What Is Known
- Pediatric-onset ulcerative colitis is often more aggressive than adult-onset disease.
- Dysregulated immune response contributes to the disease pathogenesis.
- Multidrug resistance protein 1, breast cancer resistance protein, and multidrug resistance-associated protein 1 expressions are altered in adult ulcerative colitis.
What Is New
- Pediatric- and adult-onset ulcerative colitis are characterized by mixed Th1/Th17 response, with higher cytokine expression detected in children.
- Elevated expressions of suppressor of cytokine signaling molecules may not be sufficient to restrain the overwhelming immune response.
- In both children and adults, the multidrug resistance protein 1 expression is downregulated irrespective of the disease activity, while they differ in the expressions of multidrug resistance-associated protein 1 and breast cancer resistance protein.
Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) that is mainly restricted to the colon (1). The pathogenesis includes dysregulated immune response to commensal microbiota in a genetically susceptible host, with elevated innate (tumor necrosis factor [TNF]-α; interleukin [IL]-6, IL-1β) and adaptive (interferon [IFN]-γ, IL-17) inflammatory cytokine production (2–4). It reflects a continuous stimulation of immune response or possibly inadequate negative feedback regulation mediated by suppressors of cytokine signaling (SOCS) molecules 1 and 3 (5,6). The UC has a peak incidence in young adulthood; however, 20% to 30% of patients develop a disease during childhood (7). Disease onset in children is often more aggressive compared with adults (8) and evidence points to differences in immune responses (9). Efflux transporters maintain gut homeostasis by pumping potentially harmful substances out of the cell and their expression was shown to be altered under inflammatory conditions (10,11). Because different intrinsic and extrinsic factors can affect inflammatory profile and transporter expression in UC pathogenesis, it is important to analyze their function at the earliest point of disease onset. Therefore, we chose to assess the differences in the inflammatory profile measuring cytokine (IFN-γ, IL-17A, IL-2, IL-6, IL-1β) and efflux transporter gene (multidrug resistance gene [MDR1]; multidrug resistance-associated protein 1 [MRP1]; breast cancer resistance protein [BCRP]) expression in pediatric- and adult-onset newly diagnosed/untreated patients and patients in remission. Comparing the childhood onset with adult onset could provide new data on differential molecular patterns related to age and disease pathology.
MATERIALS AND METHODS
This is a comparison study of adult and pediatric patients with IBD compared with control subjects. The Materials and Methods section and Patient Characteristics are provided as Supplemental Digital Content 1, Materials and Methods (http://links.lww.com/MPG/A783) and Supplemental Digital Content 2, Table (http://links.lww.com/MPG/A784), respectively.
Altogether 28 children were included: 9 UC-New (average age 14 ± 4.1 years), 4 UC-Remission (average age 13 ± 4.3 years), and 15 healthy controls (average age 13 ± 4.1 years). Adult cohort involved 27 individuals: 9 UC-New (average age 48 ± 20), 8 UC-Remission (average age 50 ± 13), and 10 healthy controls (average age 58 ± 17 years). The description of sample collection and methods used to analyze the samples are provided as Supplemental Digital Content 1, Materials and Methods (http://links.lww.com/MPG/A783) Patient's characteristics are presented in the Supplemental Digital Content 2, Table (http://links.lww.com/MPG/A784).
Expression of Cytokines and Suppressors of Cytokine Signaling Molecules
Comparison of the expression levels of the investigated cytokines and SOCS molecules showed no difference between child and adult healthy controls (data not shown).
Inflamed colon in UC-New children was depicted by elevated expression of IFN-γ (11-fold), IL-6 (4.6-fold), IL-1β (21-fold), and IL-17A (19-fold) mRNAs compared with controls. Similar pattern of cytokine expression was obtained for UC-New adults with upregulated IFN-γ (3.7-fold), IL-6 (6.8-fold), and IL-17A (11-fold) expression compared with control group.
