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To examine and quantify the sexual dimorphism in pathologic features manifested in the musculoskeletal and cardiopulmonary systems and incidence of mortality in the tumor necrosis factor-transgenic (TNF-Tg; Tg3647 strain) mouse model of inflammatory erosive arthritis.Kaplan-Meier survival estimates were determined in male and female Tg3647 mice and sex-matched wild-type (WT) littermate mice. Longitudinal and cross-sectional pathologic outcomes in the musculoskeletal and cardiopulmonary systems were assessed via ultrasound, micro-computed tomography, grip strength measurements, histologic and serologic analyses, flow cytometry, and skeletal muscle physiologic measures.Compared to male Tg3647 mice (n = 30), female Tg3647 mice (n = 34) had significantly shorter lifespans (P0.001) and exhibited the following pathologic features (n = 4-6 per group; P0.05 versus male Tg3647 littermates): gross deficits in body mass and muscle weight, early-onset inflammatory arthritis with severity of end-stage arthritis that was as severe as that seen in male transgenic mice, and early onset and increased severity of inflammatory interstitial lung disease (ILD). Histologically, the ILD observed in Tg3647 mice was characterized by inflammatory cell accumulation and pulmonary arteriole thickening, which was concomitant with the presence of right ventricular hypertrophy, a feature that was also more severe in the female compared to male Tg3647 mice (P0.05). No sexual dimorphisms in TNF-induced deficient grip strength, axial skeletal growth, or bone loss were found. Globally, the extent of the pathologic changes observed in female Tg3647 mice was greater than that observed in male Tg3647 mice when each group was compared to their sex-matched WT littermates.These findings indicate that TNF selectively drives the early onset of arthritis and progression of pathologic changes in the cardiopulmonary system in female Tg3647 mice. These results in the Tg3647 mouse identify it as a suitable model to better understand the mechanisms underlying sexual dimorphism and cardiopulmonary disease in the setting of inflammatory arthritis and other connective tissue diseases.

Methicillin-resistant Staphylococcus aureus (MRSA) reinfection following revision surgery remains a major orthopaedic problem. Toward the development of immunotherapy with anti-glucosaminidase monoclonal antibodies (anti-Gmd), we aimed to: (i) develop a murine 1-stage exchange model of bioluminescent MRSA (USA300LAC::lux) contaminated femoral implants; and (ii) utilize this model to demonstrate the synergistic effects of combination vancomycin and anti-Gmd therapy on reinfection and bone healing. Following an infection surgery, the original plate and two screws were removed on day 7, and exchanged with sterile implants. Mice were randomized to five groups: (i) no infection control; (ii) infected placebo; (iii) anti-Gmd; (iv) vancomycin; and (v) combination therapy. Bioluminescent imaging (BLI) was performed on days 0, 1, 3, 5, 7, 8, 10, 12, and 14. Mice were euthanized on day 14 (day 7 post-revision), and efficacy was assessed via colony forming units (CFU) on explanted hardware, micro-CT, and histology. As monotherapies, anti-Gmd inhibited Staphylococcus abscess communities, and vancomycin reduced CFU on the implants. However, only combination therapy prevented increased BLI post-revision surgery, with a significant 6.5-fold reduction on day 10 (p 0.05 vs. placebo), and achieved sterile implant levels by day 12. Synergistic effects were also apparent from reduced osteolysis and increased new bone formation around the screws only observed following combination therapy. Taken together, we find that: (i) this murine femoral plate 1-stage revision model can efficiently evaluate therapies to prevent reinfection; and (ii) immunotherapy plays a distinct role from antibiotics to reduce reinfection following revision surgery, such that synergy to achieve osseointegration is possible. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1590-1598, 2018.

Although Staphylococcus aureus osteomyelitis is considered to be incurable, the major bacterial reservoir in live cortical bone has remained unknown. In addition to biofilm bacteria on necrotic tissue and implants, studies have implicated intracellular infection of osteoblasts and osteocytes as a mechanism of chronic osteomyelitis. Thus, we performed the first systematic transmission electron microscopy (TEM) studies to formally define major reservoirs of S. aureus in chronically infected mouse (Balb/c J) long bone tissue. Although rare, evidence of colonized osteoblasts was found. In contrast, we readily observed S. aureus within canaliculi of live cortical bone, which existed as chains of individual cocci and submicron rod-shaped bacteria leading to biofilm formation in osteocyte lacunae. As these observations do not conform to the expectations of S. aureus as non-motile cocci 1.0 to 1.5 μm in diameter, we also performed immunoelectron microscopy (IEM) following in vivo BrdU labeling to assess the role of bacterial proliferation in canalicular invasion. The results suggest that the deformed bacteria: (1) enter canaliculi via asymmetric binary fission; and (2) migrate toward osteocyte lacunae via proliferation at the leading edge. Additional in vitro studies confirmed S. aureus migration through a 0.5-μm porous membrane. Collectively, these findings define a novel mechanism of bone infection, and provide possible new insight as to why S. aureus implant-related infections of bone tissue are so challenging to treat. © 2016 American Society for Bone and Mineral Research.

