Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 µm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (µSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

Relapsed boyhood tibia polymicrobial osteomyelitis linked to dermatophytosis: a case report.

BACKGROUND: Relapsed childhood polymicrobial osteomyelitis associated with dermatophytosis has not been reported in the literature. CASE PRESENTATION: Here we report on a case of a 45-year-old man who had left tibial osteomyelitis for 29 years, accompanied by skin fungal infection of the ipsilateral heel for 20 years, and underwent a second operation due to recurrence of polymicrobial infection 6 years ago. The patient had a history of injury from a rusty object, which penetrated the anterior skin of the left tibia middle segment causing subsequent bone infection, but was asymptomatic after receiving treatments in 1983. The patient was physically normal until dermatophytosis occurred on the ipsilateral heel skin in 1998. The patient complained that the dermatophytosis was gradually getting worse, and the tibial wound site became itchy, red, and swollen. The left tibial infection resurged in May 2012, leading to the patient receiving debridement and antibiotic treatment. H&E and Gram-stained histology was performed on biopsy specimens of sequestrum and surrounding inflammatory tissue. Tissue culture and microbiology examination confirmed polymicrobial infection with Staphylococcus aureus (S. aureus) and Corynebacterium and a fungus. Additionally, the patient also received potassium permanganate for dermatophytosis when he was admitted into the hospital. CONCLUSIONS: Together with longitudinal follow-up of medical history, surgical findings, histopathological and microbiology culture evidence, we conclude that boyhood tibia polymicrobial osteomyelitis with S. aureus and Corynebacterium occurred in this patient, and the fungal activation of dermatophytosis may have led to osteomyelitis relapse.

Quantitative flow chamber system for evaluating in vitro biofilms and the kinetics of S. aureus biofilm formation in human plasma media.

BACKGROUND: It has been well established that biofilm formation on orthopaedic implants is a critical event in the pathogenesis of orthopaedic infections, yet the natural history of this process with respect to bacterial adhesion, proliferation, and glycocalyx matrix production remains poorly understood. Moreover, there are no quantitative methods yet available to assess the differences in biofilm formation between different bacterial strains or implant materials. Consequently, this study aimed to investigate the natural history of S. aureus in in vitro biofilm formation in human plasma media using a flow chamber system. Bioluminescent S. aureus strains were used to better understand the bacterial growth and biofilm formation on orthopaedic materials. Also, the effects of human plasma media were assessed by loading the chamber with Tryptic Soy Broth with 10% human plasma (TSB + HP). RESULTS: Scanning electron microscopy (SEM) was utilized to assess the morphological appearance of the biofilms, revealing that S. aureus inoculation was required for biofilm formation, and that the phenotypes of biofilm production after 24 h inoculation with three tested strains (SH1000, UAMS-1, and USA300) were markedly different depending on the culture medium. Time course study of the bioluminescence intensity (BLI) and biofilm production on the implants due to the UAMS-1 and USA300 strains revealed different characteristics, whereby UAMS-1 showed increasing BLI and biofilm growth until peaking at 9 h, while USA300 showed a rapid increase in BLI and biofilm formation at 6 h. The kinetics of biofilm formation for both UAMS-1 and USA300 were also supported and confirmed by qRT-PCR analysis of the 16S rRNA gene. Biofilms grown in our flow chamber in the plasma media were also demonstrated to involve an upregulation of the biofilm-forming-related genes icaA, fnbA, and alt. The BLI and SEM results from K-wire experiments revealed that the in vitro growth and biofilm formation by UAMS-1 and USA300 on stainless-steel and titanium surfaces were virtually identical. CONCLUSION: We demonstrated a novel in vitro model for S. aureus biofilm formation with quantitative BLI and SEM outcome measures, and then used this model to demonstrate the presence of strain-specific phenotypes and its potential use to evaluate anti-microbial surfaces.

TNF-Induced Interstitial Lung Disease in a Murine Arthritis Model: Accumulation of Activated Monocytes, Conventional Dendritic Cells, and CD21+/CD23- B Cell Follicles Is Prevented with Anti-TNF Therapy.

