Lessons to be learned from serum biomarkers in psoriasis and IBD the potential role in SpA

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1 Expert Review of Clinical Immunology ISSN: X (Print) (Online) Journal homepage: Lessons to be learned from serum biomarkers in psoriasis and IBD the potential role in SpA Maureen C. Turina, Robert Landewé & Dominique Baeten To cite this article: Maureen C. Turina, Robert Landewé & Dominique Baeten (2017) Lessons to be learned from serum biomarkers in psoriasis and IBD the potential role in SpA, Expert Review of Clinical Immunology, 13:4, , DOI: / X To link to this article: The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Accepted author version posted online: 05 Oct Published online: 21 Oct Submit your article to this journal Article views: 812 View related articles View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at

2 EXPERT REVIEW OF CLINICAL IMMUNOLOGY, 2017 VOL. 13, NO. 4, REVIEW Lessons to be learned from serum biomarkers in psoriasis and IBD the potential role in SpA Maureen C. Turina, Robert Landewé and Dominique Baeten Department of Clinical Immunology and Rheumatology, Amsterdam Rheumatology and immunology Center, Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands ABSTRACT Introduction: Early diagnosis, monitoring of disease activity, prediction of treatment response, and structural outcome remain major challenges in spondyloarthritis (SpA). Biomarkers could play a role in addressing these challenges, but in SpA there is a lack of suitable biomarkers. Areas covered: As SpA is clinically and pathophysiologically closely related to psoriasis and inflammatory bowel disease (IBD), we reviewed in literature, the value of serum biomarkers in these conditions with the aim to find potential candidates for assessing SpA. Expert commentary: Candidates of interest were antimicrobial peptides, including serum human beta defensin-2 (hbd-2) and lipocalin-2 (LCN-2), and class-1 MHC molecule beta2-microglobulin. Since these biomarkers are relevant in psoriasis and/or IBD from a pathophysiological point of view, and may play a role in the pathogenesis of SpA, we recommend further exploration of their value as biomarker in the diagnosis and prognosis of SpA. ARTICLE HISTORY Received 25 September 2015 Accepted 29 September 2016 Published online 21 October 2016 KEYWORDS Spondyloarthritis; psoriasis; inflammatory bowel diseases; serum; biomarkers 1. Introduction 1.1. Unmet needs in spondyloarthritis Spondyloarthritis (SpA) is an immune-mediated inflammatory disease affecting the spine, the peripheral joints, and extraarticular tissues such as the skin, the gut, and the eye [1]. The pathology of SpA is characterized by a combination of chronic tissue inflammation, destruction of cartilage and bone, and pathological new bone formation that may lead to ankylosis. Despite major advances in the field, such as the use of magnetic resonance imaging (MRI) to detect spinal inflammation and to find appropriate patients for treatment with TNF blockers [2 8], several important challenges remain to be addressed. First, there is a huge diagnostic delay of approximately 5 10 years between the onset of SpA symptoms and the diagnosis [9 11]. This delay is due to the fact that the early clinical signs and symptoms are often non-specific, that pathognomonic features of SpA, such as radiologic sacroiliitis, only appear late in the disease course, and that well-known biological markers such as HLA-B27 lack specificity (many healthy individuals carry HLA-B27) and therefore predictive value in the diagnostic context. Second, good biological predictors for treatment response are lacking. Currently, the only informative marker is C-reactive protein (CRP). Nevertheless, CRP as a marker for treatment response falls short because only one third of the patients have elevated CRP levels prior to treatment. Third, the progression rate of new bone formation as observed on consecutive X-rays of the lumbar and cervical spine is very slow and besides only occurs in a relatively small group of patients. Currently, markers predicting which patients will develop significant new bone formation are lacking. Overall, an early diagnosis, a proper prediction of structural damage, as well as prediction of treatment response, all in individual patients, remain major challenges in SpA. Biomarkers could be of value in addressing these challenges Biomarkers in SpA A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of a normal biologic process, a pathophysiologic process, or a pharmacologic response to a therapeutic intervention [12]. A biomarker should be sensitive, specific, reproducible, and deriving the biomarker from a patient should preferably be a non-invasive procedure. Taking these criteria into account, biomarkers can have an important role to fulfill the previously defined unmet needs in the process of diagnosis, treatment response, and prognosis. Several biomarkers have already been studied in SpA. We will shortly mention the most robust serum biomarkers in SpA hereafter. Common markers of systemic inflammation such as CRP and erythrocyte sedimentation rate (ESR) have been used for a long time in the clinical care of SpA patients. Accordingly, elevated CRP is included as a criterion in the ASAS classification criteria for axial SpA criteria but not for ASAS peripheral SpA criteria [2,13]. When elevated, CRP levels may correlate with disease activity measures and decrease upon effective treatment [14 17]. Moreover, CRP appears to CONTACT Dominique Baeten d.l.baeten@amc.uva.nl Department of Clinical Immunology and Rheumatology, Amsterdam Rheumatology and immunology Center, Academic Medical Center/University of Amsterdam, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

