DNA Hypomethylation in the TNF-Alpha Gene Predicts Rheumatoid Arthritis Classification in Patients with Early Inflammatory Symptoms.

Biomarkers for the classification of rheumatoid arthritis (RA), and particularly for anti-citrullinated peptide antibody (ACPA)-negative patients, remain an important hurdle for the early initiation of treatment. Taking advantage of DNA-methylation patterns specific to early RA, quantitative methylation-specific qPCR (qMSP) offers a robust technology for the development of biomarkers. We developed assays and established their value as RA classification biomarkers. METHODS: DNA-methylation data were screened to select candidate CpGs to design qMSP assays. Eight assays were developed and tested on two early inflammatory arthritis cohorts. Logistic regression and bootstrapping were used to demonstrate the added value of the qMSP assays. RESULT: Differentially methylated CpG data were screened for candidate CpG, thereby meeting the qMSP assay requirements. The top CpG candidate was in the TNF gene, for which we successfully developed a qMSP assay. Significantly lower DNA-methylation levels were observed in RA (p < 4 × 10-9), with a high predictive value (OR < 0.54/AUC < 0.198) in both cohorts (n = 127/n = 157). Regression using both datasets showed improved accuracy = 87.7% and AUC = 0.944 over the model using only clinical variables (accuracy = 85.2%, AUC = 0.917). Similar data were obtained in ACPA-negative patients (n = 167, accuracy = 82.6%, AUC = 0.930) compared to the clinical variable model (accuracy = 79.5%, AUC = 0.892). Bootstrapping using 2000 datasets confirmed that the AUCs for the clinical+TNF-qMSP model had significant added value in both analyses. CONCLUSION: The qMSP technology is robust and can successfully be developed with a high specificity of the TNF qMSP assay for RA in patients with early inflammatory arthritis. It should assist classification in ACPA-negative patients, providing a means of reducing time to diagnosis and treatment.

Lymphocyte subset phenotyping for the prediction of progression to inflammatory arthritis in anti-citrullinated-peptide antibody-positive at-risk individuals.

OBJECTIVES: Inflammatory arthritis (IA) is considered the last stage of a disease continuum, where features of systemic autoimmunity can appear years before clinical synovitis. Time to progression to IA varies considerably between at-risk individuals, therefore the identification of biomarkers predictive of progression is of major importance. We previously reported on the value of three CD4+T-cell subsets as biomarkers of progression. Here, we aim to establish the value of 18 lymphocyte subsets (LS) for predicting progression to IA. METHODS: Participants were recruited based on a new musculoskeletal complaint and being positive for anti-citrullinated-peptide Antibody. Progression (over 10 years) was defined as the development of clinical synovitis. LS analysis was performed for lymphocyte lineages, naïve/memory subsets, inflammation-related cells (IRC), and regulatory cells (Treg/B-reg). Modelling used Logistic/Cox regressions. RESULTS: Of 210 patients included, 93 (44%) progressed to IA, 41/93 (44%) within 12 months (rapid progressors). 5/18 LS were associated with progression (Treg/CD4-naïve/IRC (adjusted p < 0.0001), CD8 (p = 0.021), B-reg (p = 0.015)) and 3 trends (NK-cells/memory-B-cells/plasmablasts).Unsupervised hierarchical clustering using these 8 subsets segregated 3 clusters of patients, one cluster being enriched (63/109(58%)) and one poor (10/45(22%)) in progressors.Combining all clinical and LS variables, forward logistic regression predicted progression with accuracy=85.7% and AUC=0.911, selecting smoking/Rheumatoid-Factor/HLA-Shared-Epitope/Tender-Joint-Count-78 and Treg/CD4-naive/CD8/NK-cells/B-reg/plasmablasts.To predict rapid progression, a Cox regression was performed resulting in a model combining smoking/rheumatoid factor and IRC/CD4-naïve/Treg/NK-cells/CD8+T-cells (AUC=0.794). CONCLUSION: Overall, progression was predicted by specific LS, suggesting potential triggers for events leading to the development of IA, while rapid progression was associated with a different set of subsets.

Development of a long-term, IL7 dependent cell death rescue assay in CD4+ T-cells.