There was, however, no statistical difference in the expression of cytokines between UC-Remission and controls, except for IFN-γ in adults (P < 0.01) (Fig. 1). In contrast to the other investigated cytokines, the IL-2 expression in the colon was elevated only in UC-Remission in both pediatric and adult patients (3.2- and 18-fold, respectively) (Fig. 1). The expression of SOCS molecules in the inflamed colon in children followed the pattern of cytokine expression with elevated levels in UC-New subgroup (6.7- and 4.5-fold for SOCS1 and SOCS3, respectively) and levels close to control group in UC-Remission. In contrast to children, the expression of SOCS1 in the inflamed mucosa of adults was elevated only in UC-Remission (6.5-fold), whereas the expression of SOCS3 was upregulated at both subgroups (5.7- and 4.2-fold, respectively).
The comparison of the cytokine/SOCS expression between UC-New children and adults in the inflamed colon showed a significantly higher expression of IFN-γ (P < 0.05), IL-17A (P < 0.05) and SOCS1 (P < 0.05) in children, whereas in UC-Remission there was a significantly higher expression of IL-2 in adults (P < 0.001).
Unexpectedly, elevated expressions of IFN-γ (6.5-fold), IL-6 (4.8-fold), IL-1β (4.4-fold), SOCS1 (2.9-fold), and SOCS3 (4.4-fold) were detected in the uninflamed ileum mucosa of UC-New children, with levels close to healthy controls in UC-Remission (Fig. 1). Similar results were found for adults, with increased levels of IL-6 (9.8-fold), IL-1β (6.2-fold), SOCS1 (2.4-fold), and SOCS3 (3.9-fold) in UC-New subgroup. The comparison of cytokine/SOCS expressions between UC-New children and adults in unaffected ileum showed no significant differences.
Expression of Efflux Transporters
In the inflamed colon, MDR1 expression was reduced in UC-New children (3.1-fold) and adults (3.4-fold), when compared with healthy control biopsy specimens (Fig. 2). Decreased MDR1 levels were also detected in the colon of UC-Remission subgroups (1.8- and 3.2-fold for children and adults, respectively). The observed MDR1 downregulation correlated with disease activity in children (r = −0.6, P = 0.015) but not in adults (r = 0.055, P = 0.79). The MRP1 expression in children was elevated in UC-New subgroup (2.6-fold), with no differences between defined groups in adults. In the case of BCRP, there were no differences in the expression for children, whereas in adults its expression was reduced in the UC-New subgroup (2.9-fold), compared with controls. Both MRP1 and BCRP expression in UC-Remission was close to the healthy control levels (Fig. 2).
In the uninflamed ileum of children, there were no differences in the MDR1 or BCRP expression between UC subgroups and controls, whereas the MRP1 expression was elevated in the UC-New subgroup (2.7-fold) similar to the inflamed colon. In adults however, the MDR1 expression in uninflamed ileum was reduced in both UC-New (2.1-fold) and UC-Remission subgroups (3.2-fold) compared with controls, whereas BCRP levels were reduced only in UC-New subgroup (2.4-fold) (Fig. 2), recapitulating findings for a diseased colon tissue.
The correlation analyses showed a negative correlation between MDR1 and IFN-γ (r = −0.71, P = 0.0005) and IL-6 (r = −0.66, P = 0.0029) expression at the inflamed colon in children. In addition, cytokines IL-6 (r = 0.73, P = 0.0057), IL-1β (r = 0.61, P = 0.013), and IL-17A (r = 0.73, P = 0.0051) as well as MDR1 (r = −0.60, P = 0.015) correlated with disease activity in pediatric UC, but not in adults IL-6 (r = 0.31, P = 0.13); IL-1β (r = 0.38, P = 0.053); IL-17A (r = 0.16, P = 0.45).
