The relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human osteoporotic and osteoarthritic bone tissues

Background

Pro-inflammatory cytokines possess osteoclastogenic or anti-osteoclastogenic activities. They influence osteoclasts directly or via the receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) system. Recent evidence suggests that inflammation may play a role in osteoporosis (OP) and osteoarthritis (OA). We aimed therefore to determine whether there is a difference between both groups: first, in the expression of the osteoclastogenic and anti-osteoclastogenic cytokines, second, in correlation of these cytokines with bone mineral density (BMD) and levels of bone turnover markers (BTM) and third, in correlation between the expression of these cytokines and osteoclast specific genes and RANK/RANKL/OPG genes.

Methods

Human bone samples from 54 age and sex matched patients with OP or OA were collected during hip arthroplasty surgery. The expression of 25 genes encoding pro-inflammatory cytokines, their receptors, osteoclast specific genes and RANK/RANKL/OPG genes was measured using quantitative real-time PCR. Total hip, femoral neck and lumbar spine BMD and BTM in blood samples were measured. The comparison between OP and OA was assessed using Student''s t-test or Mann-Whitney U test and correlations between gene expression, BMD and BTM were determined using nonparametric correlation.

Results

The results demonstrated a higher expression of interleukin (IL)-6 and IL-1α in OP, and interferon (IFN)-γ in OA (p < 0.0005). Negative correlations of total hip BMD with tumor necrosis factor-α (TNF-α) in OA and with RANKL/RANK in OP were found (p < 0.05). Significant correlations with BTM were shown for IL-1α and IFN-γ in OP (rho = 0.608 and -0.634) and for TNF-α, IL-6 and transforming growth factor-β1 (TGF-β1) in OA (rho = 0.591, -0.521 and 0.636). Results showed OP specific negative correlations (IFN-γ with ITGB3, IFN-β1 with CTSK, tartrate resistant acid phosphatase (TRAP), CALCR, RANK, RANKL, IL-1α with CTSK, OPG, IL-17A with CALCR) and positive (TGF-β1 with CTSK, TRAP, RANK), and OA specific negative (IL-1α with osteoclast associated immunoglobulin-like receptor (OSCAR), TNF-α with RANK, RANKL, OPG) and positive (IL-6 with RANK, RANKL, OPG) correlations.

Conclusions

Our results demonstrate that the relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human OP and OA bone and could present an important factor for characteristics of OP and OA bone phenotypes.     

Complex dynamics of osteoclast formation and death in long-term cultures

Background

Osteoclasts, cells responsible for bone resorption, contribute to the development of degenerative, metabolic and neoplastic bone diseases, which are often characterized by persistent changes in bone microenvironment. We aimed to investigate the dynamics of osteoclast formation and death in cultures that considerably exceeded the length of standard protocol and to design a mathematical model describing osteoclastogenesis.

Methodology/Principal Findings

RAW 264.7 monocytic cells fuse to form multinucleated osteoclasts upon treatment with pro-resorptive cytokine RANKL. We have found that in long-term experiments (15–26 days), the dynamics of changes in osteoclast numbers was remarkably complex and qualitatively variable in different experiments. Whereas 19 of 46 experiments exhibited single peak of osteoclast formation, in 27 experiments we observed development of successive waves of osteoclast formation and death. Periodic changes in osteoclast numbers were confirmed in long-term cultures of mouse bone marrow cells treated with M-CSF and RANKL. Because the dynamics of changes in osteoclast numbers was found to be largely independent of monocytes, a two-species model of ordinary differential equations describing the changes in osteoclasts and monocytes was ineffective in recapitulating the oscillations in osteoclast numbers. Following experimental observation that medium collected from mature osteoclasts inhibited osteoclastogenesis in fresh cultures, we introduced a third variable, factor f, to describe osteoclast-derived inhibitor. This model allowed us to simulate the oscillatory changes in osteoclasts, which were coupled to oscillatory changes in the factor f, whereas monocytes changed exponentially. Importantly, to achieve the experimentally observed oscillations with increasing amplitude, we also had to assume that osteoclast presence stimulates osteoclast formation.