3 334 M. C. TURINA ET AL. be a predictor for radiographic axial progression [18]. However, CRP and ESR are only elevated in 30 50% of the SpA patients with clinical signs and symptoms of active disease and inflammation [14 17] and, obviously, are not specific for SpA. The limited sensitivity and specificity explains why CRP and ESR can be useful biomarkers at the group level (such as in clinical trials) but lack sufficient predictive value to be used in individual patients. Other serum markers of inflammation that have been studied in SpA are Interleukin-6 (IL-6) and calprotectin. IL-6 is the main driver of CRP and, similar to CRP, serum IL-6 levels are significantly increased in active SpA and decrease upon clinical response after tumor necrosis factor (TNF) blockade [19,20]. The calcium-binding protein calprotectin, a heterodimer of S100A8 and S100A9, is expressed and secreted during monocyte infiltration into inflamed tissues, including macrophage infiltration in synovial tissue of SpA patients [21,22]. Calprotectin serum levels are elevated in SpA, correlate moderately well (r = ) with disease activity, and decrease upon TNF blockade [21 23]. Moreover, calprotectin serum levels are an independent predictor for radiological axial progression at the group level [24]. Two other biomarkers that were studied in SpA are more related to the pathological processes than to inflammation as such. Vascular endothelial growth factor (VEGF), a growth factor involved in neo-angiogenesis, may be involved in the pronounced hypervascularity in SpA synovitis [25,26]. Serum VEGF levels are significantly elevated in SpA versus healthy controls and significantly albeit moderately at best correlate (r = ) with disease activity, but levels do not decrease after short-term treatment with TNF blockers [20,27]. While an original report indicated that serum VEGF levels were predictive of new bone formation [28], these results could not be confirmed by others [29]. Matrix metalloproteinase-3 (MMP-3) is an enzyme involved in tissue remodeling and cartilage damage. MMP-3 expression is elevated in synovial tissue, synovial fluid and serum in SpA and this expression correlates well with disease activity [30 34] and normalized upon effective treatment [30]. With regard to predicting new bone formation in ankylosing spondylitis (AS), the prototype of axial SpA, most studies have focused on molecules involved in the regulation of osteoblast activity, including the wnt-pathway, which is involved in the process of new bone formation. Dickkopf-1 (DKK1) and sclerostin are inhibitors of this pathway, and Diarra et al. [35] found that serum DKK1 levels were decreased in AS patients versus healthy controls or rheumatoid arthritis patients and low levels were associated with radiographic axial progression as expressed by syndesmophytes formation. These results were confirmed by others [36 38]. For sclerostin, discordant results were seen regarding the relationship with radiographic axial spinal progression [36,39 42]. Finally, several studies have screened for disease-specific autoantibodies as potential diagnostic biomarker in SpA. Unfortunately, most studies did not find disease-specific autoantibodies and the few that were reported could not be independently confirmed [43 45]. Recently, one group found elevated levels of IgG autoantibodies CD74 specific for HLA class II-associated invariant chain peptide (CLIP) in SpA [46,47]. These data as well as the diagnostic value of this autoantibody should be validated in independent cohorts. In summary, despite major efforts over the last years only few biomarkers have been described and validated in SpA, and often these biomarkers are useful at the group level but lack specificity to be used in individual patients in clinical care. Therefore, we have chosen a different strategy to exploratory search for new biomarkers beyond the field of SpA that could be of interest in SpA from a pathophysiological point of view. SpA is genetically, pathophysiologically, and clinically associated with psoriasis and inflammatory bowel diseases (IBD), two conditions in which serum biomarkers have been described. Therefore, we reviewed here the value of biomarkers in these conditions and discuss their potential utility for SpA. Reviewing was done for the settings of diagnosis (i.e., difference between disease and healthy or disease and similar inflammatory diseases), with respect to correlation with disease activity and response to effective treatment, as well as for predicting structural damage (in axial SpA). We have evaluated both the pathophysiological and clinical value of these markers. After defining the most robust and reliable biomarkers in psoriasis and IBD, we compared the results with SpA and determined whether there potential role in SpA is of interest from a pathological point of view. Thus, the aim is to review biomarkers in psoriasis and IBD and, subsequently, to explore if some of these biomarkers not yet studied in SpA may be of interest to the field. 