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Plasma soluble triggering receptor expressed on myeloid cells-1 in Crohn's disease
Digestive and Liver Disease 44 (2012) 466–470
Contents lists available at SciVerse ScienceDirect
Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Alimentary Tract
Plasma soluble triggering receptor expressed on myeloid cells-1 in Crohn’s disease Vincent Billioud a,b , Sébastien Gibot c,d , Frédéric Massin e , Abderrahim Oussalah a,b , Jean-Baptiste Chevaux a,b , Nicolas Williet a,b , Jean-Pierre Bronowicki a,b , Marc-André Bigard a,b , Jean-Louis Guéant a,b , Laurent Peyrin-Biroulet a,b,∗ a
INSERM U954, Université Henri Poincaré 1, Vandoeuvre-lès-Nancy, France Department of Hepato-Gastroenterology, University Hospital of Nancy, Vandoeuvre-lès-Nancy, France c Groupe Choc, Contrat AVENIR INSERM, Université Henri Poincaré 1, Nancy, France d Department of Intensive Care Medicine, University Hospital of Nancy, Université Henri Poincaré 1, Nancy, France e Immunology Laboratory, Medical Faculty, Université Henri Poincaré 1, Vandoeuvre-lès-Nancy, France b
a r t i c l e
i n f o
Article history: Received 22 October 2011 Accepted 11 January 2012 Available online 16 February 2012 Keywords: Crohn’s disease Disease activity Triggering receptor expressed on myeloid cells-1
a b s t r a c t Background: No definite conclusions can be drawn from available data on the accuracy of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) to assess disease activity in Crohn’s disease. Aims: Plasma sTREM-1 levels were correlated with disease activity markers in Crohn’s disease. Methods: 191 consecutive patients from a single referral centre (Nancy IBD cohort) were prospectively enrolled between June 1, 2005 and December 12, 2008. Plasma sTREM-1 levels were also assessed amongst 20 healthy controls. Results: The sTREM-1 was detectable in 87 Crohn’s disease patients (46%). Plasma sTREM-1 level was higher in Crohn’s disease patients (interquartile range, 0–356) than in healthy controls (interquartile range, 0–15.1; P = 0.01). It was neither correlated with Crohn’s disease activity index (r = 0.05, P = 0.56), Creactive protein (r = 0.06, P = 0.53), nor with albumin (r = −0.041, P = 0.66). Crohn’s disease activity index, C-reactive protein and albumin median levels were similar between patients with positive sTREM-1 levels and those with undetectable sTREM-1 levels. Azathioprine (P = 0.06), infliximab (P = 0.68) and methotrexate (P = 0.56) did not influence sTREM-1 levels. Conclusion: Plasma sTREM-1 does not appear to be an accurate marker of disease activity in Crohn’s disease and cannot be recommended for assessing disease activity in these patients. © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Measurement of disease activity in patients suffering from Crohn’s disease (CD) is critical in determining therapy efficacy and guide therapeutic strategies [1]. Biological markers, such as C-reactive protein (CRP) and faecal markers provide rapid and noninvasive possibilities for the evaluation of disease activity in CD [2]. However, such laboratory markers present several limitations, such as the assessment of inflammation in ileal CD and difficulties in defining an optimal cut point [3,4]. In 2011, CRP is the only validated biological marker of CD activity [5]. To improve medical management of CD, a tight control monitoring of disease activity
∗ Corresponding author at: Department of Hepato-Gastroenterology, University Hospital of Nancy-Brabois, Université Henri Poincaré 1, Allée du Morvan, 54511 Vandoeuvre-lès-Nancy, France. Tel.: +33 3 83 15 36 31; fax: +33 3 83 15 36 33. E-mail address: [email protected] (L. Peyrin-Biroulet).
is needed [6]. In this regard, the development of new biological markers for CD activity is keenly awaited [7]. Triggering receptor expressed on myeloid cells-1 (TREM-1) belongs to the immunoglobulin super family and has been identified on both human and murine polymorphonuclear cells and mature monocytes [8]. It contains an ectodomain, a short intracellular part and a transmembrane domain [8]. Pairing with a transmembrane adapter protein, it initiates a cascade of phosphorylation events that ultimately lead to cell activation and production of pro-inflammatory chemokines and cytokines [8]. TREM-1 function is to modulate rather than to activate/initiate inflammation. TREM-1 cooperates with nucleotide-binding and oligomerization domain (Nod)-like receptors or Toll-like receptors in a synergistic way to trigger an exuberant immune response [8]. The role of TREM-1 in amplifying the inflammatory response is now well understood in acute inflammation such as during sepsis [9]. The soluble form of TREM-1 (sTREM-1) may be useful as a diagnostic tool to predict infectious pneumonia and sepsis [9,10].
