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Spondyloarthritis
Original research
Exploring complement biomarkers in suspected axial spondyloarthritis
  1. Clara Elbæk Mistegård1,2,3,
  2. Anne Troldborg1,2,3,
  3. Anne Gitte Loft1,3,
  4. Steffen Thiel2,
  5. Laura Spiller4,
  6. Mikhail Protopopov4,
  7. Valeria Rios Rodriguez4,
  8. Burkhard Muche4,5,
  9. Judith Rademacher4,6,
  10. Anne-Katrin Weber4,
  11. Susanne Lüders4,
  12. Joachim Sieper4,
  13. Denis Poddubnyy4 and
  14. Fabian Proft4
  1. 1Department of Rheumatology, Aarhus University Hospital, Aarhus, Denmark
  2. 2Department of Biomedicine, Aarhus University, Aarhus, Denmark
  3. 3Department of Clinical Medicine, Aarhus University, Aarhus, Denmark
  4. 4Department of Gastroenterology, Infectiology and Rheumatology (Including Nutrition Medicine), Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
  5. 5Department of Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany
  6. 6Berlin Institute of Health, BIH, Berlin, Germany
  1. Correspondence to Dr Fabian Proft; Fabian.Proft{at}charite.de

Abstract

Objectives To investigate lectin pathway proteins (LPPs) as biomarkers for axial spondyloarthritis (axSpA) in a cross-sectional cohort with a suspicion of axSpA, comprising newly diagnosed axSpA and chronic low back pain (cLBP) individuals.

Methods Serum samples from 515 participants within the OptiRef cohort, including 151 axSpA patients and 364 cLBP patients, were measured using immunoassays for LPPs (mannan-binding lectin (MBL), collectin liver-1 (CL-L1), M-ficolin, H-ficolin and L-ficolin, MBL-associated serine proteases (MASP)−1, –2 and –3, MBL-associated proteins (MAp19 and MAp44) and the complement activation product C3dg).

Results Serum levels of L-ficolin, MASP-2 and C3dg were elevated in axSpA patients, whereas levels of MASP-3 and CL-L1 were decreased, and this remained significant for C3dg and MASP-3 after adjustment for C reactive protein (CRP). A univariate regression analysis showed serum levels of CL-L1, MASP-2, MASP-3 and C3dg to predict the diagnosis of axSpA, and MASP-3 and C3dg remained significant in a multivariate logistic regression analysis. Assessment of the diagnostic potential showed that a combination of human leukocyte antigen B27 (HLA-B27) and measurements of L-ficolin, MASP-3 and C3dg increased the diagnostic specificity for axSpA, however, with a concomitant loss of sensitivity.

Conclusions Serum levels of complement activation, that is, C3dg, and MASP-3 differed significantly between axSpA and cLBP patients after adjustment for CRP. Although combining HLA-B27 with measurements of L-ficolin, MASP-3 and C3dg increased the diagnostic specificity for axSpA, this seems unjustified due to the concomitant loss of sensitivity. However, both C3dg and MASP-3 were associated with axSpA diagnosis in multivariate logistic regression, suggesting an involvement of complement in the inflammatory processes and possibly pathogenesis in axSpA.

  • Spondylitis, Ankylosing
  • Inflammation
  • Sensitivity and Specificity
  • Low Back Pain
  • Proteins, Complement System
  • Complement Activation
  • Lectin Complement Pathway

Data availability statement

Data are available upon reasonable request.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/rmdopen-2024-004127

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • Axial spondyloarthritis (axSpA) is a chronic inflammatory disease with largely unknown pathogenesis, and patients with axSpA often suffer from symptoms for 6–8 years prior to diagnosis, probably delaying effective treatment of the disease.

    • Complement lectin pathway proteins demonstrated diagnostic potential in a cross-sectional cohort of patients with axSpA and healthy blood donors and significant changes in a cross-sectional cohort of patients with axSpA compared with patients with chronic low back pain (cLBP) with and without SpA features.

    • It is essential to investigate if complement lectin pathway proteins are valuable for diagnostic purposes in a clinically relevant setting, to address a clinically unmet need for objective biomarkers for axSpA diagnosis and disease activity and to unravel the largely unknown pathogenesis.

    WHAT THIS STUDY ADDS

    • The cross-sectional study assessed serum levels of complement lectin pathway proteins at the time of diagnosis in 151 patients with a clinical diagnosis of axSpA and 364 patients with cLBP, the diagnostic potential of the lectin pathway proteins and the associations with disease activity and functional impairment in patients with axSpA.