Interstitial lung disease (ILD) is a well-known extra-articular manifestation of rheumatoid arthritis (RA). RA-associated ILD (RA-ILD) exists on a wide spectrum, with variable levels of inflammatory and fibrotic activity, although all subtypes are regarded as irreversible pathologic conditions. In both articular and pulmonary manifestations, TNF is a significant pathogenic factor. Whereas anti-TNF therapy alleviates joint pathologic conditions, it exacerbates fibrotic RA-ILD. The TNF-transgenic (TNF-Tg) murine model of RA develops both inflammatory arthritis and an ILD that mimics a cellular nonspecific interstitial pneumonia pattern dominated by an interstitial accumulation of inflammatory cells with minimal-to-absent fibrosis. Given the model's potential to elucidate the genesis of inflammatory RA-ILD, we aim to achieve the following: 1) characterize the cellular accumulations in TNF-Tg lungs, and 2) assess the reversibility of inflammatory ILD following anti-TNF therapy known to resolve TNF-Tg inflammatory arthritis. TNF-Tg mice with established disease were randomized to anti-TNF or placebo therapy and evaluated with imaging, histology, and flow cytometric analyses, together with wild-type controls. Flow cytometry of TNF-Tg versus wild-type lungs revealed significant increases in activated monocytes, conventional dendritic cells, and CD21+/CD23- B cells that are phenotypically distinct from the B cells in inflamed nodes, which are known to accumulate in joint-draining lymph nodes. In contrast to human RA-ILD, anti-TNF treatment significantly alleviated both joint and lung inflammation. These results identify a potential role for activated monocytes, conventional dendritic cells, and CD21+/CD23- B cells in the genesis of RA-ILD, which exist in a previously unknown, reversible, prefibrotic stage of the disease.

Avian Reticuloendotheliosis Viral Oncogene Related B Regulates Lymphatic Endothelial Cells during Vessel Maturation and Is Required for Lymphatic Vessel Function in Adult Mice.

NF-κB signals through canonical transcription factor p65 (RelA)/p50 and noncanonical avian reticuloendotheliosis viral oncogene related B (RelB)/p52 pathways. The RelA/p50 is involved in basal and inflammatory lymphangiogenesis. However, the role of RelB/p52 in lymphatic vessel biology is unknown. Herein, we investigated changes in lymphatic vessels (LVs) in mice deficient in noncanonical NF-κB signaling and the function of RelB in lymphatic endothelial cells (LECs). LVs were examined in Relb-/-, p52-/-, or control mice, and the gene expression profiles in LECs with RelB knockdown. Relb-/-, but not p52-/-, mice exhibited multiple LV abnormalities. They include the following: i) increased capillary vessel diameter, ii) reduced smooth muscle cell (SMC) coverage of mature vessels, iii) leakage, and iv) loss of active and passive lymphatic flow. Relb-/- mature LVs had thinner vessel walls, more apoptotic LECs and SMCs, and fewer LEC junctions. RelB knockdown LECs had decreased growth, survival, and adhesion, and dysregulated signaling pathways involving these cellular events. These results suggest that Relb-/- mice have abnormal LVs, mainly in mature vessels with reduced SMC coverage, leakage, and loss of contractions. RelB knockdown in LECs leads to reduced growth, survival, and adhesion. RelB plays a vital role in LEC-mediated LV maturation and function.

Exacerbated Staphylococcus aureus Foot Infections in Obese/Diabetic Mice Are Associated with Impaired Germinal Center Reactions, Ig Class Switching, and Humoral Immunity.

Obese patients with type 2 diabetes (T2D) are at an increased risk of foot infection, with impaired immune function believed to be a critical factor in the infectious process. In this study, we test the hypothesis that humoral immune defects contribute to exacerbated foot infection in a murine model of obesity/T2D. C57BL/6J mice were rendered obese and T2D by a high-fat diet for 3 mo and were compared with controls receiving a low-fat diet. Following injection of Staphylococcus aureus into the footpad, obese/T2D mice had greater foot swelling and reduced S. aureus clearance than controls. Obese/T2D mice also had impaired humoral immune responses as indicated by lower total IgG levels and lower anti-S. aureus Ab production. Within the draining popliteal lymph nodes of obese/T2D mice, germinal center formation was reduced, and the percentage of germinal center T and B cells was decreased by 40-50%. Activation of both T and B lymphocytes was similarly suppressed in obese/T2D mice. Impaired humoral immunity in obesity/T2D was independent of active S. aureus infection, as a similarly impaired humoral immune response was demonstrated when mice were administered an S. aureus digest. Isolated splenic B cells from obese/T2D mice activated normally but had markedly suppressed expression of Aicda, with diminished IgG and IgE responses. These results demonstrate impaired humoral immune responses in obesity/T2D, including B cell-specific defects in Ab production and class-switch recombination. Together, the defects in humoral immunity may contribute to the increased risk of foot infection in obese/T2D patients.

