Urinary prostanoids are elevated by anti-TNF and anti-IL6 receptor disease-modifying antirheumatic drugs but are not predictive of response to treatment in early rheumatoid arthritis.

BACKGROUND: Disease-modifying antirheumatic drugs (DMARDs) are widely used for treating rheumatoid arthritis (RA). However, there are no established biomarkers to predict a patient's response to these therapies. Prostanoids, encompassing prostaglandins, prostacyclins, and thromboxanes, are potent lipid mediators implicated in RA progression. Nevertheless, the influence of DMARDs on prostanoid biosynthesis in RA patients remains poorly understood. This study aims to assess the impact of various DMARDs on urinary prostanoids levels and to explore whether urinary prostanoid profiles correlate with disease activity or response to therapy. METHODS: This study included 152 Swedish female patients with early RA, all rheumatoid factor (RF) positive, enrolled in the NORD-STAR trial (registration number: NCT01491815). Participants were randomized into four therapeutic regimes: methotrexate (MTX) combined with (i) prednisolone (arm ACT), (ii) TNF-α blocker certolizumab pegol (arm CZP), (iii) CTLA-4Ig abatacept (arm ABA), or (iv) IL-6R blocker tocilizumab (arm TCZ). Urine samples, collected before start of treatment and at 24 weeks post-treatment, were analyzed for tetranor-prostaglandin E metabolite (tPGEM), tetranor-prostaglandin D metabolite (tPGDM), 2,3-dinor thromboxane B2 (TXBM), 2,3-dinor-6-keto prostaglandin F1a (PGIM), leukotriene E4 (LTE4) and 12-hydroxyeicosatetraenoic acid (12-HETE) using liquid chromatography-mass spectrometry (LC-MS). Generalized estimating equation (GEE) models were used to analyze the change in urinary eicosanoids and their correlations to clinical outcomes. RESULTS: Patients receiving MTX combined with CZP or TCZ exhibited significant elevations in urinary tPGEM and TXBM levels after 24 weeks of treatment. Other eicosanoids did not show significant alterations in response to any treatment. Baseline urinary eicosanoid levels did not correlate with baseline clinical disease activity index (CDAI) levels, nor with changes in CDAI from baseline to week 24. Their levels were also similar between patients who achieved CDAI remission and those with active disease at week 24. CONCLUSIONS: Treatment with anti-TNF or anti-IL6R agents in early RA patients leads to an increased systemic production of proinflammatory and prothrombotic prostanoids. However, urinary eicosanoid levels do not appear to be predictive of the response to DMARDs therapy.

Microsomal prostaglandin E synthase-1 inhibition promotes shunting in arachidonic acid metabolism during inflammatory responses in vitro.

Microsomal Prostaglandin E Synthase 1 (mPGES-1) is the key enzyme for the generation of the pro-inflammatory lipid mediator prostaglandin E2 (PGE2), which contributes to several pathological features of many diseases. Inhibition of mPGES-1 has been shown to be a safe and effective therapeutic strategy in various pre-clinical studies. In addition to reduced PGE2 formation, it is also suggested that the potential shunting into other protective and pro-resolving prostanoids may play an important role in resolution of inflammation. In the present study, we analysed the eicosanoid profiles in four in vitro inflammation models and compared the effects of mPGES-1 inhibition with those of cyclooxygenase-2 (Cox-2) inhibition. Our results showed a marked shift to the PGD2 pathway under mPGES-1 inhibition in A549 cells, RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs), whereas enhanced prostacyclin production was observed in rheumatoid arthritis synovial fibroblasts (RASFs) treated with an mPGES-1 inhibitor. As expected, Cox-2 inhibition completely suppressed all prostanoids. This study suggests that the therapeutic effects of mPGES-1 inhibition may be mediated by modulation of other prostanoids in addition to PGE2 reduction.

Microsomal prostaglandin E synthase-1 inhibition prevents adverse cardiac remodelling after myocardial infarction in mice.

