Serum interleukin 17A and interleukin 17F in children with inflammatory bowel disease

Interleukin 17A (IL-17A) and interleukin 17F (IL-17F) appear to play important role in pathogenesis of some autoimmune diseases. However, their role in inflammatory bowel disease (IBD) has not been yet fully elucidated. We aimed to determine serum IL-17A and IL-17F in children with IBD and to assess their association with IBD activity. Recruited children underwent blood tests including complete blood count, C-reactive protein, erythrocyte sedimentation rate, IL-17A and IL-17F and stool sampling for calprotectin. The study group comprised 68 children with IBD, including 43 with ulcerative colitis and 25 with Crohn’s disease. Control group included 20 healthy children. IL-17A was significantly increased in children with IBD (median: 10.95 pg/ml; range: 0.65–200.54 pg/ml) compared to controls (median: 4.09 pg/ml; range: 0.67–26.20 pg/ml) (p = 0.002). IL-17A was significantly increased in patients with active phase of ulcerative colitis (median: 14.58 pg/ml; range: 0.65–200.54 pg/ml) compared to those in ulcerative colitis remission (median: 8.13 pg/ml; range: 1.61–58.56 pg/ml) (p = 0.04). There were no significant differences in IL-17A among patients with active and inactive Crohn’s disease (p = 0.18). IL-17F did not differ significantly between children with IBD (median: 15.11 pg/ml; range: 0.09–189.84 pg/ml) and controls (median: 11.56 pg/ml; range: 0.19–32.49 pg/ml) (p = 0.33). Our study suggests that interleukin 17A may diverse active phase from remission only in ulcerative colitis but not in Crohn’s disease.

IL-17A has been shown to play a protective role in the host defence against pathogens through eliciting acute immune response at epithelial and mucosal barriers 8,9 , to take part in tissue healing after injury 8 or to maintain the epithelial tight-junction barrier during inflammation 8 . However, unrestrained and excessive activation of IL-17A is one of the potential mechanism underlying autoimmunity, chronic inflammatory conditions and neoplasms 8 . It has been shown that IL-17F is involved in maintaining host's barrier function 8 and may play role in the immunopathogenesis of some chronic disorders 8 .
Although recently there has been a substantial increase of interest of the role of IL-17 family in the development of autoimmune and inflammatory disorders, the knowledge about IL-17 cytokines contribution in the pathogenesis of inflammatory bowel disease (IBD) is limited and conflicting research results provoke discussion in that field. However, an improved understanding of the role of IL-17A and IL-17F in IBD may lead to important clinical implications. Interleukins may serve as a novel potential biomarker of the disease or may be used to design therapeutic approaches in IBD. To the best of our knowledge there are only single studies in small groups of children with IBD assessing IL-17A in paediatric IBD with conflicting results and it appears to exist no study assessing IL-17F in children with IBD 13,14 . The aim of the study was to evaluate serum IL-17 A and IL-17F concentration in children with IBD compared to healthy controls.
The disease duration ranged from 0 to 8 years with mean 1.19 ± 1.85 years. There were 25 (36.8%) treatment naïve patients. The other 43 (63.2%) patients were on IBD treatment.
The most common location of Crohn's disease was ileocolonic with the involvement of the upper gastrointestinal tract (11; 44%) and ileocolonic without the involvement of the upper gastrointestinal tract (7; 28%). Five patients (20%) had colonic Crohn's disease and the latter 2 (8%) had Crohn's disease limited to the distal 1/3 ileum.
The IBD was active in 39 (57.4%) patients (23 with ulcerative colitis and 16 with Crohn's disease). PCDAI ranged from 0 to 65 points with median 30 points and mean ± SD 28 ± 21.9 points. PUCAI ranged from 0 to 75 points with median 20 points and mean ± SD 24.6 ± 22.9 points.
In the control group there were 20 children with functional abdominal pain including 12 (60%) girls and 8 (40%) boys aged from 4.5 to 17.5 years old (median: 12.25 years; mean: 11.9 ± 3.47 years old).
Subgroup analysis, demonstrated in Table 1, revealed that IL-17A concentration was significantly higher in ulcerative colitis patients compared to controls (p = 0.003). No significant differences were found between Crohn's disease patients and controls and between children with Crohn's disease and ulcerative colitis.
The comparison of IL-17A concentration between patients in active phase of IBD and in IBD remission is presented in Fig. 1. We found that serum IL-17A was significantly increased in patients with active phase of ulcerative colitis compared to those in ulcerative colitis remission (p = 0.04). However, there were no significant differences in IL-17A among patients with active Crohn's disease and in Crohn's disease remission (p = 0.18).
