Monocyte transcriptomes from patients with axial spondyloarthritis reveal dysregulated monocytopoiesis and a distinct inflammatory imprint.

BACKGROUND: In patients with axial spondyloarthritis (axSpA), monocytes show a pre-activated phenotype. Gut inflammation is a trigger of monocyte activation and may also affect their development in the bone marrow (BM). As gut inflammation is commonly observed in axSpA patients, we performed a detailed analysis of monocyte transcriptomes of axSpA patients in two cohorts and searched for signs of activation and developmental adaptations as putative imprints of gut inflammation. METHODS: Transcriptomes of blood CD14+ monocytes of HLA-B27+ axSpA patients and healthy controls (HC) were generated by microarrays from cohort 1 and by RNA-sequencing from cohort 2. Differentially expressed genes from both analyses were subjected to gene set enrichment analysis (GSEA) and to co-expression analysis in reference transcriptomes from BM cells, blood cells and activated monocytes. As serological markers of translocation, 1,3 beta-glycan, intestinal fatty acid binding protein, and lipopolysaccharide binding protein (LBP) were determined by LAL and ELISA. RESULTS: Transcriptome analysis identified axSpA-specific monocyte signatures showing an imprint of LPS/cytokine-activated monocytes, late granulopoietic BM cells, blood neutrophils, and G-CSF-mobilized blood cells, which suggests LPS/TNF activation and more prominent BM adaptation promoting a neutrophil-like phenotype. GSEA mapped axSpA upregulated genes to inflammatory responses and TNFα signaling and downregulated probe-sets to metabolic pathways. Among translocation markers, LBP levels were significantly increased in axSpA patients vs. HC (p < 0.001). Stratified analysis by disease activity and stage identified an "active disease signature" (BASDAI ≥ 4) with an imprint of LPS/cytokine-activated monocytes and CD16+ monocyte subsets. The "AS signature" (vs. non-radiographic axSpA) showed a reinforced neutrophil-like phenotype due to deprivation of dendritic cell transcripts. CONCLUSIONS: The neutrophil-like phenotype of axSpA monocytes points towards a biased monocytopoiesis from granulocyte-monocyte progenitors. This shift in monocytopoiesis and the LPS/cytokine imprint as well as the elevated LBP levels are indicators of systemic inflammation, which may result from bacterial translocation. The BM adaptation is most prominent in AS patients while disease activity appears to be linked to activation and trafficking of monocytes.

An explorative study on deep profiling of peripheral leukocytes to identify predictors for responsiveness to anti-tumour necrosis factor alpha therapies in ankylosing spondylitis: natural killer cells in focus.

BACKGROUND: Therapeutic targeting of tumour necrosis factor (TNF)-α is highly effective in ankylosing spondylitis (AS) patients. However, since one-third of anti-TNF-treated AS patients do not show an adequate clinical response there is an urgent need for new biomarkers that would aid clinicians in their decision-making to select appropriate therapeutic options. Thus, the aim of this explorative study was to identify cell-based biomarkers in peripheral blood that could be used for a pre-treatment stratification of AS patients. METHODS: A high-dimensional, multi-parametric flow cytometric approach was applied to identify baseline predictors in 31 AS patients before treatment with the TNF blockers adalimumab (TNF-neutralisation) and etanercept (soluble TNF receptor). RESULTS: As the major result, the frequencies of natural killer (NK) cells, and in particular CD8-positive (CD8+) NK cell subsets, were most predictive for therapeutic outcome in AS patients. While an inverse correlation between classical CD56+/CD16+ NK cells and reduction of disease activity was observed, the CD8+ NK cell subset behaved in the opposite direction. At baseline, responders showed significantly increased frequencies of CD8+ NK cells compared with non-responders. CONCLUSIONS: This is the first study demonstrating that the composition of the NK cell compartment has predictive power for prediction of therapeutic outcome for anti-TNF-α blockers, and we identified CD8+ NK cells as a potential new player in the TNF-α-driven chronic inflammatory immune response of AS.

Serum levels of leptin and high molecular weight adiponectin are inversely associated with radiographic spinal progression in patients with ankylosing spondylitis: results from the ENRADAS trial.

