Baseline metabolic profiles of early rheumatoid arthritis patients achieving sustained drug-free remission after initiating treat-to-target tocilizumab, methotrexate, or the combination: insights from systems biology

Background We previously identified, in newly diagnosed rheumatoid arthritis (RA) patients, networks of co-expressed genes and proteomic biomarkers associated with achieving sustained drug-free remission (sDFR) after treatment with tocilizumab- or methotrexate-based strategies. The aim of this study was to identify, within the same patients, metabolic pathways important for achieving sDFR and to subsequently study the complex interactions between different components of the biological system and how these interactions might affect the therapeutic response in early RA. Methods Serum samples were analyzed of 60 patients who participated in the U-Act-Early trial (ClinicalTrials.gov number NCT01034137) and initiated treatment with methotrexate, tocilizumab, or the combination and who were thereafter able to achieve sDFR (n = 37); as controls, patients were selected who never achieved a drug-free status (n = 23). Metabolomic measurements were performed using mass spectrometry on oxidative stress, amine, and oxylipin platforms covering various compounds. Partial least square discriminant analyses (PLSDA) were performed to identify, per strategy arm, relevant metabolites of which the biological pathways were studied. In addition, integrative analyses were performed correlating the previously identified transcripts and proteins with the relevant metabolites. Results In the tocilizumab plus methotrexate, tocilizumab, and methotrexate strategy, respectively, 19, 13, and 12 relevant metabolites were found, which were subsequently used for pathway analyses. The most significant pathway in the tocilizumab plus methotrexate strategy was “histidine metabolism” (p < 0.001); in the tocilizumab strategy it was “arachidonic acid metabolism” (p = 0.018); and in the methotrexate strategy it was “arginine and proline metabolism” (p = 0.022). These pathways have treatment-specific drug interactions with metabolites affecting either the signaling of interleukin-6, which is inhibited by tocilizumab, or affecting protein synthesis from amino acids, which is inhibited by methotrexate. Conclusion In early RA patients treated-to-target with a tocilizumab- or methotrexate-based strategy, several metabolites were found to be associated with achieving sDFR. In line with our previous observations, by analyzing relevant transcripts and proteins within the same patients, the metabolic profiles were found to be different between the strategy arms. Our metabolic analysis further supports the hypothesis that achieving sDFR is not only dependent on predisposing biomarkers, but also on the specific treatment that has been initiated. Trial registration ClinicalTrials.gov, NCT01034137. Registered on January 2010 Electronic supplementary material The online version of this article (10.1186/s13075-018-1729-2) contains supplementary material, which is available to authorized users.


Background
Rheumatoid arthritis (RA) is a systemic disease characterized by inflammation and damage of the affected joints; although the cause is not known, both genetic as well as environmental factors are reported to be associated with the condition [1][2][3][4]. Initiating treatment, aiming for sustained remission or low disease activity early in the course of the disease, is important to preserve physical function and improve long-term prognosis [5][6][7]. Biological disease-modifying anti-rheumatic drugs (DMARDs) are to date mainly used as the second line of therapy in the management of early RA, although several studies showed their superior efficacy over traditional DMARDs in reducing disease activity and halting joint damage [8,9]. Starting biological therapy in newly diagnosed RA patients as standard care, however, still remains highly controversial considering costs and unnecessary exposure to adverse events as a proportion of patients will be over-treated when using such an approach. Therefore, predictors are not only needed for treatment response to the currently recommended conventional DMARDs, but also for identifying patients for whom it would be favorable to initiate, as first therapy, a step-down biological-based strategy (i.e. tapering and finally discontinuing treatment) as achieving remission in the early stage of the disease improves the long-term clinical outcome.
Recent developments in "omics" technologies-such as genomics, transcriptomics, proteomics, and metabolomics-made it feasible to measure a broader spectrum of disease biomarkers for prediction of disease progression and development of personalized treatment strategies in RA [10]. Metabolomics is the non-targeted study of small-molecule metabolites and has become of increased interest in recent years due to the development and accessibility of new high-throughput technologies, including nuclear magnetic resonance spectroscopy and mass spectrometry (MS) [11]. Metabolites provide, under a given set of conditions, detailed information on cellular processes that are indicative for the disease state and are considered as the final downstream product of gene expression [12]. Especially in RA, metabolites are of particular interest as widespread cytokine-mediated inflammatory processes alter the cellular metabolism, when macrophages and lymphocytes become activated [13]. The role of these compounds in biomarker discovery has also been demonstrated previously, suggesting that metabolic analysis is potentially valuable in identifying markers for treatment response in patients with RA [14][15][16][17].
The aim of this study was to identify relevant metabolites and important metabolomic pathways associated with achieving sustained drug-free remission (sDFR) after a treat-to-target tocilizumab-or methotrexate-based strategy initiated in DMARD-naïve early RA patients. We previously identified, within the same patients, networks of co-expressed genes [18] and several inflammatory proteins [19] associated with sDFR and now, in the present study, by revealing metabolic biomarkers, exploring the systems biology of these patients in more detail by also integrating the findings of our previous studies.

