2022 EULAR points to consider for the measurement, reporting and application of IFN-I pathway activation assays in clinical research and practice.

BACKGROUND: Type I interferons (IFN-Is) play a role in a broad range of rheumatic and musculoskeletal diseases (RMDs), and compelling evidence suggests that their measurement could have clinical value, although testing has not progressed into clinical settings. OBJECTIVE: To develop evidence-based points to consider (PtC) for the measurement and reporting of IFN-I assays in clinical research and to determine their potential clinical utility. METHODS: EULAR standardised operating procedures were followed. A task force including rheumatologists, immunologists, translational scientists and a patient partner was formed. Two systematic reviews were conducted to address methodological and clinical questions. PtC were formulated based on the retrieved evidence and expert opinion. Level of evidence and agreement was determined. RESULTS: Two overarching principles and 11 PtC were defined. The first set (PtC 1-4) concerned terminology, assay characteristics and reporting practices to enable more consistent reporting and facilitate translation and collaborations. The second set (PtC 5-11) addressed clinical applications for diagnosis and outcome assessments, including disease activity, prognosis and prediction of treatment response. The mean level of agreement was generally high, mainly in the first PtC set and for clinical applications in systemic lupus erythematosus. Harmonisation of assay methodology and clinical validation were key points for the research agenda. CONCLUSIONS: IFN-I assays have a high potential for implementation in the clinical management of RMDs. Uptake of these PtC will facilitate the progress of IFN-I assays into clinical practice and may be also of interest beyond rheumatology.

Serum IFNα2 levels are associated with disease activity and outperform IFN-I gene signature in a longitudinal childhood-onset SLE cohort.

OBJECTIVE: To study the association of serum IFNα2 levels measured by ultrasensitive single-molecule array (Simoa) and the IFN-I gene signature (IGS) with disease activity and determine whether these assays can mark disease activity states in a longitudinal cohort of childhood-onset SLE (cSLE) patients. METHODS: Serum IFNα2 levels were measured in 338 samples from 48 cSLE patients and 67 healthy controls using an IFNα Simoa assay. Five-gene IGS was measured by RT-PCR in paired whole blood samples. Disease activity was measured by clinical SELENA-SLEDAI and BILAG-2004. Low disease activity was defined by Low Lupus Disease Activity State (LLDAS) and flares were characterized by SELENA-SLEDAI flare index. Analysis was performed using linear mixed models. RESULTS: A clear positive correlation was present between serum IFNα2 levels and the IGS (r = 0.78, P < 0.0001). Serum IFNα2 levels and IGS showed the same significant negative trend in the first 3 years after diagnosis. In this timeframe, mean baseline serum IFNα2 levels decreased by 55.1% (Δ 201 fg/ml, P < 0.001) to a mean value of 164 fg/ml, which was below the calculated threshold of 219.4 fg/ml that discriminated between patients and healthy controls. In the linear mixed model, serum IFNα2 levels were significantly associated with both cSELENA-SLEDAI and BILAG-2004, while the IGS did not show this association. Both IFN-I assays were able to characterize LLDAS and disease flare in receiver operating characteristic analysis. CONCLUSIONS: Serum IFNα2 levels measured by Simoa technology are associated with disease activity scores and characterize disease activity states in cSLE.

Serum interferon-α2 measured by single-molecule array associates with systemic disease manifestations in Sjögren's syndrome.