1590-8658/$36.00 © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2012.01.005
V. Billioud et al. / Digestive and Liver Disease 44 (2012) 466–470
More recently, TREM-1 has also been studied in chronic inflammatory disorders such as chronic rheumatoid diseases [11,12]. Innate immune response is considered to play a key role in pathogenesis of inflammatory bowel disease (IBD) [13]. To our knowledge, only four studies have evaluated TREM-1 in patients with IBD and intestinal Behcet’s disease (BD) [14–17]. sTREM-1 levels may be increased in IBD patients and correlated with disease activity [14–17]. However, no definitive conclusions could be drawn due to small sample size. The aim of this prospective observational study was therefore to evaluate whether levels of plasma sTREM-1 correlated with disease activity markers in a large CD population. 2. Methods 2.1. Study population Between June 1, 2005 and December 12, 2008, consecutive patients seen in the outpatient clinics or in hospitalization at the University Hospital of Nancy were included in the study if they met the following inclusion criteria: adult age (≥18 years), established diagnosis of CD. Exclusion criteria for this study were coexistence of infectious or ischaemic colitis, coexistence of other localized or systemic infection. The plasma from 20 healthy volunteers was also collected in order to assess plasma sTREM-1 levels. All patients and healthy volunteers signed a consent form permitting blood samples to be used for scientific purposes. Information about the Nancy IBD cohort is reported to the Commission Nationale de l’Informatique et des Libertés (no. 1404720), which supervises the implementation of the act regarding data processing, data files and individual liberties that came into effect on January 6, 1978, and was amended on August 6, 2004, to protect the personal data of individuals. 2.2. Outcome measures 2.2.1. Assessment of clinical data, CRP and albumin The activity of CD was defined according to Crohn’s disease activity index (CDAI) as described previously [18]. CDAI was collected prospectively by a senior gastroenterologist (L.P.B.). Serum CRP and albumin levels were routinely measured at the time of inclusion. Demographic and clinical data (Montreal classification, concomitant medication, previous surgery) were recorded using the information of the Nancy IBD cohort [19]. All these data were collected through patient’s electronic charts review.
467
Software, Mariakerke, Belgium). The diagnostic accuracy of plasma sTREM-1 (pg/mL) and CRP (mg/L) for detecting active and severe forms of CD was evaluated using receiver operating characteristic (ROC) analysis according to DeLong et al. [20] using ‘CDAI > 220’ and ‘CDAI > 300’ as classification variables. 3. Results 3.1. Baseline characteristics of the 191 patients Between June 1, 2005 and December 12, 2008, a total of 191 patients could be included in the study. The baseline characteristics of the patients are shown in Table 1. The median age at inclusion was 30 years (IQR, 23.3–41.8) and the median disease duration was 37 months (IQR, 8–106.8). Most of patients were women (64.4%). According to the Montreal classification, more than half of patients presented an ileocolonic involvement (57.1%). Few patients (13.1%) suffered from a penetrating disease whilst nearly one third of them had a stricturing phenotype (38.5%). Most of patients were treated with immunomodulators at study inclusion as about two third of them received azathioprine (64.2%) and/or infliximab (68.4%). The median CDAI was 122 (IQR, 54.8–202) and the median CRP level was 13.3 mg/L (IQR, 6–32.3). Albumin serum level was normal in most of patients, with a median of 39.2 g/L (IQR, 35.6–43.3). The median plasma sTREM-1 level was 0 pg/mL (IQR, 0–318.5). The sTREM-1 was detectable in 87 CD patients (46.5%). 3.2. Comparison of sTREM-1 levels between CD patients and controls Plasma sTREM-1 level was significantly higher in the CD group than in controls (372 pg/mL versus 11 pg/mL, respectively; P = 0.01) (Fig. 1). 3.3. Correlations between plasma sTREM-1, CDAI, CRP and albumin As expected, CRP level was significantly correlated with the CDAI (r = 0.33, P = 0.01) (Fig. 2). None of the disease activity markers were correlated with the sTREM-1 levels. sTREM-1 level was not correlated with the CDAI (r = 0.05, P = 0.56) (Fig. 3). Regarding biological markers of activity, sTREM-1 level was neither correlated with CRP
2.3. Assessment of sTREM-1 Plasma concentrations of sTREM-1 were determined by ELISA (Quantikine, RnD Systems, MN, USA) according to the manufacturer’s recommendations. The detection level was 15 pg/mL. Measurements were performed in duplicate. Inter and intra-assay coefficients of variation were <7%. 2.4. Statistical analysis All quantitative variables are described as medians and percentiles [interquartile range (IQR), 25–75th percentile]. All proportions are expressed as percentages with 95% confidence intervals (95% CIs). Correlations were studied using the Spearman’s rank-order correlation coefficient. Proportions were compared using the chi-square test or the Fisher’s exact test, as appropriate. The comparison of plasma concentrations of sTREM-1 (pg/L) between subgroups was performed using the Mann–Whitney U test. All the reported P-values were two-sided, and P-values of <0.05 were considered statistically significant. Statistical analyses were performed using MedCalc Software, version 11.6.1.0 (MedCalc
Fig. 1. Comparison of plasma sTREM-1 levels between Crohn’s disease patients and healthy controls.
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V. Billioud et al. / Digestive and Liver Disease 44 (2012) 466–470
Table 1 Baseline characteristics of the 191 patients. Variable
N
Gender (male) Familial history of IBD Montreal classification B1 (non-stricturing, non-penetrating) B2 (stricturing) B3 (penetrating) L1 (isolated ileal disease) L2 (isolated colonic disease) L3 (ileocolonic disease) L4 (isolated upper disease) P (perianal disease modifier) Concomitant medication Azathioprine Methotrexate Infliximab Infliximab with immunosuppressant (azathioprine or methotrexate) Previous surgery for CD Ileal resection Colectomy
191 34
n (%) 68 (35.6) 16 (47.1)
28.8–42.5 29.4–64.7
191 191 191 191 191 191 191 191
109 (57.1) 57 (29.8) 25 (13.1) 32 (16.8) 48 (25.1) 109 (57.1) 7 (3.7) 85 (44.5)
50–64.2 23.3–36.4 8.3–17.9 11.4–22.1 18.9–31.3 50–64.2 1–6.4 37.4–51.6
179 175 177 175
115 (64.2) 10 (5.7) 121 (68.4) 90 (51.4)
57.2–71.3 2.2–9.2 61.4–75.3 44–58.9
167 169
34 (20.4) 24 (14.2)
14.2–26.5 8.9–19.5
Variable
N
Median
IQR
Age at inclusion (years) Age at diagnosis (years) Disease duration (months) CDAI Albumin (g/L) CRP (mg/L) sTREM-1 (pg/mL)
191 191 191 121 117 93 191
30 22 37 122 39.2 13.3 0
23.3–41.8 18–31 8 to106.8 54.8–202 35.6–43.3 6–32.3 0–318.5
95% CI (%)
Note. N, number of subjects; CI, confidence interval; IQR, interquartile range: 25–75th; IBD, inflammatory bowel disease; CD, Crohn’s disease; CDAI, Crohn’s disease activity index; CRP, C-reactive protein; sTREM, soluble triggering receptor expressed on myeloid cells.