We found a positive correlation between IL-17A and IL-17F in children with IBD (p = 0.03; R = 0.29). However, there were no significant correlations between IL-17F and IBD activity indices, inflammatory markers i.e. calprotectin, CRP, ESR, white blood cells count. The comparison of serum IL-17F between patients in active phase of IBD and in IBD remission is presented in Fig. 2.

Discussion
Considerable progress in the understanding of IBD pathogenesis in recent years entails substantial advances in the diagnosis and therapy of that disease 1,15-18 . However, still there is a need of reliable non-invasive or minimally invasive biomarkers which would facilitate timely recognition of IBD and would predict exacerbation of the disease. It is particularly important in children and adolescent to avoid repeated gastrointestinal tract endoscopy under anaesthesia. It appears that cytokines involved in the inflammatory process underlying IBD may become potential diagnostic tools and moreover represent a target for therapeutic interventions in IBD 6 .
One of cytokines which has been recently explored in terms of IBD pathogenesis is IL-17A. It has been suggested that in the gut antigen-stimulated dendritic cells secrete IL-23 which promotes the differentiation of naive CD4 + T cells into Th-17-cells producing IL-17A 22,23 . IL-17A upregulates intercellular adhesion molecule-1 on intestinal epithelial cells and induces release of IL-6 and IL-8 from epithelial cells and myofibroblasts 23 . It results in an augmented recruitment of neutrophils and production of pro-inflammatory mediators eventually leading to the damage of gut epithelium 22,23 .  Although several studies have been focused on the role of IL-17 family in the pathogenesis of IBD, most of them has been carried out in relatively small populations of patients giving conflicting results 13,14,[19][20][21] . Therefore, to date all the pieces of the puzzle have not been assembled.
In our study we demonstrated that serum concentration of IL-17A was significantly increased in children with IBD compared to the control group. Moreover, in a group of patients with ulcerative colitis relationship between IL-17A and clinical activity index (PUCAI) and calprotectin nearly reached a statistically significant level. On the contrary, there were no significant differences in serum concentration of IL-17F between IBD patients and controls. IL-17F revealed only a single significant correlation with IL-17A.
Our findings indicating higher levels of serum IL-17A in children with IBD than in control group are consistent with some previous studies 20, 21 . Kaplan et al. showed significant increase of serum IL-17A in adult patients with IBD compared with the control group 21 . This result ties well with a previous study by Fujino et al. 20 who presented that serum IL-17A was significantly elevated in a group of adults with IBD compared to controls and to patients with infectious colitis, or ischaemic colitis patients 20 . Present findings along with prior studies support conception of significant role of IL-17A in IBD.
In contrast to these findings Kleiner et al. presented that serum IL-17A was significantly decreased in children with IBD patients compared to healthy controls 14 .
Detailed analysis revealed that in our study group only in patients with ulcerative colitis IL-17A was higher than in controls. Cho et al. did not show any significant differences in serum IL-17A between children Crohn's disease or ulcerative colitis compared to healthy controls 13 . Moreover, study by Sahin et al. also did not revealed significant differences in serum IL-17A among patients with Crohn's disease and controls 19 . It may be explained by the fact that patients with Crohn's disease in these studies had relatively moderate activity of the disease. It might be also related with the effect of IBD therapy leading to inhibition of pro-inflammatory cytokines 19 . Different expression of IL-17A in patients with Crohn's disease and ulcerative colitis may be associated with the fact that these diseases differ in some aspect of pathogenesis 24 . Kobayashi et al. 25 found that IL-23 differentially regulates Th1/Th17 balance in ulcerative colitis and Crohn's disease enhancing production of different cytokines. Thus, ulcerative colitis may be assumed as Th17 disease with increased production of Il-17, while Crohn's disease as Th1 disease with upregulated synthesis of IFNγ 25 .
In general, conflicting results of studies evaluating IL-17A level may be caused by some discrepancies between studied populations including various disease duration, phenotype, activity, use of diverse medications and concomitant diseases.
We demonstrated that IL-17A may be associated with ulcerative colitis activity which accords with other research 20,21 . Similarly to our findings, serum concentration of IL-17A was significantly increased in active ulcerative colitis patients compared to those in the disease remission in studies conducted by Kaplan et al. and Fujino et al. 20,21 . Moreover, Ohman et al. found that serum IL-17A in newly diagnosed treatment naïve patients with ulcerative colitis correlated with disease severity and predicted the course of the disease over the following three years 26 . www.nature.com/scientificreports/ However, in contrast to these studies we did not find significant differences in IL-17A among Crohn's disease patients depending on the disease activity 20,21 . It may be explained due to various criteria of patients' qualification into active and non-active IBD group used in these studies.
