Background
Inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease (CD) are complex immune-mediated disorders associated with heterogeneous disease presentation. Diagnosis and evaluation of disease activity involves imaging and procedures such as ileocolonoscopy, magnetic resonance imaging (MRI), and computed tomography (CT) scans that may be invasive, risky, and costly [
1‐
3]. Thus, these procedures are not performed frequently, and biomarkers are more commonly used for disease activity evaluation. Approximately 10–25% of CD patients develop at least one intestinal stricture, demonstrating the need for an accurate biomarker for detection of stricture [
4]. Current IBD biomarkers include C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and the more IBD-specific fecal calprotectin (FC), which has a greater correlation to inflammatory colitis activity [
5]. However, these biomarkers do not consistently demonstrate accuracy in correlation to certain IBD parameters such as mucosal disease activity and strictures [
6]. For this reason, accurate IBD biomarkers for IBD disease activity are being sought after actively.
Antimicrobial peptides such as lactoferrin have demonstrated clinical utility as IBD biomarkers [
7]. An additional antimicrobial peptide, cathelicidin, is reported to have anti-inflammatory and anti-fibrogenic effects [
8‐
11]. Cathelicidin (also known as hCAP18) is a 18 kDa peptide consisting of 37 amino acids [
12]. Its antimicrobial functions depend on permeabilization of bacterial cell membrane with multiple cationic cathelicidin molecules [
13]. Cathelicidin is expressed broadly in various tissues such as colonic mucosa [
14], breast [
15], salivary glands [
16], and various kinds of immune cells [
17]. Human cathelicidin promoter consists of a vitamin D response element (VDRE) [
18]. Oral vitamin D administration has been demonstrated to induce skin cathelicidin expression in patients with atopic dermatitis [
19]. Another report demonstrated the association between vitamin D, activation of cathelicidin expression, and tuberculosis infection [
20]. Besides antimicrobial functions, cathelicidin possesses anti-inflammatory effect against endotoxin lipopolysaccharide [
21]. Other antimicrobial peptides such as hepcidin [
22] and beta-defensin-2 [
23] do not correlate with IBD disease activity.
Focusing on IBD, Schauber et al. show that colonic cathelicidin (
CAMP) mRNA expression is increased in ulcerative colitis patients [
24]. Animal studies also demonstrated that mice with a cathelicidin deficiency are more susceptible to DSS-mediated colitis than wild-type mice [
25]. Low serum cathelicidin levels are associated with sepsis in patients of intensive care units [
26] and mortality in cases of severe renal disease [
27], while high serum cathelicidin levels are observed in patients with autoimmune diseases such as psoriasis [
28] and vasculitis [
29]. Collectively, this evidence suggests a general correlation of circulating cathelicidin with infection, inflammation, and autoimmune diseases.
IBD is regarded as an autoimmune disease. It is possible that circulating LL-37 levels are altered in the patients of IBD. Levels of circulating cathelicidin in IBD patients have never been analyzed in literature, and the correlation between cathelicidin and IBD disease activity has not yet been determined. We hypothesize that circulating cathelicidin levels have the potential to accurately indicate IBD disease activity. In this study, we examined serum cathelicidin levels for correlation to IBD disease activity. We compared diagnostic accuracies of LL-37 alone, CRP alone, and combined LL-37 + CRP for indicating various clinical and mucosal disease activity parameters. We evaluated the prognostic capability of serum LL-37 levels in indicating the disease development 6–18 months later and determined the relative risk of intestinal stricture in CD patients with low serum cathelicidin levels.
Methods
Patients and samples
Blood was collected from control, UC, and CD patients through procedures established by UCLA. For cohort 1, IBD patients were recruited from a UCLA Gastroenterology clinic, and control samples were obtained from a UCLA Internal Medicine clinic. Serum samples from cohort 1 were prepared by UCLA Department of Pathology. Serum samples from cohort 2 were obtained from UCLA Center for Inflammatory Bowel Diseases Biobank. All blood samples were collected between 2012 and 2015. The blood samples were collected following indicated diagnostic procedures ordered by the physicians. Serum volume availability must be 100 μL or above. Specimen must be in good quality with no sign of hemolysis. For IBD patients, clinical data of follow-up visits at 6–18 months after the initial blood draw were collected for the analysis. The blood samples of cohort 1 and 2 do not overlap.