    • Serum levels of complement activation, that is, C3dg, were significantly elevated, whereas serum levels of complement proteins MASP-3 and M-ficolin were significantly decreased after adjustment for C reactive protein.

    • Complement activation marker C3dg, and complement protein MASP-3, were associated with the diagnosis of axSpA in a multivariate logistic regression analysis.

    • Assessment of the diagnostic potential showed that a combination of human leukocyte antigen B27 and measurements of L-ficolin, MASP-3 and C3dg increased the diagnostic specificity for axSpA, with a concomitant loss of sensitivity.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • The study supports an association between complement proteins and axSpA diagnosis. However, it did not show a justified diagnostic value in the current highly clinically relevant setting.

    • The association, however, prompts further investigation into complement involvement in axSpA pathogenesis and disease development, and investigations into specific extra-articular manifestations of this disease.

    Introduction

    Axial spondyloarthritis (axSpA) is a common chronic, inflammatory rheumatic disease characterised by inflammation of the sacroiliac joints and spine,1 causing pain and potentially progressive functional impairment resulting in personal and socioeconomic consequences. The disease shares certain clinical, biological and genetic features with other inflammatory diseases commonly grouped as spondyloarthritis (SpA), that is, psoriatic arthritis, arthritis with associated inflammatory bowel disease and reactive arthritis.2

    The advancement in therapeutic interventions for axSpA, including biological disease-modifying anti-rheumatic drugs (bDMARDs), such as tumour necrosis factor inhibitors, interleukin-17 inhibitors and targeted synthetic DMARDs (tsDMARDs, ie, Janus kinase inhibitors) has shown to improve symptomatic disease activity,3 thus facilitating a stronger focus on early detection of axSpA. The increased emphasis on early diagnosis is substantiated by accumulating evidence supporting these therapeutic interventions in averting the development of structural damage.4 5

    Patients with axSpA have often suffered symptoms for mean durations of 6–8 years prior to diagnosis and display structural damage at the time of diagnosis.6 7 Different strategies have been established to decrease diagnostic delay, including online self-referral tools8 9 and clinical implementation of MRI. However, MRI is limited by availability and costs and complicated by the high prevalence of acute and chronic changes demonstrated in healthy individuals, athletes, runners and postpartum women.10–12 Humoral inflammation marker C reactive protein (CRP) is within the normal range in about half of the patients with axSpA.13 Thus, it cannot be regarded as an objective measure of disease activity. Investigations of objective biomarkers for an early diagnosis of axSpA remain highly relevant and could provide further insights into the pathogenesis of the disease.

    The pathogenesis of axSpA is largely unexplained. The disease is associated with a genetic predisposition, that is, human leukocyte antigen B27 (HLA-B27) accounts for approximately 20% of axSpA heritability, whereas other genes account for 4%–7%.14 15 Furthermore, polymorphisms in other genes (eg, ERAP1) are associated with radiographic axSpA (r-axSpA) (formerly known as ankylosing spondylitis) in HLA-B27-positive patients or patients carrying another risk allele for the disease (HLA-B40).16 17 However, emerging evidence suggests the involvement of the innate immune system,18 19 and based on the current limited understanding of the pathogenesis, axSpA could be considered to have both autoimmune and autoinflammatory components.15 20

    An essential part of innate immunity is the complement system. It is based on a fundamental principle of immunological pattern recognition, where pattern recognition molecules (PRMs) identify foreign structures (eg, bacterial surfaces) or modified self-structures (eg, cellular debris) known as pathogen-associated molecular patterns and danger-associated molecular patterns (DAMPs), respectively.21

    Complement lectin pathway PRMs encompass mannan-binding lectin (MBL), collectin-LK (a heteromer of collectin liver-1 (CL-L1) and collectin kidney-1 (CL-K1)), H-ficolin, L-ficolin and M-ficolin (also termed ficolin-3, ficolin-2 and ficolin-1, respectively). These PRMs are found in complexes with three serine proteases (MBL-associated serine protease 1 (MASP-1), MASP-2 and MASP-3) and two non-enzymatic proteins (MBL-associated proteins (MAps) named MAp19 and MAp44). When activated, the complement system participates in (1) defence against invading microorganisms by direct killing through the assembling of the membrane attack complex (MAC) or opsonisation for phagocytosis, (2) generation of inflammation (ie, the release of potent anaphylatoxins C3a and C5a) and III) regulation of homeostasis and the activation of cells of the adaptive immune system (eg, B cells).22–24 Due to the intrinsic capacities of the complement system, it provides a bridge between innate and adaptive immunity, host defence and homeostasis, and hereby represents an attractive focal point in axSpA.18

    Previous studies have shown elevated complement activation (ie, soluble MAC) in axSpA25; others have found complement proteins (C5, C6 and C9) to be upregulated in proteomic analysis and serve as potential biomarkers for both diagnosis and disease activity in r-axSpA.26 We have previously identified the complement lectin pathway PRM L-ficolin as a potential axSpA biomarker in a cohort of Danish patients with axSpA.27 28 The current study investigated the diagnostic potential in a larger cohort of newly diagnosed, ts/bDMARD-naïve axSpA patients compared with clinically relevant controls suffering from other causes of chronic low back pain (cLBP).