OBJECTIVES: Type I IFN (IFN-I) activation is a prominent feature of primary SS (pSS), SLE and SSc. Ultrasensitive single-molecule array (Simoa) technology has facilitated the measurement of subfemtomolar concentrations of IFNs. Here we aimed to measure IFN-α2 in serum from pSS, SLE and SSc using a Simoa immunoassay and correlate these levels to blood IFN-stimulated gene (ISG) expression and disease activity. METHODS: Serum IFN-α2 was measured in patients with pSS (n = 85 and n = 110), SLE (n = 24) and SSc (n = 23) and healthy controls (HCs; n = 68) using an IFN-α Simoa assay on an HD-X analyser. IFN-I pathway activation was additionally determined from serum by an IFN-I reporter assay and paired samples of whole blood ISG expression of IFI44, IFI44L, IFIT1, IFIT3 and MxA by RT-PCR or myxovirus resistance protein 1 (MxA) protein ELISA. RESULTS: Serum IFN-α2 levels were elevated in pSS (median 61.3 fg/ml) compared with HCs (median ≤5 fg/ml, P < 0.001) and SSc (median 11.6 fg/ml, P = 0.043), lower compared with SLE (median 313.5 fg/ml, P = 0.068) and positively correlated with blood ISG expression (r = 0.66-0.94, P < 0.001). Comparable to MxA ELISA [area under the curve (AUC) 0.93], IFN-α2 measurement using Simoa identified pSS with high ISG expression (AUC 0.90) with 80-93% specificity and 71-84% sensitivity. Blinded validation in an independent pSS cohort yielded a comparable accuracy. Multiple regression indicated independent associations of autoantibodies, IgG, HCQ treatment, cutaneous disease and a history of extraglandular manifestations with serum IFN-α2 concentrations in pSS. CONCLUSION: Simoa serum IFN-α2 reflects blood ISG expression in pSS, SLE and SSc. In light of IFN-targeting treatments, Simoa could potentially be applied for patient stratification or retrospective analysis of historical cohorts.

Gene signature fingerprints stratify SLE patients in groups with similar biological disease profiles: a multicentre longitudinal study.

OBJECTIVES: Clinical phenotyping and predicting treatment responses in SLE patients is challenging. Extensive blood transcriptional profiling has identified various gene modules that are promising for stratification of SLE patients. We aimed to translate existing transcriptomic data into simpler gene signatures suitable for daily clinical practice. METHODS: Real-time PCR of multiple genes from the IFN M1.2, IFN M5.12, neutrophil (NPh) and plasma cell (PLC) modules, followed by a principle component analysis, was used to identify indicator genes per gene signature. Gene signatures were measured in longitudinal samples from two childhood-onset SLE cohorts (n = 101 and n = 34, respectively), and associations with clinical features were assessed. Disease activity was measured using Safety of Estrogen in Lupus National Assessment (SELENA)-SLEDAI. Cluster analysis subdivided patients into three mutually exclusive fingerprint-groups termed (1) all-signatures-low, (2) only IFN high (M1.2 and/or M5.12) and (3) high NPh and/or PLC. RESULTS: All gene signatures were significantly associated with disease activity in cross-sectionally collected samples. The PLC-signature showed the highest association with disease activity. Interestingly, in longitudinally collected samples, the PLC-signature was associated with disease activity and showed a decrease over time. When patients were divided into fingerprints, the highest disease activity was observed in the high NPh and/or PLC group. The lowest disease activity was observed in the all-signatures-low group. The same distribution was reproduced in samples from an independent SLE cohort. CONCLUSIONS: The identified gene signatures were associated with disease activity and were indicated to be suitable tools for stratifying SLE patients into groups with similar activated immune pathways that may guide future treatment choices.

Hyperresponsive cytosolic DNA-sensing pathway in monocytes from primary Sjögren's syndrome.

OBJECTIVES: Cytosolic DNA-sensing pathway stimulation prompts type I IFN (IFN-I) production, but its role in systemic IFN-I pathway activation in primary SS (pSS) is poorly studied. Here we investigate the responsiveness of pSS monocytes and plasmacytoid dendritic cells (pDCs) to stimulator of interferon genes (STING) activation in relation to systemic IFN-I pathway activation and compare this with SLE. METHODS: Expression of DNA-sensing receptors cGAS, IFI16, ZBP-1 and DDX41, signalling molecules STING, TBK1 and IRF3, positive and negative STING regulators, and IFN-I-stimulated genes MxA, IFI44, IFI44L, IFIT1 and IFIT3 was analysed in whole blood, CD14+ monocytes, pDCs, and salivary glands by RT-PCR, monocyte RNA sequencing data, flow cytometry and immunohistochemical staining. Peripheral blood mononuclear cells (PBMCs) from pSS, SLE and healthy controls (HCs) were stimulated with STING agonist 2'3'-cGAMP. STING phosphorylation (pSTING) and intracellular IFNα were evaluated using flow cytometry. RESULTS: STING activation induced a significantly higher proportion of IFNα-producing monocytes, but not pDCs, in both IFN-low and IFN-high pSS compared with HC PBMCs. Additionally, a trend towards more pSTING+ monocytes was observed in pSS and SLE, most pronounced in IFN-high patients. Positive STING regulators TRIM38, TRIM56, USP18 and SENP7 were significantly higher expression in pSS than HC monocytes, while the dual-function STING regulator RNF26 was downregulated in pSS monocytes. STING was expressed in mononuclear infiltrates and ductal epithelium in pSS salivary glands. STING stimulation induced pSTING and IFNα in pSS and SLE pDCs. CONCLUSION: pSS monocytes and pDCs are hyperresponsive to stimulation of the STING pathway, which was not restricted to patients with IFN-I pathway activation.