BACKGROUND: Previous research indicates a role of adipokines in inflammation and osteogenesis. Hence adipokines might also have a pathophysiological role in inflammation and new bone formation in patients with ankylosing spondylitis (AS). The aim of this study was to investigate the role of adipokine serum levels as predictors of radiographic spinal progression in patients with AS. METHODS: A total of 120 patients with definite AS who completed a 2-year follow up in the ENRADAS trial were included in the current study. Radiographic spinal progression was defined as: (1) worsening of the modified Stoke Ankylosing Spondylitis spine (mSASSS) score by ≥2 points and/or (2) new syndesmophyte formation or progression of existing syndesmophytes after 2 years. Serum levels of adipokines (adiponectin (APN) and its high molecular weight form (HMW-APN), chemerin, leptin, lipocalin-2, omentin, resistin, visfatin) were measured using enzyme-linked immunosorbent assays. RESULTS: There was a significant association between radiographic spinal progression and both leptin and HMW-APN. Baseline serum levels of both adipokines were lower in patients who showed radiographic spinal progression after 2 years. This association was especially evident in men; they had generally lower leptin and HMW-APN serum levels as compared to women. The inverse association between adipokines and radiographic spinal progression was confirmed in the logistic regression analysis: the odds ratios (OR) for the outcome "no mSASSS progression ≥2 points" were 1.16 (95% CI 1.03 to 1.29) and 1.17 (95% CI 0.99 to 1.38), for leptin and HMW-APN, respectively; for "no syndesmophyte formation/progression" the respective OR were 1.29 (95% CI 1.11 to 1.50) and 1.18 (95% CI 0.98 to 1.42), adjusted for the presence of syndesmophytes at baseline, C-reactive protein at baseline, sex, body mass index (BMI), non-steroidal anti-inflammatory drugs intake score over 2 years, and smoking status at baseline. CONCLUSION: Serum leptin and HMW-APN predict protection from spinal radiographic progression in patients with AS. Women generally have higher leptin and HMW-APN serum levels that might explain why they have less structural damage in the spine as compared to male patients with AS. TRIAL REGISTRATION: EudraCT: 2007-007637-39. ClinicalTrials.gov, NCT00715091 . Registered on 14 July 2008.

Synovial cell production of IL-26 induces bone mineralization in spondyloarthritis.

Spondyloarthritis (SpA) is characterized by inflammation and new bone formation and can be treated by inhibition of the proinflammatory cytokines TNF-α and IL-17A. IL-26 is considered a proinflammatory cytokine, predominantly related to Th17 cells. In the present study, we investigate IL-26 expression in SpA patients, and examine the in vitro production of IL-26 by synovial cells and the effects of IL-26 on human osteoblasts. IL-26 was measured by ELISA in plasma and synovial fluid (SF) of 15 SpA patients and in plasma samples from 12 healthy controls. Facet joints from axial SpA patients were stained for IL-26 and analyzed by fluorescence microscopy. Synovial fluid mononuclear cells, C-C motif chemokine receptor 6 memory Th17 cells, and fibroblast-like synoviocytes (FLSs) were isolated, and supernatants were analyzed for IL-26 content by ELISA. FLSs were further stained for IL-26 production and the myofibroblast marker α-smooth-muscle-actin (αSMA) and analyzed by flow cytometry. Human osteoblasts were cultured in the presence of IL-26, and the degree of mineralization was quantified. We found that IL-26 levels in SF were increased compared with plasma (P < 0.0001). Moreover, IL-26 expression was found in facet joints of axial SpA patients within the bone marrow. IL-26 secretion was primarily found in αSMA+ myofibroblasts. In contrast, Th17 cells did not produce detectable amounts of IL-26. Human osteoblasts treated with IL-26 showed increased mineralization compared with untreated osteoblasts (P = 0.02). In conclusion, IL-26 seems to be produced by myofibroblasts in the inflamed synovium and could be a possible facilitator of bone mineralization in SpA. KEY MESSAGES: IL-26 levels are higher in synovial fluid compared to plasma in spondyloarthritis. IL-26 was identified in axial facet joints of spondyloarthritis patients. Myofibroblasts from the spondyloarthritis synovium produce large amounts of IL-26. IL-26 induces bone mineralization in human osteoblasts.

Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development.