(r = 0.06, P = 0.53) nor with albumin (r = −0.041, P = 0.66) (Fig. 4 and Supplementary Fig. 1). 3.4. Comparison of patients according to the positivity of plasma sTREM-1 (Supplementary Table 1) The study population was divided into two groups according to the positivity or negativity of sTREM-1. Eighty seven patients (45.6%) presented a positive sTREM level. The same proportion of patients were treated with azathioprine (P = 0.06), infliximab (P = 0.72) or methotrexate (P = 0.99) in the two groups. There was no difference regarding disease phenotype and history of intestinal surgery according to the positivity of sTREM-1 level. The median
Fig. 2. Correlation between plasma Crohn’s disease activity index (CDAI) and Creactive protein (CRP) levels.
CDAI was 132 in patients with a positive sTREM-1 level compared to 117 in patients with undetectable sTREM-1 levels (P = 0.66). The median CRP level was 15.8 mg/L versus 11.1 mg/L (P = 0.11) and the median albumin level was 38.7 g/L versus 39.6 g/L (P = 0.54) in patients with positive or undetectable sTREM-1 levels, respectively. 3.5. Influence of medications, disease behaviour and previous intestinal surgery on plasma sTREM-1 levels and correlations with CDAI The median sTREM-1 level did not differ significantly according to disease behaviour or disease location (Table 2). Regarding
Fig. 3. Correlation between plasma soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) levels and Crohn’s disease activity index (CDAI).
V. Billioud et al. / Digestive and Liver Disease 44 (2012) 466–470
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Table 2 Comparison of serum sTREM-1 levels according to Montreal classification, concomitant treatment, and previous surgery. Item
Montreal classification B1 (non-stricturing, non-penetrating) B2 (stricturing) B3 (penetrating) L1 (isolated ileal disease) L2 (isolated colonic disease) L3 (ileocolonic disease) L4 (isolated upper disease) P (perianal disease modifier) Concomitant medication Azathioprine Methotrexate Infliximab Previous surgery for CD Ileal resection Colectomy
No
Yes
N
Median sTREM-1
IQR
82 134 166 159 143 82 184 106
0 0 0 0 0 0 0 0
0–217 0–392 0–330 0–340.5 0–371 0–230 0–313 0–290
64 165 56
0 0 0
0–174 0–307.5 0–293.5
133 145
0 0
0–356 0–353
N
P-value Median sTREM-1
IQR
109 57 25 32 48 109 7 85
0 0 0 0 0 0 0 0
0–425.5 0–129 0–250.3 0–175 0–236.5 0–396.3 0–421.3 0–364.8
0.25 0.21 0.99 0.69 0.65 0.57 0.87 0.71
115 10 121
7.4 38.5 0
0–412.8 0–1427 0–333.5
0.06 0.56 0.68
0–101 0–47.5
0.07 0.06
34 24
0 0
Note. N, number of subjects; IQR, interquartile range: 25–75th; sTREM, soluble triggering receptor expressed on myeloid cells; CD, Crohn’s disease.
previous intestinal surgery for CD, neither a history of ileal resection (P = 0.07) nor colectomy (P = 0.06) modified the median sTREM-1 level. The median sTREM-1 levels were also similar in patients who received azathioprine (P = 0.06), infliximab (P = 0.68) or methotrexate (P = 0.56) compared with those who were not receiving such therapy at study inclusion (Table 2). Correlations between sTREM-1 and CDAI were studied according to the type of ongoing immunosuppressive therapy. Plasma sTREM-1 level was not correlated with CDAI amongst patients receiving or not infliximab or immunosuppressants (Supplementary Table 2). Correlations were also performed according to disease behaviour (B1, B2, and B3 according to Montreal classification). Plasma sTREM-1 level was not correlated with CDAI amongst patients with a non-stricturing/non-penetrating (B1), a structuring (B2) or a penetrating disease (B3) (Supplementary Table 3). 3.6. Diagnostic accuracy of plasma sTREM-1 level and CRP for assessing disease activity Using ROC analysis, CRP had a significant diagnostic accuracy for diagnosing moderate to severe CD (CDAI > 220), with an AUROC of 0.68 (standard error (SE) = 0.08, P = 0.03), whereas plasma sTREM-1
Fig. 4. Correlation between plasma soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and C-reactive protein (CRP) levels.