Inclusion and exclusion criteria
Inclusion criteria: Samples were obtained from male and female subjects (age 21–67 years). IBD-specific inclusion criteria: The IBD patients sought diagnosis and/or treatment for IBD. IBD diagnosis (UC and CD) was confirmed by board-certified gastroenterologists. Exclusion criteria: No pregnant women, prisoners, or minors under age 18 were included. Patients with concurrent acute infection (CMV, C. difficile, and tuberculosis) and malignant conditions were not included. IBD patients without follow-up visits at 6–18 months after the initial blood draw were not included. Dr. Koon has already included all qualified blood samples available at the time of ELISA experiments in 2015.
Assessment of disease activity and presence of stricture
The Partial Mayo Score (PMS) and Harvey Bradshaw Index (HBI) were used to assess the clinical disease activity of UC and CD patients respectively [
30,
31]. Evaluation of PMS and HBI was performed on the same day of blood sample collection for LL-37 ELISA, CRP, and other tests. The diagnosis of stricture was determined by imaging procedures or colonoscopy occurring within 1 month of blood sample collection. Mayo Endoscopic Subscore (MES) was used to assess the mucosal disease activity of the UC patients (range 0–3) [
32]. All data were collected prospectively.
LL-37 and C-reactive protein (CRP)
Measurement of LL-37 was performed using an ELISA kit (#HK321, Fisher Scientific, Pittsburgh, PA) at Dr. Koon’s laboratory according to manufacturer’s instructions as previously described [
25]. CRP levels were determined by the UCLA Department of Pathology and their values were available at the CareConnect database.
Statistical analysis
Power analysis has shown that this study required at least 40 patients per group of control, UC, and CD patients to achieve a statistically significant difference of serum LL-37 levels between control (37 ng/ml), UC (56 ng/ml), and CD (58 ng/ml) patients with standard deviation = 21, alpha = 0.5, and power = 0.8. The combined data from two cohorts yielded 70 control, 80 UC, and 95 CD patients total that satisfied this requirement.
LL-37 protein concentration and CRP levels were arranged in order from low to high. Low LL-37 levels indicated moderate and severe disease, and high LL-37 levels indicated remission. High CRP levels indicated moderate and severe disease, and low CRP levels indicated remission. We determined the performance of multiple cut-off points of LL-37 and CRP levels at each disease parameter. The optimized universal cut-off points were shown in this study.
Prevalence of disease, sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV), with their 95% confidence intervals (CI) were calculated using a clinical calculator website:
http://vassarstats.net/clin1.html. The accuracy of a test was determined by Area under Curve (AUC) of Receiver Operating Characteristics (ROC) using:
http://www.rad.jhmi.edu/jeng/javarad/roc/JROCFITi.html. Odds ratio and relative risk were calculated using a clinical calculator website:
https://www.medcalc.org/calc/. Results were expressed as mean +/− SEM. Bar graphs and scatter plots were made using Microsoft Excel and GraphPad Prism (San Diego, CA). Unpaired Student’s
t-tests were used for intergroup comparisons of continuous data and Fisher’s exact tests were used for intergroup comparisons of category data using GraphPad Prism. Significant
p values are shown in each figure.
Discussion
This study determined the optimal LL-37 cut-off points for indicating clinical disease activity, mucosal disease activity, stricture, and future clinical activity. The LL-37 test should only be used to indicate disease activity after the IBD diagnosis is confirmed because IBD patients with moderate and severe disease activity may also have serum cathelicidin levels as low as control patients. In animal models, systemic overexpression of cathelicidin suppresses colitis while systemic deficiency of endogenous cathelicidin worsens colitis [
11,
25]. This explains why the cathelicidin level is inversely proportional to disease activity in IBD.
Colonic epithelial cells express cathelicidin, and colonic cathelicidin mRNA expression is shown to be increased in UC patients but not CD patients [
24]. However, the contribution of colonic cathelicidin expression to circulating cathelicidin levels and overall IBD disease activity is not known, and it is beyond the scope of this study. Our previous animal study demonstrated that cathelicidin genotype of bone-marrow derived cells plays a significant role in dextran sulfate-mediated colitis [
25]. This evidence suggests that immune cells may modulate colitis development via cathelicidin expression.
Although physicians can assess clinical disease activity of patients by asking questions about IBD-related symptoms, they often rely on one or more biomarkers (CRP, ESR, and FC) to provide an additional reference in the assessment of overall disease activity and drug responses. As shown in Figs.