2. Serum biomarkers in psoriasis Psoriasis is an immune-mediated, chronic inflammatory disease primarily of the skin but can also affect nails and peripheral and axial joints. The disease is characterized by abnormal differentiation and hyperproliferation of epidermal keratinocytes and infiltration of immune cells. Roughly 1 in 5 psoriasis patients will develop psoriatic arthritis (PsA), one of the phenotypical forms of SpA [48]. At the genetic level, SNPs in IL- 23R and Th-17 genes have shown to be associated with both psoriasis and SpA. In clinical practice, a diagnosis of psoriasis is based on clinical history and examination and, in the case of diagnostic uncertainty, skin biopsies. Disease activity is mainly determined by the Psoriasis Area and Severity Index (PASI) [49], which includes the percentage of affected skin area, the erythema, the induration, and scaling. Currently, serum biomarkers do not play an important role in clinical practice but are extensively studied in clinical trials (Table 1). Serum CRP levels were found to be elevated in the moderate and severe forms of psoriasis but not in patients with mild disease, when compared with the healthy individuals [50,51]. Several studies have reported serum CRP elevation in % of the psoriasis patients versus 1% in the control group [51 53]. Furthermore, serum CRP levels decreased significantly after effective treatment was started [51,95 98]. It is therefore only a potentially useful marker for disease activity in patients who did not receive systemic treatment for at least 1 month. In the treated patients, PASI is preferred to monitor disease activity. ESR levels were elevated in approximately half

4 EXPERT REVIEW OF CLINICAL IMMUNOLOGY 335 Table 1. Serum biomarkers in psoriasis: elevation in comparison with healthy controls, correlation with disease activity according to PASI, and modulation by effective treatment. Biomarker Elevation in comparison with healthy controls Correlation with disease activity measured PASI Treatment response References CRP + +/ +/ [14 16,50 53] ESR + NA NA [54] TNFα + NA + [55,56] TNFα-R1 + +/ NA [55,57 59] IFNγ + NA NA [56] IL NA [55,56,60] IL-12 +/ + NA [56] IL-17 +/ + + [55,56,61,62] IL-18* [56,63,64] IL [55,62,65] E-selectin + +/ NA [57,60,66,67] ICAM-1* + + +/ [57,60,64,66,68] VAP-1 + [69 71] S100A8/A9 + + NA [72,73] (calprotectin) LCN-2 + +/ [58,74 76] hbd / [77,78] Resistin + +/ +/ [79 81] Leptin + [79,80,82 85] Adiponectin +/ +/ [80,82 84,86,87] Ghrelin + + [80,88] RBP [76,83] VEGF + + +/ [55,65,89 91] Nitric oxide + +/ + [90,92 94] CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; hbd-2: human beta-defensin-2; ICAM-1: intracellular adhesion molecule-1; IFNγ: interferon gamma; IL: interleukin; LCN-2: lipocalin-2; NA: not applicable; RBP-4: retinol binding protein-4; TNF: tumor necrosis factor; TNFα-R1: tumor necrosis factor alpha-receptor 1; VEGF: vascular endothelial growth factor; VAP-1: vascular adhesion protein-1. *Plasma and serum of the psoriasis patients compared to healthy controls. Elevated ESR was clearly more frequent in the active state of the disease (80%) versus non-active psoriasis [54]. Serum and plasma cytokines, including interferon-gamma (IFNγ), TNF-α, TNF-R1, IL-6, IL-8, IL-12, and IL-18, IL-22, and IL- 23, have been investigated thoroughly in psoriasis. Coimbra et al. and others [55,56,60,63,65] have found that the serum and plasma levels of these cytokines were overall significantly higher in psoriasis as compared with healthy controls. But the data on the correlation with PASI were rather conflicting. Psoriasis-specific treatment including methotrexate, UV-A/B treatment, and TNF blockade did have an effect on serum levels of these cytokines [55,56,61,62]. Adhesion molecules are expressed by activated tissue cells (e.g., endothelium) as well as leukocytes and allow selective migration of distinct leukocyte subsets to specific tissues and sites of inflammation. For example, activated epithelial cells express E-selectin, which can interact with E-selectin on another epithelial cell or with alpha E beta 7 (a member of the integrin family) on CD8 positive T cells. Of the soluble adhesion molecules that can easily be measured in serum, E-selectin, ICAM-1 (in serum and plasma), and VAP-1 are best studied in