Conclusions/Significance

This study identifies the critical role for osteoclast autocrine regulation in controlling long-term dynamic of osteoclast formation and death and describes the complementary roles for negative and positive feedback mediators in determining the sharp dynamics of activation and inactivation of osteoclasts.     

Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase

Background

Phenylketonuria (PKU) is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques.

Methodology

Peripheral blood mononuclear cell (PBMC) cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). Flow cytometry was utilized to analyze osteoclast precursors (OCPs) and T cell phenotype. Tumour necrosis factor α (TNF-α), RANKL and osteoprotegerin (OPG) were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated.

Principal Findings

Several in vitro studies in PKU patients'' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS). This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia.

Conclusions

Our results indicate that PKU spontaneous osteoclastogenesis depends on the circulating OCP increase and the activation of T cells. Osteoclastogenesis correlates with clinical parameters, suggesting its value as a diagnostic tool for an early assessment of an increased bone resorption in PKU patients.     

IL-32 and IL-17 interact and have the potential to aggravate osteoclastogenesis in rheumatoid arthritis

Introduction

Interleukin (IL)-32 and IL-17 play critical roles in pro-inflammatory responses and are highly expressed in the synovium of patients with rheumatoid arthritis (RA). We investigated the relations between these two cytokines (IL-17 and IL-32) for their ability to induce each other and to stimulate osteoclasts in RA fibroblast-like synoviocytes (FLSs) and T cells.

Methods

FLSs were isolated through surgical synovectomy obtained from patients with RA or osteoarthritis (OA). Real-time PCR were performed to evaluate the expression of IL-32, IL-17 and osteoclast-related genes. Immunohistochemical staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to determine the distribution of inflammatory cytokines and the presence of osteoclastogenesis.

Results

IL-17 induced the expression of IL-32 in the FLSs from RA patients, as assessed by microarray. IL-32 production was increased by IL-17. IL-32 in the FLSs from RA patients induced the production of IL-17 in CD4⁺ T cells. IL-32 and IL-17 were colocalized near TRAP-positive areas in joint specimens. IL-17 and IL-32 synergistically induced the differentiation of osteoclasts, as demonstrated by the expression of osteoclast-related genes. IL-32 and IL-17 also could induce resorption by osteoclasts in a RANKL-dependent manner.

Conclusions

IL-17 affected the expression of IL-32 in FLSs of RA patients and IL-32 induced the production of IL-17 in CD4+ T cells. Both IL-17 and IL-32 cytokines can reciprocally influence each other''s production and amplify the function of osteoclastogenesis in the in RA synovium. Separately, IL-17 and IL-32 each stimulated osteoclastogenesis without RANKL. Together, the two cytokines synergistically amplified the differentiation of osteoclasts, independent of RANKL stimulation.     

miR-31 controls osteoclast formation and bone resorption by targeting RhoA

Introduction

Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.

Methods

miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.

Results

miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.

Conclusions

miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.     

Intermittent Hypoxia Effect on Osteoclastogenesis Stimulated by Neuroblastoma Cells

Background

Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions.

Methods

We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone.

Results

Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells.

Conclusions

Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.     

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

Background

Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown.

Methodology/Principal Findings

The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts.

Conclusions/Significance

Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.     

Osteoclasts are active in bone forming metastases of prostate cancer patients

Background

Bone forming metastases are a common and disabling consequence of prostate cancer (CaP). The potential role of osteoclast activity in CaP bone metastases is not completely explained. In this study, we investigated ex vivo whether the osteolytic activity is present and how it is ruled in CaP patients with bone forming metastases.

