There is a current imperative to reveal more precisely the molecular pathways of early onset of systemic autoimmune diseases (SADs). The investigation of newly diagnosed drug-naive SAD patients might contribute to identify novel disease-specific and prognostic markers. The multiplex analysis of 30 plasma proteins in 60 newly diagnosed drug-naive SADs, such as RA (rheumatoid arthritis,
Systemic autoimmune rheumatic diseases (SADs) including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis (SSc) are characterized by an abnormal immune system response, complement dysregulation, imbalance of cytokines production, and inflammation [
Patients were enrolled during regular visits at the Department of Rheumatology and Immunology (University of Szeged). Healthy controls were voluntary staff members of the BRC or University of Szeged. Subjects were informed about the research by a physician. Written informed consent was obtained from all subjects, and our study was reviewed and approved by an independent ethical committee of the university. Laboratory studies and interpretations were performed on coded samples lacking personal and diagnostic identifiers. The study adhered to the tenets of the most recent revision of the Declaration of Helsinki. Details about the study design and handling of biological materials were submitted to the Human Investigation Review Board of the University of Szeged under the 149/2019-SZTE Project Identification code.
The multiplex protein analysis of 60 drug-naive SAD patients, RA patients (
After the withdrawal of 20 ml blood into an EDTA vacutainer (Becton Dickinson), human peripheral blood mononuclear cells and plasma samples were purified by Leucosep tubes (Greiner Bio-One, Austria). PBMCs were used for immunophenotyping in another project. Plasma fractions were stored at -80°C in aliquots before running the assay. Luminex xMAP technology was used to determine the protein concentrations of 30 distinct cytokines/chemokines performing Procarta Plex™ Immunoassay (ThermoFisher Scientific, Waltham, MA, USA) according to the instructions of the manufacturer. The Luminex panel was designed by the authors quantifying the proteins listed in Supplementary Table
The arithmetic mean (mean), standard deviation (SD), and the standard error of the mean (SEM) of the plasma cytokine concentrations were calculated. The pairwise comparison of the concentrations of each cytokine of patients versus healthy controls (RA vs. HC; SLE vs. HC; SSc vs. HC) was carried out by one-way ANOVA (
The following 30 plasma cytokines in the custom Procarta Plex™ panel were quantified in the RA, SLE, and SSc patients and healthy controls (HCs): SDF-1a, GITRL, IL-1b, IL-2, IL-4, IL-5, IL-33, IL-10, Insulin, PYY (3-36), CCL22, IL-13, IL-17A, Gal-3, FKN, IFN-
The scatter plots of the protein concentrations of plasma proteins (pg/ml) in drug-naive RA (
The summary of the significant differences of plasma cytokine concentrations between drug-naive autoimmune patients (RA, SLE, SSc) and healthy controls (HCs) in a pairwise comparison. The arithmetic means (mean), standard deviation (SD), and the standard error of the mean (SEM) of the plasma cytokine concentrations were calculated. The pairwise comparison of the concentrations of each cytokine of patients versus healthy controls (RA vs. HC; SLE vs. HC; SSc vs. HC) was carried out by one-way ANOVA (
Cytokine | Cohort | Mean (pg/ml) | SD | SEM | One-way ANOVA ( |
CI (95%) |
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IL-12p40 | HC | 4.18 | 2.85 | 0.45 | 9.7 |
3.30-5.06 |
RA | 8.90 | 6.45 | 1.16 | 6.63-11.17 | ||
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IL-10 | HC | 4.96 | 4.66 | 0.74 | 3.2 |
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RA | 12.02 | 19.69 | 3.54 |
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IL-13 | HC | 7.25 | 15.53 | 2.46 | 9.7 |
2.43-12.06 |
RA | 41.74 | 49.74 | 8.93 | 24.23-59.25 | ||
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IFN- |
HC | 23.64 | 18.35 | 2.90 | 1.6 |
17.96-29.33 |
RA | 98.83 | 191.03 | 34.31 | 31.58-166.07 | ||
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M-CSF | HC | 27.34 | 34.09 | 5.39 | 4.8 |
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RA | 100.58 | 227.01 | 40.77 |
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IL-4 | HC | 31.14 | 69.44 | 10.98 | 1.7 |
9.62-52.66 |
RA | 121.79 | 157.07 | 28.21 | 66.50-177.09 | ||
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NTproBNP | HC | 39.24 | 47.07 | 7.44 | 24.64-53.82 | |
RA | 142.07 | 194.66 | 34.96 | 1.9 |
73.55-210.59 | |
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IL-17A | HC | 15.98 | 26.99 | 4.27 | 3.5 |
7.62-24.34 |
RA | 154.17 | 288.13 | 51.75 | 52.74-255.6 | ||
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BMP-9 | HC | 37.78 | 34.56 | 5.46 | 2.9 |
27.07-48.49 |
RA | 160.67 | 347.69 | 62.45 | 38.27-283.06 | ||
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PYY | HC | 163.96 | 91.68 | 14.50 | 4.0 |
135.55-192.37 |
RA | 361.60 | 265.77 | 47.73 | 268.05-455.16 | ||
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GITRL | HC | 110.19 | 100.94 | 15.96 | 1.4 |
78.91-141.47 |
RA | 901.22 | 1498.96 | 269.22 | 373.54-1428.89 | ||
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MMP-12 | HC | 154.26 | 278.20 | 43.99 | 7.4 |
68.05-240.48 |
RA | 833.00 | 1525.20 | 273.93 | 296.09-1369.91 | ||
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TNFRSF6 | HC | 209.45 | 574.09 | 90.77 | 3.9 |
31.54-387.36 |
RA | 1200.63 | 1998.27 | 358.90 | 497.18-1904.07 | ||
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IL-12p40 | HC | 4.18 | 2.85 | 0.45 | 5.7 |
3.29-5.06 |
SLE | 10.86 | 7.43 | 1.70 | 7.52-14.20 | ||