Innate immunity and interferons in the pathogenesis of Sjögren's syndrome.

Primary SS (pSS) is a rheumatic disease characterized by an immune-mediated exocrinopathy, resulting in severe dryness of eyes and mouth. Systemic symptoms include fatigue and joint pain and a subset of patients develop more severe disease with multi-organ involvement. Accumulating evidence points to involvement of innate immunity and aberrant activity of the type I IFN system in both the initiation and propagation of this disease. Analysis of the activity of IFN-inducible genes has evidenced that more than half of pSS patients present with a so-called 'type I IFN signature'. In this review, we examine activation of the IFN system in pSS patients and how this may drive autoimmunity through various immune cells. We further discuss the clinical value of assessing IFN activity as a biomarker in pSS patients and review novel therapies targeting IFN signalling and their potential use in pSS.

Revisiting the JOQUER trial: stratification of primary Sjögren's syndrome and the clinical and interferon response to hydroxychloroquine.

To re-analyse the clinical outcomes and interferon (IFN) activity data from the JOQUER trial, a phase III trial investigating hydroxychloroquine (HCQ) in patients with primary Sjögren's syndrome (pSS), after stratifying patients into putative pathobiological subgroups utilizing the Newcastle Sjögren's Stratification Tool (NSST) based on patient-reported symptoms of dryness, pain, fatigue, anxiety and depression. 107 patients were assigned to one of four subgroups using NSST at baseline-the high symptom burden (HSB), pain dominant with fatigue (PDF), dryness dominant with fatigue (DDF) and low symptom burden (LSB). Endpoints were re-analysed after stratification, testing for treatment differences within subgroups and adjusting for baseline differences using a repeated measures covariate model. The HSB subgroup (n = 32) showed a relative improvement in ESSPRI of 1.49 points (95% CI 0.54-2.43; p = 0.002) within 12 weeks in patients taking HCQ compared to placebo, with no further changes after 24 weeks. For the LSB subgroup (n = 14), the ESSPRI worsened in the placebo but not the HCQ arm after 12 weeks (mean difference 1.44, 95% CI 0.05-2.83, p = 0.042). Neither the HSB nor the LSB patients showed significant changes in IFN activity at 24 weeks. There were no significant differences in ESSPRI in the PDF (n = 39) and DDF (n = 22) patients taking HCQ. However, significant reductions in overall IFN score at 24 weeks were seen in both PDF (difference at 24 weeks; 6.41, 95% CI, 2.48-10.34, p = 0.002) and DDF (difference at 24 weeks; 7.23, 95% CI, 1.85-12.6, p = 0.009) without improvement in ESSPRI. Although the JOQUER trial reported no overall benefit from HCQ in pSS patients, stratification suggests that both HSB and LSB subgroups may respond to HCQ. However, these patients may benefit through mechanisms other than the reduction of IFN activities.

Hydroxychloroquine treatment downregulates systemic interferon activation in primary Sjögren's syndrome in the JOQUER randomized trial.

