Cytokine profiles in autoantibody defined subgroups of systemic lupus

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Cytokine profiles in autoantibody defined subgroups of systemic lupus erythematosus (SLE) Frida Torell, Susanna Eketjäll, Helena Idborg, Per-Johan Jakobsson, Iva Gunnarsson, Elisabet Svenungsson, and Johan Trygg J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00811 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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Journal of Proteome Research

Cytokine profiles in autoantibody defined subgroups of systemic lupus erythematosus (SLE) Frida Torell1, Susanna Eketjäll2,3, Helena Idborg4, Per-Johan Jakobsson4, Iva Gunnarsson4, Elisabet Svenungsson4, Johan Trygg1,5,* Affiliations: 1 Computational Life Science Cluster (CLiC), Department of Chemistry, Umeå University, Umeå, Sweden 2 Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Integrated Cardio Metabolic Centre (ICMC), Karolinska Institutet, Huddinge, Sweden 3 Science for Life Laboratory, Department of Clinical Neuroscience, Karolinska Institutet, Solna, Sweden 4 Rheumatology Unit, Department of Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden 5 Corporate research, Sartorius AG, Göttingen, Germany * Corresponding author (e-mail: [email protected]; ORCID: 0000-0003-3799-6094)

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Abstract 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. Based on 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 for low cytokine levels in the ANA negative group, autoantibody profiles did not discriminate between different cytokine patterns. Key words: Systemic Lupus Erythematosus, Cytokine, Multivariate data analysis, Subgrouping, HCA, OPLS-DA


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Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease, predominantly diagnosed in women [1]. The diverse clinical presentation of this autoimmune connective tissue disorder includes manifestations in many organ systems such as skin, joints, kidneys, heart and the central nervous system (CNS) [2]. The heterogeneous presentation of SLE is further complicated by the fact that SLE is characterized by periods of flares (active disease) and remissions (inactive disease) [1, 3]. The remission periods may include periods without antinuclear antibodies (ANA), resulting in an at least temporarily ANA negativity [4]. Owing to the diverse nature of the disease, diagnosing SLE is challenging. The American College of Rheumatology (ACR) [5] has presented a list of classification criteria, which are used to classify for SLE in studies. Accordingly, a patient needs to present with at least four out of the eleven criteria listed by the ACR to classify for SLE [6]. The imbalance of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor (TNF)-α and type I and type II interferons is part of the immune dysfunction observed in SLE patients and it also contributes to the tissue inflammation and organ damage [7, 8]. Interferons and interleukins regulate the B cells autoantibody secretion, seen in autoimmune diseases such as SLE [9, 10]. Thereby, SLE patients often have an overproduction of interferons and interleukins. The effects of cytokines are multifaceted, since cytokines can have both synergistic and antagonistic effects on other cytokines. Altered cytokine profiles have been associated with SLE in different ways. Increased levels of Th17-related cytokines were implicated with treatment resistant nephritis [11]. IL-6, IL-17, IL-17F, IL-21, IL-22, IL-26, Integrinα3, and TNF-α are secreted by T helper type 17 (Th17) cells [12]. In another study, the imbalance of plasma cytokines levels of cytokines associated with T-helper-cells (Th1 [IL-12, IFN-γ], Th2 [IL-4, IL-6, IL-10] and Th17 [IL-17, IL-23]) and regulatory T-cells (Treg [IL-10, TGF-β]) were investigated and IL-6, IL-10, IL-12 and IL17 were found to be significantly higher in the SLE patients [13]. IL-1β, IL-6, IL-10 and TNF-α have been found in higher levels in SLE patients compared to healthy volunteers [14, 15]. The type I interferon system is known to play an important etiopathogenic role in the pathogenesis of SLE [16]. In addition, IL6 and TNF-α have been found to be useful as biomarkers for disease activity in SLE [17, 18]. TNF- α activity and dyslipoproteinemia have been shown to be linked to each other and to cardiovascular and renal manifestations in SLE [19]. 3 ACS Paragon Plus Environment