In the present study we show that both childhood- and adult-onset UC involves elevated expressions of IFN-γ and IL-17A, emphasizing a mixed Th1 and Th17 response at the time of diagnosis. This is in agreement with previous studies in UC patients (3,12,13), and it seems that IFN-γ acts in synergy with IL-17 to promote intestinal inflammation (14). Nevertheless, of great importance is to note that UC-New children have markedly higher IFN-γ and IL-17A levels compared to adults, which may be the reason that the childhood onset UC is often more severe (8). In addition to increased Th1/Th17 cytokine response, both study groups have increased and comparable IL-6 expression at time of diagnosis, whereas only in children IL-1β expression is markedly increased. It is possible that the role of IL-1β differs between children and adults in the state of acute inflammation. IL-1β was shown to be upregulated in the inflamed UC tissue (11), but the majority of adult patients in other studies had chronic inflammation and were under therapy. Studies conducted on animal models have shown that IL-1β and inflammasome activation are necessary for the initiation of intestinal inflammation and infiltration of pathogenic Th17 cells (15,16), but may not be necessary for the maintenance of pathogenic phenotype (17). Recently, colony stimulating factor 1 (CSF-1) and IL-34 have been shown to be important for sustaining chronic inflammation in the gut (18). Increased expression of cytokines in UC-New children is accompanied by elevated expression of negative regulators SOCS1 and SOCS3. Although in UC-Remission levels of all measured cytokines (except IL-2), and corresponding regulatory SOCS molecules, are close to healthy control values in children, in adults the expressions of IFN-γ and IL-6 together with SOCS1 and SOCS3 are elevated, implicating an ongoing subclinical inflammation. In addition to the inflamed colon mucosa, higher expressions of IL-6 and IL-1β are also found in the histologically uninflamed ileum mucosa, along with high IFN-γ in children, which is in line with the study by Stronati et al. (19), but not Drastich et al (20). Levels of IFN-γ, IL-6, and IL-1β in pediatric and IL-6 and IL-1β in adult UC-Remission are, however, close to healthy control levels, following the same pattern as in inflamed colon. This also raises an important question on to why the histological inflammation in the ileum is not present. One could argue that it is the result of the high SOCS1 and SOCS3 expression, but according to our results, the fold increase of both SOCS molecules in the ileum, compared with fold increases of their matched cytokines, is not as high. Of importance is to note that we only monitored mRNA expression, which may not reflect protein levels.
Although SOCS molecules participate in a feedback regulation of the overwhelming inflammatory response (21), their upregulation in UC may just not be sufficient. SOCS1 is a negative regulator of signal transducer and activator of transcription 1 (STAT1) pathway (22) and also plays a critical role in preventing colitis in murine IBD model (23). We detected increased SOCS1 levels in colon and ileum tissue samples in children with UC, as expected due to the presence of higher IFN-γ (24). Moreover, we did not find increased SOCS1 expression in the inflamed colon of UC-New adults. Findings in both cases implicate insufficient SOCS1-dependent negative control of a STAT1 pathway and possible involvement of other SOCS1-independent regulatory mechanisms, like prostacyclin PGI2, which is elevated in IBD patients (25). We were unable to detect difference in SOCS3 expression between UC-New children and adults, which are elevated at both colon and ileum mucosa. Similar finding was shown for colon of adult UC patients (22,26). SOCS3 levels in children in remission are, however, close to control levels, whereas in adults they are increased. The exact role of SOCS3 in IBD is still inconclusive. It has been associated with the balance between mucosal wound healing (27) and exacerbation of the acute intestinal inflammation (28). SOCS3 is negative feedback regulator of IL-6 and IL-17 signaling pathway that involves STAT3 (29,30). Therefore, inadequate SOCS3 function could contribute to dysregulated STAT3 pathway and exacerbation of IL-6/IL-17-driven inflammation as observed in UC patients (13). High colon SOCS3 expression in adult UC remission was, however, associated with a shorter time until relapse (31).