2 and
5, LL-37 and CRP have similar accuracy in indicating clinical disease activity. The LL-37 ELISA experiment is inexpensive, quick to produce results, and easy to perform. Collection of blood samples for LL-37 tests may be more convenient than collection of fecal samples for calprotectin tests as patients may not be able to provide stool samples at the time of clinical visit. The LL-37 test may be a promising alternative to the CRP test.
Co-evaluation of LL-37 and CRP showed 18% improvement of accuracy in indicating UC endoscopic remission and 13% improvement in indicating moderate or severe UC endoscopic disease activity when compared to CRP alone (Fig.
3). It is common to assess mucosal disease activity using biomarkers since ileocolonoscopy is not frequently performed. As CRP testing is not highly accurate in the evaluation of mucosal disease activity, this combined test optimizes the existing CRP test. The LL-37 + CRP test may be useful when invasive ileocolonoscopy is not advisable. This LL-37 + CRP combined test (AUC = 0.8) may be more accurate than FC (AUC = 0.639) in indicating endoscopic remission (MES = 0), according to a recent study [
34]. Unfortunately, we were not able to compare the performance of LL-37 + CRP with FC side-by-side since the FC tests were not frequently performed in our cohorts. Similar to a previous study [
35], we found no correlation between HBI and CRP levels in CD patients.
Another strength of this LL-37 test is the prediction of future clinical activity. The LL-37 test is superior to the CRP test in predicting recovery within 6–18 months (Figs.
2 and
5). For IBD patients with moderate or severe clinical disease activity at the time of blood draw, patients with high LL-37 levels showed better recovery than patients with low LL-37 levels after 6–18 months. There was no association between LL-37 levels and use of medications in both cohorts (data not shown). We believe that the good recovery in high LL-37 group is independent of response to any particular kind of medication. Co-evaluation of LL-37 and CRP does not improve the predictive correlation (data not shown). The potential capability of LL-37 in predicting UC endoscopic disease activity prognosis is promising. We found a trend of good recovery from mucosal disease in high LL-37 UC group (data not shown); however, the data are insufficient for a full analysis at this moment.
There is no well-established biomarker for stricture diagnosis. The LL-37 test (low LL-37 group) can indicate an elevated risk of the presence of stricture with high specificity (Table
2a-c). Although the LL-37 test is not intended to diagnose the presence of stricture due to low sensitivity, our finding presents a novel method to help physicians identify high-risk CD patients for further screening. Based on the anti-fibrogenic effect of cathelicidin in animal models [
11], it is reasonable to expect that low circulating cathelicidin expression correlates with an elevated risk of stricture. CD stricture can be classified into inflammatory stricture or non-inflammatory fibrostenotic stricture. However, there is no clear consensus in the approach of differentiation between these two types [
36]. Unfortunately, there was no distinction between stricture types made by the physicians for the patients included in this study. We aim to reevaluate the potential use of LL-37 levels in indicating the inflammatory stricture versus fibrostenotic stricture in the future once adequate guideline have been established. In addition, LL-37 was not associated with the relative risk of fistula occurrence in CD patients at the time of blood draw (data not shown). This study already partially explored the association of LL-37 levels and stricture development. It may be interesting to determine whether LL-37 levels can predict development of other intestinal complications in the future.
This report provides novel evidence for the value of circulating cathelicidin levels as a new IBD biomarker. Circulating cathelicidin levels inversely correlate with clinical and mucosal disease activity in UC and CD patients. High LL-37 levels alone predict good clinical prognosis. Co-existence of a low LL-37 level and clinical remission indicates an elevated risk of intestinal stricture. Besides the diagnostic utility of cathelicidin in IBD patients, cathelicidin has been demonstrated to have potential as a novel therapeutic strategy against colitis [
10], intestinal fibrosis [
11], and colitis-associated colon cancer [
37]. Systemic administration of cathelicidin peptide to patients is unsafe due to its hemolytic property [
38]. For this reason, we are exploring a new therapeutic strategy involving oral administration of a non-peptide cathelicidin mimic for treating colitis and its complications.
Acknowledgement
We thank Prof. Charalabos Pothoulakis, MD for technical and financial assistance to this project.
This study is associated with a U.S. patent #14/743,909 filed on 6/18/2015. We are collaborating with a diagnostic company in the development of this cathelicidin biomarker for clinical applications. This company has conducted a separate study in another cohort in Europe and confirmed that the findings are consistent with ours (corporate confidential data not shown). The patent development for clinical applications is in progress. We continue to validate the correlation of cathelicidin with various IBD disease activity parameters multiple large cohorts of patients. We believe that cathelicidin tests may be further optimized for indicating other IBD disease parameters in the future.