On the other hand, Sahin et al. revealed no differences in IL-17A between patients with active and inactive Crohn's disease and significantly decreased level of IL-17A in patients with active Crohn's disease compared to the control group 19 .
In our study in ulcerative colitis children positive relationship between IL-17A with PUCAI and stool calprotectin showed a tendency toward significance. Kaplan et al. found in all patients with IBD a positive correlation between IL-17A and C-reactive protein, endoscopic activity index, and Crohn's disease activity index 21 . These findings imply that IL-17A may be considered as a marker for inflammatory activity in IBD patients.
Moreover, we found a negative relationship between IL-17A with haemoglobin, haematocrit, and medium cell value in children with ulcerative colitis. It may be explained by the fact that has IL-17 cytokine family members are suggested to be inhibitors of haematopoiesis 27 . IL-17A downregulates erythropoiesis through inhibition of late stage erythroid progenitors (colony-forming unit-erythroid, CFU-E) 28 .
Interleukin 17F is closely related to IL-17A and its role in the development of inflammatory bowel disease remains not well-established. Seiderer et al. found that intestinal IL-17F mRNA expression was increased in active CD but not in ulcerative colitis 29 32 . In our study there were no significant differences in serum IL-17F levels between children with IBD and healthy controls. However, there was a positive correlation between IL-17A and IL-17F in children with IBD. This may suggest that although both cytokines share high level of homology at the amino acid level, cellular sources and some functions, IL-17A more strongly than IL-17F is involved in mediating autoimmunity and chronic inflammation, which has been observed previosuly 33 .
The main limitation of our study is relatively homogenic study population with rather moderate clinical activity of the disease. Another limitation may be the fact that patients with ulcerative colitis prevailed in our study group. Moreover, our results should be interpreted with caution since we evaluated the concentration of IL-17A and IL-17F only in patients' serum. Future research may consider determining the expression of IL-17 in mucosal samples from patients to fully understand correlation between IL-17 family members and IBD activity.
In conclusion we demonstrated that serum IL-17A levels, but not IL-17F, are significantly elevated in children with IBD compared to controls. These results may imply that IL-17A plays critical role in the pathogenesis of paediatric IBD. Moreover, we presented that IL-17A was significantly increased in patients with active ulcerative colitis compared to those in ulcerative colitis remission. Our findings may suggest that IL-17A may be a marker of ulcerative colitis clinical activity in children. Nevertheless, further studies are needed to fully elucidate the contribution of IL-17 family in the pathogenesis of paediatric IBD.

Methods
Study population. We recruited consecutive children with IBD hospitalized at the Department of Paediatrics and Gastroenterology, Medical University of Lublin, Poland between June 2017 and October 2019. IBD was diagnosed according to the revised Porto criteria 2 . Disease phenotype was established based on the Paris Classification 34 . Clinical activity in children with Crohn's disease was determined by Paediatric Crohn's Disease Activity Index (PCDAI) and in children with ulcerative colitis by Paediatric Ulcerative Colitis Activity Index (PUCAI) 35,36 . Exclusion criteria from the study group were lack of informed consent of parents and/or patient aged ≥ 16 years old, any clinical or laboratory signs of acute infection at the time of enrolment, a history of a surgery within the 4 weeks preceding the recruitment.
In the same study period healthy children with functional abdominal pain were enrolled to the control group. The diagnosis of functional abdominal pain was established on the basis of Rome IV Diagnostic Criteria for Functional Gastrointestinal Disorders 37 . Exclusion criteria were as follows lack of informed consent of parents and/or patient aged ≥ 16 years old, any concomitant chronic disease, any clinical or laboratory signs of acute or chronic inflammation at the time of enrolment, a history of a surgery within the 4 weeks preceding the recruitment.
Methods. Children underwent routine blood testing including complete blood count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and stool sampling for calprotectin according to standard laboratory practice. Serum concentration interleukin 17A and 17F was measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Human IL-17A and Human IL-17F ELISA kits) according to the manufacturer's recommendations (Diaclone SAS Besancon Cedex, France). According to the instruction IL-17A standard concentration using the standard curve ranged from 0 to 100 pg/ml, while IL17F from 0-500 pg/ml. Following the instructions of the protocol the concentration from the standard curve was multiplied by the appropriate dilution factors. Statistical analysis. The statistical analysis was performed using Statistica v. 13 software (StatSoft, Poland).
The data are shown as mean and standard deviation or median and range 38,39 . Non-parametric tests for analysis considering skewed distribution of variables (W Shapiro-Wilk test) and inhomogeneity of variance (F-Fisher test) were carried out 38,39 . Differences between two groups were tested with the use of Mann-Whitney U-rank test, while differences between more than two means for more than two groups with The H Kruskal-Wallis