psoriasis [57,60,64,66 71,99]. All of these molecules showed elevated serum levels in PsO versus healthy controls. Soluble serum E-selectin and ICAM-1 (serum and plasma) levels, however, were consistently (but only weakly to moderately) associated with disease activity as expressed by PASI scores (r = 0.44 and r = , respectively). Accordingly, only serum E-selectin levels decreased significantly and consistently upon effective treatment [60,66,67,69,99]. Among the antimicrobial peptides and proteins (AMP), serum calprotectin (S100A8/A9 proteins), lipocalin-2 (LCN-2, also called neutrophil gelatinase-associated lipocalin [NGAL]), and human β-defensin-2 (hbd-2) were associated with psoriasis. Highly up-regulated serum calprotectin levels were found in a proteomic analysis of the epidermis from psoriasis patients [72]. In addition, Benoit et al. [73] showed that serum calprotectin levels in serum were significantly elevated when compared to healthy controls and that patients with a high PASI (>15) score also had higher levels of calprotectin. Regarding LCN-2 serum levels, several groups consistently found elevated levels in psoriasis patients when compared with healthy individuals. However, absent or only weak (r = 0.27) correlations were found between LCN-2 levels and PASI scores, and serum levels were not normalized by effective treatment [58,74 76]. Serum levels of another AMP, hbd-2, were elevated consistently when compared with healthy donors. Jansen et al. [77] reported a high correlation with PASI (r = 0.82), whereas another group did not see such a strong correlation (r < 0.1, p > 0.05) [78]. Adipokines are produced by fat (adipose) tissue and play a role in metabolic processes, but these proteins are also associated with inflammation. Extensively studied adipokines in serum and plasma were resistin, leptin, adiponectin, ghrelin, and retinol binding protein-4 (RBP-4). With the exception of adiponectin, all serum and plasma adipokine levels were significantly higher in psoriasis versus healthy individuals [76,79,82 86,88]. Only serum resistin levels correlated weakly with PASI (r 2 = 0.027) and none of the adipokines consistently showed a normalization in levels after effective treatment [76,80,81,83,86,87] Growth factors are involved in angiogenesis and hypervascularity in psoriasis and are well studied as biomarkers. Of these growth factors, VEGF was the most evaluated markers. Serum VEGF was the most promising in psoriasis, since there were differences in serum levels between psoriasis patients and healthy controls and there was a strong correlation (R = 0.65) with PASI [55,89,90]. However, serum levels did not normalize upon effective treatment [55,65,91]. Nitric oxide (NO) is a free radical and is secreted by various cell types, including fibroblasts and endothelial cells. Tekin et al. [92] performed a study in 22 psoriasis samples versus 21 healthy controls and found significantly higher serum levels in the former group. These results were reproduced by others in serum and plasma [90,93,94]. Furthermore, NO serum and plasma levels decreased after effective treatment [92]. In summary, serum CRP, ESR, IL-18 (in serum and plasma), IL-22, ICAM-1 (in serum and plasma), calprotectin, LCN-2, hbd- 2, VEGF, RBP-4, and NO (in serum and plasma) were the most informative biomarkers since these markers are elevated in psoriasis patients with the disease when compared to healthy controls, show weak to moderate correlations with disease activity, and/or decrease upon effective treatment at the

5 336 M. C. TURINA ET AL. group level, indicating that at least from a pathophysiological point of view these are interesting markers. In spite of this, though, the value of these markers as biomarker in clinical practice (at the individual patient level) seems if investigated modest at best. In psoriasis, serum CRP and ESR were the only serum biomarkers used in clinical practice for the evaluation of treatment, despite the disappointing results mentioned earlier. The most interesting serum and plasma biomarkers in psoriasis from a pathophysiological point of view are the antimicrobial peptides hbd-2 and LCN- 2: the expression of both peptides is driven by IL-17 and may therefore reflect a key pathophysiological process in psoriasis skin, although the role in clinical practice remains inconclusive. 