Interfering with signalling pathways by targeting cell surface proteins has become an important strategy in the development of novel therapeutic agents. Notably, interfering with cytokine signalling revolutionised the treatment of chronic diseases. Cytokines can induce a range of effects that are not always accounted for in assays detecting cytokine binding to cell surface receptors and/or proximal signalling interference. Hence, robust assays are needed to characterise the activity of potential drug candidates targeting such effects. We chose interleukin-7 (IL7) as a cytokine model due to its long-term effect on T-cells. In this report we describe the development and refinement of an in vitro assay for measuring the long-term effect of IL7, more specifically on CD4+ T-cells, while the assay could be adapted to look at CD8+ T-cells. PBMCs and/or purified CD4+ T-cells stained with VPD450 (cell cycle dye) were expanded for 5 days using the mitogen Phytohemagglutinin and/or CD3/CD28 agonists. This resulted in cell proliferation (VPD450 dilution) and activation-induced cell death (7-AAD uptake) which was rescued by the addition of IL7, resulting in cell survival over a further 5 days. JAK-inhibitor (Tofactinib) or a blocking anti-IL7Rα antibody (clone R34.34) abolished cell survival suggesting antagonism, while another antibody (clone A019D5) displayed an agonist effect. These results were confirmed at the proximal signalling level using an IL7/STAT5-luciferase reporter assay. This novel assay for a biological long term effect may be useful for the characterisation of potential therapeutic drugs targeting the IL7/IL7R in CD4+ T-cells.

T-cell subset abnormalities predict progression along the Inflammatory Arthritis disease continuum: implications for management.

The presence of a disease continuum in inflammatory arthritis (IA) is a recognised concept, with distinct stages from at-risk stage (presence of anti citrullinated-peptide autoantibody) to diagnosis of rheumatoid arthritis (RA), including therapy-induced remission. Despite T-cell dysregulation being a key feature of RA, there are few reports of T-cell phenotyping along the IA-continuum. We investigated the disturbances of naïve, regulatory and inflammation related cell (IRC) CD4+ T-cell subsets in 705 individuals across the IA-continuum, developing a simple risk-score (summing presence/absence of a risk-associated with a subset) to predict progression from one stage to the next. In 158 at-risk individuals, the 3 subsets had individual association with progression to IA and the risk-score was highly predictive (p < 0.0001). In evolving IA patients, 219/294 developed RA; the risk-score included naïve and/or Treg and predicted progression (p < 0.0001). In 120 untreated RA patients, the risk-score for predicting treatment-induced remission using naïve T-cells had an odds ratio of 15.4 (p < 0.0001). In RA patients in treatment-induced remission, a score using naïve T-cells predicted disease flare (p < 0.0001). Evaluating the risk of progression using naïve CD4+ T-cells was predictive of progression along the whole IA-continuum. This should allow identification of individuals at high-risk of progression, permitting targeted therapy for improved outcomes.

Defining remission in rheumatoid arthritis: does it matter to the patient? A comparison of multi-dimensional remission criteria and patient reported outcomes.

OBJECTIVES: In a cross-sectional study, we evaluated the prevalence of 'multi-dimensional remission' (MDR) and its component parameters, assessed using objective measures in a cohort of RA patients in treatment-induced DAS28-remission, and their relationship with patient-reported outcome measures. We sought to confirm the feasibility and face validity of the MDR construct, providing a platform for future longitudinal studies in which its clinical utility might be further established. METHODS: 605 patients were selected from an inflammatory arthritis register using DAS28(CRP)<2.6. Demographic, clinical and patients reported outcomes (PRO) data were collected. Ultrasound power doppler synovitis (n = 364) and T-cell subsets (n = 297) were also measured. Remission using clinical parameters was defined as: tender and swollen joint count (TJC/SJC) and CRP all ⩽1; ultrasound remission: total power doppler = 0 and T cell remission: positive normalized naïve T-cell frequency. MDR was defined as the achievement of all three dimensions. RESULTS: Overall, only 53% (321/605) of the patients achieved clinical parameters, failures being mainly due to raised CRP (52%), TJC (28)>1 (37%) or SJC (28)>1 (16%). 211/364 (58%) of patients achieved ultrasound remission and 193/297 (65%) patients showed T-cell remission. Complete data were available for 231 patients. MDR was observed in only 35% and was associated with the best (lower) PRO scores (all P ⩽ 0.05 vs non-MDR) when compared with the other definitions of remission assessed. The MDR rate was similar in early and established RA patients on b-DMARDs; however, it was lower in established RA patients who received multiple cs-DMARDs (P = 0.011). CONCLUSIONS: In this study, MDR, which may represent a state closer to normality, was found to occur in about a third of DAS28-remission patients and was associated with better patient-reported outcome measures. MDR could be a novel optimal treatment target, notably from a patient's perspective. The relevance of these findings needs further assessment.