showed an AUROC of 0.52 that did not reach statistical significance (SE = 0.06, P = 0.76) (Supplementary Table 4). Similarly, CRP had a significant diagnostic accuracy for diagnosing severely active CD (CDAI > 300), with an AUROC of 0.78 (SE = 0.09, P = 0.002), whereas plasma sTREM-1 showed an AUROC of 0.56 that did not reach statistical significance (SE = 0.09, P = 0.53) (Supplementary Table 4). 4. Discussion This is the largest study investigating the potential contribution of sTREM-1 measurement in assessing disease activity in CD patients. The role of TREM-1 in the pathogenesis of IBD has first been assessed in animals [14]. Schenk et al. nicely demonstrated that antagonistic TREM-1-derived peptide decreased inflammation and TNF mRNA production in experimental colitis in mice [14]. This elegant study provides promising data from 18 patients with active IBD who revealed an approximately 5-fold increase in the number of TREM-1-expressing macrophages on intestinal biopsies when compared with normal control tissues [14]. TREM-1 expression in intestinal mucosa seemed to be related to disease activity as TREM1 mRNA was expressed at high levels in affected segments whereas it was substantially reduced in tissue sample with inactive disease [14]. Overall, few studies have evaluated the relevance of measuring TREM-1 levels in patients suffering from IBD [14–17]. Tzivras et al. found that serum sTREM-1 levels were higher amongst 39 IBD patients compared with controls [16]. They also showed significantly higher sTREM-1 level in ulcerative colitis (UC) patients with a greater clinical or endoscopic activity index and in CD patients with a CDAI > 400 compared to patients in remission [16]. These results have to be interpreted with caution as only 8 CD patients were included in this study [16]. Consistent results were found amongst 88 patients suffering from intestinal BD [17]. Serum sTREM-1 level was more tightly correlated with disease activity than was the erythrocyte sedimentation rate (ESR) or CRP levels, suggesting the usefulness of sTREM-1 as a potential marker of intestinal BD disease activity [17]. In the present study, we found a significant correlation between CRP and CDAI and higher sTREM-1 levels in CD patients compared to healthy controls, showing that our study population was appropriate to investigate a possible correlation between CDAI and sTREM-1 levels from a statistical point of view. Importantly, we did not find any correlation between sTREM-1 levels and the clinical
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V. Billioud et al. / Digestive and Liver Disease 44 (2012) 466–470
activity index or biological activity markers in CD patients. Taking sTREM-1 as a dichotomous variable (positive or negative) led to similar findings. Interestingly, our results are in line with a previous study by Park et al. including 53 IBD patients [15]. Amongst the 31 UC patients, sTREM-1 was more accurately correlated with disease activity than was ESR or CRP. However, the authors failed to show a significant correlation of sTREM-1 level with disease activity in the 22 CD patients, even though they presented significantly increased sTREM-1 levels than healthy controls [15]. This data indicates that sTREM-1 could be useful in evaluating disease activity in UC, but not in CD. This will require further investigation. Recently, a genetic study showed that three TREM-1 single nucleotide polymorphisms were associated with BD, but not with UC and CD [21]. It has been shown that engagement of TREM-1 in combination with Toll-like receptor activation synergistically increased the production of proinflammatory cytokines including TNF-␣ [22]. Importantly, our study allowed inclusion of patients treated with immunomodulators, which was usually considered as an exclusion criterion in previous observations [15–17]. We found similar sTREM-1 levels irrespective of the administration of immunomodulator and anti-TNF therapy. In addition, we failed to find a correlation between sTREM-1 and CDAI amongst patients with or without ongoing immunosuppressive therapy. Hence, such treatments could not be responsible for the absence of correlation between sTREM-1 and disease activity markers in our CD cohort. One of the main goals of non invasive disease activity evaluation is to enable iterative assessment of therapeutic efficacy and to predict relapse amongst CD treated patients. Therefore, biological markers should be validated and reliable in such a situation as already observed for CRP and faecal calprotectin [23,24]. There are some limitations inherent to our study. First, information on mucosal healing and biopsy samples were not available in this cohort of CD patients. In addition, no UC patients were included in this study. Finally, some clinical data were lacking due to the retrospective study design of our study. The strengths of the study are the large number of CD patients included (n = 191), the inclusion of consecutive CD patients, and the prospective assessment of disease activity markers. In conclusion, the present study failed to show a significant correlation between sTREM-1 levels and clinical or biological activity markers. Taken together our data indicate that plasma sTREM-1 cannot be recommended for assessing disease activity amongst CD patients. Conflict of interest statement The authors declare no conflicts of interest Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.dld.2012.01.005.
References [1] Dignass A, Van Assche G, Lindsay JO, et al. The second European evidencebased Consensus on the diagnosis and management of Crohn’s disease: current management. J Crohns Colitis 2010;4:28–62. [2] Vermeire S, Van Assche G, Rutgeerts P. Laboratory markers in IBD: useful, magic, or unnecessary toys. Gut 2006;55:426–31. [3] Franchimont D. C-reactive protein: informative or misleading marker of Crohn’s disease. Inflamm Bowel Dis 2007;13:501–2. [4] Lewis JD. The utility of biomarkers in the diagnosis and therapy of inflammatory bowel disease. Gastroenterology 2011;140:1817–26, e1812. [5] Van Assche G, Dignass A, Panes J, et al. The second European evidence-based Consensus on the diagnosis and management of Crohn’s disease: definitions and diagnosis. J Crohns Colitis 2010;4:7–27. [6] Bruining DH, Sandborn WJ. Do not assume symptoms indicate failure of antitumor necrosis factor therapy in Crohn’s disease. Clin Gastroenterol Hepatol 2011;9:395–9. [7] Li X, Conklin L, Alex P. New serological biomarkers of inflammatory bowel disease. World J