    Materials and methods

    Materials

    Patients were consecutively recruited from the specialised outpatient clinic of Charité—Universitätsmedizin Berlin, Germany, as a part of the Optimal Referral Strategy for Early Diagnosis of axSpA (OptiRef) project.8 9 Briefly, patients with cLBP (duration ≥3 months) and onset before 45 years of age with suspicion of axSpA were either referred by a physician using the Berlin referral tool (requiring the additional presence of inflammatory back pain, HLA-B27 positivity or sacroiliitis on imaging)29 30 or fulfilled the requirements of an online screening tool (www.bechterew-check.de) that expects the presence of at least one additional parameter from a list of 13 SpA parameters.8 Physician-referred and online-screened patients underwent a systematic evaluation by a rheumatologist, encompassing standardised assessment of clinical manifestations, laboratory assessments (including CRP levels and HLA-B27 status) and imaging modalities (using X-ray examinations and when deemed clinically pertinent, MRI). After an interdisciplinary assessment by the team, including rheumatologists and specialised radiologists, a final diagnosis of ‘axSpA’ or ‘no-axSpA’ (=cLBP) was made.

    Methods

    Blood samples were collected at inclusion and handled according to local instructions, which included centrifuging at 3000 RPM for 10 min at 4°C, subsequently frozen at –80°C.

    Immunoassays were employed to quantify the complement lectin pathway proteins (LPPs), namely MBL, H-ficolin, L-ficolin, M-ficolin, CL-L1, MASP-1, MASP-2, MASP-3, MAp19 and MAp44. Furthermore, complement activation marker C3dg was determined. Except for L-ficolin, which was assessed using a commercially available ELISA kit (Hycult Biotechnology, Uden, the Netherlands), all other proteins were quantified through time-resolved immunofluorometric assays (TRIFMAs). The specific antibodies used in TRIFMAs were custom developed and manufactured in-house. Comprehensive details of the assay procedures are provided elsewhere.31–39 Sample dilution and loading on microtitre plates were automated using a pipetting robot (JANUS, PerkinElmer, Hamburg, Germany). All analyses were performed in duplicate. Measurements were repeated if the duplicate analyses’ coefficient of variation (CV) was above 15%. Inter-assay CV based on internal controls was also determined for each protein (all CVs were below 15%). The levels of LPP in serum are reported in ng/mL, whereas C3dg is reported in U/mL. Protein measurements were performed without knowledge of clinical data and diagnosis, ensuring a blinded approach to the measurements.

    Patient and public involvement

    Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

    Statistics

    Patient demographics were characterised by median values with IQRs for continuous variables and numbers with percentages for categorical variables due to non-Gaussian distributions. Statistical comparisons for continuous variables were conducted using the Mann-Whitney U test, while categorical variables were compared using the χ2 test. Serum concentrations of LPPs were assessed through t-tests, and subsequent covariance analysis was performed after adjusting for CRP levels.

    Elevated levels of L-ficolin and C3dg were defined as exceeding the 75th percentile of serum levels observed in patients with cLBP; decreased levels of MASP-3 were defined as below the 25th percentile of serum levels in patients with cLBP. The diagnostic potential of high L-ficolin levels, high C3dg levels and low MASP-3 levels in combination with the established biomarker HLA-B27 was evaluated based on sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratios (LRs). Univariate logistic regression analysis assessed the predictive capacity of LPP levels for diagnosing axSpA, followed by corresponding multivariate analysis.

    Spearman’s correlation examined associations between LPP levels and variables such as CRP, disease activity (ie, Ankylosing Spondylitis Disease Activity Score; ASDAS-CRP and Bath Ankylosing Spondylitis Disease Activity Index; BASDAI) and functional impairment (ie, Bath Ankylosing Spondylitis Functional Index; BASFI). Differences in the levels of LPPs significantly associated with the correlation analysis (L-ficolin and C3dg) were assessed based on the presence of high disease activity (ASDAS-CRP >2.1 or BASDAI ≥4) and high functional impairment (BASFI >2.3, ie, the median) using the Mann-Whitney U test.