BACKGROUND AND PURPOSE: Heart failure with reduced ejection fraction (HFrEF) is a major consequence of myocardial infarction (MI). The microsomal prostaglandin E synthase-1 (mPGES-1)/PGE2 pathway has been shown to constrain reperfusion injury after acute myocardial ischaemia. However, it is unknown whether pharmacological inhibition of mPGES-1, a target with lower risk of thrombosis compared with selective inhibition of cyclooxygenase-2, affects chronic cardiac remodelling after MI. EXPERIMENTAL APPROACH: Mice were subjected to left anterior descending coronary artery ligation, followed by intraperitoneal treatment with the mPGES-1 inhibitor compound III (CIII) or 118, celecoxib (cyclooxygenase-2 inhibitor) or vehicle, once daily for 28 days. Urinary prostanoid metabolites were measured by liquid chromatography-tandem mass spectrometry. KEY RESULTS: Chronic administration of CIII improved cardiac function in mice after MI compared with vehicle or celecoxib. CIII did not affect thrombogenesis or blood pressure. In addition, CIII reduced infarct area, augmented scar thickness, decreased collagen I/III ratio, decreased the expression of fibrosis-related genes and increased capillary density in the ischaemic area. Shunting to urinary metabolites of PGI2 , not thromboxane B2 or PGD2 , after inhibition of mPGES-1 was positively correlated with cardiac function after MI. CIII administration significantly increased urinary PGI2 /PGE2 metabolite ratio compared to vehicle or celecoxib. The PGI2 /PGE2 metabolite ratio correlated positively with ejection fraction, fractional shortening and scar thickness. Treatment with 118 also improved cardiac function. CONCLUSION AND IMPLICATIONS: Inhibition of mPGES-1 prevented chronic adverse cardiac remodelling via an augmented PGI2 /PGE2 metabolite ratio and therefore represents a potential therapeutic strategy for development of HFrEF after MI.

Autoantigenic properties of the aminoacyl tRNA synthetase family in idiopathic inflammatory myopathies.

OBJECTIVES: Autoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory myopathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to known myositis-specific autoantibodies. We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM. METHODS: Plasma samples from 217 patients with IIM according to 2017 EULAR/ACR criteria, including 50 patients with ASSD, 165 without, and two with unknown ASSD status were identified retrospectively, as well as age and gender-matched sera from 156 population controls, and 219 disease controls. Patients with previously documented ASSD had to test positive for at least one of the five most common anti-aaRS autoantibodies (anti-Jo1, -PL7, -PL12, -EJ, and -OJ) and present with one or more of the following clinical manifestations: interstitial lung disease, myositis, arthritis, Raynaud's phenomenon, fever, or mechanic's hands. Demographics, laboratory, and clinical data of the IIM cohort (ASSD and non-ASSD) were compared. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 117 recombinant protein variants, representing 33 myositis-related proteins, including all nineteen cytoplasmic aaRS. Prospectively collected clinical data for the IIM cohort were retrieved and compared between groups within the IIM cohort and correlated with the results of the autoantibody screening. Principal component analysis was used to analyze clinical manifestations between ASSD, non-ASSD groups, and individuals with novel anti-aaRS autoantibodies. RESULTS: We identified reactivity towards 16 aaRS in 72 of the 217 IIM patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four previously seronegative patients for myositis-specific autoantibodies and eight with previously detected myositis-specific autoantibodies. IIM individuals with novel anti-aaRS autoantibodies (n = 12) all had signs of myositis, and they had either muscle weakness and/or muscle enzyme elevation, 2/12 had mechanic's hands, 3/12 had interstitial lung disease, and 2/12 had arthritis. The individuals with novel anti-aaRS and a pathological muscle biopsy all presented widespread up-regulation of major histocompatibility complex class I. The reactivities against novel aaRS could be confirmed in ELISA and western blot. Using the multiplex bead array assay, we could confirm previously known reactivities to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n = 45)) and identified patients positive for anti-Zo, -KS, and -HA (n = 10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was also detected in controls. CONCLUSION: Our results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.

Urine Galectin-3 binding protein reflects nephritis activity in systemic lupus erythematosus.