Gastroenterol 2008;14:5115–24. [8] Klesney-Tait J, Turnbull IR, Colonna M. The TREM receptor family and signal integration. Nat Immunol 2006;7:1266–73. [9] Derive M, Massin F, Gibot S. Triggering receptor expressed on myeloid cells1 as a new therapeutic target during inflammatory diseases. Self Nonself 2010;1:225–30. [10] Gibot S, Cravoisy A, Levy B, et al. Soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia. N Engl J Med 2004;350:451–8. [11] Collins CE, La DT, Yang HT, et al. Elevated synovial expression of triggering receptor expressed on myeloid cells 1 in patients with septic arthritis or rheumatoid arthritis. Ann Rheum Dis 2009;68:1768–74. [12] Kuai J, Gregory B, Hill A, et al. TREM-1 expression is increased in the synovium of rheumatoid arthritis patients and induces the expression of pro-inflammatory cytokines. Rheumatology (Oxford) 2009;48:1352–8. [13] Abraham C, Medzhitov R. Interactions between the host innate immune system and microbes in inflammatory bowel disease. Gastroenterology 2011;140:1729–37. [14] Schenk M, Bouchon A, Seibold F, Mueller C. TREM-1-expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases. J Clin Invest 2007;117:3097–106. [15] Park JJ, Cheon JH, Kim BY, et al. Correlation of serum-soluble triggering receptor expressed on myeloid cells-1 with clinical disease activity in inflammatory bowel disease. Dig Dis Sci 2009;54:1525–31. [16] Tzivras M, Koussoulas V, Giamarellos-Bourboulis EJ, et al. Role of soluble triggering receptor expressed on myeloid cells in inflammatory bowel disease. World J Gastroenterol 2006;12:3416–9. [17] Jung YS, Kim SW, Yoon JY, et al. Expression of a soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) correlates with clinical disease activity in intestinal Behcet’s disease. Inflamm Bowel Dis 2011;17:2130–7. [18] Best WR, Becktel JM, Singleton JW, Kern Jr F. Development of a Crohn’s disease activity index. National Cooperative Crohn’s Disease Study. Gastroenterology 1976;70:439–44. [19] Peyrin-Biroulet L, Oussalah A, Williet N, et al. Impact of azathioprine and tumour necrosis factor antagonists on the need for surgery in newly diagnosed Crohn’s disease. Gut 2011;60:930–6. [20] DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 1988;44:837–45. [21] Jung ES, Kim SW, Moon CM, et al. Relationships between genetic polymorphisms of triggering receptor expressed on myeloid cells-1 and inflammatory bowel diseases in the Korean population. Life Sci 2011;89:289–94. [22] Bleharski JR, Kiessler V, Buonsanti C, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J Immunol 2003;170:3812–8. [23] Jurgens M, Mahachie John JM, Cleynen I, et al. Levels of C-reactive protein are associated with response to infliximab therapy in patients with Crohn’s disease. Clin Gastroenterol Hepatol 2011;9:421–7, e421. [24] Sipponen T, Bjorkesten CG, Farkkila M, et al. Faecal calprotectin and lactoferrin are reliable surrogate markers of endoscopic response during Crohn’s disease treatment. Scand J Gastroenterol 2010;45:325–31.
Contents lists available at SciVerse ScienceDirect
Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Alimentary Tract
Plasma soluble triggering receptor expressed on myeloid cells-1 in Crohn’s disease Vincent Billioud a,b , Sébastien Gibot c,d , Frédéric Massin e , Abderrahim Oussalah a,b , Jean-Baptiste Chevaux a,b , Nicolas Williet a,b , Jean-Pierre Bronowicki a,b , Marc-André Bigard a,b , Jean-Louis Guéant a,b , Laurent Peyrin-Biroulet a,b,∗ a
INSERM U954, Université Henri Poincaré 1, Vandoeuvre-lès-Nancy, France Department of Hepato-Gastroenterology, University Hospital of Nancy, Vandoeuvre-lès-Nancy, France c Groupe Choc, Contrat AVENIR INSERM, Université Henri Poincaré 1, Nancy, France d Department of Intensive Care Medicine, University Hospital of Nancy, Université Henri Poincaré 1, Nancy, France e Immunology Laboratory, Medical Faculty, Université Henri Poincaré 1, Vandoeuvre-lès-Nancy, France b
a r t i c l e
i n f o
Article history: Received 22 October 2011 Accepted 11 January 2012 Available online 16 February 2012 Keywords: Crohn’s disease Disease activity Triggering receptor expressed on myeloid cells-1
a b s t r a c t Background: No definite conclusions can be drawn from available data on the accuracy of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) to assess disease activity in Crohn’s disease. Aims: Plasma sTREM-1 levels were correlated with disease activity markers in Crohn’s disease. Methods: 191 consecutive patients from a single referral centre (Nancy IBD cohort) were prospectively enrolled between June 1, 2005 and December 12, 2008. Plasma sTREM-1 levels were also assessed amongst 20 healthy controls. Results: The sTREM-1 was detectable in 87 Crohn’s disease patients (46%). Plasma sTREM-1 level was higher in Crohn’s disease patients (interquartile range, 0–356) than in healthy controls (interquartile range, 0–15.1; P = 0.01). It was neither correlated with Crohn’s disease activity index (r = 0.05, P = 0.56), Creactive protein (r = 0.06, P = 0.53), nor with albumin (r = −0.041, P = 0.66). Crohn’s disease activity index, C-reactive protein and albumin median levels were similar between patients with positive sTREM-1 levels and those with undetectable sTREM-1 levels. Azathioprine (P = 0.06), infliximab (P = 0.68) and methotrexate (P = 0.56) did not influence sTREM-1 levels. Conclusion: Plasma sTREM-1 does not appear to be an accurate marker of disease activity in Crohn’s disease and cannot be recommended for assessing disease activity in these patients. © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Measurement of disease activity in patients suffering from Crohn’s disease (CD) is critical in determining therapy efficacy and guide therapeutic strategies [1]. Biological markers, such as C-reactive protein (CRP) and faecal markers provide rapid and noninvasive possibilities for the evaluation of disease activity in CD [2]. However, such laboratory markers present several limitations, such as the assessment of inflammation in ileal CD and difficulties in defining an optimal cut point [3,4]. In 2011, CRP is the only validated biological marker of CD activity [5]. To improve medical management of CD, a tight control monitoring of disease activity
∗ Corresponding author at: Department of Hepato-Gastroenterology, University Hospital of Nancy-Brabois, Université Henri Poincaré 1, Allée du Morvan, 54511 Vandoeuvre-lès-Nancy, France. Tel.: +33 3 83 15 36 31; fax: +33 3 83 15 36 33. E-mail address: [email protected] (L. Peyrin-Biroulet).