The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA+ CD4+ Tmem cells from blood and CD69+ Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.

Imprinting of Skin/Inflammation Homing in CD4+ T Cells Is Controlled by DNA Methylation within the Fucosyltransferase 7 Gene.

E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4+ T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4+ effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites.

Core 2 ß1,6-N-acetylglucosaminyltransferase-I, crucial for P-selectin ligand expression is controlled by a distal enhancer regulated by STAT4 and T-bet in CD4+ T helper cells 1.

P-selectin ligands (P-ligs) support the recruitment of lymphocytes into inflamed tissues. Binding to P-selectin is mediated by oligosaccharide groups synthesized by means of several glycosyltransferases including core 2 ß1,6-N-acetylglucosaminyltransferase-I (C2GlcNAcT-I), encoded by the gene Gcnt1. Using Gcnt1(-/-) Th1 cells, we show that C2GlcNAcT-I is crucial for inflammatory T cell homing in vivo. To understand the molecular regulation of Gcnt1 in CD4(+) T helper cells, we performed ChIP-on-chip experiments across the Gcnt1 locus assessing the chromatin structure in P-lig-expressing versus non-expressing CD4(+) T cells. This identified a distal region about 20kb upstream of the promoter where the presence of a H3K27me3 mark correlated with Gcnt1 repression. This region possessed IL-12-dependent enhancer activity in reporter assays, in accordance with preferential IL-12-dependent induction of Gcnt1 in vitro. STAT4 and T-bet cooperated in control of the enhancer activity. Deficiency in either one resulted in drastically reduced Gcnt1 mRNA expression in differentiated Th1 cells. While both STAT4 and T-bet were bound to the enhancer early after activation only T-bet binding persisted throughout the expansion phase after TCR signal cessation. This suggests sequential action of STAT4 and T-bet at the enhancer. In summary, we show that Gcnt1 transcription and subsequent P-lig induction in Th1 cells is governed by binding of STAT4 and T-bet to a distal enhancer and further regulated by epigenetic marks such as H3K27me3.

Granulation Tissue Eroding the Subchondral Bone Also Promotes New Bone Formation in Ankylosing Spondylitis.

OBJECTIVE: We previously suggested that fibroblast-rich granulation tissue eroding the subchondral bone is instrumental in the joint remodeling that occurs in ankylosing spondylitis (AS). The purpose of this study was to determine if this granulation tissue also carries bone-forming capabilities, which we approached by searching for bone-forming cells (hypertrophic chondrocytes, osteoblasts) in its vicinity. We also assessed adipogenic tissue transformation, which has been suggested to be an intermediate feature in AS bone formation based on imaging studies. METHODS: The facet joints of AS patients, osteoarthritis (OA) patients, and autopsy subjects (controls) were screened for subchondral granulation tissue. We searched for hypertrophic chondrocytes by assessing RUNX-2, type X collagen, and matrix metalloproteinase 13 (MMP-13) expression, for osteoblasts by analyzing RUNX-2, CD56, and type I collagen expression, as well as for signs of new bone formation. Adipocytes and lipid accumulation were assessed in Safranin O-stained sections. RESULTS: In the joints of AS and OA patients, RUNX-2-positive cells were found to be lining the granulation tissue. These cells coexpressed type I collagen but lacked type X collagen and MMP-13 expression, confirming their osteoblastic nature. In 91% of AS joints and in 20% of OA joints (P < 0.05), we observed foci of new bone formation at contact zones between the granulation tissue and the cartilage. Joints containing bony spots showed greater replacement of the adjacent bone marrow by granulation tissue than did joints without bone formation (P < 0.05). The granulation tissue often contained adipocytes and lipid accumulations. Replacement of the subchondral bone marrow by fat tissue was also frequently found but was not associated with new bone formation. CONCLUSION: The subchondral granulation tissue carries osteoblasts, which promote new bone formation, leading to intraarticular ankylosis of the facet joints in AS.

Anti-RANKL treatment inhibits erosive joint destruction and lowers inflammation but has no effect on bone formation in the delayed-type hypersensitivity arthritis (DTHA) model.