BACKGROUND: Lupus nephritis (LN) is a major and severe organ involvement in systemic lupus erythematosus (SLE), whose diagnosis and treatment necessitate to perform kidney biopsy, which is an invasive procedure. Non-invasive urine biomarkers are an active area of investigation to support LN diagnosis and management. OBJECTIVE: To investigate the role of urinary galectin-3 binding protein (u-Gal-3BP) as a candidate biomarker of renal disease in biopsy proven LN. PATIENTS AND METHODS: Levels of u-Gal-3BP were investigated in a cross-sectional fashion by ELISA in 270 subjects: 86 LN patients, 63 active SLE patients with no kidney involvement, 73 SLE patients with inactive disease and 48 age and sex-matched population-based controls (PBC). Moreover, urine samples were analysed separately by ELISA for additional markers of kidney pathology: neutrophil gelatinase-associated lipocalin (NGAL), osteopontin (OPN), kidney injury molecule-1 (KIM-1) and galectin-3 (Gal-3). The concentrations of all studied molecules were normalized to urine creatinine levels. In 10 patients, post-treatment levels of the biomarkers were measured. RESULTS: Normalized u-Gal-3BP levels were higher in LN patients compared to the other groups (p < .0001). Comparing different LN classes, u-Gal-3BP levels were higher among patients with proliferative (class III/IV) and membranous (class V) as compared to mesangial (class II) forms (p = .04). In proliferative forms, u-Gal-3BP levels correlated with the activity index in renal biopsies (r = 0.42, p = .004). Moreover, in a subset of 10 patients with repeated kidney biopsy and urine sampling before and after induction treatment, a significant decrease of u-Gal-3BP was observed (p = .03). Among the other markers, KIM-1 was also able to discriminate LN from the other groups, while NGAL, OPN and Gal-3 could not in this cohort. CONCLUSION: Given its ability to discriminate LN patients from active non-renal and inactive SLE patients, the observed correlation with the activity index in renal biopsies, and its levels declining following treatment, u-Gal-3BP shows promise as a non-invasive urinary biomarker to help detecting and to monitor renal involvement in SLE patients and should be validated in larger cohorts.

Biosynthesis of prostaglandin 15dPGJ2 -glutathione and 15dPGJ2-cysteine conjugates in macrophages and mast cells via MGST3.

Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.

Prostanoid Metabolites as Biomarkers in Human Disease.

Prostaglandins (PGD2, PGE2, PGF2α), prostacyclin (PGI2), and thromboxane A2 (TXA2) together form the prostanoid family of lipid mediators. As autacoids, these five primary prostanoids propagate intercellular signals and are involved in many physiological processes. Furthermore, alterations in their biosynthesis accompany a wide range of pathological conditions, which leads to substantially increased local levels during disease. Primary prostanoids are chemically instable and rapidly metabolized. Their metabolites are more stable, integrate the local production on a systemic level, and their analysis in various biological matrices yields valuable information under different pathological settings. Therefore, prostanoid metabolites may be used as diagnostic, predictive, or prognostic biomarkers in human disease. Although their potential as biomarkers is great and extensive research has identified major prostanoid metabolites that serve as target analytes in different biofluids, the number of studies that correlate prostanoid metabolite levels to disease outcome is still limited. We review the metabolism of primary prostanoids in humans, summarize the levels of prostanoid metabolites in healthy subjects, and highlight existing biomarker studies. Since analysis of prostanoid metabolites is challenging because of ongoing metabolism and limited half-lives, an emphasis of this review lies on the reliable measurement and interpretation of obtained levels.

Cytokines as Biomarkers in Systemic Lupus Erythematosus: Value for Diagnosis and Drug Therapy.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease. The disease is characterized by activation and dysregulation of both the innate and the adaptive immune systems. The autoimmune response targets self-molecules including cell nuclei, double stranded DNA and other intra and extracellular structures. Multiple susceptibility genes within the immune system have been identified, as well as disturbances in different immune pathways. SLE may affect different organs and organ systems, and organ involvement is diverse among individuals. A universal understanding of pathophysiological mechanism of the disease, as well as directed therapies, are still missing. Cytokines are immunomodulating molecules produced by cells of the immune system. Interferons (IFNs) are a broad group of cytokines, primarily produced by the innate immune system. The IFN system has been observed to be dysregulated in SLE, and therefore IFNs have been extensively studied with a hope to understand the disease mechanisms and identify novel targeted therapies. In several autoimmune diseases identification and subsequent blockade of specific cytokines has led to successful therapies, for example tumor necrosis factor-alpha (TNF-α) inhibition in rheumatoid arthritis. Authors of this review have sought corresponding developments in SLE. In the current review, we cover the actual knowledge on IFNs and other studied cytokines as biomarkers and treatment targets in SLE.

Urinary prostaglandin D2 and E2 metabolites associate with abdominal obesity, glucose metabolism, and triglycerides in obese subjects.