is needed [6]. In this regard, the development of new biological markers for CD activity is keenly awaited [7]. Triggering receptor expressed on myeloid cells-1 (TREM-1) belongs to the immunoglobulin super family and has been identified on both human and murine polymorphonuclear cells and mature monocytes [8]. It contains an ectodomain, a short intracellular part and a transmembrane domain [8]. Pairing with a transmembrane adapter protein, it initiates a cascade of phosphorylation events that ultimately lead to cell activation and production of pro-inflammatory chemokines and cytokines [8]. TREM-1 function is to modulate rather than to activate/initiate inflammation. TREM-1 cooperates with nucleotide-binding and oligomerization domain (Nod)-like receptors or Toll-like receptors in a synergistic way to trigger an exuberant immune response [8]. The role of TREM-1 in amplifying the inflammatory response is now well understood in acute inflammation such as during sepsis [9]. The soluble form of TREM-1 (sTREM-1) may be useful as a diagnostic tool to predict infectious pneumonia and sepsis [9,10].
1590-8658/$36.00 © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2012.01.005
V. Billioud et al. / Digestive and Liver Disease 44 (2012) 466–470
More recently, TREM-1 has also been studied in chronic inflammatory disorders such as chronic rheumatoid diseases [11,12]. Innate immune response is considered to play a key role in pathogenesis of inflammatory bowel disease (IBD) [13]. To our knowledge, only four studies have evaluated TREM-1 in patients with IBD and intestinal Behcet’s disease (BD) [14–17]. sTREM-1 levels may be increased in IBD patients and correlated with disease activity [14–17]. However, no definitive conclusions could be drawn due to small sample size. The aim of this prospective observational study was therefore to evaluate whether levels of plasma sTREM-1 correlated with disease activity markers in a large CD population. 2. Methods 2.1. Study population Between June 1, 2005 and December 12, 2008, consecutive patients seen in the outpatient clinics or in hospitalization at the University Hospital of Nancy were included in the study if they met the following inclusion criteria: adult age (≥18 years), established diagnosis of CD. Exclusion criteria for this study were coexistence of infectious or ischaemic colitis, coexistence of other localized or systemic infection. The plasma from 20 healthy volunteers was also collected in order to assess plasma sTREM-1 levels. All patients and healthy volunteers signed a consent form permitting blood samples to be used for scientific purposes. Information about the Nancy IBD cohort is reported to the Commission Nationale de l’Informatique et des Libertés (no. 1404720), which supervises the implementation of the act regarding data processing, data files and individual liberties that came into effect on January 6, 1978, and was amended on August 6, 2004, to protect the personal data of individuals. 2.2. Outcome measures 2.2.1. Assessment of clinical data, CRP and albumin The activity of CD was defined according to Crohn’s disease activity index (CDAI) as described previously [18]. CDAI was collected prospectively by a senior gastroenterologist (L.P.B.). Serum CRP and albumin levels were routinely measured at the time of inclusion. Demographic and clinical data (Montreal classification, concomitant medication, previous surgery) were recorded using the information of the Nancy IBD cohort [19]. All these data were collected through patient’s electronic charts review.
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Software, Mariakerke, Belgium). The diagnostic accuracy of plasma sTREM-1 (pg/mL) and CRP (mg/L) for detecting active and severe forms of CD was evaluated using receiver operating characteristic (ROC) analysis according to DeLong et al. [20] using ‘CDAI > 220’ and ‘CDAI > 300’ as classification variables. 3. Results 3.1. Baseline characteristics of the 191 patients Between June 1, 2005 and December 12, 2008, a total of 191 patients could be included in the study. The baseline characteristics of the patients are shown in Table 1. The median age at inclusion was 30 years (IQR, 23.3–41.8) and the median disease duration was 37 months (IQR, 8–106.8). Most of patients were women (64.4%). According to the Montreal classification, more than half of patients presented an ileocolonic involvement (57.1%). Few patients (13.1%) suffered from a penetrating disease whilst nearly one third of them had a stricturing phenotype (38.5%). Most of patients were treated with immunomodulators at study inclusion as about two third of them received azathioprine (64.2%) and/or infliximab (68.4%). The median CDAI was 122 (IQR, 54.8–202) and the median CRP level was 13.3 mg/L (IQR, 6–32.3). Albumin serum level was normal in most of patients, with a median of 39.2 g/L (IQR, 35.6–43.3). The median plasma sTREM-1 level was 0 pg/mL (IQR, 0–318.5). The sTREM-1 was detectable in 87 CD patients (46.5%). 3.2. Comparison of sTREM-1 levels between CD patients and controls Plasma sTREM-1 level was significantly higher in the CD group than in controls (372 pg/mL versus 11 pg/mL, respectively; P = 0.01) (Fig. 1). 3.3. Correlations between plasma sTREM-1, CDAI, CRP and albumin As expected, CRP level was significantly correlated with the CDAI (r = 0.33, P = 0.01) (Fig. 2). None of the disease activity markers were correlated with the sTREM-1 levels. sTREM-1 level was not correlated with the CDAI (r = 0.05, P = 0.56) (Fig. 3). Regarding biological markers of activity, sTREM-1 level was neither correlated with CRP
2.3. Assessment of sTREM-1 Plasma concentrations of sTREM-1 were determined by ELISA (Quantikine, RnD Systems, MN, USA) according to the manufacturer’s recommendations. The detection level was 15 pg/mL. Measurements were performed in duplicate. Inter and intra-assay coefficients of variation were <7%. 2.4. Statistical analysis All quantitative variables are described as medians and percentiles [interquartile range (IQR), 25–75th percentile]. All proportions are expressed as percentages with 95% confidence intervals (95% CIs). Correlations were studied using the Spearman’s rank-order correlation coefficient. Proportions were compared using the chi-square test or the Fisher’s exact test, as appropriate. The comparison of plasma concentrations of sTREM-1 (pg/L) between subgroups was performed using the Mann–Whitney U test. All the reported P-values were two-sided, and P-values of <0.05 were considered statistically significant. Statistical analyses were performed using MedCalc Software, version 11.6.1.0 (MedCalc
Fig. 1. Comparison of plasma sTREM-1 levels between Crohn’s disease patients and healthy controls.