BACKGROUND: The aims of the present study were to determine the relationship between bone destruction and bone formation in the delayed-type hypersensitivity arthritis (DTHA) model and to evaluate the effect of receptor activator of nuclear factor κB ligand (RANKL) blockade on severity of arthritis, bone destruction, and bone formation. METHODS: DTHA was induced in C57BL/6 mice. Inflammation, erosive joint damage, and new bone formation were semiquantitatively scored by histology. Osteoclast activity was assessed in vivo, and messenger RNA (mRNA) expression of mediators of bone destruction and bone formation were analyzed by mRNA deep sequencing. Serum concentrations of tartrate-resistant acid phosphatase 5b, carboxy-terminal telopeptide I (CTX-I), matrix metalloproteinase 3 (MMP3), and serum amyloid P component (SAP) were determined by enzyme-linked immunosorbent assay. Anti-RANKL monoclonal antibody treatment was initiated at the time of immunization. RESULTS: Bone destruction (MMP3 serum levels, cathepsin B activity, and RANKL mRNA) peaked at day 3 after arthritis induction, followed by a peak in cartilage destruction and bone erosion on day 5 after arthritis induction. Periarticular bone formation was observed from day 10. Induction of new bone formation indicated by enhanced Runx2, collagen X, osteocalcin, MMP2, MMP9, and MMP13 mRNA expression was observed only between days 8 and 11. Anti-RANKL treatment resulted in a modest reduction in paw and ankle swelling and a reduction of serum levels of SAP, MMP3, and CTX-I. Destruction of the subchondral bone was significantly reduced, while no effect on bone formation was seen. CONCLUSIONS: Anti-RANKL treatment prevents joint destruction but does not prevent new bone formation in the DTHA model. Thus, although occurring sequentially during the course of DTHA, bone destruction and bone formation are apparently not linked in this model.

Effect of continuous versus on-demand treatment of ankylosing spondylitis with diclofenac over 2†years on radiographic progression of the spine: results from a randomised multicentre trial (ENRADAS).

BACKGROUND: To date, only a single controlled trial provided evidence that non-steroidal anti-inflammatory drugs (NSAIDs) given continuously reduce radiographic progression compared with an on-demand therapy over 2 years in patients with ankylosing spondylitis (AS). In the current study, we tested whether such an effect of NSAIDs could be confirmed in another randomised trial. METHODS: Patients with AS were randomised for treatment with either continuous (150 mg/day) or on-demand diclofenac for 2 years. Tumour necrosis factor-blocker treatment was not allowed during the entire study period. The primary outcome was the difference in radiographic progression in the spine as measured by the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) scored by two readers blinded to treatment arm and time point. RESULTS: 62 of 85 patients enrolled in the continuous arm and 60 of 82 enrolled in the on-demand arm completed the study. The mSASSS progression was numerically higher in the continuous group (1.28 (0.7 to 1.9) vs 0.79 (0.2 to 1.4)) (p=0.39). If only patients were analysed who were either C reactive protein positive or had syndesmophytes at baseline, there was again a higher radiographic progression in the continuous versus the on-demand group: 1.68 (0.7 to 2.6) vs 0.96 (0.0 to 1.9) and 2.11 (1.1 to 3.1) vs 0.95 (0.0 to 1.9), respectively. There was no difference between the two treatment groups regarding adverse events. CONCLUSIONS: In our study, continuous treatment with diclofenac over 2 years did not reduce radiographic progression compared with on-demand treatment in AS. TRIAL REGISTRATION NUMBERS: EudraCt-no 2007-007637-39; ClinicalTrials.gov NCT00715091.

Cartilage in facet joints of patients with ankylosing spondylitis (AS) shows signs of cartilage degeneration rather than chondrocyte hypertrophy: implications for joint remodeling in AS.