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SLE patients are often misdiagnosed since there is a great overlap in symptomatology between SLE and other systemic autoimmune disease. The overlap does not only involve clinical manifestations but also patterns of inflammation and autoantibodies as well as recently described susceptibility genes [20, 21]. In addition, the SLE manifestations range from subtle symptoms to life-threatening conditions. The heterogeneity is probably one important reason for the fact that we lack general biomarkers that can be used to measure outcomes in clinical trials [22]. We hypothesized that by subgrouping SLE, we might get a better opportunity to find valid and reliable biomarkers to monitor periods of flares and remission, or to predict them. Different strategies have been applied to subgroup SLE patients. The autoantibody profiles of SLE patients have been used to identify subgroups or clusters within the SLE diagnosis. Five clusters were identified in the study by Arti-Essen et al.: one associated with antibodies against anti-cardiolipin (aCL) and with positivity in the lupus anticoagulant (LA), resulting in an antiphospholipid positive subgroup, one associates with antibodies against Smith antibodies (Sm) and ribonuclear proteins (RNP-A and RNP-68) antibodies, resulting in a Sm/RNP subgroup, one with autoantibodies against SSA/Ro52 and SSA/Ro60 as well as SSB/La resulting in a Sjögren antigen positive subgroup, one pure anti-dsDNA-only cluster and finally one cluster with patients not belonging to any of the groups [23]. Li et al. detected three clusters representing one anti-dsDNA subgroup, one anti-Smith, anti-RNP and aPL-subgroup and one antiRo/anti-La subgroup in an SLE study focusing on autoantibody profiles [24]. In another study Ching et al. found two clusters, one with autoantibodies against Sm/anti-RNP antigens and one against Ro/La antigens [25]. These autoantibody profiles have, however, not been correlated with cytokine profiles. Even though, it is well known that cytokines are involved in the disease pathogenesis of SLE by contributing to systemic inflammation, local tissue damage, and immunomodulation [17]. In the present study, the aim was to study and evaluate five autoantibody based subgroups; ANA neg, aPL, Sm/RNP, SS-like subgroups, and other SLE, based on their cytokine profile. Disease activity in the SLE samples compared to the control samples included in this study has recently been characterized by Idborg et al [26]. Cytokines were detected using Mesoscale Discovery (MSD) multiplex analysis. The analyzed samples were used to establish a cytokine profile for SLE compared to controls and to study the cytokine profile associated with the five autoantibody based subgroups. 4 ACS Paragon Plus Environment

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Experimental Section Patients In the present study, samples from 310 SLE patients, with established SLE (American College of Rheumatology criteria [5]) and 310 matched controls were analysed. Building on the autoantibody status at the time of sampling five antibody based subgroups were defined among the patients; 1) the ANA negative group was negative for all measured ANA specificities and aPL. 2) the antiphospholipid positive (aPL) patients tested positive for at least two of anti-cardiolipin antibodies (aCL IgG/M/A) and anti-β-2glycoprotein-1 antibodies (2GPI IgG/M/A) or for anticoagulant (LA), 3) anti-Sm/RNP patients tested positive for at least two of the following: anti-Sm) and/or anti-ribonucleoprotein antibodies (RNP A, RNP 68), 4) the Sjögren’s syndrome antigen positive subgroup (SS) tested positive for at least two antibodies against Sjögren’s syndrome antigen A (Ro60 or Ro52) and Sjögren’s syndrome antigen B (La), 5) the sub group “other SLE” included the patients having autoantibodies that fulfilled criteria for more than one group. These autoantibody based subgroups were the most pronounced in our patient cohort, while other autoantibodies appeared more sporadically. To make the size of the subgroups more similar and still keep the majority of the biological variation, a design based sample selection was applied. The subgroups and sample selections have been described by Surowiec et al. [27]. In short, the sample selection was performed using principal component analysis (PCA) where five samples were selected from each corner and with three centre points for each subgroup and controls, resulting in n=23 x 6.With the exception of aPL where only 22 samples were available. Separate PCA was calculated, for each subgroup and controls, using the available meta data. The meta data available for all patients included SLE Disease Activity Index (SLEDAI) [28], Systemic Lupus Activity Measure (SLAM) [29] and the damage index Systemic Lupus International Collaborating Clinics Damage Index (SLICC-DI) [30, 31] features, as well as autoantibody profiles, immunology data and routine blood tests. Thereby, 20+3 people from each group were selected in a fashion that span multivariate space for each autoantibody based subgroup, as well as the population based control group. The selection was also adjusted to satisfy for: gender (all female), age (same age span and mean for all groups) and disease duration (matched disease duration). Selected clinical variables including complement proteins and C-reactive protein (CRP) levels are shown as well as treatment with immunosuppressive drugs are described in the 5 ACS Paragon Plus Environment


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table below, see Table 1. The same type of data for the whole cohort is available as Supporting information (Table S1). Table 1 Selected clinical variables describing the five groups of SLE patients. Parameter