Obesity is associated with low-grade chronic inflammation, which contributes to the development of the metabolic syndrome and its associated complications, such as insulin resistance and type-2 diabetes. Limited data from animal and human studies support local generation of pro-inflammatory prostanoid lipid mediators in white adipose tissue. However, the link between systemic prostanoid levels and parameters characterizing the metabolic syndrome is missing in human obesity. Therefore, we performed a targeted lipidomic analysis using urine samples from obese human subjects (n = 45) and show for the first time in humans that urinary prostanoid levels correlate with metabolic parameters that indicate a dysregulated glucose and triglyceride metabolism. We identified tetranor-PGDM and tetranor-PGEM as the two major urinary prostanoid metabolites in obese subjects with levels of 247 ±â€¯31 and 23.3 ±â€¯4.0 pmol/mg creatinine, respectively. Tetranor-PGDM was significantly associated with serum triglycerides, while tetranor-PGEM was associated with abdominal obesity as defined by an increased waist-to-hip ratio (WHR), with glycated hemoglobin (HbA1c), and with impaired oral glucose tolerance. These results confirm the previously established notion of low-grade chronic inflammation in obesity and further identify an association of the prostanoid pathway with obesity-associated dyslipidemia, abdominal obesity, and insulin resistance.

Inhibition of mPGES-1 or COX-2 Results in Different Proteomic and Lipidomic Profiles in A549 Lung Cancer Cells.

Pharmacological inhibition of microsomal prostaglandin E synthase (mPGES)-1 for selective reduction in prostaglandin E2 (PGE2) biosynthesis is protective in experimental models of cancer and inflammation. Targeting mPGES-1 is envisioned as a safer alternative to traditional non-steroidal anti-inflammatory drugs (NSAIDs). Herein, we compared the effects of mPGES-1 inhibitor Compound III (CIII) with the cyclooxygenase (COX)-2 inhibitor NS-398 on protein and lipid profiles in interleukin (IL)-1ß-induced A549 lung cancer cells using mass spectrometry. Inhibition of mPGES-1 decreased PGE2 production and increased PGF2α and thromboxane B2 (TXB2) formation, while inhibition of COX-2 decreased the production of all three prostanoids. Our proteomics results revealed that CIII downregulated multiple canonical pathways including eIF2, eIF4/P70S6K, and mTOR signaling, compared to NS-398 that activated these pathways. Moreover, pathway analysis predicted that CIII increased cell death of cancer cells (Z = 3.8, p = 5.1E-41) while NS-398 decreased the same function (Z = -5.0, p = 6.5E-35). In our lipidomics analyses, we found alterations in nine phospholipids between the two inhibitors, with a stronger alteration in the lysophospholipid (LPC) profile with NS-398 compared to CIII. Inhibition of mPGES-1 increased the concentration of sphinganine and dihydroceramide (C16:0DhCer), while inhibition of COX-2 caused a general decrease in most ceramides, again suggesting different effects on cell death between the two inhibitors. We showed that CIII decreased proliferation and potentiated the cytotoxic effect of the cytostatic drugs cisplatin, etoposide, and vincristine when investigated in a live cell imaging system. Our results demonstrate differences in protein and lipid profiles after inhibition of mPGES-1 or COX-2 with important implications on the therapeutic potential of mPGES-1 inhibitors as adjuvant treatment in cancer. We encourage further investigations to illuminate the clinical benefit of mPGES-1 inhibitors in cancer.

Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann-Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10-9, 3 × 10-6, and 5 × 10-6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

Urinary PGE2 metabolite levels in hospitalised infants with infections compared to age-matched controls.

AIM: To determine the urinary tetranor-prostaglandin E2 metabolite in healthy infants and in hospitalised infants with upper and lower respiratory tract as well as gastrointestinal infections. METHODS: A prospective cross-sectional study to determine baseline concentrations of urinary tetranor-prostaglandin E2 metabolite was conducted in 81 healthy infants aged one week to one year and in 142 hospitalised infants with infections. Prostaglandin metabolite levels were measured by liquid chromatography tandem mass spectrometry. RESULTS: In healthy infants, urinary prostaglandin E2 metabolite levels decreased with age and did not differ between girls and boys. Infections of the lower respiratory (n = 78) and gastrointestinal tract (n = 12) correlated with increased levels of the prostaglandin E2 metabolite. In contrast, infants hospitalised with upper respiratory tract infections (n = 23) exhibited similar levels as healthy, age-matched controls. Lower prostaglandin E2 levels were found after treatment with acetaminophen in hospitalised children. Prostaglandin E2 metabolite levels did not correlate with length of hospitalisation or need for respiratory support. CONCLUSION: This study first provides normal levels of urinary prostaglandin E2 metabolite in infants and secondly demonstrates elevated levels in hospitalised children with lower respiratory tract and gastrointestinal infections.

miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1ß and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

Cytokine Profiles in Autoantibody Defined Subgroups of Systemic Lupus Erythematosus.