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M-CSF | HC | 27.34 | 34.09 | 5.39 | 6.8 |
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SLE | 63.06 | 63.94 | 14.67 |
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IL-4 | HC | 31.14 | 69.44 | 10.98 | 5.2 |
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SLE | 70.16 | 73.03 | 16.75 |
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GITRL | HC | 110.19 | 100.94 | 15.96 | 1.3 |
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SLE | 217.58 | 222.56 | 51.06 |
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NTproBNP | HC | 39.24 | 47.07 | 7.44 | 1.9 |
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SLE | 315.89 | 727.91 | 166.99 |
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NTproBNP | HC | 39.235 | 47.07 | 7.44 | 3.8 |
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SSc | 174.206 | 272.76 | 86.25 |
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PYY | HC | 163.9605 | 91.68 | 14.50 | 3.1 |
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SSc | 232.962 | 68.12 | 21.54 |
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MMP-12 | HC | 154.2605 | 278.20 | 43.99 | 2.1 |
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SSc | 514.748 | 798.52 | 252.52 |
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The protein concentrations were the following (RA
Five cytokines, IL-12p40, M-CSF, IL-4, GITRL, and NTproBNP, were significantly elevated in SLE
The scatter plots of the protein concentrations (pg/ml) of SLE (
Three cytokines, NTproBNP, PYY (3-36), and MMP-12, were significantly increased in SSc
The scatter plots of the protein concentrations (pg/ml) of SSc (
We are the first to report the cleaved peptide tyrosine tyrosine PYY (3-36) is an early marker of drug-naive RA
Thirteen cytokines showed significantly elevated concentrations in the plasma of SAD patients, distinguishing RA (Figure
The concentrations of plasma cytokines (pg/ml) in the ascending order of (a) RA (
Better identification of the specific molecular players of the early stage of SADs may contribute to the recognition of novel prognostic markers and could facilitate the pathogenesis-appropriate timing of therapeutic interventions. In summary, we have quantified plasma proteins in early SAD patients, prior to therapeutic modification of the disease pathology. A characteristic pattern of soluble mediators was revealed distinguishing early diagnosed therapy-naive RA, SLE, or SSc from HCs. These eleven markers with nonoverlapping CI (95%) were associated with RA: IL-12p40, IL-10, IL-13, IFN-
There were no significant correlations in PYY (3-36) concentration (pg/ml values) compared to CRP, ESR, RF, aMCV, and DAS28 scores in RA, separately. However, the concentration of two cytokines in the plasma of drug-naive RA patients, the IL-4 or GITRL concentrations, showed correlation with bioactive PYY (3-36) level, respectively. (Supplementary Figure
We are the first to report PYY (3-36) as a specific plasma marker of drug-naive RA. Additionally, the multiplex analysis of 30 plasma proteins supported a disease-specific cytokine pattern in RA, SLE, and SSc, respectively. Based on these data, we could delineate a prototype model for the machine-learning automated classification of drug-naive RA, SLE, and SSc.
Additional data are in the Supplementary Files, or raw data can be requested from the corresponding author.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This research was funded by the following grants: GINOP-2.3.2-15-2016-00001 (BRC), GINOP-2.3.2-15-2016-00030 (BRC and UoS), 2017-1.3.1-VKE-2017-00028 (Avicor), and 2018-1.3.1-VKE-2018-00024 (BRC and Avidin) from the National Research, Development and Innovation Office (NKFI), Hungary. This study was prepared with the professional support of the doctoral student scholarship program of the cooperative doctoral program of the Ministry of Innovation and Technology financed by the National Research, Development and Innovation Fund for Jozsef A. Balog (KDP-17-4/PALY-2021, 1000464).
Supplementary Table 1: clinical characteristics of RA study participants. The median DAS28 disease activity score was 6.01, and Q1-Q3 interquartiles were 5.4-6.5. Several clinical and immunoserological parameters were present at the time of diagnosis of RA including RF: rheumatoid factor; MCV: mutated citrullinated vimentin; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate. Data are expressed as median and interquartile range (Q1, Q3) for continuous variables and as number (
Supplementary Table 2: clinical characteristics of SLE study participants. The median SLEDAI-2K activity score was 16, and Q1-Q3 interquartiles were 10-21. Several clinical and immunoserological parameters were present at the time of diagnosis of SLE including ANA (antinuclear antibodies); anti-DNA antibody; LA (lupus anticoagulant) (activated partial thromboplastin
Supplementary Table 3: clinical characteristics of SSc study participants. Several clinical and immunoserological parameters were present at the time of diagnosis of SSc including ANA (antinuclear antibodies); anti-Scl-70, anti-Scl-70 antibodies; ACA (anticentromere antibodies); and anti-RNA polymerase III antibodies. Data are expressed as median and interquartile range (Q1, Q3) for continuous variables and as number (
Supplementary Table 4: the list of the proteins measured in the plasma of the human subjects enrolled in the study. The lower range of the detection corresponds to the threshold of the sensitivity of the assay. The Procarta Plex™ panel was designed by the authors, and the assay was loaded on a MAGPIX Luminex instrument.
Supplementary Figure 1: the Pearson correlations of the plasma concentrations of IL-4