    All statistical tests were two sided, and significance was defined as p values <0.05. Statistical analyses were performed using Stata V.17 software (StataCorp, Texas), while graphical representations were generated using GraphPad Prism V.14 software (GraphPad Software, California).

    Ethics

    The study was approved by the local ethics committee (Charite—Universitätsmedizin Berlin, Berlin, Germany), approval number EA4/161/15, and conducted in accordance with the declaration of Helsinki and Good Clinical Practice. Written informed consent was obtained from all patients prior to study enrolment.

    Results

    Demographics

    Patient demographics are shown in table 1. The entire study population exhibited a median age of 36 years, comprised of 47% men, and 41% were found to be HLA-B27 positive. The median symptom duration was 6 years, inflammatory back pain was found in 59%, and 18% exhibited elevated CRP levels (>5 mg/L). Good response to non-steroid anti-inflammatory drugs (NSAID) was reported in 73% of the patients. All patients were naïve to cs/ts/bDMARDs. Extra-articular manifestations such as uveitis, psoriasis and inflammatory bowel disease were found in 7%, 10% and 3%, respectively. Peripheral manifestations (arthritis, dactylitis or enthesitis) were found in one of four of the study populations. In total, 151 patients had axSpA, and 364 patients had cLBP.

    View this table:
    Table 1

    Patient characteristics of the included patients from the OptiRef cohort

    Median age was 33 and 38 in the two patient groups, significantly lower in axSpA patients compared with cLBP patients (p<0.001). The distribution of biological sex varied between the two patient groups, with men comprising 55% of axSpA patients and 43% of cLBP patients (p=0.014). The prevalence of HLA-B27 positivity differed in the two groups. It was 83% in axSpA patients and 23% in cLBP patients (p<0.001). Lifestyle factors such as body mass index (BMI) and smoking prevalence did not differ in the two groups. The median symptom duration was 5 and 7 years in axSpA and cLBP patients, respectively (p=0.027). A variety of SpA features, as defined in the ASAS 2009 criteria, including inflammatory back pain, good response to NSAID, elevated CRP and anterior uveitis, were more pronounced in axSpA patients compared with cLBP patients (all p≤0.002). In contrast, no significant differences were observed regarding psoriasis, inflammatory bowel disease and peripheral manifestations (ie, arthritis, dactylitis or enthesitis). Regarding disease activity at diagnosis, the axSpA patients showed a median ASDAS-CRP of 2.7 and a median BASDAI of 4.6, indicating high disease activity. In total, n=77 (51%) of the axSpA patients were classified as r-axSpA.

    Serum levels of LPP

    The levels of L-ficolin, MASP-2 and C3dg were significantly elevated in axSpA patients compared with cLBP patients in the unadjusted analysis (figure 1), whereas levels of CL-L1 and MASP-3 were decreased in axSpA patients. Estimated differences in serum levels are provided in online supplemental table S1. No significant differences were observed for MBL, H-ficolin, MASP-1, MAp44 and MAp19 (online supplemental figure S1). Additionally, no significant differences in serum levels were observed among axSpA patients grouped according to radiographic or non-radiographic disease manifestations (online supplemental figure S2). After adjustment for CRP, complement LPPs MASP-3 and C3dg remained significant, and M-ficolin was significantly decreased in axSpA patients compared with cLBP patients (table 2).

    Figure 1
    Figure 1

    Differences in lectin pathway protein serum levels were compared by t-tests. P values are indicated in graphs. Bars indicate median and IQR. One cLBP patient sample has an extraordinarily high level of L-ficolin of 24 053.16 ng/mL and is not included in the graphic representation but was included in all analyses. axSpA, axial spondyloarthritis; cLBP, chronic low back pain.

    View this table:
    Table 2

    Differences in lectin pathway protein serum levels adjusted for CRP

    Diagnostic potential of LPP

    The diagnostic potential was evaluated for L-ficolin, which has previously been shown to be elevated in axSpA patients, and for MASP-3 and C3dg, which were found to be decreased and elevated, respectively, in axSpA patients compared with cLBP patients in the current study.