While it is well known that Staphylococcus aureus establishes chronic implant-associated osteomyelitis by generating and persisting in biofilm, research to elucidate pathogen, and host specific factors controlling this process has been limited due to the absence of a quantitative in vivo model. To address this, we developed a murine tibia implant model with ex vivo region of interest (ROI) imaging analysis by scanning electron microscopy (SEM). Implants were coated with Staphylococcus aureus strains (SH1000, UAMS-1, USA300LAC) with distinct in vitro biofilm phenotypes, were used to infect C57BL/6 or Balb/c mice. In contrast to their in vitro biofilm phenotype, results from all bacteria strains in vivo were similar, and demonstrated that biofilm on the implant is established within the first day, followed by a robust proliferation phase peaking on Day 3 in Balb/c mice, and persisting until Day 7 in C57BL/6 mice, as detected by SEM and bioluminescent imaging. Biofilm formation peaked at Day 14, covering ∼40% of the ROI coincident with massive agr-dependent bacterial emigration, as evidenced by large numbers of empty lacunae with few residual bacteria, which were largely culture negative (80%) and PCR positive (87.5%), supporting the clinical relevance of this implant model.

Contrast-enhancement magnetic resonance imaging (CE-MRI) of synovial volume is the radiographic gold standard to quantify joint inflammation; however, cost limits its use. Therefore, we examined if power Doppler-ultrasound (PD-US) outcomes of synovitis in tumor necrosis factor transgenic (TNF-Tg) mice correlate with CE-MRI. TNF-Tg mice underwent PD-US of their knees to measure the joint space volume (JSV) and power Doppler volume (PDV), and the results were correlated with synovial volume determined by CE-MRI. Immunohistochemistry for CD31 was performed to corroborate the PD signal. Synovial volume strongly correlated with both JSV and PDV (p0.01). CD31(+) blood vessels were observed in inflamed synovium proximal to the joint surface, which corresponded to areas of intense PD signals. JSV and PDV are valid measures of joint inflammation that correlate with synovial volume determined by CE-MRI and are associated with vascularity. Given the emergence of PD-US as a nonquantitative outcome of joint inflammation, we find JSV and PDV to be feasible and highly cost-effective for longitudinal studies in animal models. Furthermore, given the increasing use of PD-US in standard clinical practice, JSV and PDV could be translated to better quantify joint flare and response to therapy in patients with rheumatoid arthritis (RA).

Towards the development of a methicillin-resistant Staphylococcus aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus a placebo and a Gmd-deficient isogenic strain (ΔGmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis, and histomorphometry of abscess numbers (p 0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies ΔGmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in ΔGmd cultures or 1C11 treated cultures of a protein A-deficient strain (ΔSpa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p 0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of Staphylococcal abscess communities (SAC) and opsonophagocytosis of megaclusters. .

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with episodic flares. In TNF-Tg mice, a model of inflammatory–erosive arthritis, the popliteal lymph node (PLN) enlarges during the pre-arthritic ‘expanding’ phase, and then ‘collapses’ with adjacent knee flare associated with the loss of the intrinsic lymphatic pulse. As the mechanisms responsible are unknown, we developed in vivo methods to quantify lymph viscosity and pressure in mice with wild-type (WT), expanding and collapsed PLN. While no differences in viscosity were detected via multiphoton fluorescence recovery after photobleaching (MP-FRAP) of injected FITC-BSA, a 32.6% decrease in lymph speed was observed in vessels afferent to collapsed PLN (P

B cell depletion therapy ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritis flare in tumor necrosis factor (TNF)-transgenic mice is associated with efferent lymph node (LN) "collapse," triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. The aim of this study was to examine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells.We used contrast-enhanced magnetic resonance imaging, indocyanine green near-infrared imaging, and intravital immunofluorescence imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-transgenic mice. We conducted tests to determine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic draining and cell egress from arthritic joints.Unlike active lymphatics to normal and prearthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescence imaging demonstrated that CD11b+ monocyte/macrophages in lymphatic vessels afferent to expanding LNs travel at high velocity (mean±SD 186±37 μm/second), while these cells are stationary in lymphatic vessels afferent to collapsed popliteal LNs. B cell depletion therapy for arthritis flares in TNF-transgenic mice significantly decreased knee synovium volume (by 50% from the baseline level) and significantly increased lymphatic clearance compared with placebo (P0.05). This increased lymphatic drainage restored macrophage egress from inflamed joints without recovery of the lymphatic pulse.These results support a novel mechanism in which B cell depletion therapy for joint arthritis flares lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.

