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1.  Tie2 Signaling Cooperates with TNF to Promote the Pro-Inflammatory Activation of Human Macrophages Independently of Macrophage Functional Phenotype 
PLoS ONE  2014;9(1):e82088.
Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 –differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 –differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.
doi:10.1371/journal.pone.0082088
PMCID: PMC3880273  PMID: 24404127
2.  FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis 
Arthritis Research & Therapy  2013;15(6):R209.
Introduction
The FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.
Methods
The levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.
Results
Flt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68+ and CD163+ macrophages, CD55+ fibroblast-like synoviocytes (FLS), CD31+ endothelial cells or infiltrating monocytes and CD19+ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.
Conclusions
The Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.
doi:10.1186/ar4403
PMCID: PMC3978611  PMID: 24314260
3.  The Rho-GEF Trio regulates a novel pro-inflammatory pathway through the transcription factor Ets2 
Biology Open  2013;2(6):569-579.
Summary
Inflammation is characterized by endothelium that highly expresses numerous adhesion molecules to trigger leukocyte extravasation. Central to this event is increased gene transcription. Small Rho-GTPases not only control the actin cytoskeleton, but are also implicated in gene regulation. However, in inflammation, it is not clear how this is regulated. Here, we show that the guanine-nucleotide exchange factor Trio expression is increased upon inflammatory stimuli in endothelium. Additionally, increased Trio expression was found in the vessel wall of rheumatoid arthritis patients. Trio silencing impaired VCAM-1 expression. Finally, we excluded that Trio-controlled VCAM-1 expression used the classical NFκB or MAP-kinase pathways, but rather acts on the transcriptional level by increasing phosphorylation and nuclear translocalization of Ets2. These data implicate Trio in regulating inflammation and provide novel targets for therapeutic purposes to treat inflammatory diseases such as rheumatoid arthritis.
doi:10.1242/bio.20134382
PMCID: PMC3683159  PMID: 23789107
Endothelium; GEF; GTPase; Inflammation; Signal transduction
4.  Signal Transduction Pathways in Chronic Inflammatory Autoimmune Disease: Small GTPases 
Ras superfamily small GTPases represent a wide and diverse class of intracellular signaling proteins that are highly conserved during evolution. These enzymes serve as key checkpoints in coupling antigen receptor, growth factor, cytokine and chemokine stimulation to cellular responses. Once activated, via their ability to regulate multiple downstream signaling pathways, small GTPases amplify and diversify signaling cascades which regulate cellular proliferation, survival, cytokine expression, trafficking and retention. Small GTPases, particularly members of the Ras, Rap, and Rho family, critically coordinate the function and interplay of immune and stromal cells during inflammatory respones, and increasing evidence indicates that alterations in small GTPase signaling contribute to the pathological behavior of these cell populations in human chronic inflammatory diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Here, we review how Ras, Rap, and Rho family GTPases contribute to the biology of cell populations relevant to human chronic inflammatory disease, highlight recent advances in understanding how alterations in these pathways contribute to pathology in RA and SLE, and discuss new therapeutic strategies that may allow specific targeting of small GTPases in the clinic.
doi:10.2174/1874312901206010259
PMCID: PMC3460313  PMID: 23028410
GTPases; systemic lupus erythematosus; matrix metalloproteinases; farnesyltransferase inhibitors.
5.  Why CCR2 and CCR5 Blockade Failed and Why CCR1 Blockade Might Still Be Effective in the Treatment of Rheumatoid Arthritis 
PLoS ONE  2011;6(7):e21772.
Background
The aim of this study was to provide more insight into the question as to why blockade of CCR1, CCR2, and CCR5 may have failed in clinical trials in rheumatoid arthritis (RA) patients, using an in vitro monocyte migration system model.