    The presence of only one LPP (ie, high L-ficolin, high C3dg or low MASP-3) biomarker resulted in specificities of 75%, sensitivities ranging between 36% and 40%, PPV ranging between 37 and 40, and NPV ranging between 74 and 75; thus overall inferior performance compared with HLA-B27 positivity only in this preselected cohort according to the inclusion criteria (table 3). Combinations of HLA-B27 and one LPP biomarker resulted in increased specificities ranging between 93% and 95% but concomitantly decreasing sensitivities to 29%–33%. PPVs increased to 63–72, and NPVs increased marginally to 76–77 compared with only one LPP biomarker (74–75). The positive LR+ increased to 4.16–6.31 compared with either HLA-B27 positivity (3.57) or one LPP biomarker (either high L-ficolin, high C3dg or low MASP-3) only (1.43–1.62). Furthermore, prevalence of high levels of LPPs was assessed according to HLA-B27 status (figure 2).

    Figure 2
    Figure 2

    Frequency of high levels of L-ficolin and C3dg, and low levels of MASP-3 in the study population. Heat map of the serum levels of L-ficolin (ficolin 2), C3dg, MASP-3 and HLA-B27 results from 151 axSpA patients and 364 cLBP patients. Green colour represents levels below the 25th percentile; light green represents levels from 25th to 50th percentile, pink represents levels from 50th to 75th percentile; and red represents the highest levels above the 75th percentile. For HLA-B27, red represents HLA-B27 positive; green represents HLA-B27 negative; grey represents unknown HLA-B27 status. The percentiles are based on the serum levels measured in cLBP patients. axSpA, axial spondyloarthritis; cLBP, chronic low back pain; HLA-B27, human leukocyte antigen B27; MASP-3, MBL-associated serine protease 3.

    View this table:
    Table 3

    The diagnostic potential of complement proteins, either alone or in combination with a positive HLA-B27

    Prediction of axSpA diagnosis

    Univariate logistic regression analyses assessed the predictive value of continuous complement lectin pathways protein levels in relation to axSpA diagnosis (table 4). Serum levels of CL-L1 and MASP-3 were negatively associated with an axSpA diagnosis, and the disclosed OR were 0.99758 (0.99529; 0.99987) and 0.99985 (0.99976; 0.99993) and corresponding p=0.038 and p=0.001, respectively. Serum levels of MASP-2 and C3dg were positively associated with an axSpA diagnosis, and the disclosed OR were 1.00100 (1.00009; 1.00190) and 1.00080 (1.00041; 1.00120), p=0.031 and p<0.001, respectively. In a multivariate logistic regression analysis (table 4), MASP-3 and C3dg remained significant.

    View this table:
    Table 4

    Univariate and multivariate logistic regression model to predict axSpA diagnosis

    Correlation between complement LPPs and clinical outcome measurements

    Serum levels of L-ficolin correlated positively with ASDAS-CRP, BASFI and CRP (ρ=0.283, 0.256, and 0.303, all p<0.05) (table 5). M-ficolin correlated positively with CRP (0.362, p<0.05). MASP-3 correlated negatively with CRP (−0.205, p<0.05), whereas no significant correlations were observed for ASDAS-CRP, BASDAI or BASFI. Complement activation, that is, C3dg, correlated positively with ASDAS-CRP, BASFI and CRP (ρ=0.273, 0.273 and 0.364, respectively, all p<0.05).

    View this table:
    Table 5

    Correlations between lectin pathway protein levels and disease activity in axSpA patients

    Based on the observed correlations with clinical data, the serum levels of L-ficolin and C3dg were assessed according to a dichotomous measurement of high disease activity (ASDAS-CRP >2.1 and BASDAI ≥4) and high degree of functional impairment above the median-reported BASFI in the current study population (BASFI >2.3) (figure 3). Serum levels of L-ficolin differed significantly according to high disease activity for ASDAS-CRP and BASDAI (both p ≤ 0.04). In contrast, no significant differences were observed for complement activation, that is, C3dg (data not shown).

    Figure 3
    Figure 3

    (A) L-ficolin serum levels and high disease activity measured by ASDAS-CRP. (B) L-ficolin serum levels and high disease activity measured by BASDAI. (C) L-ficolin serum levels and large functional impairment measured by BASFI. Serum levels were compared by Mann-Whitney U test and adjacent p values are indicated in the figures. axSpA, axial spondyloarthritis; ASDAS-CRP, Ankylosing Spondylitis Disease Activity Score. BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index.

    Discussion

    In this study, we investigated serum levels of complement LPPs in a cross-sectional clinically well-characterised cohort of 515 individuals referred with the suspicion of axSpA to an academic expert centre, comprising 151 patients newly diagnosed with axSpA and 364 patients with cLBP. The primary aim was to assess the diagnostic potential of these proteins as biomarkers for axSpA in a clinically relevant context. Our results revealed that axSpA patients exhibited elevated levels of complement LPPs L-ficolin, MASP-2 and the complement activation marker C3dg compared with patients with cLBP. Conversely, we observed decreased CL-L1 and MASP-3 levels in axSpA patients. After adjustment for CRP, significant differences were evident for M-ficolin, MASP-3 and C3dg.