Glucocorticoid (GC) therapy is associated with increased risk of fracture in patients with rheumatoid arthritis (RA). To elucidate the cause of this increased risk, we examined the effects of chronic erosive inflammatory arthritis and GC treatment on bone quality, structure, and biomechanical properties in a murine model.Mice with established arthritis and expressing human tumor necrosis factor α (TNFα) transgene (Tg) and their wild-type (WT) littermates were continually treated with GC (prednisolone 5 mg/kg/day via subcutaneous controlled-release pellet) or placebo for 14, 28, or 42 days. Microstructure, biomechanical properties, chemical composition, and morphology of the tibiae and lumbar vertebral bodies were assessed by micro-computed tomography, biomechanical testing, Raman spectroscopy, and histology, respectively. Serum markers of bone turnover were also determined.TNF-Tg and GC treatment additively decreased mechanical strength and stiffness in both the tibiae and the vertebral bodies. GC treatment in the TNF-Tg mice increased the ductility of tibiae under torsional loading. These changes were associated with significant alterations in the biochemical and structural composition of the mineral and organic components of the bone matrix, a decrease in osteoblast activity and bone formation, and an increase in osteoclast activity.Our findings indicate that the concomitant decrease in bone strength and increase in bone ductility associated with chronic inflammation and GC therapy, coupled with the significant changes in the bone quality and structure, may increase the susceptibility of the bone to failure under low-energy loading. This may explain the mechanism of symptomatic insufficiency fractures in patients with RA receiving GC therapy who do not have radiographic manifestations of fracture.

Bone autografts are considered the gold standard for cranioplasty, although they lead to co-morbidity. Bone allografts are more easily obtained but have low osteogenic potential and fail to integrate into healthy bone. Previously, we showed that, by coating long-bone allografts with freeze-dried recombinant adeno-associated virus (rAAV) vector encoding for an osteogenic gene, enhanced osteogenesis and bone integration were achieved. In this study our aim was to evaluate the bone repair potential of calvarial autografts and allografts coated with either single-stranded rAAV2 vector (SS-rAAV-BMP2) or self-complementary pseudotyped vector (SC-rAAV-BMP2) encoding for bone morphogenetic protein (BMP)2 in a murine cranioplasty model. The grafts were implanted into critical defects in the calvariae of osteocalcin/luciferase (Oc/Luc) transgenic mice, which allowed longitudinal monitoring of osteogenic activity using bioluminescence imaging (BLI). Our results showed that the bioluminescent signal of the SC-rAAV-BMP2-coated allografts was 40% greater than that of the SS-rAAV-BMP2-coated allografts (p0.05) and that the bioluminescent signal of the SS-rAAV-BMP2-coated allografts was not significantly different from the signals of the autografts or uncoated allografts. Micro-computed tomography (μCT) confirmed the significant increase in osteogenesis in the SC-rAAV-BMP2 group compared with the SS-rAAV-BMP2 group (p0.05), indicating a significant difference in bone formation when compared with the other grafts tested. In addition, histological analysis revealed extensive remodelling of the autografts. Collectively, these results demonstrate the feasibility of craniofacial regeneration using SC-rAAV-BMP2-coated allografts, which may be an attractive therapeutic solution for repair of severe craniofacial bone defects.

CD23(+)CD21(high)CD1d(high) B cells in inflamed nodes (Bin cells) accumulate in the lymph nodes (LNs) draining inflamed joints of the TNF-α-transgenic mouse model of rheumatoid arthritis and are primarily involved in the significant histological and functional LN alterations that accompany disease exacerbation in this strain. In this study, we investigate the origin and function of Bin cells. We show that adoptively transferred GFP(+) sorted mature follicular B (FoB) cells home preferentially to inflamed LNs of TNF-α-transgenic mice where they rapidly differentiate into Bin cells, with a close correlation with the endogenous Bin fraction. Bin cells are also induced in wild-type LNs after immunization with T-dependent Ags and display a germinal center phenotype at higher rates compared with FoB cells. Furthermore, we show that Bin cells can capture and process Ag-immune complexes in a CD21-dependent manner more efficiently than can FoB cells, and they express greater levels of MHC class II and costimulatory Ags CD80 and CD86. We propose that Bin cells are a previously unrecognized inflammation-induced B cell population with increased Ag capture and activation potential, which may facilitate normal immune responses but may contribute to autoimmunity when chronic inflammation causes their accumulation and persistence in affected LNs.