Patient selection
From the U-Act-Early strategy trial, patients were selected who achieved sDFR, defined as being drug-free for ≥ 3 months until end of the study, after initiating tocilizumab, step-up methotrexate, or tocilizumab plus methotrexate therapy. As controls, we selected patients who never achieved a drug-free status at any time point during the study. A detailed description of the study design has been reported previously [20]. Briefly, DMARD-naïve patients with early RA were randomized (1:1:1) to one of the three strategy arms and treated to the target of sustained remission, defined as disease activity score assessing 28 joints (DAS28) < 2.6 with ≤ 4 swollen joints for ≥ 24 weeks. Tocilizumab was administered intravenously every four weeks at a dose of 8 mg/kg with a maximum of 800 mg. Methotrexate (oral) was given every week with a starting dose of 10 mg and was increased to 30 mg (or maximum tolerable dose) with steps of 5 mg every four weeks until remission was reached. If the treatment target was achieved, medication was tapered stepwise and finally discontinued, if remission persisted.

Metabolomic platforms
Baseline serum samples were measured on oxidative stress, amines, and oxylipins MS platforms, which have been applied previously and are validated [21][22][23]. The oxidative stress platform covers various isoprostane classes, signaling lipids from the sphingosine and sphinganine classes and their phosphorylated forms, as well as three classes of lysophosphatidic acids: lysophosphatidic acids, alkyl-lysophosphatidic acids, and cyclic-lysophosphatidic acids (all in the chain length species range of C14-C22). The amine platform covers amino acids and biogenic amines and the oxylipin platform covers classical and non-classical eicosanoids from different polyunsaturated fatty acids. In total, 263 metabolites were measured for each sample on the different platforms: 57 signaling lipid mediators, 128 oxylipins, and 78 amines. Serum samples were thawed on ice and vortexed before preparation procedures following inner standard protocols [22,23]; extra samples were pooled for internal quality control (QC). For each platform, QC samples were added and the relative standard deviation (RSD) per metabolite was calculated. Only those metabolites that complied with the acceptance criteria (RSD < 15% for amines; RSD < 30% for oxidative stress and oxylipins) were selected for further data analysis. Additional information about the metabolite profiling on the different platforms is provided in Additional file 1. All metabolite analyses were performed by the Biomedical Metabolomics Facility Leiden department at Leiden University.

Data pre-processing
For peak determination and integration, signaling lipid mediators profiled by the oxidative stress platform were pre-processed by LabSolutions (Shimadzu, Version 5.65); peak-picking of oxylipins was performed with Agilent MassHunter Quantitative Analysis software (Agilent, Version B.05.01) and amines with MultiQuant Software for Quantitative Analysis (AB SCIEX, Version 3.0.2). For all metabolites, raw data correction was accomplished using selected internal standards by calculating the ratio of peak area of the target compound to the peak area of assigned internal standard from which a response ratio for each analyte was obtained. QC samples were used for evaluating the quality of the targeted compounds according to the in-house written protocol and the data were hereafter ready to be used for statistical analyses.