    Complement is a multifaceted system organised in a proteolytic cascade and activated through one of three pathways: the classical, the lectin and the alternative pathway. The specific role of complement proteins, such as the compiled arsenal of substrate recognition due to differences in PRMs, the hierarchical organisation of the entire system and the different genes encoding the various proteins, allows various effects of increases and decreases in specific protein levels.

    The elevated levels of L-ficolin in the unadjusted analysis support the results of our previous studies.27 28 L-ficolin is a well-established PRM and activator of the lectin pathway of the complement system.40 The binding of a PRM to a fitting molecular structure leads to inflammation through the release of potent anaphylatoxins (C3a and C5a), deposition of complement fragments (ie, C3b) and opsonisation for phagocytosis, and potentially assembling of the MAC. The locally induced inflammation in the sacroiliac joints present in axSpA potentially results in a release of DAMPs. Even though the current knowledge of L-ficolin substrates is limited,40 one might speculate if L-ficolin, due to its capacities as a C-type lectin PRM, binds DAMPs41 in the sacroiliac joints and causes complement activation.

    However, the significance of L-ficolin differences between axSpA and cLBP patients was lost when we adjusted for CRP in the current study. The reason for this discrepancy might be attributable to distinct control groups. In the present study, CRP levels and prevalence of elevated CRP (>5 mg/L) differed significantly between axSpA patients and cLBP patients, whereas the prevalence of adjacent inflammatory disease manifestations within the SpA spectrum, including psoriasis, peripheral arthritis, and inflammatory bowel disease, did not differ. The prevalence of peripheral manifestations was relatively high in accordance with the applied referral algorithm. Additionally, HLA-B27 positivity among cLBP patients was 23%, thus exceeding HLA-B27 positivity of 8% usually seen in populations of European descent.1

    The cLBP patients hereby serve to constitute a clinically pertinent control group in the pursuit of a diagnostic biomarker for axSpA. The choice is justified by the fact that cLBP patients represent a differential diagnosis for individuals suspected of axSpA, given that a subset of patients initially suspected of having axSpA might already have or might ultimately receive a diagnosis of another disease within the SpA spectrum, for example, psoriasis. It is widely acknowledged that a considerable clinical, biological and genetic overlap exists between the various disease entities in the SpA spectrum.2 42 Thus, if the complement lectin pathway is associated with (ax)SpA pathogenesis, applying complement proteins as diagnostic biomarkers of axSpA might be challenging. This could explain the smaller differences in L-ficolin levels observed in the present study compared with a previous study of axSpA patients and healthy blood donors.27

    Furthermore, the present study investigated L-ficolin levels in serum samples compared with our previous studies of L-ficolin levels in EDTA samples. The difference in sample material might influence the results, as L-ficolin is decreased in serum samples.43 However, measurements of L-ficolin in serum and EDTA exhibited a well-defined correlation (R2=0.6454, p<0.001), and tube type seems to primarily affect the numerical values obtained (shown in online supplemental figure S3).

    Complement protease MASP-2 and complement activation marker C3dg were also elevated in axSpA patients in the current study. C3dg remained significant in the analysis adjusted for CRP, whereas MASP-2 did not. Complement activation in axSpA is still controversial. Previous studies have shown complement activation to be elevated in r-axSpA patients,25 whereas others have not.44 Previous studies (including the latter) are, however, troubled by the expanding understanding of MRI findings, for example, the presence of changes on MRI according to ASAS 2009 criteria45 in athletes,46 and postpartum women,11 which has only been acknowledged in the recent years. This might have resulted in misclassification of patients in clinical cohorts encompassing both r-axSpA and non-radiographic axSpA (nr-axSpA). The involvement of complement activation in pathologic bone formation is underscored by observations in animal models, where the administration of a complement inhibitor mitigates structural changes in r-axSpA.47 The specific mechanisms involved still need to be fully understood. However, in vitro studies have demonstrated that complement activation can enhance the differentiation of osteoclasts,48 potentially contributing to the complex tissue remodelling observed in axSpA, resulting in coexisting osteoporosis and osteoproliferation, that is, ankylosis.49

    MASP-3 was decreased in axSpA patients compared with cLBP patients in both the raw and adjusted analyses. MASP-3 is an enzyme that acts as a maturase for complement factor D, contributing to the formation of the C3 convertase within the alternative pathway of the complement system.22 50 The alternative pathway serves as an amplification loop of complement activation. Unlike the majority of enzymes in humans, MASP-3 predominantly circulates in an enzymatically active form,51 suggesting the regulatory mechanisms preventing harmful side effects to be located at (or after) factor D level in the complement cascade. The lower levels of MASP-3 observed in axSpA patients may be due to additional regulation serving as a negative feedback mechanism caused by constitutively high levels of complement activation.