OBJECTIVE: HCQ is frequently used to treat primary SS (pSS), but evidence for its efficacy is limited. HCQ blocks IFN activation, which is present in half of the pSS patients. The effect of HCQ treatment on the expression of IFN-stimulated genes (ISGs) was studied in pSS. Furthermore, HCQ-treated patients were stratified based on IFN activation and differences in disease activity and clinical parameters were studied. METHODS: Expression of ISGs and IFN scores was determined in 77 patients, who were previously enrolled in the placebo-controlled JOQUER trial. Patients were treated for 24 weeks with 400 mg/d HCQ or placebo. RESULTS: HCQ treatment reduced IFN scores and expression of ISGs compared with the placebo-treated group. HCQ reduced ESR, IgG and IgM levels independently of the patients' IFN activation status. No differences in EULAR SS disease activity index or EULAR SS patient reported index scores were observed after HCQ treatment, even after IFN stratification. CONCLUSION: Treatment for 24 weeks with HCQ significantly reduced type I IFN scores and ISG-expression compared with the placebo-treated group. HCQ reduced several laboratory parameters, but failed to improve clinical response. This suggests that in pSS, type I IFN is associated to some laboratory parameters abnormalities, but not related to the clinical response.

Associations of cigarette smoking with disease phenotype and type I interferon expression in primary Sjögren's syndrome.

Several studies have shown a negative association between smoking and primary Sjögren's syndrome (pSS), and smoking may interfere with the immune response. The purpose of this study was to investigate if smoking affects disease activity and disease phenotype in pSS. In this cross-sectional study, consecutive pSS patients filled out the EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) form and a structured questionnaire regarding smoking habits. EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) scores were calculated and blood samples were analysed for type I interferon signature using RT-PCR. Of 90 patients (93% women, median age 66.5 years), 72% were type I IFN signature positive and 6, 42 and 53% were current, former and never smokers, respectively. No significant differences by smoking status were found regarding ESSDAI total score, activity in the ESSDAI domains or type I IFN signature. Patients with a higher cumulative cigarette consumption (≥ median) had higher scores in ESSPRI total [5.0 (3.0-6.3) vs 8.0 (6.0-8.3); p < 0.01] and ESSPRI sicca and pain domains. Comparing type I IFN signature negative and positive patients, the latter had significantly lower activity in ESSDAI articular domain (7/25 vs 3/64; p < 0.01) and lower scores in ESSPRI total [7.7 (5.2-8.2) vs 6.0 (4.0-7.7); p = 0.04]. Smoking was not associated with disease phenotype although patients with a higher cumulative cigarette consumption had worse symptoms in some disease domains. Current smokers were few making it difficult to draw any firm conclusions about associations to current smoking.

TBK1: A key regulator and potential treatment target for interferon positive Sjögren's syndrome, systemic lupus erythematosus and systemic sclerosis.

OBJECTIVE: Upregulation of type I interferons (IFN-I) is a hallmark of systemic autoimmune diseases like primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). Expression of IFN-I is induced by three different receptor families: Toll-like receptors (TLRs), RIG-like receptors (RLRs) and DNA-sensing receptors (DSRs). TANK-binding kinase (TBK1) is an important signaling hub downstream of RLRs and DSRs. TBK1 activates IRF3 and IRF7, leading to IFN-I production and subsequent induction of interferon stimulated genes (ISGs). The objective of this study was to explore the potential of BX795, an inhibitor of TBK1, to downregulate IFN-I activation in pSS, SLE and SSc. METHODS: TBK1, IRF3, IRF7 and STAT1 were determined by RT-PCR in PAXgene samples and phosphorylated-TBK1 (pTBK1) was analyzed by flowcytometry in plasmacytoid dendritic cells (pDCs) from IFN-I positive (IFNpos) patients. Peripheral blood mononuclear cells (PBMCs) of pSS, SLE and SSc patients and TLR7 stimulated PBMCs of healthy controls (HCs) were cultured with the TBK1 inhibitor BX795, followed by analysis of ISGs. RESULTS: Increased gene expression of TBK1, IRF3, IRF7 and STAT1 in whole blood and pTBK1 in pDCs was observed in IFNpos pSS, SLE and SSc patients compared to HCs. Upon treatment with BX795, PBMCs from IFNpos pSS, SLE, SSc and TLR7-stimulated HCs downregulated the expression of the ISGs MxA, IFI44, IFI44L, IFIT1 and IFIT3. CONCLUSIONS: TBK1 inhibition reduced expression of ISGs in PBMCs from IFNpos patients with systemic autoimmune diseases indicating TBK1 as a potential treatment target.