The aim of this study was to evaluate how the cytokine profiles differed between autoantibody based subgroups of systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disease, characterized by periods of flares (active disease) and remission (inactive disease). The disease can affect many organ systems, e.g., skin, joints, kidneys, heart, and the central nervous system (CNS). SLE patients often have an overproduction of cytokines, e.g., interferons, chemokines, and interleukins. The high cytokine levels are part of the systemic inflammation, which can lead to tissue injury. In the present study, SLE patients were divided into five groups based on their autoantibody profiles. We thus defined these five groups: ANA negative, antiphospholipid (aPL) positive, anti-Sm/anti-RNP positive, Sjögren's syndrome (SS) antigen A and B positive, and patients positive for more than one type of autoantibodies (other SLE). Cytokines were measured using Mesoscale Discovery (MSD) multiplex analysis. On the basis of the cytokine data, ANA negative patients were the most deviating subgroup, with lower levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-12/IL-23p40, and interferon gamma-induced protein (IP)-10. Despite low cytokine levels in the ANA negative group, autoantibody profiles did not discriminate between different cytokine patterns.

Mass spectrometry-based analysis of cerebrospinal fluid from arthritis patients-immune-related candidate proteins affected by TNF blocking treatment.

BACKGROUND: Signs of inflammation in cerebrospinal fluid (CSF) of rheumatoid arthritis patients correlate positively with fatigue, a central nervous system (CNS)-related symptom that can be partially suppressed by TNF blockade. This suggests a possible role for CNS inflammation in arthritis that may be affected by TNF blockade. We therefore investigated the effects of TNF blockade on the arthritis CSF proteome and how candidate proteins related to clinical measures of disease activity and inflammation. METHODS: Mass spectrometry-based quantitative proteomic analysis was performed on CSF from seven polyarthritis patients before and during infliximab treatment. Treatment-associated proteins were identified using univariate (Wilcoxon signed rank test) and multivariate (partial least squares discriminant analysis (PLS-DA)) strategies. Relations between selected candidate proteins and clinical measures were investigated using the Spearman correlations. Additionally, selected proteins were cross-referenced to other studies investigating human CSF in a thorough literature search to ensure feasibility of our results. RESULTS: Univariate analysis of arthritis CSF proteome revealed a decrease of 35 proteins, predominantly involved in inflammatory processes, following TNF blockade. Seven candidate proteins, Contactin-1 (CNTN1), fibrinogen gamma chain (FGG), hemopexin (HPX), cell adhesion molecule-3 (CADM3), alpha-1B-glycoprotein (A1BG), complement factor B (CFB), and beta-2-microglobulin (B2M), were selected for further studies based on identification by both univariate and multivariate analyses and reported detection in human CSF and known associations to arthritis. Decreased levels of FGG and CFB in CSF after treatment showed strong correlations with both erythrocyte sedimentation rate and disability scores, while CNTN1 and CADM3 were associated with pain. CONCLUSION: Several immune-related proteins in the CSF of arthritis patients decreased during TNF blockade, including FGG and CFB that both correlated strongly with systemic inflammation. Our findings stress that also intrathecal inflammatory pathways are related to arthritis symptoms and may be affected by TNF blockade.

Two subgroups in systemic lupus erythematosus with features of antiphospholipid or Sjögren's syndrome differ in molecular signatures and treatment perspectives.