    Finally, complement PRMs CL-L1 and M-ficolin were altered in the comparative analyses of serum levels. CL-L1 was decreased in axSpA patients in the raw analysis but was insignificant after adjustment for CRP. Contrarily, M-ficolin was found to be significantly reduced in axSpA patients after adjustment for CRP. M-ficolin is unlike the remaining PRMs of the lectin pathway, primarily produced from monocytes and granulocytes,33 and has been shown to correlate with DAS-28 in rheumatoid arthritis patients52 and to correlate with CRP in patients with suspected immunodeficiency.53 No significant differences in protein levels were found among the remaining complement proteins (online supplemental figure S1).

    As for assessing complement proteins as diagnostic biomarkers, based on our previous findings27 and the findings presented in the current study, the diagnostic potential of high L-ficolin, low MASP-3 and high C3dg was assessed. The use of high and low levels of specific complement proteins is justified by the various functional aspects of the complement system described previously. Adding either one of the three LPP biomarkers to HLA-B27 positivity increased the LR+ compared with HLA-B27 positivity only. However, a concordant loss of sensitivity was observed, and the improvement in LR+ does not seem justified. In a univariate logistic regression analysis, MASP-2 and complement activation marker C3dg were positively associated with an axSpA diagnosis, whereas MASP-3 and CL-L1 were negatively associated with an axSpA diagnosis. In the multivariate logistic regression analysis, only complement activation marker C3dg and MASP-3 remained significant.

    Complement protein level associations with disease activity and functional impairment were assessed to address an unmet clinical need for objective disease activity measurements in axSpA. L-ficolin and complement activation marker C3dg correlated positively with disease activity determined by ASDAS-CRP. However, this might be due to correlation with CRP, as no significant correlations were observed for BASDAI. On the other hand, positive correlations were also observed with functional impairment determined by BASFI. One might speculate if higher levels of L-ficolin and complement activation might be associated with severe radiographic changes, progressive functional impairment and higher BASFI at the time of diagnosis. As for the patients, everyday clinical practice assessment of disease activity is often considered more dichotomous as ASDAS-CRP >2.1 and BASDAI ≥4.0. In this dichotomous approach, serum levels of L-ficolin were significantly elevated in patients with high disease activity. No significant differences were observed for BASFI. However, no established cut-off for high BASFI has been validated, and hence, median BASFI in the current cohort was applied, making the observation more speculative. Furthermore, BASFI represents a measurement of current functional capacity and is influenced by longstanding disease, which might be less relevant in this cohort of newly diagnosed axSpA patients.

    The strengths of the current study include the standardised diagnostic approach for axSpA according to a prespecified protocol at an ASAS expert centre.9 The size of the clinical cohort (n=515), including well-characterised axSpA patients and cLBP patients serving as relevant clinical controls, further strengthens the external validity of our study. Furthermore, all patients were cs/ts/bDMARD-naïve, which renders no possible influence of biological treatment on complement signalling and resulting up-regulation/down-regulation of protein levels. Moreover, axSpA patients and cLBP patients were comparable according to life style factors relevant for inflammatory processes such as BMI and smoking habits. Another strength includes the complete measurement of all 10 proteins involved in the complement lectin pathway and complement activation marker C3dg. This work represents the most extensive investigations of complement proteins in newly diagnosed axSpA patients and clinically relevant controls with cLBP. Our study has some limitations to consider. MRI of the sacroiliac joint and spine was only performed if deemed clinically relevant and not systematically as a part of the standardised protocol for the clinical workup. Additionally, the applied analyses for complement measurements are externally validated laboratory analyses, but not all commercially or clinically available, currently limiting the clinical impact of our results. Furthermore, measurements were performed on serum samples, which is not ideal for measurements of L-ficolin.43 However, we have tested the specific set-up and found correlations of R2=0.6454, p<0.001, with levels found in EDTA plasma.