Systemic interferon type I and type II signatures in primary Sjögren's syndrome reveal differences in biological disease activity.

Objective: To assess the relationships between systemic IFN type I (IFN-I) and II (IFN-II) activity and disease manifestations in primary SS (pSS). Methods: RT-PCR of multiple IFN-induced genes followed by principal component analysis of whole blood RNA of 50 pSS patients was used to identify indicator genes of systemic IFN-I and IFN-II activities. Systemic IFN activation levels were analysed in two independent European cohorts (n = 86 and 55, respectively) and their relationships with clinical features were analysed. Results: Three groups could be stratified according to systemic IFN activity: IFN inactive (19-47%), IFN-I (53-81%) and IFN-I + II (35-55%). No patient had isolated IFN-II activation. IgG levels were highest in patients with IFN-I + II, followed by IFN-I and IFN inactive patients. The prevalence of anti-SSA and anti-SSB was higher among those with IFN activation. There was no difference in total-EULAR SS Disease Activity Index (ESSDAI) or ClinESSDAI between the three subject groups. For individual ESSDAI domains, only the biological domain scores differed between the three groups (higher among the IFN active groups). For patient reported outcomes, there were no differences in EULAR Sjögren's syndrome patient reported index (ESSPRI), fatigue or dryness between groups, but pain scores were lower in the IFN active groups. Systemic IFN-I but not IFN-I + II activity appeared to be relatively stable over time. Conclusions: Systemic IFN activation is associated with higher activity only in the ESSDAI biological domain but not in other domains or the total score. Our data raise the possibility that the ESSDAI biological domain score may be a more sensitive endpoint for trials targeting either IFN pathway.

Contrasting expression pattern of RNA-sensing receptors TLR7, RIG-I and MDA5 in interferon-positive and interferon-negative patients with primary Sjögren's syndrome.

OBJECTIVE: The interferon (IFN) type I signature is present in over half of patients with primary Sjögren's syndrome (pSS) and associated with higher disease-activity and autoantibody presence. Plasmacytoid dendritic cells (pDCs) are considered as the main source of enhanced IFN type I expression. The objective of this study was to unravel the molecular pathways underlying IFN type I bioactivity in pDCs of patients with pSS. METHODS: Blood samples from 42 healthy controls (HC) and 115 patients with pSS were stratified according to their IFN type I signature. CD123+BDCA4+ pDCs and CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMCs). Genome-wide microarray analysis was conducted on sorted pDCs in a small sample set, followed by validation of differentially expressed genes of interest in pDCs and monocytes. RESULTS: We found an upregulation of endosomal toll-like receptor (TLR) 7, but not TLR9, in IFN-positive (IFNpos) pDCs (p<0.05) and monocytes (p=0.024). Additionally, the downstream signalling molecules MyD88, RSAD2 and IRF7 were upregulated, as were the cytoplasmic RNA-sensing receptors DDX58/retinoic acid inducible gene-I (RIG-I) and IFIH1/melanoma differentiation associated gene-5 (MDA5). In vitro triggering of the TLR7-pathway in HC PBMCs induced upregulation of DDX58/RIG-I and IFIH1/MDA5, and downregulated TLR9. The upregulation of TLR7, its downstream signalling pathway, DDX58/RIG-I and IFIH1/MDA5 were confined to patients with IFN-positive pSS. IFN-negative patients had a contrasting expression pattern-TLR7 normal, and decreased TLR9, RIG-I and MDA5. CONCLUSIONS: Here we conclude a contrasting expression pattern of the RNA-sensing receptors TLR7, RIG-I and MDA5 in pDCs and monocytes of patients with IFNpos pSS. This profile could explain the pathogenic IFN production and might reveal novel therapeutic targets in these patients.