BACKGROUND: Previous studies and own clinical observations of patients with systemic lupus erythematosus (SLE) suggest that SLE harbors distinct immunophenotypes. This heterogeneity might result in differences in response to treatment in different subgroups and obstruct clinical trials. Our aim was to understand how SLE subgroups may differ regarding underlying pathophysiology and characteristic biomarkers. METHODS: In a cross-sectional study, including 378 well-characterized SLE patients and 316 individually matched population controls, we defined subgroups based on the patients' autoantibody profile at inclusion. We selected a core of an antiphospholipid syndrome-like SLE (aPL+ group; positive in the lupus anticoagulant (LA) test and negative for all three of SSA (Ro52 and Ro60) and SSB antibodies) and a Sjögren's syndrome-like SLE (SSA/SSB+ group; positive for all three of SSA (Ro52 and Ro60) and SSB antibodies but negative in the LA test). We applied affinity-based proteomics, targeting 281 proteins, together with well-established clinical biomarkers and complementary immunoassays to explore the difference between the two predefined SLE subgroups. RESULTS: The aPL+ group comprised 66 and the SSA/SSB+ group 63 patients. The protein with the highest prediction power (receiver operating characteristic (ROC) area under the curve = 0.89) for separating the aPL+ and SSA/SSB+ SLE subgroups was integrin beta-1 (ITGB1), with higher levels present in the SSA/SSB+ subgroup. Proteins with the lowest p values comparing the two SLE subgroups were ITGB1, SLC13A3, and CERS5. These three proteins, rheumatoid factor, and immunoglobulin G (IgG) were all increased in the SSA/SSB+ subgroup. This subgroup was also characterized by a possible activation of the interferon system as measured by high KRT7, TYK2, and ETV7 in plasma. In the aPL+ subgroup, complement activation was more pronounced together with several biomarkers associated with systemic inflammation (fibrinogen, α-1 antitrypsin, neutrophils, and triglycerides). CONCLUSIONS: Our observations indicate underlying pathogenic differences between the SSA/SSB+ and the aPL+ SLE subgroups, suggesting that the SSA/SSB+ subgroup may benefit from IFN-blocking therapies while the aPL+ subgroup is more likely to have an effect from drugs targeting the complement system. Stratifying SLE patients based on an autoantibody profile could be a way forward to understand underlying pathophysiology and to improve selection of patients for clinical trials of targeted treatments.

Kynurenine pathway is altered in patients with SLE and associated with severe fatigue.

OBJECTIVE: Fatigue has been reported as the most disturbing symptom in a majority of patients with SLE. Depression is common and often severe. Together these symptoms cause significant morbidity and affect patients with otherwise relatively mild disease. Tryptophan and its metabolites in the kynurenine pathway are known to be important in several psychiatric conditions, for example, depression, which are often also associated with fatigue. We therefore investigated the kynurenine pathway in patients with SLE and controls. METHODS: In a cross-sectional design plasma samples from 132 well-characterised patients with SLE and 30 age-matched and gender-matched population-based controls were analysed by liquid chromatography tandem mass spectrometry to measure the levels of tryptophan and its metabolites kynurenine and quinolinic acid. Fatigue was measured with Fatigue Severity Scale and depression with Hospital Anxiety and Depression Scale. SLE disease activity was assessed with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). RESULTS: The kynurenine/tryptophan ratio, as a measure of indoleamine 2,3-dioxygenase (IDO) activity, was increased in patients with SLE. Patients with active disease (SLEDAI ≥6) showed lower tryptophan levels compared with controls (54 µM, SD=19 vs 62 µM, SD=14, p=0.03), although patients with SLE overall did not differ compared with controls. Patients with SLE had higher levels of tryptophan metabolites kynurenine (966 nM, SD=530) and quinolinic acid (546 nM, SD=480) compared with controls (kynurenine: 712 nM, SD=230, p=0.0001; quinolinic acid: 380 nM, SD=150, p=0.001). Kynurenine, quinolinic acid and the kynurenine/tryptophan ratio correlated weakly with severe fatigue (rs =0.34, rs =0.28 and rs =0.24, respectively) but not with depression. CONCLUSIONS: Metabolites in the kynurenine pathway are altered in patients with SLE compared with controls. Interestingly, fatigue correlated weakly with measures of enhanced tryptophan metabolism, while depression did not. Drugs targeting enzymes in the kynurenine pathway, for example, IDO inhibitors or niacin (B12) supplementation, which suppresses IDO activity, merit further investigation as treatments in SLE.

TNF-α and plasma albumin as biomarkers of disease activity in systemic lupus erythematosus.