To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor beta (TGFbeta) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation.Joint disease in Smad3-knockout (Smad3(-/-)) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3(-/-) mice.In Smad3(-/-) mice, an end-stage OA phenotype gradually developed. TGFbeta-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3(-/-) mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3(-/-) cells restored the normal p38 response to TGFbeta. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3(-/-) chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms alpha, beta, and gamma, but not delta.Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3-phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.

In contrast to rheumatoid arthritis (RA), the joint inflammation referred to as Jaccoud's arthritis that occurs in systemic lupus erythematosus (SLE) is nonerosive. Although the mechanism responsible is unknown, the antiosteoclastogenic cytokine interferon-alpha (IFNalpha), whose transcriptome is present in SLE monocytes, may be responsible. This study was undertaken to examine the effects of IFNalpha and lupus on osteoclasts and erosion in the (NZB x NZW)F(1) mouse model of SLE with K/BxN serum-induced arthritis.Systemic IFNalpha levels in (NZB x NZW)F(1) mice were elevated by administration of AdIFNalpha. SLE disease was marked by anti-double-stranded DNA (anti-dsDNA) antibody titer and proteinuria, and Ifi202 and Mx1 expression represented the IFNalpha transcriptome. Microfocal computed tomography was used to evaluate bone erosions. Flow cytometry for CD11b and CD11c was used to evaluate the frequency of circulating osteoclast precursors (OCPs) and myeloid dendritic cells (DCs) in blood.Administration of AdIFNalpha to (NZB x NZW)F(1) mice induced osteopetrosis. (NZB x NZW)F(1) mice without autoimmune disease were fully susceptible to focal erosions in the setting of serum-induced arthritis. However, (NZB x NZW)F(1) mice with high anti-dsDNA antibody titers and the IFNalpha transcriptome were protected against bone erosions. AdIFNalpha pretreatment of NZW mice before K/BxN serum administration also resulted in protection against bone erosion (r(2) = 0.4720, P0.01), which was associated with a decrease in the frequency of circulating CD11b+CD11c- OCPs and a concomitant increase in the percentage of CD11b+CD11c+ cells (r(2) = 0.6330, P0.05), which are phenotypic of myeloid DCs.These findings suggest that IFNalpha in SLE shifts monocyte development toward myeloid DCs at the expense of osteoclastogenesis, thereby resulting in decreased bone erosion.

The effects of antiresorptive agents (e.g., alendronate [Aln], osteoprotegerin [OPG]) on bone infection are unknown. Thus, their effects on implant-associated osteomyelitis (OM) were investigated in mice using PBS (placebo), gentamycin, and etanercept (TNFR:Fc) controls. None of the drugs affected humoral immunity, angiogenesis, or chronic infection. However, the significant (P0.05 vs. PBS) inhibition of cortical osteolysis and decreased draining lymph node size in Aln- and OPG-treated mice was associated with a significant (P0.05) increase in the incidence of high-grade infections during the establishment of OM. In contrast, the high-grade infections in TNFR:Fc-treated mice were associated with immunosuppression, as evidenced by the absence of granulomas and presence of Gram(+) biofilm in the bone marrow. Collectively, these findings indicate that although antiresorptive agents do not exacerbate chronic OM, they can increase the bacterial load during early infection by decreasing lymphatic drainage and preventing the removal of necrotic bone that harbors the bacteria.

This study was undertaken to investigate the effect of lymphatic inhibition on joint and draining lymph node (LN) pathology during the course of arthritis progression in mice.Tumor necrosis factor (TNF)-transgenic mice were used as a model of chronic inflammatory arthritis. Mice were subjected to contrast-enhanced magnetic resonance imaging to obtain ankle and knee joint synovial volumes and draining popliteal LN volumes before and after 8 weeks of treatment with vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody, VEGFR-2 neutralizing antibody, or isotype IgG. Animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining popliteal LNs. Histologic, immunohistochemical, and reverse transcriptase-polymerase chain reaction analyses were used to examine lymphatic vessel formation and the morphology of joints and popliteal LNs.Compared with IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of popliteal LNs, the number of lymphatic vessels in joints and popliteal LNs, lymphatic drainage from paws to popliteal LNs, and the number of VEGF-C-expressing CD11b+ myeloid cells in popliteal LNs. However, it increased the synovial volume and area of inflammation in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation.These findings indicate that lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.