3. Serum biomarkers in IBD IBD includes Crohn s disease (CD) and ulcerative colitis (UC). Peripheral arthritis and sacroiliitis are seen in 7 16% [ ] and 2 32% [100,102,103] of the IBD patients, respectively. Similarly, 60 70% of the SpA patients show subclinical signs of gut inflammation on biopsies obtained by ileocolonscopy [104,105], but only 7% of these patients will eventually develop clinically overt IBD over time. The link between SpA and IBD is corroborated by genetic studies, with the best example being polymorphisms in the interleukin 23 receptor (IL23 R) gene which are associated both with IBD (OR 0.38 and 0.73 for CD and UC, respectively) and AS (OR = ) [ ]. The diagnosis of IBD in clinical practice is based on the combination of clinical history, physical examination, stool findings (i.e., calprotectin and lactoferrin), serological antibodies, inflammatory markers in blood, and colonoscopy [110]. The major clinical outcome measure in CD, the Crohn s Disease Activity Index (CDAI) [111], includes clinical and laboratory findings to determine disease activity. In UC, several scores are used to determine disease activity including clinical and endoscopic scores. A frequently used clinical score is Simple Clinical Colitis Activity Index (SCCAI) [112]. The Mayo activity index is based on clinical as well as endoscopic findings [113]. Of the acute phase reactants, serum CRP was found to be a biomarker for IBD since the serum levels were elevated in CD and to a lesser extent in UC when compared with healthy controls or functional bowel disorders [114,115]. Elevated serum levels were correlated with a higher disease activity [ ]. Louis et al. [118] studied a group of 226 CD patients receiving TNF-inhibitors and a clinical response was associated with a decrease in CRP with similar results in a larger cohort. There were discrepant data about the ability of serum CRP to predict disease relapse [119,120]. Other well studied inflammatory markers were ESR, serum procalcitonin, and serum YKL-40. All three markers correlated with the disease activity but only ESR and serum procalcitonin differentiated between IBD and healthy individuals or other gastrointestinal diseases [ ]. Moreover, a trend toward elevated serum levels of YKL-40 was found in more severe CD patients having strictures (i.e., narrowing of the intestinal lumina) [124], but since the majority of active patients do not have elevated levels, in clinical practice serum levels of YKL-40 is clinically of little value. Cytokines measured in serum did not seem to be important markers of disease in IBD, despite their role in the inflammatory process [129,130]. The only potential marker is serum IL-6, the main driver of CRP. Levels of IL-6 in serum were increased in active CD compared with healthy controls but correlation with disease activity was weak [ ]. Of the soluble adhesion molecules, serum ICAM-1 was the most promising serum and plasma marker as the levels were elevated when compared with healthy controls. Although discrepant data are available on correlation with disease activity, effective treatment down-regulates ICAM-1 levels [ ]. E-selectin levels in serum or plasma were significantly higher in IBD than in healthy controls. However, there was no significant association between disease activity and treatment [ ]. A few studies were published on vascular cell adhesion molecule (VCAM). The serum or plasma levels in IBD were not consistently elevated versus healthy controls and correlations to disease activity were poor [ ]. The adipokines adiponectin, leptin, ghrelin, resistin, and vaspin had been investigated thoroughly in IBD. Only serum levels of resistin and vaspin were consistently elevated in IBD versus healthy individuals. Moreover, strong correlations with PASI were found for ghrelin (in serum and plasma), resistin (in serum and plasma), and vaspin levels with disease activity [ ]. Serum and plasma ghrelin and resistin levels were decreased upon effective treatment but this was not studied for the other adipokines [148,149]. Of the S100 proteins, S100A12 levels in serum were elevated in IBD when compared with healthy controls, but there are conflicting data about the correlation with active disease activity [150,151]. Serum levels of S100A8/S100A9, calprotectin, were elevated in IBD versus healthy individuals andcorrelatedwiththecdai[ ]. Similar results were seen in children using pediatric CDAI indices [155]. Furthermore, calprotectin serum levels decreased upon treatment with TNF inhibition [154,156]. Fecal calprotectin is one of the most extensively studied stool markers in IBD. Calprotectin levels in stool can differentiate between IBD and healthy controls or irritable bowel syndrome (IBS). One meta-analysis of Von Roon et al. [157] included 30 studies with 1210 IBD patients, 697 colorectal cancer, 697 IBS, and 3393 healthy controls and reported that fecal calprotectin is a useful biomarker to differentiate between IBD and non- IBD patients. The overall weighted area under curve (AUC) was with (using a cut-off level of 50 ug/g and 100 ug/g, respectively) exceptionally high. Moreover, differentiating CD from IBS by fecal calprotectin showed an AUC of Van Rheenen et al. [158] determined that fecal calprotectin could be used as screening tool in suspected clinical IBD, thereby decreasing the use of invasive colonoscopyby67%,butatthecostoffalse-negativeresultsin6% of the cases. Results of a meta-analysis that included 13 studies (1471 IBD patients) showed that fecal calprotectin levels are also good marker for disease activity based on the Mayo activity index [159]. Moreover, fecal calprotectin

