only occasionally associated with osteoblasts. Although the morphology of budding and immature particles appeared to be identical with that of typical C-type viruses, most of the mature forms of particles showed atypical structure and size. The electron-dense core was very large and not clearly defined, measuring approximately 70-130 nm in diameter. This diffuse core sometimes completely filled the space within the envelope of the particles. The diameters of the pleomorphic mature C-type particles ranged from approximately 90 to 150 nm. The possible association between the production of pleomorphic C-type virus particles by bone cells and spontaneous osteomagenesis in 101 and (C3H X 101)F1 mice is discussed.

Mode of C-type virus production by bone cells of C3H/Fg mice.

Electron microscopy of bone from the distal ends of femurs of six 3-month-old male C3H/Fg mice revealed varying modes and quantities of C-type virus production by cells in the preosteoblast to osteocyte developmental gradient. Production occurred by budding from the cell surface, either from very short pedicles or from longer microvillous-like processes. Although apparently absent in preosteoblasts, limited numbers of budding viruses (90-110 nM) were seen in 6 of 53 osteoblasts examined. Examination of 200 osteocytes revealed evidence of virus production in all but eight. In these cells, budding occurred from the cell body and its processes with release of viruses into the pericellular lacunar space. Budding from the longer microvillous-like processes sometimes resulted in binding together of viruses in bead-like fashion by thin strands of limiting membrane. In a few cases budding was also seen on the cell processes within the canaliculi. The quantities of viruses present within the lacunae usually increased with increasing age of the osteocytes, indicating continual virus production. Most of the viruses observed, however, were pre C-types along with lesser numbers of mature C particles. Thus, factors contributing to the production of mature C particles may be diminished in osteocytes of mice at this age.

A possible non-oncogenic effect of C type bone cell virus on serum calcium levels of potentially leukemic mice.

In bone of C3H/Fg mice, particles structurally identical to C-type leukemia virus arise from membranes of osteocytes and osteoblasts. Although these virus apparently do not induce morphologic or neoplastic change in bone they may have other, more subtle, effects. Thus, comparison of sera from male C3H/Fg mice, a high leukemia-prone strain, with C57BL and C3H/HeJ mice, low leukemia strains which do not contain C-type virus in bone, reveals that serum calcium levels are significantly lower in the former than in the latter. Further, when C3H/Fg mice develop frank leukemia there is a corresponding increase in virus particles while the serum calcium concentration levels fall to even lower values. The presence of leukemia itself appears not to be the cause as indicated by the failure of implanted lymphocytic leukemic cells in C3H/Fg mice to significantly affect serum calcium concentration. It is postulated that the effects of the virus could be due either to increased osteo blastic activity or to inhibition of osteocytic osteolytic activity or to both.