Regulatory T (Treg) cells play a role in the immune tolerance of the gut (32). We show that IL-2 expression is elevated only in UC-Remission and is significantly higher in adults than in children. This finding may imply the protective role for IL-2 and possibly Tregs (33), which is the main objective of an ongoing clinical trial using low dose IL-2 to promote the selective expansion of Tregs (www.clinicaltrials.gov/ct2/show/NCT02200445).
Because efflux transporters contribute to gut homeostasis, the changes in their expression and/or function could have an impact on UC pathogenesis. Here we demonstrate a reduction in MDR1 expression at colon mucosa of both children and adults irrespective of the disease subgroup, which is in agreement with previously published articles (11,34). This could be the result of the methylation of MDR1 promotor (35) and emphasize an epigenetic dysregulation in IBD. Moreover, our results show a downregulation in MDR1 expression in uninflamed ileum mucosa in adults, but not children, although no macroscopic signs of an inflammation could be detected. This implicates an overall downregulation of MDR1 expression along the entire intestine in adults. Many environmental factors present in adults and absent in children could compromise the MDR1 expression along the entire intestine. Although well-known efflux transporter function is an efflux of a potentially harmful compounds, it was also shown that MDR1 interferes with bacterial adhesion to enterocytes and reduces the risk of gastrointestinal disorders (36). In line with that, the observed downregulation of MDR1 could emphasize its involvement in IBD pathogenesis possibly through dysregulation of interactions with commensals, as shown in mdr1a(−/−) mice (37) who also show an enhanced infiltration of inflammatory cells, IL-1β production and myeloperoxidase activity (38) in spontaneous intestinal inflammation (39). Additionally, the absence of MDR1 in Tregs alters activation and their suppression potential leading to an inability to suppress colonic inflammation (40). This is probably not the case in UC as we have detected higher IL-2 levels in a remission phase. Whether the long-term exposure of Tregs to diseased tissue microenvironment changes their MDR1 expression and suppressive capacity, however, remains to be elucidated. Another difference between childhood- and adult-onset UC is in BCRP expression. Whereas in children the BCRP expression is unchanged at inflamed and noninflamed intestine mucosa, in adults its expression is downregulated in inflamed colon mucosa and close to control values in remission. It is known that BCRP participates in the maintenance of intracellular redox status (41) and reduced BCRP expression in inflamed and unaffected mucosa in adult patients may affect efflux of reactive oxygen species outside epithelial cells rendering them more sensitive to apoptosis by Fas ligand (42). The reduced MDR1 and/or BCRP expression in UC was not a part of an overall reduction in efflux transporter expression because we found increased MRP1 expression only in children at both inflamed and uninflamed intestine mucosa. This is partly in agreement with the study by Blokzijl et al (43) who proposed that elevated MRP1 may serve a protective role in the IBD pathogenesis, which is in contrast to leukotriene C4 (LTC4) transporter activity and augmentation of Th17 inflammation (44), especially at the early UC onset but it needs to be further investigated. Results of correlation analysis indicate that in children MDR1 negatively correlated not only with the levels of IFN-γ and IL-6 but also with the disease severity score, which was also previously shown for adults (45).
We are aware that, in addition to monitoring only mRNA levels of selected genes, the low number of patients is a limitation of this study. We, however, included well-defined subgroups of children and adults with UC. In comparison to adults, pediatric patients provide the insight into the initial immune response due to relative lack of confounding factors present in adults (46). Another limitation of the study is the Truelove and Witts scoring system for adult UC, which is not comparable to Pediatric Ulcerative Colitis Activity Index. Applying another scoring system for adults that is comparable to Pediatric Ulcerative Colitis Activity Index, such as simple clinical colitis activity index, would be more appropriate for future studies. To our knowledge, this is the first study showing decreased MDR1 and increased MRP1 expression in pediatric UC patients. The expression of MDR1 in UC seems to depend on disease activity and negatively correlates with severity of the disease and inflammatory cytokine levels.
