Lab Joosten

Proteome-wide Analysis and CXCL4 as a Biomarker in Systemic Sclerosis

BACKGROUNDnPlasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon.nnnMETHODSnWe isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo.nnnRESULTSnProteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis.nnnCONCLUSIONSnLevels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Type I interferons might form the link between Toll-like receptor (TLR) 3/7 and TLR4-mediated synovial inflammation in rheumatoid arthritis (RA)

Background: Rheumatoid arthritis (RA) has been associated with an increased risk of infections, but the underlying pathways have not yet been identified. Toll-like receptors (TLR) probably play a role in synovial inflammation and may also contribute to the understanding of the role of infections in RA. Objectives: To investigate if the synovial expression of TLR3 and TLR7 in RA correlates with that of inflammatory cytokines, and to assess whether this has functional consequences for local cytokine production and to study potential links between the TLR3/7 axis and TLR4 in RA synovium. Methods: Immunohistochemistry was used to study the expression of TLR3, TLR7, interferon α (IFNα), tumour necrosis factor α (TNFα) and interleukins IL1β, IL12, IL17 and IL18 in RA synovium obtained by arthroscopy from 34 patients with RA. Monocytes, monocyte-derived dendritic cells (MoDCs) and RA synovial fibroblasts were stimulated via TLR3 (poly-IC) and TLR7 (loxorubin), after which IL1β, IL6 and TNFα were measured by Luminex bead array technology. Following preincubation with IFNα, IL1β and IL18, TLR3 and TLR7 mRNA expression was assessed using real-time PCR. Cytokine production after preincubation with IFNα and subsequent TLR stimulation was measured. Results: Synovial TLR3/7 expression was co-expressed with IFNα, IL1β and IL18, but not with TNFα, IL12 and IL17. Stimulation of TLR3/TLR7 on monocytes, MoDCs or synovial fibroblasts led to secretion of type I IFN but no biologically active IL1β or IL18 could be detected. Type I IFNα increased TLR3/7 mRNA expression whereas IL1β and IL18 did not. In spite of the fact that the mRNA level of TLR4 remained unchanged, IFNα enhanced the response to TLR4 agonists, a phenomenon that was clearly more marked in patients with RA. Conclusion: Type I interferons are highly co-expressed with TLR3/TLR7 in RA synovium. They enhance TLR3/TLR7-mediated cytokine production and also TLR4-mediated responses.

An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint.

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1β enhancer region (−3690 to −2720) upstream of the human IL-6 promoter region (−163 to +12) was essential in mounting a robust response in HIG-82 synovial fibroblasts and in RAW 264,7 macrophages. A replication-deficient adenovirus was engineered with luciferase (Luc) controlled by the IL-1/IL-6 promoter (Ad5.IL-1/IL-6-Luc). LPS caused a 23- and 4.6-fold induction of Luc. activity in RAW cells infected with Ad5.IL-1/IL-6-Luc or the conventional Ad5.CMV-Luc construct, respectively. Next, adenoviruses (106u2009ffu) were injected into the knees of C57Bl/6 mice. An intra-articular injection of zymosan, 3 days after Ad5.IL-1/IL-6-Luc, increased Luc. activity by 39-fold but had no effect in the Ad5.CMV-Luc joints. The constitutive CMV promoter was rapidly silenced and could not be reactivated in vivo. In contrast, the IL-1/IL-6 promoter could be reactivated by Streptococcal cell wall (SCW)-induced arthritis up to 21 days after infection. Next the IL-1/IL-6 promoter was compared to the C3-Tat/HIV-LTR two-component system in wild-type, IL-6−/− and IL-1−/− gene knockout mice. Both systems responded well to LPS-, zymosan- and SCW-induced arthritis. However, the basal activity of the IL-1/IL-6 promoter was lower and IL-6 independent. This study showed that the IL-1/IL-6 promoter is feasible to achieve disease-regulated transgene expression for treatment of arthritis.