Methodology/Principal Findings
Monocytes from healthy donors (HD; n = 8) or from RA patients (for CCR2 and CCR5 antibody n = 8; for CCR1 blockade n = 13) were isolated from peripheral blood and pre-incubated with different concentrations of either anti-CCR1, anti-CCR2, or anti-CCR5 blocking antibodies (or medium or isotype controls). In addition, a small molecule CCR1 antagonist (BX471) was tested. Chemotaxis was induced by CCL2/MCP-1 (CCR2 ligand), CCL5/RANTES (CCR1 and CCR5 ligand), or by a mix of 5 RA synovial fluids (SFs), and cellular responses compared to chemotaxis in the presence of medium alone. Anti-CCR2 antibody treatment blocked CCL2/MCP-1-induced chemotaxis of both HD and RA monocytes compared to isotype control. Similarly, anti-CCR5 antibody treatment blocked CCL5/RANTES-induced chemotaxis of RA monocytes. While neither CCR2 nor CCR5 blocking antibodies were able to inhibit SF-induced monocyte chemotaxis, even when both receptors were blocked simultaneously, both anti-CCR1 antibodies and the CCR1 antagonist were able to inhibit SF-induced monocyte chemotaxis.
Conclusions/Significance
The RA synovial compartment contains several ligands for CCR1, CCR2, and CCR5 as well as other chemokines and receptors involved in monocyte recruitment to the site of inflammation. The results suggest that CCR2 and CCR5 are not critical for the migration of monocytes towards the synovial compartment in RA. In contrast, blockade of CCR1 may be effective. Conceivably, CCR1 blockade failed in clinical trials, not because CCR1 is not a good target, but because very high levels of receptor occupancy at all times may be needed to inhibit monocyte migration in vivo.
doi:10.1371/journal.pone.0021772
PMCID: PMC3128605  PMID: 21747955
6.  Apoptosis induced by overall metabolic stress converges on the Bcl-2 family proteins Noxa and Mcl-1 
Apoptosis  2011;16(7):708-721.
Apoptosis provoked by glucose shortage in dividing T cells is mediated via the BH3-only protein Noxa and inhibition of its binding partner Mcl-1. It is unknown how signals from cellular metabolism can affect the balance between Mcl-1 and Noxa and to what extent other Bcl-2 members are involved in this apoptosis cascade. Here, we defined the mechanism underlying apoptosis in relation to various types of metabolic stress. First, we established that the Noxa/Mcl-1 balance is regulated by glucose deprivation as well as by general metabolic stress, via changes in proteasome-mediated degradation of Mcl-1. Second, in contrast with cytokine-deprivation, no transcriptional modulation of Mcl-1, Puma, Bim or Noxa was observed during glucose deprivation. Third, no changes in PKB or GSK3 activity occurred and no clear role for AMPK was detected. Fourth, apoptosis triggered by nutrient deprivation was executed without signs of overt autophagy and independent of ROS production or p38 MAP kinase activity. Lastly, apoptosis under nutrient limitation could also be delayed by knock-down of Bim or overexpression of Bcl-2. In conclusion, Noxa functions in a specific apoptotic pathway that integrates overall nutrient stress, independent from attenuated PI3K/PKB signaling and without clear involvement of autophagy.
doi:10.1007/s10495-011-0599-8
PMCID: PMC3098366  PMID: 21516346
Apoptosis; Glucose; Nutrient deprivation; Noxa
7.  Histone deacetylases in RA: epigenetics and epiphenomena 
Reduced synovial expression of histone deacetylases (HDACs) is proposed to contribute to pathology in rheumatoid arthritis (RA) by enhancing histone-dependent access of transcription factors to promoters of inflammatory genes. In the previous issue of Arthritis Research & Therapy, Kawabata and colleagues provided independent evidence that HDAC activity is increased in the synovium and fibroblast-like synoviocytes (FLSs) of patients with RA and is paralleled by increased HDAC1 expression and synovial tumor necrosis factor-alpha (TNFα) production. Remarkably, stimulation of RA FLSs with TNFα specifically increases HDAC activity and HDAC1 expression, suggesting that changes in synovial HDAC activity and expression may be secondary to local inflammatory status.
doi:10.1186/ar3137
PMCID: PMC2991004  PMID: 20959025
8.  A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis 
Introduction
The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA).
Methods
CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests.
Results
Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor α, interferon γ and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help.
Conclusions
The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.
doi:10.1186/ar2900
PMCID: PMC2875627  PMID: 20053277
9.  The Ras guanine nucleotide exchange factor RasGRF1 promotes matrix metalloproteinase-3 production in rheumatoid arthritis synovial tissue 
Arthritis Research & Therapy  2009;11(4):R121.