Restrictive lung disease in TNF-transgenic mice: correlation of pulmonary function testing and micro-CT imaging.

Purpose: Micro-computed tomography (µCT) is increasingly being used on animal models as a minimally-invasive longitudinal outcome measure of pulmonary disease progression. However, while imaging can elucidate macroscopic structural changes over the whole lung, µCT is unable to describe the mechanical changes and functional impairments imposed by progressive disease, which can only be measured via pulmonary function tests (PFTs). The tumor necrosis factor-transgenic (TNF-Tg) mouse model of rheumatoid arthritis (RA) develops pulmonary pathology that mimics many aspects of the inflammatory interstitial lung disease (ILD) seen in a subset of patients with RA. Prior studies using µCT imaging of these mice found increased pulmonary density, characteristic of restrictive disease; however, there have been conflicting reports in the literature regarding the obstructive versus restrictive phenotype of this model. Our study looks to 1) define the functional impairments and 2) characterize the restrictive/obstructive nature of the disease found in this model. Materials and Methods: In this study, we performed PFTs at end-stage ILD, and paired these findings with µCT results, correlating radiology to functional parameters. TNF-Tg and WT littermates of both sexes underwent µCT imaging and PFT testing at 5.5 months-old. Spearman's correlation analyses were performed comparing lung tissue volume (LTV) to PFT parameters of gas exchange and tissue stiffness. Results: Compared to WT, TNF-Tg mice had impaired gas exchange capacity, increased respiratory resistance, and reduced lung compliance, elastance, and inspiratory capacity, indicating increased tissue stiffness and compromised pulmonary function. LTV was also consistently higher in TNF-Tg lungs. Conclusions: These findings demonstrate that: 1) TNF-Tg mice display a restrictive pathology, and 2) in vivo µCT is a valid outcome measure to infer changes in pulmonary mechanical and functional parameters.

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.

Tracking Anti-Staphylococcus aureus Antibodies Produced In Vivo and Ex Vivo during Foot Salvage Therapy for Diabetic Foot Infections Reveals Prognostic Insights and Evidence of Diversified Humoral Immunity.

Management of foot salvage therapy (FST) for diabetic foot infections (DFI) is challenging due to the absence of reliable diagnostics to identify the etiologic agent and prognostics to justify aggressive treatments. As Staphylococcus aureus is the most common pathogen associated with DFI, we aimed to develop a multiplex immunoassay of IgG in serum and medium enriched for newly synthesized anti-S. aureus antibodies (MENSA) generated from cultured peripheral blood mononuclear cells of DFI patients undergoing FST. Wound samples were collected from 26 DFI patients to identify the infecting bacterial species via 16S rRNA sequencing. Blood was obtained over 12 weeks of FST to assess anti-S. aureus IgG levels in sera and MENSA. The results showed that 17 out of 26 infections were polymicrobial and 12 were positive for S. aureus While antibody titers in serum and MENSA displayed similar diagnostic potentials to detect S. aureus infection, MENSA showed a 2-fold-greater signal-to-background ratio. Multivariate analyses revealed increases in predictive power of diagnosing S. aureus infections (area under the receiver operating characteristic curve [AUC] > 0.85) only when combining titers against different classes of antigens, suggesting cross-functional antigenic diversity. Anti-S. aureus IgG levels in MENSA decreased with successful FST and rose with reinfection. In contrast, IgG levels in serum remained unchanged throughout the 12-week FST. Collectively, these results demonstrate the applicability of serum and MENSA for diagnosis of S. aureus DFI with increased power by combining functionally distinct titers. We also found that tracking MENSA has prognostic potential to guide clinical decisions during FST.

Osteoblast-osteoclast interactions.

Bone homeostasis depends on the resorption of bones by osteoclasts and formation of bones by the osteoblasts. Imbalance of this tightly coupled process can cause diseases such as osteoporosis. Thus, the mechanisms that regulate communication between osteoclasts and osteoblasts are critical to bone cell biology. It has been shown that osteoblasts and osteoclasts can communicate with each other through direct cell-cell contact, cytokines, and extracellular matrix interaction. Osteoblasts can affect osteoclast formation, differentiation, or apoptosis through several pathways, such as OPG/RANKL/RANK, RANKL/LGR4/RANK, Ephrin2/ephB4, and Fas/FasL pathways. Conversely, osteoclasts also influence formation of bones by osteoblasts via the d2 isoform of the vacuolar (H+) ATPase (v-ATPase) V0 domain (Atp6v0d2), complement component 3a, semaphorin 4D or microRNAs. In addition, cytokines released from the resorbed bone matrix, such as TGF-ß and IGF-1, also affect the activity of osteoblasts. Drugs could be developed by enhancing or restricting some of these interactions. Several reviews have been performed on the osteoblast-osteoclast communication. However, few reviews have shown the research advances in the recent years. In this review, we summarized the current knowledge on osteoblast-osteoclast communication.