Methodology

Forty-six patients affected by newly diagnosed CaP and healthy controls were enrolled. At diagnosis, 37 patients had a primary tumour only, while 9 had primary tumour and concomitant bone forming metastases. In all patients there was no evidence of metastasis to other non-bone sites. For all patients and controls we collected blood and urinary samples. We evaluated patients'' bone homeostasis; we made peripheral blood mononuclear cell (PBMC) cultures to detect in vitro osteoclastogenesis; we dosed serum expression of molecules involved in cancer induced osteoclatogenesis, such as RANKL, OPG, TNF-alpha, DKK-1 and IL-7. By Real-Time PCR, we quantified DKK-1 and IL-7 gene expression on micro-dissected tumour and healthy tissue sections.

Principal Findings

CaP bone metastatic patients showed bone metabolism disruption with increased bone resorption and formation compared to non-bone metastatic patients and healthy controls. The CaP PBMC cultures showed an enhanced osteoclastogenesis in bone metastatic patients, due to an increase of RANKL/OPG ratio. We detected increased DKK-1 serum levels and tissue gene expression in patients compared to controls. IL-7 resulted high in patients'' sera, but its tissue gene expression was comparable in patients and controls.

Conclusions

We demonstrated ex vivo that osteoclastogenesis is an active mechanism in tumour nesting of bone forming metastatic cancer and that serum DKK-1 levels are increased in CaP patients, suggesting to deeply investigate its role as tumour marker.     

Flavonoids isolated from Tridax procumbens (TPF) inhibit osteoclasts differentiation and bone resorption

Background

The Tridax procumbens flavonoids (TPF), are well known for their medicinal properties among local natives. The TPF are traditionally used for dropsy, anaemia, arthritis, gout, asthma, ulcer, piles, and urinary problems. It also used in treating gastric problems, body pain, and rheumatic pains of joints. The TPF have been reported to increase osteogenic functioning in mesenchymal stem cells. However, their effects on osteoclastogenesis remain unclear. The TPF isolated from T. procumbens and investigated the effects of the TPF inhibit on osteoclast differentiation and bone resorption activities using primary osteoclastic cells. Osteoclast formation was assessed by counting the number of tartrate resistant acid phosphatase (TRAP) positive multinucleated cells and by measuring both TRAP activities.

Results

The TPF significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in primary osteoclastic cells. The TPF also decreased the expression of mRNAs related to osteoclast differentiation, including Trap, Cathepsin K, Mmp-9, and Mmp-13 in primary osteoclastic cells. The treatment of primary osteoclastic cells with the TPF decreased Cathepsin K, Mmp-9, and Mmp-13 proteins expression in primary osteoclastic cells.

Conclusion

These results indicated that TPF inhibit osteoclastogenesis and pits formation activities. Our results suggest that the TPF could be a potential anti-bone resorptic agent to treat patients with bone loss-associated diseases such as osteoporosis.     

Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults

Background

An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.

Methods

Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.

Results

Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.

Conclusions

CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.     

Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-β knock-out mice

Background

CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-β in the immunomodulatory effects of CpG-ODN is not known.

Objective

Here, we aimed to elucidate the role of IFN-β in the anti-allergic effect of CpG motifs.

Methods

We assessed the immune response in OVA-primed/OVA-challenged IFN-β knockout (-/-) mice compared to wild type (WT) control, after intranasal and systemic treatment with synthetic CpG motifs.

Results

Vaccination with CpG-ODN reduced the number of cells in airways of OVA-sensitized WT but not IFN-β-/- mice. Although airway eosinophilia was reduced in both treated groups, they were significantly higher in IFN-β^-/- mice. Other inflammatory cells, such as lymphocytes and macrophages were enhanced in airways by CpG treatment in IFN-β-/- mice. The ratio of IFN-γ/IL-4 cytokines in airways was significantly skewed to a Th1 response in WT compared to IFN-β^-/- group. In contrast, IL-4 and IgE were reduced with no differences between groups. Ag-specific T-cell proliferation, Th1-cytokines such as IFN-γ, IL-2 and also IL-12 were significantly lower in IFN-β-/- mice. Surprisingly, we discovered that intranasal treatment of mice with CpG-ODN results in mild synovitis particularly in IFN-β-/- mice.