INTRODUCTION: In ankylosing spondylitis (AS), joint remodeling leading to joint ankylosis involves cartilage fusion. Here, we analyzed whether chondrocyte hypertrophy is involved in cartilage fusion and subsequent joint remodeling in AS. METHODS: We assessed the expression of chondrocyte hypertrophy markers runt-related transcription factor 2 (Runx2), type X collagen (COL10), matrix metalloproteinase 13 (MMP13), osteocalcin and beta-catenin and the expression of positive bone morphogenic proteins (BMPs) and negative regulators (dickkopf-1 (DKK-1)), sclerostin, (wingless inhibitory factor 1 (wif-1)) of chondrocyte hypertrophy in the cartilage of facet joints from patients with AS or osteoarthritis (OA) and from autopsy controls (CO) by immunohistochemistry. Sex determining region Y (SRY)-box 9 (Sox9) and type II collagen (COL2) expression was assessed as indicators of chondrocyte integrity and function. RESULTS: The percentage of hypertrophic chondrocytes expressing Runx2, COL10, MMP13, osteocalcin or beta-catenin was significantly increased in OA but not in AS joints compared to CO joints. Frequencies of sclerostin-positive and DKK-1-positive chondrocytes were similar in AS and CO. In contrast, wif-1- but also BMP-2- and BMP-7-expressing and Sox9-expressing chondrocytes were drastically reduced in AS joints compared to CO as well as OA joints whereas the percentage of COL2-expressing chondrocytes was significantly higher in AS joints compared to CO joints. CONCLUSIONS: We found no evidence for chondrocyte hypertrophy within hyaline cartilage of AS joints even in the presence of reduced expression of the wnt inhibitor wif-1 suggesting that chondrocyte hypertrophy is not a predominant pathway involved in joint fusion and remodeling in AS. In contrast, the reduced expression of Sox9, BMP-2 and BMP-7 concomitantly with induced COL2 expression rather point to disturbed cartilage homeostasis promoting cartilage degeneration in AS.

In vivo pre-activation of monocytes in patients with axial spondyloarthritis.

INTRODUCTION: Innate immune responses, including monocyte functions, seem to play an important role in the pathogenesis of axial spondyloarthritis (axSpA). Therefore, we characterized the phenotype and functional state of monocytes of patients with axSpA. METHODS: Fifty-seven patients with axSpA, 11 patients with rheumatoid arthritis (RA), and 29 healthy controls were included in the study. We determined the percentage of classic, intermediate, and non-classic monocytes according to CD14 and CD16 expression and the expression of Toll-like receptor (TLR) 1, 2, and 4 in whole blood by flow cytometry. The percentage of monocytes producing interleukin (IL)-1beta, IL-6, tumor necrosis factor alpha (TNFα), IL-12/23p40, and IL-1 receptor antagonist (IL-1ra) was detected by flow cytometry after stimulation of whole blood without and with different TLR and nucleotide-binding oligomerization domain ligands-i.e., lipopolysaccharide (LPS), fibroblast-stimulating lipopeptid-1, PAM3CSK4, and muramyl dipeptide (MDP)-for 5 h. IL-10 production was measured after 18 h of stimulation in supernatants by enzyme-linked immunosorbent assay. RESULTS: In patients with axSpA but not patients with RA, we found higher frequencies of classic monocytes than in controls (median of 90.4% versus 80.4%, P < 0.05), higher frequencies of monocytes spontaneously producing IL-1beta and IL-1ra (P < 0.05), and a higher percentage of monocytes producing IL-1beta after MDP stimulation (P < 0.05). Elevated cytokine production was confined to axSpA patients under conventional therapy (non-steroidal anti-inflammatory drugs) and not found in patients under TNFα inhibitor treatment. The LPS-induced production of IL-6 and IL-10 was lower in axSpA patients compared with controls (P < 0.05). Monocytic TLR expression was unaffected in patients with axSpA. CONCLUSION: Enhanced spontaneous and MDP-induced cytokine secretion by monocytes suggests in vivo pre-activation of monocytes in axSpA patients under conventional therapy which is reverted under TNF inhibitor treatment.

Serum adipokine levels in patients with ankylosing spondylitis and their relationship to clinical parameters and radiographic spinal progression.

OBJECTIVE: Adipokines have metabolic and inflammatory functions but can also affect bone metabolism. The purpose of this study was to determine the relationship between serum levels of adiponectin, resistin, and visfatin and markers of inflammation, disease activity, and radiographic spinal progression in patients with ankylosing spondylitis (AS). METHODS: Levels of adiponectin, resistin, and visfatin in the serum of 86 AS patients and 25 healthy controls were measured by enzyme-linked immunosorbent assay at baseline. Radiographic spinal progression was determined by the scoring of radiographs of the spine obtained at baseline and after 2 years. RESULTS: Mean (±SD) baseline levels of resistin and visfatin were significantly higher in AS patients than in healthy controls (11.6 ± 10.6 ng/ml versus 6.6 ± 3.2 ng/ml [P = 0.01] for resistin, and 20.9 ± 48.3 ng/ml versus 3.4 ± 2.6 ng/ml [P = 0.001] for visfatin). Adipokine serum levels did not correlate with disease activity or functional indices. Only resistin serum levels correlated with markers of inflammation. Baseline levels of visfatin, but not resistin or adiponectin, were significantly higher in patients with worsening of the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) by ≥2 units after 2 years (n = 19) as compared to patients without mSASSS worsening (37.7 ± 57.8 ng/ml versus 16.1 ± 44.6 ng/ml; P = 0.029) and in patients with syndesmophyte formation/progression (n = 22) as compared to patients without such progression (37.1 ± 55.3 ng/ml versus 15.3 ± 44.8 ng/ml; P = 0.023). Visfatin levels of >8 ng/ml at baseline were predictive of subsequent radiographic spinal progression (adjusted odds ratio 3.6 for mSASSS progression and 5.4 for syndesmophyte formation/progression). CONCLUSION: Serum levels of resistin and visfatin are elevated in AS patients. Elevated visfatin levels at baseline are predictive of subsequent progression of radiographic damage in AS patients.

Identification of two regulatory elements controlling Fucosyltransferase 7 transcription in murine CD4+ T cells.

Fucosyltransferase VII encoded by the gene Fut7 is essential in CD4(+) T cells for the generation of E- and P-selectin ligands (E- and P-lig) which facilitate recruitment of lymphocytes into inflamed tissues and into the skin. This study aimed to identify regulatory elements controlling the inducible Fut7 expression in CD4(+) T cells that occurs upon activation and differentiation of naive T cells into effector cells. Comparative analysis of the histone modification pattern in non-hematopoetic cells and CD4(+) T cell subsets revealed a differential histone modification pattern within the Fut7 locus including a conserved non-coding sequence (CNS) identified by cross-species conservation comparison suggesting that regulatory elements are confined to this region. Cloning of the CNS located about 500 bp upstream of the Fut7 locus, into a luciferase reporter vector elicited reporter activity after transfection of the αß-WT T cell line, but not after transfection of primary murine CD4(+) Th1 cells. As quantification of different Fut7 transcripts revealed a predominance of transcripts lacking the first exons in primary Th1 cells we searched for an alternative promoter. Cloning of an intragenic region spanning a 1kb region upstream of exon 4 into an enhancer-containing vector indeed elicited promoter activity. Interestingly, also the CNS enhanced activity of this intragenic minimal promoter in reporter assays in primary Th1 cells suggesting that both elements interact in primary CD4(+) T cells to induce Fut7 transcription.

Histomorphologic and histomorphometric characteristics of zygapophyseal joint remodeling in ankylosing spondylitis.

OBJECTIVE: To unravel the mechanisms that control bony ankylosis in ankylosing spondylitis (AS). METHODS: Histomorphologic and histomorphometric analyses were performed on zygapophyseal joints obtained from 18 patients with AS, 9 patients with osteoarthritis (OA), and 10 cadaver donors without a rheumatic disease (controls). The proteoglycan content of the cartilage was determined by Safranin O staining and the chondrocyte apoptosis according to caspase 3 expression. RESULTS: AS joints were categorized into 3 groups according to the morphology of the joint surfaces and joint space: group 1 were joints with an open joint space, group 2 were joints with cartilaginous fusion, and group 3 were joints with bony fusion of the joint surfaces. Progressive loss of the joint space from group 1 joints to group 3 joints suggests that this grouping corresponds to sequential stages of joint remodeling. Cartilage thickness and subchondral bone plate thickness declined from group 1 to group 3 (P < 0.01). Increased chondrocyte apoptosis rates were found in groups 1 and 2 (P < 0.05), while in group 3, a reduction in the proteoglycan content was found (P < 0.001). Bone marrow replacement and invasion of the subchondral bone plate by fibrous tissue was found predominantly in AS joints in group 2. CONCLUSION: Cartilage degeneration, indicated by cartilage thinning, enhanced chondrocyte apoptosis, and proteoglycan loss, and subchondral bone thinning, promoted by invasion of the subchondral bone plate by a fibrous tissue originating from the bone marrow, are hallmarks of joint remodeling in AS.