Quantifying circulating Th17 cells by qPCR: potential as diagnostic biomarker for rheumatoid arthritis.

OBJECTIVE: The diagnosis of RA patients remains a challenge, especially in ACPA-negative disease. Novel T-cell subsets, particularly Th17 may be useful, although data on Th17 frequency using flow cytometry in RA are conflicting. We investigated whether a novel epigenetic qPCR assay for the quantification of Th17 could differentiate patients with RA from those with symptoms evolving towards an alternative diagnosis. METHODS: We used a qPCR assay measuring the extent of the methylation at a key position in the IL-17 and CD4 genes. Assays were performed on whole blood from 49 healthy controls (HC) and 165 early arthritis clinic patients. Flow cytometry was further used to detect the expression of CXCR4 on Th17 cells. RESULTS: In 75 inflammatory arthritis patients who progressed to RA, the qPCR assays showed significantly fewer Th17 cells compared with 90 patients who did not (P<0.0001). Regression models demonstrated a high predictive value for RA development (75.8% correct prediction), and particularly for the ACPA-negative group (n = 125) where Th17 and swollen joint count (SJC) were the only predictors (73% correct prediction). The chemokine receptor CXCR4 had significantly higher expression on Th17 from early RA patients (n = 11) compared with HC (n = 15). CONCLUSION: The results of the epigenetic qPCR assay showed that low levels of Th17 cells were predictive of developing RA, particularly in the ACPA-negative patients. This could have value for insights into pathogenesis and management. The results suggest the recruitment of Th17 to the inflammatory disease site, consistent with high CXCR4 expression.

A tissue-specific promoter derived from a SINE retrotransposon drives biallelic expression of PLAGL1 in human lymphocytes.

The imprinted gene PLAGL1 is an important regulator of apoptosis and cell cycle arrest. Loss of its expression has been implicated in tumorigenesis in a range of different cancers, and overexpression during fetal development causes transient neonatal diabetes mellitus (TNDM). PLAGL1 lies within an imprinted region of chromosome 6q24, and monoallelic expression from the major, differentially methylated promoter (P1) occurs in most human tissues. However, in peripheral blood leukocytes, the active promoter (P2) is non-imprinted and drives biallelic transcription. We report here a novel PLAGL1 promoter (P5) derived from the insertion of a primate-specific, MIR3 SINE retrotransposon. P5 is highly utilized in lymphocytes, particularly in T cells, and like P2, directs biallelic transcription. Our results show that it is important to consider P5 in relation to PLAGL1 function in T cells when investigating the dysregulation of this gene.

Modulation of peripheral T-cell function by interleukin-7 in rheumatoid arthritis.