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Table 1 Baseline characteristics of the 191 patients. Variable
N
Gender (male) Familial history of IBD Montreal classification B1 (non-stricturing, non-penetrating) B2 (stricturing) B3 (penetrating) L1 (isolated ileal disease) L2 (isolated colonic disease) L3 (ileocolonic disease) L4 (isolated upper disease) P (perianal disease modifier) Concomitant medication Azathioprine Methotrexate Infliximab Infliximab with immunosuppressant (azathioprine or methotrexate) Previous surgery for CD Ileal resection Colectomy
191 34
n (%) 68 (35.6) 16 (47.1)
28.8–42.5 29.4–64.7
191 191 191 191 191 191 191 191
109 (57.1) 57 (29.8) 25 (13.1) 32 (16.8) 48 (25.1) 109 (57.1) 7 (3.7) 85 (44.5)
50–64.2 23.3–36.4 8.3–17.9 11.4–22.1 18.9–31.3 50–64.2 1–6.4 37.4–51.6
179 175 177 175
115 (64.2) 10 (5.7) 121 (68.4) 90 (51.4)
57.2–71.3 2.2–9.2 61.4–75.3 44–58.9
167 169
34 (20.4) 24 (14.2)
14.2–26.5 8.9–19.5
Variable
N
Median
IQR
Age at inclusion (years) Age at diagnosis (years) Disease duration (months) CDAI Albumin (g/L) CRP (mg/L) sTREM-1 (pg/mL)
191 191 191 121 117 93 191
30 22 37 122 39.2 13.3 0
23.3–41.8 18–31 8 to106.8 54.8–202 35.6–43.3 6–32.3 0–318.5
95% CI (%)
Note. N, number of subjects; CI, confidence interval; IQR, interquartile range: 25–75th; IBD, inflammatory bowel disease; CD, Crohn’s disease; CDAI, Crohn’s disease activity index; CRP, C-reactive protein; sTREM, soluble triggering receptor expressed on myeloid cells.
(r = 0.06, P = 0.53) nor with albumin (r = −0.041, P = 0.66) (Fig. 4 and Supplementary Fig. 1). 3.4. Comparison of patients according to the positivity of plasma sTREM-1 (Supplementary Table 1) The study population was divided into two groups according to the positivity or negativity of sTREM-1. Eighty seven patients (45.6%) presented a positive sTREM level. The same proportion of patients were treated with azathioprine (P = 0.06), infliximab (P = 0.72) or methotrexate (P = 0.99) in the two groups. There was no difference regarding disease phenotype and history of intestinal surgery according to the positivity of sTREM-1 level. The median
Fig. 2. Correlation between plasma Crohn’s disease activity index (CDAI) and Creactive protein (CRP) levels.
CDAI was 132 in patients with a positive sTREM-1 level compared to 117 in patients with undetectable sTREM-1 levels (P = 0.66). The median CRP level was 15.8 mg/L versus 11.1 mg/L (P = 0.11) and the median albumin level was 38.7 g/L versus 39.6 g/L (P = 0.54) in patients with positive or undetectable sTREM-1 levels, respectively. 3.5. Influence of medications, disease behaviour and previous intestinal surgery on plasma sTREM-1 levels and correlations with CDAI The median sTREM-1 level did not differ significantly according to disease behaviour or disease location (Table 2). Regarding
Fig. 3. Correlation between plasma soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) levels and Crohn’s disease activity index (CDAI).
V. Billioud et al. / Digestive and Liver Disease 44 (2012) 466–470
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Table 2 Comparison of serum sTREM-1 levels according to Montreal classification, concomitant treatment, and previous surgery. Item
Montreal classification B1 (non-stricturing, non-penetrating) B2 (stricturing) B3 (penetrating) L1 (isolated ileal disease) L2 (isolated colonic disease) L3 (ileocolonic disease) L4 (isolated upper disease) P (perianal disease modifier) Concomitant medication Azathioprine Methotrexate Infliximab Previous surgery for CD Ileal resection Colectomy
No
Yes
N
Median sTREM-1
IQR
82 134 166 159 143 82 184 106
0 0 0 0 0 0 0 0
0–217 0–392 0–330 0–340.5 0–371 0–230 0–313 0–290
64 165 56
0 0 0
0–174 0–307.5 0–293.5
133 145
0 0
0–356 0–353
N
P-value Median sTREM-1
IQR
109 57 25 32 48 109 7 85
0 0 0 0 0 0 0 0
0–425.5 0–129 0–250.3 0–175 0–236.5 0–396.3 0–421.3 0–364.8
0.25 0.21 0.99 0.69 0.65 0.57 0.87 0.71
115 10 121
7.4 38.5 0
0–412.8 0–1427 0–333.5
0.06 0.56 0.68
0–101 0–47.5
0.07 0.06
34 24
0 0
Note. N, number of subjects; IQR, interquartile range: 25–75th; sTREM, soluble triggering receptor expressed on myeloid cells; CD, Crohn’s disease.