6 EXPERT REVIEW OF CLINICAL IMMUNOLOGY 337 decreased upon treatment [ ] and may predict disease relapse [120,164,165]. Several AMPs have been investigated in IBD. Serum LCN-2 (NGAL) levels were elevated in IBD when compared with healthy controls and IBS [ ]. HBD-2 seemed a potential marker since expression levels were increased in inflamed mucosa versus non-inflamed mucosa of the colon [169]. However, Yamaguchi et al. found no differences in hbd-2 plasma levels in UC or CD when compared to healthy controls [170]. Beta-2-microglobulin (β2-m), which forms a part of the major histocompatibility complex class 1 (MHC 1), had been investigated in two independent studies. In 2001, Zissis et al. [171] showed that serum levels were elevated in CD versus healthy controls, and that serum levels were higher when patients had a higher disease activity, but they did not see similar results for UC. Yilmaz et al. [172] recently showed similar in serum levels but now in the whole IBD population. Of the serological antibodies studied in IBD [ ], anti-saccharomyces cerevisiae antibodies (ASCA) and perinuclear anti-neutrophil cytoplasmic antibodies (panca) showed the highest diagnostic accuracy. Sensitivity and specificity for ASCA were 60% and 91% and for panca 50% and 95%, respectively, when distinguishing IBD from healthy controls. Sensitivity and specificity for distinguishing between CD and UC were for ASCA (CD): 60% and 86%, and for panca (UC): 50% and 94%, respectively [178]. Similar results were seen in other studies [179,180]. Also, when discriminating CD from UC or when comparing with other gastrointestinal diseases, including celiac disease, the specificity was too low for these antibodies [ ]. Both autoantibodies had a positive predictive value of 80 90% and a negative predictive value of 50 80%. The number of false-positive and false-negative individuals was too high to recommend these autoantibodies for routine diagnostic procedures. Ester et al. [181] found no association of ASCA or panca with response to infliximab treatment, but ASCA positivity was associated with worse disease progression: a more severe disease type (from inflammation to structuring (or stenosis) and penetrating) disease in CD [182]. Recently, Bonneau et al. [183] published a systematic review in which anti-glycan, anti-gp2, and anti-gm-csf antibodies were also described as having low sensitivity but high specificity to CD when compared with UC but the contribution of these antibodies to diagnosis is not yet clear. To summarize, serum CRP and ESR, the S100 family proteins S100A12 and S100A8/A9 (calprotectin), the antimicrobial peptides LCN-2 and hbd-2 (in serum and plasma), the MHC class 1 molecule β2-m, and ASCA antibodies are of interest as biomarkers in IBD from a pathophysiological point of view. In clinical practice however, only a few of these markers are used, including serum CRP, ESR, and fecal calprotectin. Since serum levels of CRP correlate with disease activity and serum CRP levels normalize upon effective treatment, evaluation of therapy by CRP in serum is often applied in general practice. Fecal calprotectin, but not serum calprotectin, is extensively used in clinical practice to identify IBD patients with intestinal inflammation, providing high sensitivity and specificity in detecting IBD. Nevertheless, the ileocolonoscopy remains the gold standard. With regard to antibodies, ASCA antibodies are able to differentiate between CD and UC and are used in clinical practice although not as a screening diagnostic procedure. Other markers in IBD that are of pathophysiological interest are the IL-17 driven LCN-2, hbd-2, and β2-m, that forms part of the MHC class-i molecule (Table 2). Table 2. Serum biomarkers in IBD levels: elevation in comparison with healthy controls, correlation with disease activity according to CDAI, SCCAI, or endoscopic activity, modulation by effective treatment, and outcome of structural damage. Biomarker Elevation In comparison with healthy controls Correlation with disease activity measured by CDAI or SCCAI or endoscopic activity Treatment response Outcome structural damage References CRP [ ] ESR + + NA NA [114,128] Procalcitonin + +/ NA NA [ ] YKL-40 +/ + NA +/ [ ] IL-6 + +/ NA NA [ ,156] ICAM-1 +/ + NA [ ] E-selectin + NA [ ] VCAM +/ NA [ ] Adiponectin + NA [ ,149,184] Leptin +/ +/ NA [145,146,148,149,184] Ghrelin* / [ ,146] Resistin* + +/ + NA [142,143,146,149] Vaspin +/ NA NA [144] S100A12 + +/ + NA [150,151] S100A8/A9 + NA NA + [ ] LCN NA NA [ ] hbd-2* NA NA [169,170] β2-m + + NA NA [171,172] ASCA [173,175,177,178,181,182] panca + + [ ,181,182] ASCA: anti-saccharomyces cerevisiae antibodies; β2-m: β2-microglobulin; CDAI: Crohn s disease activity index; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; hbd-2: human beta-defensin-2; HNP: human neutrophil peptides; IL: interleukin; LCN-2: lipocalin-2; NA: not applicable; panca: perinuclear antineutrophil cytoplasmic antibodies; SCCAI: simple clinical colitis activity index; VCAM: vascular cell adhesion molecule; VEGF: vascular endothelial growth factor. *Plasma and serum.