INTRODUCTION: Interleukin-7 (IL-7) is a cytokine essential for T-cell lymphopoiesis, survival and polarization with an emerging role in autoimmunity. We previously demonstrated reduced levels of circulating IL-7 in rheumatoid arthritis (RA), although high amounts are expressed in joints, suggesting differences between systemic and synovial effects. We observed healthy levels of IL-7 in 48% of RA patients in clinical remission (CR) and aimed to investigate the consequences of IL-7 deficiency on T-cell responses. METHODS: We used RA patients with active disease and in CR presenting various levels of IL-7, to investigate its modulatory effects on T cells by analysing responses to phyto-haemagglutinin (PHA), expression of polarization or survival factors, or suppression by regulatory T cells (Tregs). RESULTS: IL-7 levels were normal (>10 pg/ml) in 48% of RA patients in CR. Amongst 63 CR patients followed up for 18 months, lack of IL-7 recovery was observed in 13 out of 15 (86%) patients experiencing relapse but only 11 out of 48 (23%) of those who did not (P = 0.0002). Binary regressions showed high significance for below normal IL-7 levels for self-reported maternal family history of arthritis (odds ratio (OR): 7.66, P = 0.006) and a trend for smoking (OR: 3.33, P = 0.068) with no further demographic or clinical associations. Serum IL-7 correlated with restored CD4+T-cell response to PHA (rho = 0.879); this was not related to an increase in T-cell proliferation capacity or expression of survival factors B-cell lymphoma 2 (BCL2) and BCL2-associated protein X (BAX). Expression of Th1 polarization factor (TBET) was also dependent on exposure to IL-7 in vivo (rho = 0.600). In contrast CD25highTregs' response to PHA was not affected by in vivo IL-7, but their suppression capabilities were related to circulating IL-7 (rho = 0.589). Co-stimulation with IL-7 (mimicking the joint environment) increased responsiveness of CD4+T-cells to PHA, lowering the ability of CD25highTregs to suppress them. CONCLUSIONS: Our data demonstrate that IL-7 has a critical role in modulating T-cell function in vivo, possibly explaining opposing effects observed systemically and in the joint. Lack of IL-7 recovery in CR by maintaining a suppressed immune system may be a determinant factor in the occurrence of relapse.

An immunological biomarker to predict MTX response in early RA.

OBJECTIVES: The therapeutic goal for patients with rheumatoid arthritis (RA) is clinical remission. This is best achieved by early diagnosis and appropriate therapeutic intervention. RA is associated with dysregulation of T-cell subsets (naïve, regulatory (Treg) and inflammation-related cells (IRC)) early in the disease. Our aim was to test the hypothesis that T-cell subset quantification can predict the achievement of clinical remission with early treatment in RA. METHODS: T-cell subsets were quantified in 108 drug-naïve, early RA patients commencing methotrexate (MTX) or MTX+antitumor necrosis factor (anti-TNF) and in 105 healthy controls (HC). The primary outcome assessed was remission (DAS28<2.6). A pilot study used frozen cells (38 patients and 35 HCs, see online supplementary material) and was validated with fresh blood (70 patients and 70 HCs). RESULTS: Immune dysregulation in early RA was confirmed with an association between age and reduced naïve cells compared with HCs (p=0.006), a lower age-adjusted Treg and higher IRC frequency (p=0.001). Anticitrullinated peptide antibody (ACPA) positivity was associated with lower naïve (p=0.031) and Treg frequencies (p=0.039). In 50 patients treated with MTX, ACPA/age-adjusted analysis demonstrated that higher naïve cell frequency (relative to HC) was associated with remission (OR 5.90 (1.66 to 20.98), p=0.006, sensitivity/specificity 62%/79%, Positive Predictive Value (PPV)/Negative Predictive Value (NPV) 66%/76%). Remission with MTX+anti-TNF (n=20) was not found to be associated with naïve cell frequency, and for patients with reduced naïve cells the remission rate increased from 24% (MTX) to 42% (MTX+anti-TNF). CONCLUSIONS: Baseline T-cell subset analysis has a value in predicting early RA remission with first therapy with MTX. Immunological analysis could be used in conjunction with clinical/serological features to predict response to MTX and help select the most appropriate therapy at disease presentation.

Progression to rheumatoid arthritis in early inflammatory arthritis is associated with low IL-7 serum levels.