ANA neg

aPL

Other SLE

Sm/RNP

SS

23

22

23

23

23

23:0

22:0

23:0

23:0

23:0

37 (24-49)

32 (20-42)

32 (22-38)

31 (23-37)

30 (19-39)

14 (6-23)

14 (2-26)

16 (5-24)

12 (3-15)

19 (6-32)

3 (0-10)

6 (0-16)

5 (0-16)

6 (0-18)

5 (0-14)

0

22

23

23

23

103 (69-200)

101 (33-154)

97 (20-139)

96 (10-143)

98 (7-158)

C3 (mg/mL) b)

0.98 (0.73-1.31)

0.78 (0.27-1.05)

0.90 (0.38-1.68)

0.84 (0.36-1.31)

0.84 (0.29-1.37)

C4 (mg/mL) b)

0.20 (0.10-0.41)

0.11 (0.02-0.18)

0.15 (0.05-0.29)

0.14 (0.03-0.26)

0.13 (0.03-0.32)

2.15 (0.15-9.7)

10.38 (0.13-101)

5.64 (0.15-38.8)

3.50 (0.18-12.4)

3.29 (0.18-18.8)

12

16

14

17

16

ASA

4

11

4

5

2

Warfarin

4

5

3

3

2

Blood pressure treat.

11

10

5

11

8

Blood lipid treat.

6

4

4

2

1

Hydroxychloroquine

2

6

8

7

10

Azathioprine

5

7

2

7

5

Mycofenolate mofetil

0

0

4

3

2

Methotrexate

0

1

1

2

1

Cyclophosphamide

2

3

2

5

0

Mathera/Rituximab

1

1

2

2

0

General parameters N Gender (female:male) Age at onset a) Disease duration (year) a) SLEDAI total b) Positive ANA Complement protein C1q (%) b)

CRP (mg/L)b) Treatment (ever) Prednisolone

a)

Median (25th and 75th percentile).

b)

Median ± range.

All individuals in the study gave a written informed consent. The study was approved by the regional ethics committee at the Karolinska Institute, Stockholm, Sweden.


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Mesoscale Discovery (MSD) multiplex analysis The whole cohort of patients with established SLE as well as controls, without SLE, were analysed using the Mesoscale Discovery (MSD) V-PLEX™ Human Cytokine 30-plex kit. Plasma (EDTA) samples were analyzed on the MSD V-PLEX™ Human Cytokine 30-plex kit (K15054D; Mesoscale Discovery, Gaithersburg, MD) according to manufactures instruction, as previously described by Idborg et al. [26]. In short the plasma samples were thawed at room temperature and just before analysis diluted twice for proinflammatory and cytokine and four times for chemokine analysis in sample diluents. The diluted samples were incubated on the MSD plates for two hours at room temperature with shaking. Plates were washed and incubate an additional two hours with detection antibodies (as above). After washing 2x with Read buffer T was added to each well and the plate analyzed in a Sector Imager 6000. Calibrator and plasma samples were analyzed in duplicates. Using the MSD Workbench software the response of the calibrator concentrations were plotted as log signal unit on the vertical (Y) axis versus log concentration on the horizontal (X) axis. A weighted four parameter logistic fit (4PL) equation was used for curve fitting and back calculation of plasma sample concentrations. Multiplex Testing with the BioPlex® 2200 System Antibodies to specific nuclear antigens (dsDNA, SSA-Ro52, SSA-Ro60, SSB-La, Sm, Sm/RNP, RNP A, RNP 68, Jo-1, nucleosome, centromere) and phospholipids (cardiolipin IgG, IgM, IgA and β2-glycoprotein1 IgG, IgM, IgA) were analyzed by multiplexed bead technology (Luminex) using BioPlex 2200 system (Bio-Rad, Hercules, CA, USA) according to the specifications of the manufacturer. Results were handled as continuous variables. The cut-off for anti-cardiolipin (aCL) and anti-β2-glycoprotein1 (aβ2GP1) fulfills the 99th percentile as described [32]. For aCL antibodies IgG, IgM and IgA as well as anti-β2GPI antibodies IgG, IgM and IgA results were reported in the ranges between 1.9-160 U/mL. The multiplex assays are regarded as positive if anti aCL antibodies IgG, IgM and IgA as well as anti-β2GPI antibodies IgG, IgM and IgA ≥ 20 U/mL. Serum levels of antibodies to double stranded DNA were reported in ranges between 4-300 U/mL (anti-dsDNA; reference values

Cytokine profiles in autoantibody defined subgroups of systemic lupus

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