7 338 M. C. TURINA ET AL. 4. Lessons to be learned from serum biomarkers in psoriasis and IBD the potential role in SpA Several biomarkers studied in psoriasis and IBD may be informative in SpA as well. Serum CRP, ESR, calprotectin, and VEGF are interesting in psoriasis and IBD. These markers have already been studied in SpA (as mentioned above). While they may be discriminatory at the group level, sensitivity and/or specificity are too low to be used in clinical practice for diagnosis, disease activity measurement, evaluation of treatment response, or prediction of axial spinal progression. Some of the biomarkers of interest in psoriasis and IBD have also been studied in SpA. Negative or variable results were found for serum and plasmail-18 [185], ICAM-1 (serum and plasma) [ ], serum RBP-4 [189,190], and NO (serum and plasma) [191,192]. ASCA antibodies can help to discriminate CD from UC, have already been studied in SpA. The levels of ASCA antibodies were found to be higher in SpA patients than in healthy controls, as reported by different studies [193,194]. One study determined these antibodies in non-ibd SpA patients, and 30% of them appeared to be ASCA-positive. Nevertheless, levels did not correlate with disease activity [195]. The AMPs are a new family of relevant biomarkers in psoriasis and IBD as was found in several studies. AMPs have not yet been extensively studied in SpA. The most interesting members of this family are LCN-2 and hbd-2, both of which are driven by IL-17 and are reflecting active disease in psoriasis as well as IBD. And because the reliable measurement of IL-17 in serum remains a challenge, these biomarkers may be used as a proxy for IL-17-mediated tissue inflammation in psoriasis and IBD. As SpA is also an IL-17-driven disease [ ], these markers certainly deserve further study in this condition in a clinical setting. It should be noted, however, that these AMPs are mainly produced by epithelial cells and may thus be more useful in skin and gut diseases than in other IL-17- related inflammatory conditions. Along the same line, IL-22 is an IL-10 cytokine family member which is produced by Th17 cells under the control of IL-23. It was shown that plasma IL-22 levels were increased in SpA versus healthy individuals, but no correlation was found with Bath ankylosing spondylitis disease activity index (BASDAI) [201]. The reliability of serum IL-22 measurement and the validity of these findings require replication in independent studies. Another biomarker which was identified in IBD and may be directly related to the pathological processes in SpA is β2-m. β2-m is a component of class I MHC molecules, including HLA-B27, which is (still) the major genetic risk factor for SpA. Although the exact role of HLA-B27 in the disease pathogenesis is not yet clear, several hypotheses suggest that the level of expression of β2-m may modulate the pathological role of HLA-B27. One hypothesis proposes that β2-m-free HLA-B27 heavy chain forms disulfidelinked homodimers which can be recognized by the KIR3DL2 receptors on NK-cells and T-cells [202]. The second hypothesis concerns HLA-B27 heavy chain misfolding prior to binding of β2- M and peptide, resulting in unfolded protein responses (UPR), which subsequently may lead to an inflammatory cytokine release [200,203]. Interestingly, modulation of β2-m expression levels in the HLA-B27 transgenic rat model of SpA did profoundly affect the phenotype of the disease [ ], confirming the potential importance of this molecule in SpA. Therefore, serum β2-m expression levels should be further explored as potential biomarker in SpA. 5. Expert commentary Major efforts have been performed in the search for new biomarkers, since biomarkers that are acquired from blood, are the most easily accessible, cheap, and noninvasive manner to answer several issues regarding early diagnosis, association to disease activity, prediction of treatment effect, and prediction of clinical outcome. Nevertheless, the classical search approach has not successfully led to robust serum biomarkers. Therefore we have chosen a new and risky approach to search for new markers. In this review, we determined which serum biomarkers were described in psoriasis and IBD, with the ultimate goal to find identify novel and yet unexplored biomarkers for use in SpA. To clarify, it was not the goal to list the known biomarkers in SpA. The three diseases are related to each other at genetic, clinical, and pathophysiological levels. Although psoriasis and/or IBD are only found in a fraction of patients with SpA and therefore the value in the biomarker exploration in SpA as a whole entity could be questioned, psoriasis and IBD are also found in up to 10% each of patients with AS [207]. This reflects the concept that the old phenotypic classification of SpA in AS, PsA, IBD-related SpA, undifferentiated SpA and reactive SpA, does probably not reflect pathogenetic distinct entities [208]. Accordingly, it is fair to explore if biomarkers in psoriasis or IBD may also be relevant for SpA. Ideally, these biomarkers would not be restricted to the subtypes of PsA and IBD-associated SpA, but would be more globally applicable to SpA as a whole entity as this would genuinely reflect the pathophysiology of SpA. Here we can conclude that serum hbd-2, LCN-2, and beta2- microglobulin are relevant in psoriasis and/or IBD from a pathophysiological point of view, and may also play a role in the pathogenesis of SpA. Since we do not know if these markers are of value in clinical practice, we strongly recommend to further explore the role of these biomarkers in clinical practice in SpA. Serum LCN-2 and especially HBD-2 (serum and plasma) levels are the best by proxy measures for IL-17 levels since we are not able to measure serum IL-17 levels in an accurate manner: although some reports mentioned elevated IL-17 levels in serum of psoriasis, IBD, and SpA patients [209,210], these data remain controversial as most laboratories were unable to reproduce reliable measurement of IL-17 family cytokines in serum (which is also true for IL-23) [211]. These by proxy markers might be of interest since the IL-23/IL-17 is the most important pathogenic pathway in SpA. Nevertheless, these two markers are expressed in epithelial cells and therefore might not be of interest in SpA from a pathological standpoint. β2-microglobulin is part of the HLA-B27 molecule. Since this molecule is the major genetic risk factor in SpA, it is potentially interesting to determine its biomarker value in clinical care.