Here we show a mixed Th1/Th17 colonic cytokine profile in both age groups with higher levels observed in children. It could be linked to dysregulated STAT1 and STAT3 pathways irrespective of SOCS1 and SOCS3 gene expression. A high IL-2 level in remission are, however, emphasizing possible protective role through Tregs.
1. 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
2. Autschbach F, Giese T, Gassler N, et al. Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis
and Crohn's disease. Virchows Arch
3. Fujino S, Andoh A, Bamba S, et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut
4. Niessner M, Volk BA. Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT-PCR). Clin Exp Immunol
5. Horino J, Fujimoto M, Terabe F, et al. Suppressor of cytokine signaling-1 ameliorates dextran sulfate sodium-induced colitis in mice. Int Immunol
6. Qin H, Holdbrooks AT, Liu Y, et al. SOCS3 deficiency promotes M1 macrophage polarization and inflammation. J Immunol
7. Heyman MMB, Kirschner BBS, Gold BDB, et al. Children with early-onset inflammatory bowel disease (IBD): analysis of a pediatric IBD consortium registry. J Pediatr
8. Castro M, Papadatou B, Baldassare M, et al. Inflammatory bowel disease in children and adolescents in Italy: data from the pediatric national IBD register (1996–2003). Inflamm Bowel Dis
9. Nieuwenhuis EES, Escher JC. Early onset IBD: what's the difference? Dig Liver Dis
10. Dietrich CG. ABC of oral bioavailability: transporters as gatekeepers in the gut. Gut
11. Englund G, Jacobson A, Rorsman F, et al. Efflux transporters in ulcerative colitis
decreased expression of BCRP (ABCG2) and Pgp (ABCB1). Inflamm Bowel Dis
12. Verdier J, Begue B, Cerf-Bensussan N, et al. Compartmentalized expression of Th1 and Th17 cytokines in pediatric inflammatory bowel diseases. Inflamm Bowel Dis
13. Hölttä V, Klemetti P, Salo HM, et al. Interleukin-17 immunity in pediatric Crohn disease and ulcerative colitis
. J Pediatr Gastroenterol Nutr
14. Olsen T, Rismo R, Cui G, et al. TH1 and TH17 interactions in untreated inflamed mucosa of inflammatory bowel disease, and their potential to mediate the inflammation. Cytokine
15. Coccia M, Harrison OJ, Schiering C, et al. IL-1β mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4+Th17 cells. J Exp Med
16. Zhang J, Fu S, Sun S, et al. Inflammasome activation has an important role in the development of spontaneous colitis. Mucosal Immunol
17. Shaw MH, Kamada N, Kim Y-G, et al. Microbiota-induced IL-1β, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine. J Exp Med
18. Zwicker S, Martinez GL, Bosma M, et al. Interleukin 34: a new modulator of human and experimental inflammatory bowel disease. Clin Sci
19. Stronati L, Negroni A, Pierdomenico M, et al. Altered expression of innate immunity genes in different intestinal sites of children with ulcerative colitis
. Dig Liver Dis
20. Drastich P, Frolova-Brizova L, Zanvit P, et al. Spontaneous in vitro IL-6 production in various intestinal segments in patients with inflammatory bowel disease. Folia Microbiol
21. Hanada T, Kinjyo I, Inagaki-Ohara K, et al. Negative regulation of cytokine signaling by CIS/SOCS family proteins and their roles in inflammatory diseases. Rev Physiol Biochem Pharmacol
22. Schreiber S, Rosenstiel P, Hampe J, et al. Activation of signal transducer and activator of transcription (STAT) 1 in human chronic inflammatory bowel disease. Gut
23. Chinen T, Kobayashi T, Ogata H, et al. Suppressor of cytokine signaling-1 regulates inflammatory bowel disease in which both IFN-γ
and IL-4 are involved. Gastroenterology