The effect of the ATG16L1 Thr300Ala polymorphism on susceptibility and outcome of patients with epithelial cell-derived thyroid carcinoma

Epithelial cell-derived thyroid carcinoma (TC) is the most common endocrine malignancy. Increasing evidence suggests that autophagy, a complex process of autodigestion in conditions of cellular stress, plays an important role in the pathophysiology of the TC malignant process. One of the main mammalian autophagy proteins is autophagy related 16-like 1 (ATG16L1), which is essential for autophagosome formation, induction of autophagy, and modulation of inflammation (Saitoh et al. 2008). Subsequently, defective autophagy in ATG16L1 knockout mice results in an increased production of the proinflammatory cytokine interleukin 1b (IL1b; Saitoh et al. 2008) that is also known to affect the growth and differentiation of different malignant cell types (Apte & Voronov 2002). Considering the potential role of both autophagy and IL1b in the pathology of TC, we hypothesized that genetic variation in ATG16L1 influences the susceptibility for and the outcome of TC. One single nucleotide polymorphism of the ATG16L1 gene (c.898AOG, Thr300Ala, rs2241880) has been shown to affect the autophagy process (Cooney et al. 2010) and also to modulate production of IL1b in human cells (Plantinga et al. 2011). We investigated whether this ATG16L1 polymorphism is associated with the susceptibility or clinical outcome of TC. One hundred and thirty nine patients (75% women, mean age 39G13 (S.D.) years) with histologically confirmed TC (papillary (70%), follicular TC (24%), or both (6%)), who visited the outpatient clinic at the Department of Endocrinology of our centre, were included. Primary treatment consisted of (near-) total thyroidectomy in all patients and modified radical neck dissections in patients with confirmed nodal metastases, followed by ablation with radioactive iodine (I, RAI) of residual thyroid tissue. Initial cure is

Citrullination of synovial proteins in murine models of rheumatoid arthritis

OBJECTIVEnAntibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)-induced arthritis and collagen-induced arthritis (CIA), respectively.nnnMETHODSnSynovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase-polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen.nnnRESULTSnIn both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected.nnnCONCLUSIONnCitrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients.

Coxiella burnetii infection (Q fever) in rheumatoid arthritis patients with and without anti-TNFα therapy

Q fever is a zoonosis caused by the intracellular bacterium Coxiella burnetii. The Netherlands experienced a major Q fever outbreak between 2007 and 2010, with an estimate of more than 40u2005000 infected individuals.1 Initial infection is asymptomatic in over 50% of the infected individuals or causes a mostly self-limiting febrile disease.2 However, chronic Q fever may develop months to years after initial infection. This serious, life-threatening condition presents mostly as endocarditis or infection of an aortic aneurysm or vascular prosthesis, and is accompanied by high IgG antibody titres against phase I C burnetii .3 Individuals most at risk for chronic Q fever are those with pre-existing valvulopathy, vascular aneurysm or prosthesis and yet undefined types of immune suppression.4 ,5 Tumour necrosis factor-α (TNFα) plays an important role in the defence against intracellular bacteria such as C burnetii . In vitro studies show that TNFα is involved in internalisation and intracellular killing of C burnetii in monocytes.6 ,7 In addition, C burnetii- infected TNFα knockout mice develop early bacteraemia and severe …

Chondrocyte unresponsiveness to insulin-like growth factor-1. A novel pathogenetic mechanisms for cartilage destruction in experimental arthritis

Inhibition of chondrocyte proteoglycan synthesis is one of the mechanisms leading to cartilage destruction in inflammatory joint diseases [1]. Prolonged depletion of cartilage matrix, resulting from proteoglycan degradation and inadequate repair, weakens the ability of articular cartilage to withstand compressive and tensile forces, which ultimately leads to irreversible tissue damage. Degradation of matrix proteoglycans is thought to be caused by proteolytic enzymes either from inflammatory cells [2] or from the chondrocyte itself by interleukin-1 driven processes [3]. In this study we used an ex vivo system of intact articular cartilage to study the effect of inflammation on chondrocyte metabolism. Both normal and arthritic cartilage was tested for its response to insulin-like growth factor-1 (IGF-1), a major cartilage growth factor. It is shown that inflammation induces a state of non-responsiveness towards IGF-1 in the chondrocyte which ultimately leads to destruction of articular cartilage.