8 EXPERT REVIEW OF CLINICAL IMMUNOLOGY 339 To test these three potential biomarkers, we propose to first screen their usefulness in SpA by determining whether there are any differences in serum levels between full blown SpA and healthy controls. The total costs for determining the levels of the three potential biomarkers would be 29,61 per sample (performed in duplicate). This is a relatively low price since we will be able to determine difference already in a small group size of 20 patients versus 20 controls. If one or more of the potential biomarkers do show any significant differences, we recommend to validate the results in a larger group of patients and in different settings (i.e., early SpA cohort, disease activity, treatment response, and clinical outcome). For the upcoming years, we expect the field to go into 2 different ways: (1) the field will continue to search for serum biomarkers, partly based on this review; and (2) the field will shift to finding biomarkers in a more specific tissue compartment rather than in the systemic circulation. Molecular imaging diagnostics able to reflect the target tissue compartments is an upcoming hot topic in SpA. 6. Five-year view In SpA, inflammation might be restricted to specific tissue compartments and does not extend to the systemic circulation and/or lymphoid organs. Therefore, we expect that novel serum biomarkers could be found in molecules directly related to the local tissue immunopathology of SpA, rather than molecules reflecting systemic inflammation, including serum CRP and ESR. These biomarkers could predict early diagnosis, treatment response, and/or outcome of the disease. This hypothesis, however, requires that molecules contributing to the local tissue pathology in SpA diffuse to the systemic circulation and can be measured in serum or plasma. Based on our own research, indicating that pathogenic signatures found in SpA synovitis are not reflected in peripheral blood, one could also propose that no significant serum biomarkers will be found in SpA. If this statement is indeed correct, the search for biomarkers should rather focus on non-invasive measurements of tissue pathology (including MRI and PET-CT) instead of on serum biomarkers. Furthermore, the primary focus of this review was to find potential (serum and plasma) biomarkers in SpA. We did not evaluate the technologies to assess these biomarkers. Nevertheless, the different emerging multiplex technologies now allow to measure multiple proteins in small volumes and may thus open new avenues. Key issues Spondyloarthritis is an immune-mediated inflammatory disease affecting the spine, the peripheral joints and extraarticular tissues such as the skin, the gut, and the eye. Serum biomarkers could address several challenges in spondyloarthritis (diagnosis, disease activity monitoring, treatment response, and outcome). Serum biomarkers well studied in spondyloarthritis are CRP, ESR, IL-6, calprotectin, VEGF, MMP-3, DKK-1, sclerostin, and a few biomarkers. Although the aforementioned biomarkers are useful at the group level in spondyloarthritis, these markers lack specificity for the use in individual patients in clinical care. Spondyloarthritis is a clinically and pathophysiologically closely related to psoriasis and inflammatory bowel disease. In psoriasis, CRP, ESR, IL-18, IL-22, ICAM-1, calprotectin, LCN- 2, hbd-2, VEGF, RBP-4, and NO were the most interesting markers from a pathophysiological standpoint. In IBD, CRP and ESR, LCN-2, hbd-2, and ASCA antibodies were of interest from a pathophysiological point of view. HBD-2, LCN-2, and β2-microglobulin are the most interesting candidates from a pathophysiological standpoint to be further explored in SpA for clinical purposes. Acknowledgments We would like to thank KL Germar for proof reading the article. Funding MC Turina was supported by an unrestricted fellowship from Janssen. DL Baeten was supported by a VICI grant from The Netherlands Organization for Scientific Research (NWO), by a grant from the Dutch Arthritis Foundation (Reumafonds), and by a grant of the European Research Council (ERC). Declaration of interest R Landewé is a member of the OMERACT biomarker working group. All authors were involved in drafting the article and approved the final version for publication. 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