Statistical analyses
Baseline clinical characteristics are described as mean (standard deviation [SD]), median (interquartile range [IQR]) or as proportions (%); between-group differences (sDFR versus controls) were tested within each strategy arm using independent t test, Mann-Whitney U test, or Pearson χ 2 test, respectively. A linear mixed model with a random intercept and baseline DAS28, week of visit, and group (sDFR versus controls) as fixed effects was built to evaluate, within the strategy arms, differences in disease activity over time. As metabolite concentrations are influenced by a variety of factors, we performed principal component analyses (PCA) to identify possible confounders. The following parameters were considered: age; body mass index, gender, ethnicity, disease duration, smoking, alcohol consumption, seropositivity for rheumatoid factor (RF) or cyclic citrullinated peptide (CCP), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). Thereafter, supervised partial least square discriminant analyses (PLSDA) were performed for each class (lipids, amines, and oxylipins) to identify relevant metabolites within each strategy arm. Several multivariate discrimination techniques currently exist but the main advantage of PLSDA is the handling of collinearity and noisy data (i.e. more observations than samples), both common in metabolomics experiments [24]. Data were first normalized (natural log-transformed) and then standardized (z-score) to ensure that all metabolite scores are comparable by giving them equal weight. The variable importance on projection (VIP) was used for metabolite selection; this measure accumulates the importance of each variable, whereas a higher VIP score shows that it is more relevant to predict the outcome [25]. Metabolites with VIP > 1 in the first component were considered important as the squared sum of all VIP values is equal to "1", i.e. the average VIP. Thereafter, the Mann-Whitney U test (sDFR versus controls) was performed within these selected metabolites to identify those who are most relevant (p < 0.10) within each strategy arm (not corrected for multiple testing), which were subsequently used for pathway analysis in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Pathways were considered relevant when p ≤ 0.05. In addition, integrative analyses between the previously identified transcripts [18] and proteins [19] and relevant metabolites were performed by calculating statistically significant (p ≤ 0.05) transcriptprotein and protein-metabolite correlations (Pearson correlation coefficients [PCC]). These network analyses were visualized using VisANT 5.0 software [26,27]. All other analyses were performed using the web-based tool MetaboAnalyst version 4.0 [28] and R version 3.4.3 (R Foundation for Statistical Computing, Vienna, Austria).