24. Alexander WS, Starr R, Fenner JE, et al. SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell
25. Strassheim D, Riddle SR, Burke DL, et al. Prostacyclin inhibits IFN-gamma-stimulated cytokine expression by reduced recruitment of CBP/p300 to STAT1 in a SOCS-1-independent manner. J Immunol
26. Suzuki A, Hanada T, Mitsuyama K, et al. CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation. J Exp Med
27. Tebbutt NC, Giraud AS, Inglese M, et al. Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice. Nat Med
28. Thagia I, Shaw EJ, Smith E, et al. Intestinal epithelial suppressor of cytokine signaling (SOCS) 3 enhances microbial induced inflammatory TNFα, contributing to epithelial barrier dysfunction. Am J Physiol Gastrointest Liver Physiol
29. Chen Z, Laurence A, Kanno Y, et al. Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells. Proc Natl Acad Sci
30. Croker BA, Krebs DL, Zhang J-G, et al. SOCS3 negatively regulates IL-6 signaling in vivo. Nat Immunol
31. Li Y, Nuij VJAA, Baars JE, et al. Increased suppressor of cytokine signaling-3 expression predicts mucosal relapse in ulcerative colitis
. Inflamm Bowel Dis
32. Mohammadnia-Afrouzi M, Zavaran Hosseini A, Khalili A, et al. Decrease of CD4(+) CD25(+) CD127(low) FoxP3(+) regulatory T cells with impaired suppressive function in untreated ulcerative colitis
33. Amado IF, Berges J, Luther RJ, et al. IL-2 coordinates IL-2-producing and regulatory T cell interplay. J Exp Med
34. Blokzijl H, Vander Borght S, Bok LIH, et al. Decreased P-glycoprotein (P-gp/MDR1) expression in inflamed human intestinal epithelium is independent of PXR protein levels. Inflamm Bowel Dis
35. Tahara T, Shibata T, Nakamura M, et al. Effect of MDR1 gene promoter methylation in patients with ulcerative colitis
. Int J Mol Med
36. Crowe A. The role of P-glycoprotein and breast cancer resistance protein (BCRP) in bacterial attachment to human gastrointestinal cells. J Crohns Colitis
37. Collett A, Higgs NB, Gironella M, et al. Early molecular and functional changes in colonic epithelium that precede increased gut permeability during colitis development in mdr1a(−/−) mice. Inflamm Bowel Dis
38. Masunaga Y, Noto T, Suzuki K, et al. Expression profiles of cytokines and chemokines in murine MDR1a−/− colitis. Inflamm Res
39. Panwala CM, Jones JC, Viney JL. A novel model of inflammatory bowel disease: mice deficient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis. J Immunol
40. Tanner SM, Staley EM, Lorenz RG. Altered generation of induced regulatory T cells in the FVB mdr1a-/- mouse model of colitis. Mucosal Immunol
41. Rothnie A, Conseil G, Lau AYT, et al. Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport. Mol Pharmacol
42. Wang L, Azad N, Kongkaneramit L, et al. The Fas death signaling pathway connecting reactive oxygen species generation and FLICE inhibitory protein down-regulation. J Immunol
43. Blokzijl H, van Steenpaal A, Vander Borght S, et al. Up-regulation and cytoprotective role of epithelial multidrug resistance-associated protein 1 in inflammatory bowel disease. J Biol Chem
44. Alvarez C, Amaral MM, Langellotti C, et al. Leukotriene C(4) prevents the complete maturation of murine dendritic cells and modifies interleukin-12/interleukin-23 balance. Immunology
45. Ufer M, Häsler R, Jacobs G, et al. Decreased sigmoidal ABCB1 (P-glycoprotein) expression in ulcerative colitis
is associated with disease activity. Pharmacogenomics
46. Bousvaros A, Sylvester F, Kugathasan S, et al. Challenges in pediatric inflammatory bowel disease. Inflamm Bowel Dis