OBJECTIVE: Early diagnosis of rheumatoid arthritis (RA) remains a challenge. Interleukin (IL)-7 is a pleiotropic cytokine that plays a central role in the development and maintenance of T-cells and has been associated with T-cell dysfunction in RA. Serum levels of IL-7 are reduced in both early and established disease. The aim of this study was to determine whether serum IL-7 can identify patients with very early inflammatory joint symptoms who will progress to RA, and to examine whether IL-7 levels predict disease persistence and radiographic progression. METHODS: Patients with inflammatory joint symptoms<6 months followed over 5 years for progression to RA and 80 healthy controls were studied. Baseline IL-7 levels were measured by ELISA. RESULTS: Of 250 patients, 108 developed RA (ACR 1987- criteria). IL-7 at inclusion was reduced significantly in RA compared with non-RA patients (p=0.009). IL-7 was categorised using the lower limit of the healthy control distribution (10 pg/ml). In multivariate analysis, independent predictors of RA development were: antibodies against citrullinated peptides (ACPA) positivity (p=0.001), IL-7<10 pg/ml (p=0.003) and swollen joint count (p=0.050). In the ACPA-negative subgroup (n=199), the only predictors were: DAS-44 (p=0.001), IL-7<10 pg/ml (p=0.010) and radiographic erosions (p=0.050). At 1-year follow-up, remission (DAS<1.6) was only predicted by ACPA negativity (p=0.019) and IL-7>17 pg/ml at recruitment (p=0.013). CONCLUSION: These data demonstrate that low IL-7 levels in patients with recent onset of symptoms may have value as a diagnostic biomarker predicting the progression to RA, particularly in ACPA-negative disease, as well as being related to RA progression.

Multiplexing immunoassays for cytokine detection in the serum of patients with rheumatoid arthritis: lack of sensitivity and interference by rheumatoid factor.

OBJECTIVES: Accurately measuring cytokines in clinical material remains an important challenge in the development of biomarkers. Enzyme-linked immunoabsorbent assays (ELISAs) are considered 'gold standard'; however, their use is limited by the relatively large sample volume required for multiple analyte testing. Several alternatives (including membrane or bead-ELISA) have been developed particularly to enable multiplexing. Concerns were raised regarding their use in rheumatology due to interference by heterophilic antibodies, notably rheumatoid factor (RF). In this report, we compared several multiplex assays using serum from rheumatoid arthritis (RA) patients with respect to the presence of residual RF following attempted removal employing commonly used procedures. METHODS: Healthy control and RF-positive/negative RA sera were used to compare 4 multiplex assays with ELISA: bead-based 'Luminex' immunoassay, cytometric bead assays (CBAs), membrane-based and Mosaic™ ELISAs. Sera were tested following Ig blockade (mixed species serum) or removal (using PEG6000 or sepharose-L). RESULTS: Ig removal was only partially efficient and residual RF was detected in most sera. RF had no impact on cytokine measurement by ELISA. In single and multiplex Luminex, cytokine levels associated with false positive results correlated directly with RF titres. Following Ig-blockade/removal, these relationship remained suggesting false positivity was still associated with the presence of residual RF. Conversely, detection of cytokines in multiplex membrane-based or Mosaic- ELISA were not affected by the presence of RF; however, levels of cytokines readily detected by ELISA were often below the detection threshold of these assays. CBA assays were also low on sensitivity but unaffected by RF. CONCLUSIONS: False positivity, due to the presence of heterophilic antibodies, mainly affected Luminex assays. Other assays however remained limited in their sensitivity. Multiplexing of cytokine measurement remains a challenge, particularly in rheumatological pathologies, until assays of adequate sensitivity are developed. ELISA remains the gold standard.

Patients with RA in remission on TNF blockers: when and in whom can TNF blocker therapy be stopped?

OBJECTIVES: Combination therapy with methotrexate (MTX) and tumour necrosis factor (TNF) blockade has increased remission rates in patients with rheumatoid arthritis. However, there are no guidelines regarding cessation of therapy. There is a need for markers predictive of sustained remission following cessation of TNF blocker therapy. METHODS: Patients in remission (DAS28 <2.6) treated with a TNF blocker and MTX as initial or delayed therapy were recruited. Joints were assessed for grey scale synovitis and power Doppler (PD) activity. Immunological assessment involved advanced six-colour flow cytometry. RESULTS: Of the 47 patients recruited, 27 had received initial treatment and 20 delayed treatment with TNF blocking drugs. Two years after stopping TNF blocker therapy, the main predictor of successful cessation was timing of treatment; 59% of patients in the initial treatment group sustained remission compared with 15% in the delayed treatment group (p=0.003). Within the initial treatment group, secondary analysis showed that the only clinical predictor of successful cessation of treatment was shorter symptom duration before receiving treatment (median 5.5 months vs 9 months; p=0.008). No other clinical features were associated with successful cessation of therapy. Thirty-five per cent of patients had low PD activity but levels were not informative. Several immunological parameters were significantly associated with sustained remission including abnormal differentiation subset of T cells and regulatory T cells. Similar non-significant trends were observed in the delayed treatment group. CONCLUSION: In patients in remission with low levels of imaging synovitis receiving combination treatment with a TNF blocker and MTX, immunological parameters and short duration of untreated symptoms were associated with successful cessation of TNF blocker therapy.