Discussion
We identified several small-molecule metabolites, by using high-throughput MS, associated with achieving sDFR after treatment with tocilizumab-or methotrexate-based strategies in newly diagnosed RA patients. In line with our previous observations, by measuring transcripts and proteins within the same patients, different metabolic profiles were found between the treatment strategies, further supporting the hypothesis that achieving sDFR is likely dependent on pre-treatment concentrations of specific biomarkers as no differences in clinical characteristics could be found. Although we did find different metabolic pathways between the treatment strategies when using the identified metabolites, the pathways within each strategy arm were found to be specific for the respective treatment, which shows the possibility of selecting biomarkers for prediction of a good treatment-specific response. An important metabolic pathway within the tocilizumab plus methotrexate strategy was "sphingolipid metabolism," in which ceramide synthases is closely associated with the consumption of both sphingosine, a lipid signaling molecule stimulating several cellular processes important in RA, such as cell growth, differentiation, and migration [32], and its derivative sphinganine [33]. These metabolites have been found to induce IL-6 production and may thus influence the treatment response to tocilizumab, a humanized monoclonal antibody against the IL-6 receptor. In the present analysis, lower baseline levels of sphingosine and sphinganine were found in the sDFR group of the tocilizumab plus methotrexate strategy arm when compared to controls although the opposite was expected (i.e. less inhibition resulting in higher disease activity); a possible explanation for these findings could be that patients with higher levels of IL-6 are more likely to respond to tocilizumab. To test this hypothesis, we evaluated within the same patients the absolute IL-6 concentrations in baseline (i.e. pre-treatment) serum, as was measured previously using Luminex® [19], but found no statistically significant difference between those who would later achieve sDFR and the controls (mean 65 versus 70 pg/mL, respectively). This result confirms the findings of others showing that baseline levels of IL-6 are not predictive of clinical outcomes of tocilizumab treatment [34]. Further studies are required to elucidate on how metabolite levels are altered and how these pathophysiological changes eventually affect the response to specific therapies. Ceramide synthase 6 (CerS6), an enzyme also important in sphingolipid biosynthesis, is mediated by methotrexate [35], which might indicate a role of sphingolipids in the treatment response to a tocilizumab plus methotrexate-based strategy in early RA.
In the tocilizumab strategy arm, several involved metabolic pathways were found to be associated with signaling of the IL-6 protein whereas increased levels of prostaglandins and isoprostanes were observed in the sDFR group when compared to controls. These metabolites are involved in the metabolism of arachidonic acid, a polyunsaturated fatty acid reported to be a key intermediate promoting inflammation. Prostaglandins are known to have a stimulating effect on IL-6 and could thus influence the response to tocilizumab therapy [36]. Other important metabolites involved in IL-6 signaling are L-pipecolic acid, which is produced during the degradation of lysine, and cysteine, which is synthesized from serine. Both lysine and serine are essential amino acids in humans as they are being used in the biosynthesis of proteins whereas lysine has been reported to downregulate the release of IL-6 [37].
In the methotrexate strategy, important metabolites were proline and arginine for which higher concentrations were observed in those achieving sDFR when compared to controls. Arginine, apart from its role in protein synthesis, serves as the precursor of proline and glutamate; glutamates are involved in the generation of glutamine, which stimulates purine and pyrimidine formation that is required for cell proliferation. These organic compounds thus seem to play a crucial role in the direct treatment response to methotrexate as this drug antagonizes folic acid [38], which is important for purine and pyrimidine formation, and inhibits via this pathway the synthesis of nucleic acids and subsequently protein synthesis [39]. One of the other important amino acids for protein synthesis is histidine, of which the post-translational modified product, 3-methylhistidine, was found to have a decreased metabolism in the present study in those achieving sDFR in the methotrexate strategy. Histidine is also involved in the local immune response as it is a precursor of histamine, a compound that induces permeability of capillaries allowing, for example, leukocytes and pro-inflammatory molecules to elicit an immune response. These amino acids thus might directly affect the treatment response to methotrexate therapy.
There are some limitations to this study. First, although not uncommon in metabolomic studies, the number of samples measured was relatively small, enhancing the likelihood of false-negative findings (i.e. type II error). To minimize this risk, we used for detecting relevant metabolites analyses suitable for handling such datasets consisting of more markers than samples. Second, before the samples were measured, serum was pre-processed according to usual guidelines, which differed for each platform, and compounds were, after analyses, corrected using internal standards. These variety of factors could potentially impair replicating or external validation of findings. Third, for defining remission, and thus tapering medication, we used DAS28 criteria which is highly dependent of acute phase response and might not always reflect an inflammation-free state of the patient. To minimize this risk, no more than four swollen joints were allowed during the remission period, enhancing the likelihood that patients were also clinically in remission. Nevertheless, American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) Boolean-based remission criteria might have been a more reliable tool for assessing disease activity as it is more stringent in assessing inflammation [40].

Conclusions
We have identified several relevant metabolites in baseline serum related to achieving sDFR after treatment with tocilizumab-or methotrexate-based strategies in DMARD-naïve RA patients. In line with our previous work on the analyses of transcripts and proteins, performed within the same patients, the identified metabolic pathways were shown to be specific for the treatment that was initiated. These results might provide further insight into the role of predisposing biomarkers for eventually achieving sDFR in early RA. Signature metabolite biomarkers have been identified which could potentially serve as key prognostic factors for developing personalized care but need to be validated in large replication studies. Further studies are also warranted to elucidate on the drug metabolism in those patients with refractory disease to, by initiating other therapies based on pharmacogenomics, achieve better treatment outcomes.

Additional files
Additional file 1: Additional information regarding the metabolite profiling on the three platforms. (DOCX 15 kb) Additional file 2: Overview of the pathway analysis in the (a) tocilizumab plus methotrexate, (b) tocilizumab, and (c) methotrexate strategy arms. The top three most relevant pathways in the tocilizumab plus methotrexate arm were (1) "histidine metabolism," (2) "sphingolipid metabolism," and (3) "arachidonic acid metabolism;" in the tocilizumab arm, these were (1) "arachidonic acid metabolism," (2) "lysine degradation," and (3) "cysteine and methionine metabolism;" in the methotrexate arm, these were (1) "arginine and proline metabolism," (2) "histidine metabolism," and (3) "aminocyl-tRNA biosynthesis." KEGG Kyoto Encyclopedia of Genes and Genomes, tRNA transfer ribonucleic acid. The colors of the nodes, varying from yellow to red, indicates the level of significance with red being highly significant; the size of the nodes depicts the impact of the pathway with larger nodes illustrating a higher impact. (DOCX 54 kb)