The role of the lymphatic system in inflammatory-erosive arthritis.

Rheumatoid arthritis (RA) is a prevalent inflammatory joint disease with enigmatic flares, which causes swelling, pain, and irreversible connective tissue damage. Recently, it has been demonstrated in murine models of RA that the popliteal lymph node (PLN) is a biomarker of arthritic flare, as it "expands" in size and contrast enhancement during a prolonged asymptomatic phase, prior to when it "collapses" with accelerated synovitis and joint erosion. This PLN collapse is associated with adjacent knee flare, decreases in PLN volume and contrast enhancement, lymphatic pulse and pumping pressure, and an increase in PLN pressure. Currently, it is known that PLN collapse is accompanied by a translocation of B cells from the follicles to the sinuses, effectively clogging the lymphatic sinuses of the PLN, and that B cell depletion therapy ameliorates arthritic flare by eliminating these B cells and restoring passive lymphatic flow from inflamed joints. Here we review the technological advances that have launched this area of research, describe future directions to help elucidate the potential mechanism of PLN collapse, and speculate on clinical translation towards new diagnostics and therapies for RA.

Adaptive Upregulation of Clumping Factor A (ClfA) by Staphylococcus aureus in the Obese, Type 2 Diabetic Host Mediates Increased Virulence.

Obesity and associated type 2 diabetes (T2D) are important risk factors for infection following orthopedic implant surgery. Staphylococcus aureus, the most common pathogen in bone infections, adapts to multiple environments to survive and evade host immune responses. Whether adaptation of S. aureus to the unique environment of the obese/T2D host accounts for its increased virulence and persistence in this population is unknown. Thus, we assessed implant-associated osteomyelitis in normal versus high-fat-diet obese/T2D mice and found that S. aureus infection was more severe, including increases in bone abscesses relative to nondiabetic controls. S. aureus isolated from bone of obese/T2D mice displayed marked upregulation of four adhesion genes (clfA, clfB, bbp, and sdrC), all with binding affinity for fibrin(ogen). Immunostaining of infected bone revealed increased fibrin deposition surrounding bacterial abscesses in obese/T2D mice. In vitro coagulation assays demonstrated a hypercoagulable state in obese/T2D mice that was comparable to that of diabetic patients. S. aureus with an inactivating mutation in clumping factor A (clfA) showed a reduction in bone infection severity that eliminated the effect of obesity/T2D, while infections in control mice were unchanged. In infected mice that overexpress plasminogen activator inhibitor-1 (PAI-1), S. aureusclfA expression and fibrin-encapsulated abscess communities in bone were also increased, further linking fibrin deposition to S. aureus expression of clfA and infection severity. Together, these results demonstrate an adaptation by S. aureus to obesity/T2D with increased expression of clfA that is associated with the hypercoagulable state of the host and increased virulence of S. aureus.

Increased numbers of CD23(+) CD21(hi) Bin-like B cells in human reactive and rheumatoid arthritis lymph nodes.

A unique population of CD23(+) CD21(high) B cells in inflamed nodes (Bin) has been shown to accumulate in lymph nodes (LNs) draining inflamed joints of TNF-transgenic (TNF-tg) mice. Bin cells contribute to arthritis flare in mice by distorting node architecture and hampering lymphatic flow, but their existence in human inflamed LNs has not yet been described. Here, we report the characterization of resident B-cell populations in fresh popliteal lymph nodes (PLNs) from patients with severe lower limb diseases (non-RA) and rheumatoid arthritis (RA) patients, and from banked, cryopreserved reactive and normal human LN single cell suspension samples. Bin-like B cells were shown to be significantly increased in reactive LNs, and strikingly elevated (>30% of total) in RA samples. Histopathology and immunofluorescence analyses were consistent with B follicular hyperplasia and histological alterations in RA vs. non-RA PLNs. This is the first description of Bin-like B cells in human inflamed LNs. Consistent with published mouse data, this population appears to be associated with inflammatory arthritis and distortion of LN architecture. Further analyses are necessary to assess the role of CD23(+) CD21(hi) Bin-like B cells in RA pathogenesis and arthritic flare.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

A humoral immune defect distinguishes the response to Staphylococcus aureus infections in mice with obesity and type 2 diabetes from that in mice with type 1 diabetes.