Conclusion

Our results indicate that induction of Th1 response by therapy with CpG-ODN is only slightly and partially dependent on IFN-β, while IFN-β is not an absolute requirement for suppression of airway eosinophilia and IgE. Furthermore, our finding of mild synovitis is a warning for possible negative effects of CpG-ODN vaccination.     

SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model

Objective

SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model.

Methods

CIA was induced in wild-type (Sh3bp2^+/+) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2^KI/+) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures.

Results

Sh3bp2^KI/+ mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2^KI/+ mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ mice. In vitro experiments showed that TNF-α production in Sh3bp2^KI/+ BMMs is elevated compared with Sh3bp2^+/+ BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2^KI/+ BMMs associated with increased NFATc1 nuclear localization.

Conclusion

Gain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.     

Synovial membrane immunohistology in early-untreated rheumatoid arthritis reveals high expression of catabolic bone markers that is modulated by methotrexate

Introduction

We aimed to investigate the expression and therapeutic modulation of the receptor activator of the NF-κB ligand (RANKL) system in early-untreated rheumatoid arthritis (RA).

Methods

In this study, 15 patients with newly diagnosed RA (median symptom duration 7 months) were started on methotrexate (MTX) 20 mg weekly. Synovial biopsies were obtained by needle arthroscopy at baseline and 8 weeks after initiation of therapy. X-rays of the hands and feet were obtained at baseline and 1 year after diagnosis. Immunohistochemistry was performed to detect RANKL, receptor activator of nuclear factor-κB (RANK) and osteoprotegerin (OPG) in the synovial biopsies. The in vitro effect of MTX was tested on RA-derived primary fibroblasts and the osteoblasts-like osteosarcoma cell line (rtPCR, Western blot and ELISA) and in osteoclasts (tartrate-resistant acid phosphatase staining and dentine pit formation assay).

Results

MTX decreased synovial cellularity as well as RANK expression and the RANKL/OPG ratio. We confirmed this effect by a decrease of the mRNA and protein RANKL/OPG ratio in synovial-derived fibroblasts and osteoblasts-like tumoral cells exposed in vitro to methotrexate. Supernatants from MTX treated osteoblasts-like tumoral cells prevented pre-osteoclast formation in the absence of exogenous RANKL. Furthermore, MTX blocked osteoclastogenesis from peripheral blood mononuclear cells despite the presence of macrophage colony stimulating factor and RANKL, which indicates that MTX directly inhibits osteoclastogenesis.

Conclusions

The synovial membrane of early-untreated RA is characterized by a high RANKL/OPG ratio that can be reversed by methotrexate.     

IFN-β Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

Purpose

It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-β on its expression.

Methods

EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161–180 (IRBP_161–180). IFN-β was administered subcutaneously to IRBP_161–180 immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-β or PBS were stimulated with anti-CD3/CD28 or IRBP_161–180 for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-β for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-β for 3 days. IFN-β-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA.

Results

IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-β in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-β suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-β also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-β-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells.

Conclusion

IL-9 expression is increased during EAU which could be suppressed by IFN-β.     

Salmonella Induced IL-23 and IL-1β Allow for IL-12 Production by Monocytes and M?1 through Induction of IFN-γ in CD56+ NK/NK-Like T Cells

Background

The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-γ. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain.

Methodology/Principal Findings

We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mϕ1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1β and IL-18, but not IL-12. Supernatants of Salmonella-infected Mϕ1 contained more IL-18 and IL-1β as compared with supernatants of Mϕ1 stimulated with isolated TLR agonists, and induced IFN-γ production in human CD56⁺ cells in an IL-23 and IL-1β-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1β led to the production of GM-CSF in CD56⁺ cells. Both IFN-γ and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production.

Conclusions/Significance

The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-γ and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-γ production in the type-1 cytokine pathway.     

Molecular targeting of hepatocyte growth factor by an antagonist,NK4, in the treatment of rheumatoid arthritis

Introduction

Hepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice.

Methods

Arthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Results

The intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Conclusions

These results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4⁺ T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA.