Rachel Bayley

The autoimmune-associated genetic variant PTPN22 R620W enhances neutrophil activation and function in patients with rheumatoid arthritis and healthy individuals

Objectives A genetic variant of the leukocyte phosphatase PTPN22 (R620W) is strongly associated with autoimmune diseases including rheumatoid arthritis (RA). Functional studies on the variant have focussed on lymphocytes, but it is most highly expressed in neutrophils. We have investigated the effects of the variant on neutrophil function in health and in patients with RA. Methods Healthy individuals and patients with RA were genotyped for PTPN22 (R620W) and neutrophils isolated from peripheral blood. Neutrophil adhesion and migration across inflamed endothelium were measured. Calcium (Ca2+) release and reactive oxygen species (ROS) production in response to fMLP stimulation were also assessed. Results Expression of R620W enhanced neutrophil migration through cytokine activated endothelium (non-R620W=24%, R620W=45% migrating cells, p<0.001). Following fMLP stimulation, neutrophils that were heterozygous and homozygous for R620W released significantly more Ca2+ when compared to non-R620W neutrophils, in healthy individuals and patients with RA. fMLP stimulation, after TNF-α priming, provoked more ROS from neutrophils heterozygous for R620W in patients with RA (non-R620W vs R620W=∼1.75-fold increase) and healthy individuals (non-R620W vs R620W=fourfold increase) and this increase was statistically significant in healthy individuals (p<0.001) but not in patients with RA (p<0.25). Conclusions Expression of PTPN22 (R620W) enhanced neutrophil effector functions in health and RA, with migration, Ca2+ release and production of ROS increased. Neutrophils are found in large numbers in the RA joint, and this hyperactivity of R620W cells may directly contribute to the joint damage, as well as to the initiation and perpetuation of the chronic immune-mediated inflammatory processes driving the disease.

Differential expression of CD148 on leukocyte subsets in inflammatory arthritis

IntroductionMonocytic cells play a central role in the aetiology of rheumatoid arthritis, and manipulation of the activation of these cells is an approach currently under investigation to discover new therapies for this and associated diseases. CD148 is a transmembrane tyrosine phosphatase that is highly expressed in monocytes and macrophages and, since this family of molecules plays an important role in the regulation of cell activity, CD148 is a potential target for the manipulation of macrophage activation. For any molecule to be considered a therapeutic target, it is important for it to be increased in activity or expression during disease.MethodsWe have investigated the expression of CD148 in two murine models of arthritis and in joints from rheumatoid arthritis (RA) patients using real-time PCR, immunohistochemistry, and studied the effects of proinflammatory stimuli on CD148 activity using biochemical assays.ResultsWe report that CD148 mRNA is upregulated in diseased joints of mice with collagen-induced arthritis. Furthermore, we report that in mice CD148 protein is highly expressed in infiltrating monocytes of diseased joints, with a small fraction of T cells also expressing CD148. In human arthritic joints both T cells and monocytes expressed high levels of CD148, however, we show differential expression of CD148 in T cells and monocytes from normal human peripheral blood compared to peripheral blood from RA and both normal and RA synovial fluid. Finally, we show that synovial fluid from rheumatoid arthritis patients suppresses CD148 phosphatase activity.ConclusionsCD148 is upregulated in macrophages and T cells in human RA samples, and its activity is enhanced by treatment with tumour necrosis factor alpha (TNFα), and reduced by synovial fluid or oxidising conditions. A greater understanding of the role of CD148 in chronic inflammation may lead to alternative therapeutic approaches to these diseases.

Metabolomics in the Analysis of Inflammatory Diseases

Inflammation is a normal and extraordinarily important component of responses to infection and injury. The cardinal features of swelling, redness, stiffness and increasing temperature are strong indicators of the significant changes in tissue metabolism and the ingress of immune cells into the tissues. The increase in blood flow which underlies many of these changes may result in changes to the supply of nutrients and in particular the level of oxygen in the tissues. Inward migration of immune cells, which is also enabled by the increased blood flow, will put further stress on the metabolic environment of the tissues. The activity of macrophages and neutrophils in clearing infection and repairing tissue damage also have significant metabolic consequences particularly because of the production of cytokines and cytotoxic molecules such as reactive oxygen species and reactive nitrogen species, which are required to kill invading organisms. Production of these molecules will consume considerable quantities of oxygen, ATP and NADPH. These antimicrobial agents put considerable stress on host cells in the surrounding and distal tissues and can lead to significant loss of protective metabolites such as glutathione.

Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

Summary Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks.

Measuring the specific activity of the protein tyrosine phosphatase Lyp

Altered function of the protein tyrosine phosphatase (PTP) Lyp (PTPN22) has been implicated in the pathogenesis of a number of human diseases, and so accurate assessment of its functional activity is needed to further our understanding of its biology. We have developed an in vitro method to measure the specific catalytic activity of the Lyp phosphatase. Lyp is captured from cell lysates using an anti-Lyp monoclonal antibody coated 96-well plate, and activity measured by dephosphorylation of a fluorescent substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). The amount of protein is measured using an anti-Lyp HRP conjugate, with reference to a standard curve generated with purified Lyp. These two measurements are then used to calculate the specific phosphatase activity. We used this assay to show that the specific activity of the Lyp phosphatase is decreased by H(2)O(2) in Jurkat T cells and primary CD4+ T cells. We also modified this assay to measure the specific activity of CD45, the other main PTP regulating T cell receptor (TCR) signalling, in order to compare the relative susceptibility of CD45 and Lyp to oxidation by H(2)O(2). By measuring specific activity in Jurkat T cells and primary CD4+ T cells, we demonstrated that CD45 is more susceptible to oxidation by H(2)O(2) when compared to Lyp. Reduced function of CD45 and Lyp has been associated with human immune mediated inflammatory diseases, and a differential susceptibility to oxidation could be an important regulatory mechanism associated with both physiological and pathological changes in signalling through the TCR.

Does oxidative inactivation of CD45 phosphatase in rheumatoid arthritis underlie immune hyporesponsiveness

The protein tyrosine phosphatase (PTP) CD45 is critical in regulating the earliest steps in T-cell-receptor signaling but, similar to all PTPs, it is susceptible to oxidative inactivation. Given the widely reported effects of oxidant damage associated with rheumatoid arthritis (RA), we examined whether CD45 phosphatase activity was altered in CD4(+) T cells from RA patients and related this to CD4(+) T-cell function and redox status. CD45 phosphatase specific activity in T cells from RA peripheral blood (PB) and synovial fluid was 56% and 59% lower than in healthy control (HC) PB, respectively. In contrast, CD45 activity in T cells from disease controls (DSC) was not significantly different from HC. Both reduced glutathione (GSH) (p<0.001) and oxidized glutathione (GSSG) (p<0.05) were significantly lower in RA PB T cells compared with HC PB T cells. Treatment of RA PB T cells with the GSH precursor N-acetyl cysteine increased CD45 phosphatase activity and proliferation, while it decreased Lck kinase phosphorylation, which is regulated by CD45. Our observations lead to the hypothesis that the largely reversible oxidative inactivation of the CD45 phosphatase may underlie the decreased signaling efficiency and functional responsiveness which are characteristic of RA PB CD4(+) T cells.

On your marks, get SET(D1A): the race to protect stalled replication forks

ABSTRACT We recently identified that methylation of lysine 4 of histone H3 (H3K4) by SETD1A (SET domain containing 1A) maintains genome stability by protecting newly-replicated DNA from degradation. Mechanistically, SETD1A-dependent histone methylation regulates nucleosome mobilisation by FANCD2 (FA complementation group D2), a crucial step in maintaining genome integrity with important implications in chemo-sensitivity.

MYBL2 Supports DNA Double Strand Break Repair in Hematopoietic Stem Cells

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by blood cytopenias that occur as a result of somatic mutations in hematopoietic stem cells (HSC). MDS leads to ineffective hematopoiesis, and as many as 30% of patients progress to acute myeloid leukemia (AML). The mechanisms by which mutations accumulate in HSC during aging remain poorly understood. Here we identify a novel role for MYBL2 in DNA double-strand break (DSB) repair in HSC. In patients with MDS, low MYBL2 levels associated with and preceded transcriptional deregulation of DNA repair genes. Stem/progenitor cells from these patients display dysfunctional DSB repair kinetics after exposure to ionizing radiation (IR). Haploinsufficiency of Mybl2 in mice also led to a defect in the repair of DSBs induced by IR in HSC and was characterized by unsustained phosphorylation of the ATM substrate KAP1 and telomere fragility. Our study identifies MYBL2 as a crucial regulator of DSB repair and identifies MYBL2 expression levels as a potential biomarker to predict cellular response to genotoxic treatments in MDS and to identify patients with defects in DNA repair. Such patients with worse prognosis may require a different therapeutic regimen to prevent progression to AML.Significance: These findings suggest MYBL2 levels may be used as a biological biomarker to determine the DNA repair capacity of hematopoietic stem cells from patients with MDS and as a clinical biomarker to inform decisions regarding patient selection for treatments that target DNA repair.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5767/F1.large.jpg Cancer Res; 78(20); 5767-79. ©2018 AACR.

THU0009 Differential expression of DKK1 in synovial fibroblasts from resolving and early rheumatoid arthritis

Background Mechanisms preventing the resolution of inflammation and promoting joint damage underlie the persistence of RA and the development of damage early in disease. Dickkopf related protein 1 (DKK1) is an inhibitor of the Wnt signalling pathway involved in osteoblast growth and differentiation and cell adhesion and invasion in health and disease. In cancer, increased plasma levels of DKK1 are associated with osteolytic lesions in multiple myeloma and with solid tumour invasion and progression. In rheumatoid arthritis (RA), increased DKK1 plasma levels correlate with inflammation and the presence of bone erosions Objectives We aimed to identify differences in expression of key stromal genes including DKK1 in synovial fibroblasts from patients from three different clinical outcome groups: very early RA, established RA and spontaneously resolving arthritis Methods Synovial tissue biopsies were obtained from 12 early synovitis patients who developed RA (according to 1987 ACR criteria within 18 month follow-up) and 8 early synovitis patients with self-limiting disease. All samples were collected within 12 weeks of the onset of any musculoskeletal symptom attributed to inflammatory arthritis. In addition, 9 patients with established RA (according to 1987 ACR criteria with a symptom duration of >3 months at sample collection), were studied. Synovial fibroblasts were cultured and expanded to passage 3 using established methods. mRNA expression for a range of stromal genes was quantified using real-time quantitative PCR. Values were expressed as 2-dCt relative to GAPDH. DKK1 levels were measured in the supernatants of cultured cells using a commercial ELISA kit (R&D sytems) Results Amongst differentially expressed genes, DKK1 expression was significantly higher in synovial fibroblasts from patients with early RA than in those from patients with resolving arthritis (0.024 vs. 0.009, p<0.005). Differential expression was also observed at the protein level (23.2 ng/ml (11.1-48.5) vs. 6.6 ng/ml (4.2-23.6), p=0.07). There was no differential expression of DKK1 in early RA and established RA groups. Expression levels of DKK1 mRNA or protein did not correlate with disease duration, or with clinical indices Conclusions DKK1 is an inhibitor of the Wnt signalling pathway that has been shown to promote cell invasion and a pro-destruction imbalance of osteoblast and osteoclast activity. Our data suggest that expression of DKK1 by fibroblasts cultured from treatment naive patients discriminates between persistent and resolving disease, and occurs early in the disease process in RA Disclosure of Interest None Declared