OBJECTIVES: Composite criteria/indices are presently used to diagnose and monitor patients with systemic lupus erythematosus (SLE). Biomarkers for these purposes would be helpful in clinical practice. We therefore evaluated a large panel of cytokines and basic laboratory tests and investigated their performance as discriminators versus controls and as biomarkers of disease activity (DA). METHODS: We examined 437 patients with SLE, fulfilling American College of Rheumatology-82 criteria, and 322 matched controls. DA was assessed according to both SLE DA Index 2000 (SLEDAI-2K) and SLE Activity Measure (SLAM). British Isles Lupus Activity Group (BILAG) was used to assess renal DA. Additionally, 132 patients self-assessed their Global Disease Activity (PtGDA). Mesoscale Discovery 30-plex cytokine assay and routine blood chemistry was performed on fasting EDTA-plasma. RESULTS: Of 26 tested biomarkers, we identified TNF-α as the superior discriminator between patients with SLE and controls (median=4.5 pg/mL, IQR=3.1-6.2 vs median=2.3 pg/mL, IQR=2.0-2.8). The strongest correlations to SLEDAI-2K and SLAM were obtained with TNF-α (Spearman rho (ρ)=0.32 and ρ=0.34, respectively), partly driven by the nephritis subgroup, and with p-albumin (ρ=-0.33 and ρ=-0.31, respectively). P-albumin was decreased and TNF-α was increased in patients with kidney involvement (renal BILAG A/B vs C/D/E, p=4×10-16 and p=6×10-9 respectively). IP-10 was increased in patients with joint involvement (SLAM item 24≥2 vs ≤1, p=0.0005) but did not differ when comparing patients with active/inactive kidney involvement. The most powerful correlations to PtGDA was observed with p-albumin (ρ=-0.42), IL-6 (ρ=0.30) and TNF-α (ρ=0.29). CONCLUSION: TNF-α and p-albumin both performed well as discriminators between patients with SLE and controls and as proxies for DA according to both rheumatologists' and patients' assessments. In particular, renal DA was well reflected by TNF-α. We propose that the TNF-α and p-albumin merit further investigations as clinically useful biomarkers in SLE. We also observed that the pattern of activated cytokines varies with organ involvement.

Targeted lipidomics analysis identified altered serum lipid profiles in patients with polymyositis and dermatomyositis.

BACKGROUND: Polymyositis (PM) and dermatomyositis (DM) are severe chronic autoimmune diseases, characterized by muscle fatigue and low muscle endurance. Conventional treatment includes high doses of glucocorticoids and immunosuppressive drugs; however, few patients recover full muscle function. One explanation of the persistent muscle weakness could be altered lipid metabolism in PM/DM muscle tissue as we previously reported. Using a targeted lipidomic approach we aimed to characterize serum lipid profiles in patients with PM/DM compared to healthy individuals (HI) in a cross-sectional study. Also, in the longitudinal study we compared serum lipid profiles in patients newly diagnosed with PM/DM before and after immunosuppressive treatment. METHODS: Lipidomic profiles were analyzed in serum samples from 13 patients with PM/DM, 12 HI and 8 patients newly diagnosed with PM/DM before and after conventional immunosuppressive treatment using liquid chromatography tandem mass spectrometry (LC-MS/MS) and a gas-chromatography flame ionization detector (GC-FID). Functional Index (FI), as a test of muscle performance and serum levels of creatine kinase (s-CK) as a proxy for disease activity were analyzed. RESULTS: The fatty acid (FA) composition of total serum lipids was altered in patients with PM/DM compared to HI; the levels of palmitic (16:0) acid were significantly higher while the levels of arachidonic (20:4, n-6) acid were significantly lower in patients with PM/DM. The profiles of serum phosphatidylcholine and triacylglycerol species were changed in patients with PM/DM compared to HI, suggesting disproportionate levels of saturated and polyunsaturated FAs that might have negative effects on muscle performance. After immunosuppressive treatment the total serum lipid levels of eicosadienoic (20:2, n-6) and eicosapentaenoic (20:5, n-3) acids were increased and serum phospholipid profiles were altered in patients with PM/DM. The correlation between FI or s-CK and levels of several lipid species indicate the important role of lipid changes in muscle performance and inflammation. CONCLUSIONS: Serum lipids profiles are significantly altered in patients with PM/DM compared to HI. Moreover, immunosuppressive treatment in patients newly diagnosed with PM/DM significantly affected serum lipid profiles. These findings provide new evidence of the dysregulated lipid metabolism in patients with PM/DM that could possibly contribute to low muscle performance.