Clinical and molecular phenotype of Aicardi-Goutieres syndrome.

Aicardi-Goutieres syndrome (AGS) is a genetic encephalopathy whose clinical features mimic those of acquired in utero viral infection. AGS exhibits locus heterogeneity, with mutations identified in genes encoding the 3'-->5' exonuclease TREX1 and the three subunits of the RNASEH2 endonuclease complex. To define the molecular spectrum of AGS, we performed mutation screening in patients, from 127 pedigrees, with a clinical diagnosis of the disease. Biallelic mutations in TREX1, RNASEH2A, RNASEH2B, and RNASEH2C were observed in 31, 3, 47, and 18 families, respectively. In five families, we identified an RNASEH2A or RNASEH2B mutation on one allele only. In one child, the disease occurred because of a de novo heterozygous TREX1 mutation. In 22 families, no mutations were found. Null mutations were common in TREX1, although a specific missense mutation was observed frequently in patients from northern Europe. Almost all mutations in RNASEH2A, RNASEH2B, and RNASEH2C were missense. We identified an RNASEH2C founder mutation in 13 Pakistani families. We also collected clinical data from 123 mutation-positive patients. Two clinical presentations could be delineated: an early-onset neonatal form, highly reminiscent of congenital infection seen particularly with TREX1 mutations, and a later-onset presentation, sometimes occurring after several months of normal development and occasionally associated with remarkably preserved neurological function, most frequently due to RNASEH2B mutations. Mortality was correlated with genotype; 34.3% of patients with TREX1, RNASEH2A, and RNASEH2C mutations versus 8.0% RNASEH2B mutation-positive patients were known to have died (P=.001). Our analysis defines the phenotypic spectrum of AGS and suggests a coherent mutation-screening strategy in this heterogeneous disorder. Additionally, our data indicate that at least one further AGS-causing gene remains to be identified.

Heterozygous mutations in TREX1 cause familial chilblain lupus and dominant Aicardi-Goutieres syndrome.

TREX1 constitutes the major 3'-->5' DNA exonuclease activity measured in mammalian cells. Recently, biallelic mutations in TREX1 have been shown to cause Aicardi-Goutieres syndrome at the AGS1 locus. Interestingly, Aicardi-Goutieres syndrome shows overlap with systemic lupus erythematosus at both clinical and pathological levels. Here, we report a heterozygous TREX1 mutation causing familial chilblain lupus. Additionally, we describe a de novo heterozygous mutation, affecting a critical catalytic residue in TREX1, that results in typical Aicardi-Goutieres syndrome.

Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus.

Aicardi-Goutières syndrome (AGS) presents as a severe neurological brain disease and is a genetic mimic of the sequelae of transplacentally acquired viral infection. Evidence exists for a perturbation of innate immunity as a primary pathogenic event in the disease phenotype. Here, we show that TREX1, encoding the major mammalian 3' --> 5' DNA exonuclease, is the AGS1 gene, and AGS-causing mutations result in abrogation of TREX1 enzyme activity. Similar loss of function in the Trex1(-/-) mouse leads to an inflammatory phenotype. Our findings suggest an unanticipated role for TREX1 in processing or clearing anomalous DNA structures, failure of which results in the triggering of an abnormal innate immune response.

Mutations in genes encoding ribonuclease H2 subunits cause Aicardi-Goutières syndrome and mimic congenital viral brain infection.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive neurological disorder, the clinical and immunological features of which parallel those of congenital viral infection. Here we define the composition of the human ribonuclease H2 enzyme complex and show that AGS can result from mutations in the genes encoding any one of its three subunits. Our findings demonstrate a role for ribonuclease H in human neurological disease and suggest an unanticipated relationship between ribonuclease H2 and the antiviral immune response that warrants further investigation.