Elevated serum level of the vascular endothelial growth factor predicts radiographic spinal progression in patients with axial spondyloarthritis.

OBJECTIVE: To investigate the role of serum vascular endothelial growth factor (VEGF) as a predictor of radiographic spinal progression in patients with axial spondyloarthritis (axSpA). METHODS: Altogether, 172 patients with definite axSpA (95 with ankylosing spondylitis and 77 with non-radiographic axSpA) were included in this study. Spinal radiographs obtained at baseline and after 2 years of follow-up were scored independently by two trained readers in a concealed and randomly selected order according to the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) scoring system and for the presence of syndesmophytes. Radiographic spinal progression after 2 years was defined as (1) mSASSS worsening by ≥2 units, and (2) new syndesmophyte formation or formation of a bridging syndesmophyte from two single syndesmophytes. Serum VEGF levels were detected at baseline. RESULTS: Mean baseline VEGF values were significantly higher in patients with mSASSS worsening by ≥2 units after 2 years (n=22) than in those without progression (562±357 vs 402±309 pg/mL, respectively, p=0.027) and in patients with syndesmophyte formation (n=18) again as compared with those without new bone formation (579±386 vs 404±307 pg/mL, respectively, p=0.041). VEGF as a predictor of radiographic spinal progression performed especially well in patients who were already at high risk for such a progression due to the presence of syndesmophytes at baseline (n=48). In these patients, a VEGF serum level of >600 pg/mL had a sensitivity of 53%, a specificity of 97% and an OR=36.6 (95% CI 3.9 to 341.5) as a predictor of mSASSS worsening by ≥2 units. For syndesmophyte formation, elevated VEGF demonstrated a sensitivity of 47%, a specificity of 94% and an OR=13.6 (95% CI 2.4 to 78.3). CONCLUSIONS: An elevated serum level of VEGF (>600 pg/mL) is highly specific as a predictor of radiographic spinal progression in patients with axSpA, especially in patients who are at high risk for further progression due to the presence of syndesmophytes.

Regulation and migratory role of P-selectin ligands during intestinal inflammation.

Dendritic cells from mesenteric lymph nodes (MLN) can convert retinal to retinoic acid (RA), which promotes induction of the gut-specific homing receptor α4ß7. In contrast, priming within peripheral lymph nodes leads to upregulation of E- and P-selectin ligands (E- and P-lig). Apart from its α4ß7 promoting effect, RA was shown to suppress E- and P-lig induction in vitro. However, enhanced frequencies of P-lig(+) CD4(+) T cells were reported during intestinal inflammation. To understand this contradiction, we first determined whether location of intestinal inflammation, that is, ileitis or colitis, affects P-lig induction. Both conditions promoted P-lig expression on CD4(+) T cells; however, P-lig expressed on T cells facilitated Th1 cell recruitment only into the inflamed colon but not into inflamed small intestine induced by oral Toxoplasma gondii infection. A majority of P-lig(+)CD4(+) T cells found within MLN during intestinal inflammation co-expressed α4ß7 confirming their activation in the presence of RA. Mesenteric P-lig(+)CD4(+) cells co-expressed the 130 kDa isoform of CD43 which requires activity of core 2 (beta)1,6-N-acetyl-glycosaminyltransferase-I (C2GlcNAcT-I) suggesting that C2GlcNAcT-I contributes to P-lig expression under these conditions. To test whether inflammatory mediators can indeed overrule the inhibitory effect of RA on P-lig expression we stimulated CD4(+) T cells either polyclonal in the presence of IL-12 and IFNγ or by LPS-activated MLN-derived dendritic cells. Both conditions promoted P-lig induction even in the presence of RA. While RA impeded the induction of fucosyltransferase-VII it did not affect IL-12-dependent C2GlcNAcT-I induction suggesting that C2GlcNAcT-I can support P-lig expression even if fucosyltransferase-VII mRNA upregulation is dampened.

In situ analysis of interleukin-23- and interleukin-12-positive cells in the spine of patients with ankylosing spondylitis.