    In the present study, axSpA patients show higher levels of complement proteins L-ficolin, MASP-2 and complement activation (C3dg) compared with cLBP patients. In contrast, CL-L1 and MASP-3 levels were decreased in axSpA patients. Only differences in M-ficolin, MASP-3 and C3dg levels were significant after adjustment for CRP. Investigations of the diagnostic potential in this clinically relevant setting revealed that the combination of HLA-B27 positivity with either L-ficolin, MASP-3 and C3dg increased diagnostic specificity for axSpA, compared with HLA-B27 only, but seemed unjustified due to a concomitant loss of sensitivity.

    However, the associations of PRM L-ficolin, complement proteases MASP-2, and MASP-3, crucial to complement activation through the lectin pathway and alternative pathway, respectively, and complement activation, that is, C3dg, and axSpA diagnosis render further investigations of complement in axSpA pathogenesis and various disease manifestations, for example, psoriasis, within the SpA spectrum.

    Data availability statement

    Data are available upon reasonable request.

    Ethics statements

    Patient consent for publication

    Ethics approval

    The study was approved by the local ethics committee (Charite ́–Universitätsmedizin Berlin, Berlin, Germany), approval number EA4/161/15, and conducted in accordance with the declaration of Helsinki and Good Clinical Practice. Participants gave informed consent to participate in the study before taking part.

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    Supplementary materials

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    Footnotes

    • X @TroldborgAnne, @mprotopopov, @ProftDr

    • Contributors CEM, AT, AGL, ST, DP and FP conceptualised the study. LS, MP, VR, BM, JR, A-KW, SL, JS, DP and FP contributed to acquiring data. CEM conducted laboratory experiments for measurements of lectin pathway proteins. CEM, AT, AGL, ST and FP handled analysis of data, statistical evaluations and writing of the manuscript. CEM and ST obtained the grants for the study. All authors revised and approved the final version of the manuscript. FP acts as the guarantor of this work.

    • Funding The study was funded by donations from the Danish National Research Foundation through the Center for Cellular Signal Patterns (CellPAT) (DNRF135), the Danish Rheumatism Association (A6093 and A6462), Aase and Ejnar Danielsen Foundation and the Graduate School of HEALTH, Aarhus University. The collaboration has been facilitated by a EULAR scientific training grant for young fellows for CEM.

    • Competing interests CEM, AT, ST, AKW declare no conflicts of interest. AGL has received consulting fees from Novartis, UCB, Pfizer and Janssen; Honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Novartis, UCB, Pfizer; Support for attending meetings from Janssen and Novartis; Participation on a Data Safety Monitoring Board or Advisory Board for Novartis, Pfizer, UCB, Janssen. LS has received support for attending meetings and/or travel from Abbvie and MSD. MP has received support for attending meetings and/or travel from UCB and Jannsen; Receipt of equipment, materials, drugs, medical writing, gifts or other services from Novartis. VRR has received honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Abbvie; Support for attending meetings and/or travel from Pfizer; Participation on a Data Safety Monitoring Board or Advisory Board for Takeda. BM has received support for attending meetings and/or travel from UCB, Amgen and Galapagos Biopharma. JR has received support for attending meetings and/or travel from Abbvie, Jannsen, Novartis and UCB. SL has received support for attending meetings and/or travel from MSD and Abbvie. JS has received consulting fees from UCB; Honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Abbvie, MSD, Novartis, UCB. DP has received research grants from AbbVie, Eli Lilly, MSD, Novartis, and Pfizer; Consulting fees from AbbVie, Biocad, Bristol-Myers Squibb, Eli Lilly, Janssen, Moonlake, Novartis, Pfizer, and UCB; Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from AbbVie, Canon, DKSH, Eli Lilly, Janssen, MSD, Medscape, Novartis, Peervoice, Pfizer, and UCB; Participation on Advisory Board for AbbVie, Biocad, Bristol-Myers Squibb, Eli Lilly, Janssen, Moonlake, Novartis, Pfizer, and UCB; Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: ASAS, GRAPPA. FP has received research grants from Novartis, Eli Lily, UCB; Consulting fees from AMGEN, AbbVie, BMS, Celgene, Janssen, MSD, Novartis, Pfizer, Roche, UCB, Medscape, Galapagos, Hexal; Support for attending meetings and/or travel from Celgene, Janssen, Pfizer, Novartis, UCB; Participation on a Data Safety Monitoring Board or Advisory Board for AbbVie, Celgene, Janssen, Novartis, UCB; Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: ASAS, Y-ASAS, GRAPPA, Y-GRAPPA, EMEUNET, EULAR, DGRh, DEGUM, Rheumazentrum Berlin; Receipt of study materials from Aidian Oy.

    • Provenance and peer review Not commissioned; externally peer-reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.