previous intestinal surgery for CD, neither a history of ileal resection (P = 0.07) nor colectomy (P = 0.06) modified the median sTREM-1 level. The median sTREM-1 levels were also similar in patients who received azathioprine (P = 0.06), infliximab (P = 0.68) or methotrexate (P = 0.56) compared with those who were not receiving such therapy at study inclusion (Table 2). Correlations between sTREM-1 and CDAI were studied according to the type of ongoing immunosuppressive therapy. Plasma sTREM-1 level was not correlated with CDAI amongst patients receiving or not infliximab or immunosuppressants (Supplementary Table 2). Correlations were also performed according to disease behaviour (B1, B2, and B3 according to Montreal classification). Plasma sTREM-1 level was not correlated with CDAI amongst patients with a non-stricturing/non-penetrating (B1), a structuring (B2) or a penetrating disease (B3) (Supplementary Table 3). 3.6. Diagnostic accuracy of plasma sTREM-1 level and CRP for assessing disease activity Using ROC analysis, CRP had a significant diagnostic accuracy for diagnosing moderate to severe CD (CDAI > 220), with an AUROC of 0.68 (standard error (SE) = 0.08, P = 0.03), whereas plasma sTREM-1
Fig. 4. Correlation between plasma soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and C-reactive protein (CRP) levels.
showed an AUROC of 0.52 that did not reach statistical significance (SE = 0.06, P = 0.76) (Supplementary Table 4). Similarly, CRP had a significant diagnostic accuracy for diagnosing severely active CD (CDAI > 300), with an AUROC of 0.78 (SE = 0.09, P = 0.002), whereas plasma sTREM-1 showed an AUROC of 0.56 that did not reach statistical significance (SE = 0.09, P = 0.53) (Supplementary Table 4). 4. Discussion This is the largest study investigating the potential contribution of sTREM-1 measurement in assessing disease activity in CD patients. The role of TREM-1 in the pathogenesis of IBD has first been assessed in animals [14]. Schenk et al. nicely demonstrated that antagonistic TREM-1-derived peptide decreased inflammation and TNF mRNA production in experimental colitis in mice [14]. This elegant study provides promising data from 18 patients with active IBD who revealed an approximately 5-fold increase in the number of TREM-1-expressing macrophages on intestinal biopsies when compared with normal control tissues [14]. TREM-1 expression in intestinal mucosa seemed to be related to disease activity as TREM1 mRNA was expressed at high levels in affected segments whereas it was substantially reduced in tissue sample with inactive disease [14]. Overall, few studies have evaluated the relevance of measuring TREM-1 levels in patients suffering from IBD [14–17]. Tzivras et al. found that serum sTREM-1 levels were higher amongst 39 IBD patients compared with controls [16]. They also showed significantly higher sTREM-1 level in ulcerative colitis (UC) patients with a greater clinical or endoscopic activity index and in CD patients with a CDAI > 400 compared to patients in remission [16]. These results have to be interpreted with caution as only 8 CD patients were included in this study [16]. Consistent results were found amongst 88 patients suffering from intestinal BD [17]. Serum sTREM-1 level was more tightly correlated with disease activity than was the erythrocyte sedimentation rate (ESR) or CRP levels, suggesting the usefulness of sTREM-1 as a potential marker of intestinal BD disease activity [17]. In the present study, we found a significant correlation between CRP and CDAI and higher sTREM-1 levels in CD patients compared to healthy controls, showing that our study population was appropriate to investigate a possible correlation between CDAI and sTREM-1 levels from a statistical point of view. Importantly, we did not find any correlation between sTREM-1 levels and the clinical
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activity index or biological activity markers in CD patients. Taking sTREM-1 as a dichotomous variable (positive or negative) led to similar findings. Interestingly, our results are in line with a previous study by Park et al. including 53 IBD patients [15]. Amongst the 31 UC patients, sTREM-1 was more accurately correlated with disease activity than was ESR or CRP. However, the authors failed to show a significant correlation of sTREM-1 level with disease activity in the 22 CD patients, even though they presented significantly increased sTREM-1 levels than healthy controls [15]. This data indicates that sTREM-1 could be useful in evaluating disease activity in UC, but not in CD. This will require further investigation. Recently, a genetic study showed that three TREM-1 single nucleotide polymorphisms were associated with BD, but not with UC and CD [21]. It has been shown that engagement of TREM-1 in combination with Toll-like receptor activation synergistically increased the production of proinflammatory cytokines including TNF-␣ [22]. Importantly, our study allowed inclusion of patients treated with immunomodulators, which was usually considered as an exclusion criterion in previous observations [15–17]. We found similar sTREM-1 levels irrespective of the administration of immunomodulator and anti-TNF therapy. In addition, we failed to find a correlation between sTREM-1 and CDAI amongst patients with or without ongoing immunosuppressive therapy. Hence, such treatments could not be responsible for the absence of correlation between sTREM-1 and disease activity markers in our CD cohort. One of the main goals of non invasive disease activity evaluation is to enable iterative assessment of therapeutic efficacy and to predict relapse amongst CD treated patients. Therefore, biological markers should be validated and reliable in such a situation as already observed for CRP and faecal calprotectin [23,24]. There are some limitations inherent to our study. First, information on mucosal healing and biopsy samples were not available in this cohort of CD patients. In addition, no UC patients were included in this study. Finally, some clinical data were lacking due to the retrospective study design of our study. The strengths of the study are the large number of CD patients included (n = 191), the inclusion of consecutive CD patients, and the prospective assessment of disease activity markers. In conclusion, the present study failed to show a significant correlation between sTREM-1 levels and clinical or biological activity markers. Taken together our data indicate that plasma sTREM-1 cannot be recommended for assessing disease activity amongst CD patients. Conflict of interest statement The authors declare no conflicts of interest Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.dld.2012.01.005.
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