Synovial macrophages promote TGF-β signaling and protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidized low-density lipoproteins

OBJECTIVEnLow-density lipoproteins (LDL) in inflamed synovium is oxidized and taken-up by synoviocytes. In this study, we investigate whether direct injection of oxidized LDL (oxLDL) into a normal murine knee joint induces joint pathology and whether synovial macrophages are involved in that process.nnnDESIGNnSynovium was obtained from end-stage osteoarthritis (OA) patients in order to analyze LDL-uptake. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (phosphate buffered saline (PBS)). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes 7xa0days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production.nnnRESULTSnSynovial tissue of OA patients showed extensive accumulation of apolipoprotein B. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to increased synovial thickening in combination with significantly upregulated protein and RNA levels of CCL2 and CCL3. FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in TGF-β concentrations was measured in macrophage-depleted joints.nnnCONCLUSIONSnOxLDL can affect joint pathology, since synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity combined with increased influx of monocytes and neutrophils.

inflammation-inducible intra-articular production of human IL-1 receptor antagonist results in a more efficient inhibition of collagen-induced arthritis than does constitutive expression of the same transgene

Background Achieving biologically effective yet safe levels of recombinant anti-inflammatory proteins by gene therapy may require three regulatory features: 1) a basal level that is sufficiently low to avoid or minimise chronic immunosuppression 2) transcriptional regulation over a wide dynamic range, and 3) induced expression levels that are sufficiently high to achieve the desired biological effects. The C3-Tat/HIV promoter construct seems to have these properties.1 In this two-component expression system the complement factor 3 (C3) promoter regulates production of the HIV transactivator of transcription, and the Tat protein then stimulates expression of the desired transgene, which is regulated by the HIV-LTR promoter. Both C3 and HIV LTR promoters are inducible by proinflammatory cytokines. Objectives To achieve disease-inducible expression of recombinant anti-inflammatory proteins in order to allow autoregulation of drug dose by natural homeostatic mechanisms. Methods We compared a disease-inducible, two component expression system (C3-Tat/HIV) with the constitutive immediate early cytomegalovirus (ieCMV) promoter in the polyarticular Collagen-Induced Arthritis (CIA) model in mice. DBA/I mice were immunised with bovine type II collagen and boostered on day 22. On day 22, mice without any clinical signs of arthritis were selected and adenoviral vectors (Ad. CMV-Luc, Ad. CMV-IL-1Ra, or Ad. C3-Tat/HIV-IL-1Ra) that contained luciferase or the human IL-1Ra gene under control of one of the two promoters were used to transfect the synovial lining of both knees. The injected knee joints and ipsilateral paws were then scored for signs of arthritis and at the end histology was taken. Results Inducible promoter-driven IL-1Ra expression resulted in significantly improved inhibition of CIA than did CMV-driven IL-1Ra production. Moreover, overexpression of IL-1Ra in the knee joints also prevented CIA in the ipsilateral paws. Conclusion Our data (1) demonstrate the feasibility of an inducible expression system for producing a recombinant transgene for treatment of arthritis and (2) show that this system is more effective than strong, constitutive transgene expression for preventing collagen-induced arthritis in mice. Reference Varley AW, Geiszler SM, Gaynor RB, Munford RS. A two-component expression system that responds to inflammatory stimuli in vivoin vivo. Nat Biotechnol. 1997;15(10):1002–6

IL-18 blockade is a potential disease-modifying therapy for rheumatoid arthritis

Interleukin-18 (IL-18) has been demonstrated as promoting the development of a TH1 response in vivo in synergy with IL-12. Significant levels of IL-18 and IL-12 have been detected in the joints of patients with rheumatoid arthritis (RA).