The interferon type I signature is present in systemic sclerosis before overt fibrosis and might contribute to its pathogenesis through high BAFF gene expression and high collagen synthesis.

BACKGROUND: Interferon (IFN) signature has been reported in definite systemic sclerosis (SSc) but it has not been characterised in early SSc (EaSSc). We aim at characterising IFN type I signature in SSc before overt skin fibrosis develops. METHODS: The expression of 11 IFN type I inducible genes was tested in whole-blood samples from 30 healthy controls (HCs), 12 subjects with primary Raynaud's phenomenon (RP), 19 patients with EaSSc, 7 patients with definite SSc without cutaneous fibrosis, 21 limited cutaneous SSc and 10 diffuse cutaneous SSc subjects. The correlation between IFN activity in monocytes, B cell activating factor (BAFF) mRNA expression and type III procollagen N-terminal propeptide (PIIINP) serum levels was tested. RESULTS: In all the SSc groups, higher IFN scores were observed compared with HC. An IFN score ≥7.09 discriminated HCs from patients with SSc (sensitivity=0.7, specificity=0.88, area under receiving operating characteristic (AUROC)=0.82); the prevalence of an elevated IFN score was: HC=3.3%; RP=33.3%, EaSSc=78.9%, definite SSc=100%, limited cutaneous SSc=42.9%, diffuse cutaneous SSc=70.0%. In monocytes an IFN score ≥4.12 distinguished HCs from patients with fibrotic SSc (sensitivity=0.62, specificity=0.85, AUROC=0.76). Compared with IFN-negative subjects, IFN-positive subjects had higher monocyte BAFF mRNA levels (19.7±5.2 vs 15.20±4.0, p=2.1×10(-5)) and serum PIIINP levels (median=6.0 (IQR 5.4-8.9) vs median=3.9 (IQR 3.3-4.7), p=0.0004). CONCLUSIONS: An IFN type I signature is observed in patients with SSc from the earliest phases of the disease, even before overt skin fibrosis. The presence of IFN type I signature in monocytes is correlated with BAFF mRNA expression and serum PIIINP levels, supporting a contribution in the pathogenesis and progression of SSc.

Bone Mineral Density in Sjögren Syndrome Patients with and Without Distal Renal Tubular Acidosis.

Primary Sjögren's syndrome (pSS) can be complicated by distal renal tubular acidosis (dRTA), which may contribute to low bone mineral density (BMD). Our objective was to evaluate BMD in pSS patients with and without dRTA as compared with healthy controls. BMD of lumbar spine (LS) and femoral neck (FN) was measured in 54 pSS patients and 162 healthy age- and sex-matched controls by dual-energy X-ray absorptiometry (DXA). dRTA was defined as inability to reach urinary pH <5.3 after an ammonium chloride (NH4Cl) test. LS- and FN-BMD were significantly higher in pSS patients compared with controls (1.18 ± 0.21 g/cm(2) for patients vs. 1.10 ± 0.18 g/cm(2) for controls, P = 0.008 and 0.9 ± 0.16 g/cm(2) for patients vs. 0.85 ± 0.13 g/cm(2) for controls, P = 0.009, respectively). After adjustment for BMI and smoking, the LS- and FN-BMD remained significantly higher. Patients with dRTA (N = 15) did not have a significantly different LS- and FN-BMD compared with those without dRTA (N = 39) after adjustment for BMI, age, and gender. Thirty-seven (69 %) pSS patients were using hydroxychloroquine (HCQ). Unexpectedly, pSS patients had a significantly higher LS- and FN-BMD compared with healthy controls. Patients with dRTA had similar BMD compared with patients without dRTA. We postulate that an explanation for the higher BMD in pSS patients may be the frequent use of HCQ.

Exaggerated Increases in Microglia Proliferation, Brain Inflammatory Response and Sickness Behaviour upon Lipopolysaccharide Stimulation in Non-Obese Diabetic Mice.