Introduction
Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS.
Methods
Expression of RasGRF1, MMP-1, MMP-3, and IL-6 was detected in synovial tissue by immunohistochemistry and stained sections were evaluated by digital image analysis. Expression of RasGRF1 in FLS and synovial tissue was also assessed by immunoblotting. Double staining was performed to detect proteins in specific cell populations, and cells producing MMP-1 and MMP-3. RasGRF1 expression was manipulated in RA FLS by cDNA transfection and gene silencing, and effects on MMP-1, TIMP-1, MMP-3, IL-6, and IL-8 production measured by ELISA.
Results
Expression of RasGRF1 was significantly enhanced in RA synovial tissue, and detected in FLS and synovial macrophages in situ. In cultured FLS and synovial biopsies, RasGRF1 was detected by immunoblotting as a truncated fragment lacking its negative regulatory domain. Production of MMP-1 and MMP-3 in RA but not non-RA synovial tissue positively correlated with expression of RasGRF1 and co-localized in cells expressing RasGRF1. RasGRF1 overexpression in FLS induced production of MMP-3, and RasGRF1 silencing inhibited spontaneous MMP-3 production.
Conclusions
Enhanced expression and post-translational modification of RasGRF1 contributes to MMP-3 production in RA synovial tissue and the semi-transformed phenotype of RA FLS.
doi:10.1186/ar2785
PMCID: PMC2745805  PMID: 19678938
10.  Targeting histone deacetylase activity in rheumatoid arthritis and asthma as prototypes of inflammatory disease: should we keep our HATs on? 
Cellular activation, proliferation and survival in chronic inflammatory diseases is regulated not only by engagement of signal trans-duction pathways that modulate transcription factors required for these processes, but also by epigenetic regulation of transcription factor access to gene promoter regions. Histone acetyl trans-ferases coordinate the recruitment and activation of transcription factors with conformational changes in histones that allow gene promoter exposure. Histone deacetylases (HDACs) counteract histone acetyl transferase activity through the targeting of both histones as well as nonhistone signal transduction proteins important in inflammation. Numerous studies have indicated that depressed HDAC activity in patients with inflammatory airway diseases may contribute to local proinflammatory cytokine production and diminish patient responses to corticosteroid treatment. Recent observations that HDAC activity is depressed in rheumatoid arthritis patient synovial tissue have predicted that strategies restoring HDAC function may be therapeutic in this disease as well. Pharmacological inhibitors of HDAC activity, however, have demonstrated potent therapeutic effects in animal models of arthritis and other chronic inflammatory diseases. In the present review we assess and reconcile these outwardly paradoxical study results to provide a working model for how alterations in HDAC activity may contribute to pathology in rheumatoid arthritis, and highlight key questions to be answered in the preclinical evaluation of compounds modulating these enzymes.
doi:10.1186/ar2489
PMCID: PMC2592777  PMID: 18983693
11.  Treatment with recombinant interferon-β reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis 
Arthritis Research & Therapy  2004;6(3):R239-R249.
We investigated the therapeutic potential and mechanism of action of IFN-β protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-β or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-κB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-β. We also examined the effect of IFN-β on NF-κB activity. IFN-β, at 0.25 μg/injection and higher, significantly reduced disease severity in two experiments, each using 8–10 mice per treatment group. IFN-β-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-κB ligand and c-Fos. Tumor necrosis factor α and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-β treatment. IFN-β reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-κB activity. The data support the view that IFN-β is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.
doi:10.1186/ar1165
PMCID: PMC416442  PMID: 15142270
antibodies; cytokines; inflammation; rheumatoid arthritis
12.  The Small Gtpase, Rap1, Mediates Cd31-Induced Integrin Adhesion 
The Journal of Cell Biology  2000;148(6):1151-1158.
Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via β1 (VLA-4) and β2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.