Obesity and diabetes are among the greatest risk factors for infection following total joint arthroplasty. However, the underlying mechanism of susceptibility is unclear. We compared orthopedic implant-associated Staphylococcus aureus infections in type 1 (T1D) versus type 2 (T2D) diabetic mouse models and in patients with S. aureus infections, focusing on the adaptive immune response. Mice were fed a high-fat diet to initiate obesity and T2D. T1D was initiated with streptozotocin. Mice were then given a trans-tibial implant that was precoated with bioluminescent Xen36 S. aureus. Although both mouse models of diabetes demonstrated worse infection severity than controls, infection in T2D mice was more severe, as indicated by increases in bioluminescence, S. aureus CFU in tissue, and death within the first 7 days. Furthermore, T2D mice had an impaired humoral immune response at day 14 with reduced total IgG, decreased S. aureus-specific IgG, and increased IgM. These changes were not present in T1D mice. Similarly, T2D patients and obese nondiabetics with active S. aureus infections had a blunted IgG response to S. aureus. In conclusion, we report the first evidence of a humoral immune deficit, possibly due to an immunoglobulin class switch defect, in obesity and T2D during exacerbated S. aureus infection which may contribute to the increased infection risk following arthroplasty in patients with T2D and obesity.

TGF-ß1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair.

Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, Transforming Growth Factor Beta 1(TGF-ß1), but the effects of TGF-ß1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-ß1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-ß1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-ß1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-ß1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-ß1 on protease activity were completely abolished in tendon cells from homozygous plasminogen activator inhibitor 1 (PAI-1) knockout (KO) mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-ß1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries.

TAK1 regulates SOX9 expression in chondrocytes and is essential for postnatal development of the growth plate and articular cartilages.

TAK1 is a MAP3K that mediates non-canonical TGF-ß and BMP signaling. During the embryonic period, TAK1 is essential for cartilage and joint development as deletion of Tak1 in chondro-osteo progenitor cells leads to severe chondrodysplasia with defects in both chondrocyte proliferation and maturation. We have investigated the role of TAK1 in committed chondrocytes during early postnatal development. Using the Col2a1-CreER(T2); Tak1(f/f) mouse model, we induced deletion of Tak1 at postnatal day 7 and characterized the skeletal phenotypes of these mice at 1 and 3 months of age. Mice with chondrocyte-specific Tak1 deletion exhibited severe growth retardation and reduced proteoglycan and type II collagen content in the extracellular matrix of the articular cartilage. We found reduced Col2a1 and Acan expression, but increased Mmp13 and Adamts5 expression, in Tak1-deficient chondrocytes along with reduced expression of the SOX trio of transcription factors, SOX9, SOX5 and SOX6. In vitro, BMP2 stimulated Sox9 gene expression and Sox9 promoter activity. These effects were reduced; however, following Tak1 deletion or treatment with a TAK1 kinase inhibitor. TAK1 affects both canonical and non-canonical BMP signal transduction and we found that both of these pathways contribute to BMP2-mediated Sox9 promoter activation. Additionally, we found that ATF2 directly binds the Sox9 promoter in response to BMP signaling and that this effect is dependent upon TAK1 kinase activity. These novel findings establish that TAK1 contributes to BMP2-mediated Sox9 gene expression and is essential for the postnatal development of normal growth plate and articular cartilages.

TNF signals are dispensable for the generation of CD23+ CD21/35-high CD1d-high B cells in inflamed lymph nodes.

Tumor necrosis factor (TNF) is a key cytokine in rheumatoid arthritis (RA) pathogenesis, as underscored by the clinical effectiveness of TNF antagonists. While several of TNF's key targets in RA are well understood, its many pleiotropic effects remain to be elucidated. TNF-transgenic mice develop inflammatory-erosive arthritis associated with disruption of draining lymph node histology and function, and accumulation of B cells with unique phenotypic and functional features consistent with contribution to pathogenesis (B cells in inflamed nodes, Bin). Bin cell induction depends on the inflamed microenvironment, but the specific signals are unknown. Using anti-TNF treatment and TNF-receptor-deficient mice, here we show that Bin cells are induced and maintained independently of B cell-intrinsic TNF signals.