The non-obese diabetic (NOD) mouse, an established model for autoimmune diabetes, shows an exaggerated reaction of pancreas macrophages to inflammatory stimuli. NOD mice also display anxiety when immune-stimulated. Chronic mild brain inflammation and a pro-inflammatory microglial activation is critical in psychiatric behaviour. OBJECTIVE: To explore brain/microglial activation and behaviour in NOD mice at steady state and after systemic lipopolysaccharide (LPS) injection. METHODS: Affymetrix analysis on purified microglia of pre-diabetic NOD mice (8-10 weeks) and control mice (C57BL/6 and CD1 mice, the parental non-autoimmune strain) at steady state and after systemic LPS (100 µg/kg) administration. Quantitative PCR was performed on the hypothalamus for immune activation markers (IL-1ß, IFNγ and TNFα) and growth factors (BDNF and PDGF). Behavioural profiling of NOD, CD1, BALB/c and C57BL/6 mice at steady state was conducted and sickness behaviour/anxiety in NOD and CD1 mice was monitored before and after LPS injection. RESULTS: Genome analysis revealed cell cycle/cell death and survival aberrancies of NOD microglia, substantiated as higher proliferation on BrdU staining. Inflammation signs were absent. NOD mice had a hyper-reactive response to novel environments with some signs of anxiety. LPS injection induced a higher expression of microglial activation markers, a higher brain pro-inflammatory set point (IFNγ, IDO) and a reduced expression of BDNF and PDGF after immune stimulation in NOD mice. NOD mice displayed exaggerated and prolonged sickness behaviour after LPS administration. CONCLUSION: After stimulation with LPS, NOD mice display an increased microglial proliferation and an exaggerated inflammatory brain response with reduced BDNF and PDGF expression and increased sickness behaviour as compared to controls.

Prevalence of distal renal tubular acidosis in primary Sjögren's syndrome.

OBJECTIVES: Our objectives were to analyse the prevalence of distal renal tubular acidosis (dRTA) in primary SS (pSS) and to compare a novel urinary acidification test with furosemide and fludrocortisone (FF) with the gold standard ammonium chloride (NH4Cl) to detect dRTA. METHODS: Urinary acidification was assessed in 57 pSS patients using NH4Cl and FF. A urinary acidification defect was defined as an inability to reach a urinary pH of <5.3 after NH4Cl. RESULTS: The prevalence of complete dRTA (urinary acidification defect with acidosis) was 5% (3/57). All three patients had positive SSA/Ro and SSB/La autoantibodies and impaired kidney function. The prevalence of incomplete dRTA (urinary acidification defect without acidosis) was 25% (14/57). Compared with patients without dRTA, patients with incomplete dRTA had significantly lower venous pH and serum bicarbonate and higher urinary pH. SSB/La antibodies were more prevalent in the dRTA groups (P < 0.05). Compared with NH4Cl, the positive and negative predictive values of FF were 46% and 82%, respectively. Vomiting occurred more often during the urinary acidification test with NH4Cl than with FF (9 vs 0, P < 0.05). CONCLUSION: Incomplete dRTA is common in pSS and causes mild acidaemia and higher urinary pH, which may contribute to bone demineralization and kidney stone formation. FF cannot replace NH4Cl in testing urinary acidification in pSS, but may be considered as a screening tool, given its reasonable negative predictive value and better tolerability.

MxA as a clinically applicable biomarker for identifying systemic interferon type I in primary Sjogren's syndrome.