PMCID: PMC2174316  PMID: 10725328
guanine nucleotide exchange factor; extravasation; leukocyte function-associated antigen 1; integrin-mediated adhesion; lymphocyte
13.  Selective involvement of ERK and JNK mitogen-activated protein kinases in early rheumatoid arthritis (1987 ACR criteria compared to 2010 ACR/EULAR criteria): a prospective study aimed at identification of diagnostic and prognostic biomarkers as well as therapeutic targets 
Annals of the Rheumatic Diseases  2011;71(3):415-423.
Objectives
To investigate the expression and activation of mitogen-activated protein kinases in patients with early arthritis who are disease-modifying antirheumatic drug (DMARD) naïve.
Methods
A total of 50 patients with early arthritis who were DMARD naïve (disease duration <1 year) were prospectively followed and diagnosed at baseline and after 2 years for undifferentiated arthritis (UA), rheumatoid arthritis (RA) (1987 American College of Rheumatology (ACR) and 2010 ACR/European League Against Rheumatism (EULAR) criteria), or spondyloarthritis (SpA). Synovial biopsies obtained at baseline were examined for expression and phosphorylation of p38, extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunohistochemistry and digital analysis. Synovial tissue mRNA expression was measured by quantitative PCR (qPCR).
Results
ERK and JNK activation was enhanced at inclusion in patients meeting RA criteria compared to other diagnoses. JNK activation was enhanced in patients diagnosed as having UA at baseline who eventually fulfilled 1987 ACR RA criteria compared to those who remained UA, and in patients with RA fulfilling 2010 ACR/EULAR criteria at baseline. ERK and JNK activation was enhanced in patients with RA developing progressive joint destruction. JNK activation in UA predicted 1987 ACR RA classification criteria fulfilment (R2=0.59, p=0.02) after follow-up, and disease progression in early arthritis (R2=0.16, p<0.05). Enhanced JNK activation in patients with persistent disease was associated with altered synovial expression of extracellular matrix components and CD44.
Conclusions
JNK activation is elevated in RA before 1987 ACR RA classification criteria are met and predicts development of erosive disease in early arthritis, suggesting JNK may represent an attractive target in treating RA early in the disease process.
doi:10.1136/ard.2010.143529
PMCID: PMC3277721  PMID: 21953337
14.  Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay 
Annals of the Rheumatic Diseases  2011;71(3):424-431.
Background
Histone deacetylase inhibitors (HDACi) display potent therapeutic efficacy in animal models of arthritis and suppress inflammatory cytokine production in rheumatoid arthritis (RA) synovial macrophages and tissue.
Objectives
To determine the molecular mechanisms contributing to the suppressive effects of HDACi on RA synovial cell activation, using interleukin 6 (IL-6) regulation as a model.
Methods
RA fibroblast-like synoviocytes (FLS) and healthy donor macrophages were treated with IL-1β, tumour necrosis factor (TNF)α, lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C)) in the absence or presence of the HDACi trichostatin A (TSA) or ITF2357 (givinostat). IL-6 production and mRNA expression was measured by ELISA and quantitative PCR (qPCR), respectively. Protein acetylation and the activation of intracellular signalling pathways were assessed by immunoblotting. The DNA-binding activity of nuclear factor κB (NFκB) and activator protein 1 (AP-1) components was measured by ELISA-based assays.
Results
HDACi (0.25–1.0 μM) suppressed RA FLS IL-6 production induced by IL-1β, TNFα and Toll-like receptor ligands. Phosphorylation of mitogen-activated protein kinases and inhibitor of κBα (IκBα) following IL-1β stimulation were unaffected by HDACi, as were AP-1 composition and binding activity, and c-Jun induction. TSA induced a significant reduction in nuclear retention of NFκB in FLS 24 h after IL-1β stimulation, but this did not reduce NFκB transcriptional activity or correlate temporally with reductions in IL-6 mRNA accumulation. HDACi significantly reduced the stability of IL-6 mRNA in FLS and macrophages.
Conclusions
Our study identifies a novel, shared molecular mechanism by which HDACi can disrupt inflammatory cytokine production in RA synovial cells, namely the promotion of mRNA decay, and suggests that targeting HDAC activity may be clinically useful in suppressing inflammation in RA.
doi:10.1136/ard.2011.154211
PMCID: PMC3277722  PMID: 21953341

Results 1-14 (14)