OBJECTIVE: The interleukin-12 (IL-12) family of cytokines has been suggested to play a critical role in inflammatory autoimmune diseases, and recent studies analyzing peripheral blood and synovial fluid from patients with spondyloarthritides suggest that IL-23 might be a proinflammatory factor in these disorders. This study was undertaken to investigate the presence and source of IL-23 in the spines of patients with ankylosing spondylitis (AS). METHODS: The frequency of IL-23-positive and IL-12-positive cells within the subchondral bone marrow and within fibrous tissue replacing normal bone marrow in facet joints of patients with AS was analyzed by immunohistochemistry. The origin of IL-23-positive cells was determined by double staining of CD163+ macrophages, CD68+ macrophages, CD1a+ dendritic cells, tryptase-positive mast cells, myeloperoxidase-positive cells, CD20+ B cells, and CD3+ T cells. Findings in 28 facet joints from 22 AS patients were compared with those in 20 facet joints from 13 patients with osteoarthritis (OA) and 10 normal control specimens. RESULTS: The frequency of IL-23-positive cells in subchondral bone marrow from the joints of AS patients (mean ± SD 42.50 ± 32.81/high-power field [hpf]) was significantly increased compared to that in subchondral bone marrow from OA patients (OA 15.63 ± 29.90/hpf) (P = 0.0017) or controls (19.36 ± 16.8/hpf) (P = 0.03). Myeloperoxidase-positive cells and, to a lesser extent, macrophages and dendritic cells were found to be the origin of IL-23 in the bone marrow. In AS and OA patients, the frequency of IL-23-positive cells was significantly higher than that of IL-12-positive cells (P < 0.001 in both patient groups). Within fibrous tissue from AS and OA facet joints, IL-23 was predominantly produced by CD163+ macrophages (mean ± SD 0.64 ± 0.59/hpf and 4.36 ± 3.4/hpf, respectively) and CD68+ macrophages (2.3 ± 0.65/hpf and 6.54 ± 4.1/hpf, respectively). CONCLUSION: IL-23 is expressed in the subchondral bone marrow and in fibrous tissue replacing bone marrow in facet joints of patients with AS. It might have a role in inflammatory processes and in chronic changes in AS joints, which makes it an interesting potential therapeutic target in this disease.

Differential regulation and impact of fucosyltransferase VII and core 2 ß1,6-N-acetyl-glycosaminyltransferase for generation of E-selectin and P-selectin ligands in murine CD4+ T cells.

Ligands for E-selectin and P-selectin (E-lig and P-lig) are induced on CD4+ T cells upon differentiation into effector T cells. Glycosyltransferases, especially α 1,3-fucosyltransferase VII (FucT-VII) and core 2 ß1,6-N-acetyl-glycosaminyltransferase I (C2GlcNAcT-I), are critical for their synthesis. We here analysed the signals that control the expression of E-lig, P-lig and mRNA coding for FucT-VII and C2GlcNAcT-I. In line with previous reports, we found that P-lig expression correlates with the regulation of C2GlcNAcT-I, whereas E-lig expression can occur at low levels of C2GlcNAcT-I mRNA but requires high FucT-VII mRNA expression. Interestingly, the two enzymes are regulated by different signals. Activation-induced C2GlcNAcT-I up-regulation under permissive (T helper type 1) conditions was strongly reduced by cyclosporin A (CsA), suggesting the involvement of T-cell receptor-dependent, calcineurin/NFAT-dependent signals in combination with interleukin-12 (IL-12) -mediated signals in the regulation of C2GlcNAcT-I. In contrast, expression of FucT-VII mRNA was not significantly inhibited by CsA. Interleukin-4 inhibited the expression of FucT-VII but IL-2 and IL-7 were found to support induction of FucT-VII and E-lig. E-selectin, P-selectin and their ligands initially appeared to have rather overlapping functions. These findings however, unravel striking differences in the regulation of E-lig and P-lig expression, dictated by the dominance of FucT-VII and C2GlcNAcT-I, respectively, and their dependency on signals from either promiscuous or homeostatic cytokines (FucT-VII) or a strong T-cell receptor signal in combination with inflammatory cytokines in case of C2GlcNAcT-I.