OBJECTIVE: To establish an easy and practical assay for identifying systemic interferon (IFN) type I bioactivity in patients with primary Sjögren's syndrome (pSS). The IFN type I signature is present in over half of the pSS patients and identifies a subgroup with a higher disease activity. This signature is currently assessed via laborious expression profiles of multiple IFN type I-inducible genes. METHODS: In a cohort of 35 pSS patients, myxovirus-resistance protein A (MxA) was assessed as a potential biomarker for type I IFN activity, using an enzyme immunoassay (EIA) on whole-blood and flow cytometric analyses (fluorescence-activated cell sorting, FACS) of isolated CD14 monocytes. In addition, potential biomarkers such as CD64, CD169 and B cell-activating factor (BAFF) were simultaneously analysed in CD14 monocytes using FACS. The IFNscore, a measure for total type I IFN bioactivity, was calculated using expression values of the IFN type I signature genes--IFI44, IFI44L, IFIT3, LY6E and MX1--in CD14 monocytes, determined by real-time quantitative PCR. RESULTS: IFNscores correlated the strongest with monocyte MxA protein (r=0.741, p<0.001) and whole-blood MxA levels (r=0.764, p<0.001), weaker with CD169 (r=0.495, p<0.001) and CD64 (r=0.436, p=0.007), and not at all with BAFF protein. In particular, whole blood MxA levels correlated with EULAR Sjögren's Syndrome Disease Activity Index scores and numerous clinical pSS parameters. Interestingly, patients on hydroxychloroquine showed reduced MxA levels (EIA, p=0.04; FACS p=0.001). CONCLUSIONS: The MxA assays were excellent tools to assess IFN type I activity in pSS, MxA-EIA being the most practical. MxA levels associate with features of active disease and are reduced in hydroxychloroquine-treated patients, suggesting the clinical applicability of MxA in stratifying patients according to IFN positivity.

Abnormalities of dendritic cell precursors in the pancreas of the NOD mouse model of diabetes.

The non-obese diabetic (NOD) mouse is a widely used animal model for the study of human diabetes. Before the start of lymphocytic insulitis, DC accumulation around islets of Langerhans is a hallmark for autoimmune diabetes development in this model. Previous experiments indicated that an inflammatory influx of these DCs in the pancreas is less plausible. Here, we investigated whether the pancreas contains DC precursors and whether these precursors contribute to DC accumulation in the NOD pancreas. Fetal pancreases of NOD and control mice were isolated followed by FACS using ER-MP58, Ly6G, CD11b and Ly6C. Sorted fetal pancreatic ER-MP58(+) cells were cultured with GM-CSF and tested for DC markers and antigen processing. CFSE labeling and Ki-67 staining were used to determine cell proliferation in cultures and tissues. Ly6C(hi) and Ly6C(low) precursors were present in fetal pancreases of NOD and control mice. These precursors developed into CD11c(+) MHCII(+) CD86(+) DCs capable of processing DQ-OVA. ER-MP58(+) cells in the embryonic and pre-diabetic NOD pancreas had a higher proliferation capacity. Our observations support a novel concept that pre-diabetic DC accumulation in the NOD pancreas is due to aberrant enhanced proliferation of local precursors, rather than to aberrant "inflammatory infiltration" from the circulation.

Prevalence of interferon type I signature in CD14 monocytes of patients with Sjogren's syndrome and association with disease activity and BAFF gene expression.

OBJECTIVE: To determine the prevalence of upregulation of interferon (IFN) type I inducible genes, the so called 'IFN type I signature', in CD14 monocytes in 69 patients with primary Sjögren's syndrome (pSS) and 44 healthy controls (HC) and correlate it with disease manifestations and expression of B cell activating factor (BAFF). METHODS: Expression of IFI44L, IFI44, IFIT3, LY6E and MX1 was measured using real time quantitative PCR in monocytes. Expression values were used to calculate IFN type I scores for each subject. pSS patients positive for the IFN type I signature (IFN score≥10) and patients negative for the signature (IFN score<10) were then compared for clinical disease manifestations and BAFF expression. A bioassay using a monocytic cell line was performed to study whether BAFF mRNA expression was inducible by IFN type I activity in serum of patients with pSS. RESULTS: An IFN type I signature was present in 55% of patients with pSS compared with 4.5% of HC. Patients with the IFN type I signature showed: (a) higher EULAR Sjögren's Syndrome Disease Activity Index scores; higher anti-Ro52, anti-Ro60 and anti-La autoantibodies; higher rheumatoid factor; higher serum IgG; lower C3, lower absolute lymphocyte and neutrophil counts; (b)higher BAFF gene expression in monocytes. In addition, serum of signature-positive patients induced BAFF gene expression in monocytes. CONCLUSIONS: The monocyte IFN type I signature identifies a subgroup of patients with pSS with a higher clinical disease activity together with higher BAFF mRNA expression. Such patients might benefit from treatment blocking IFN type I production or activity.