Expression of interleukin (IL)-19 and IL-24 in inflammatory bowel disease patients: a cross-sectional study

Abstract

Interleukin (IL)-19 and IL-24 belong to the IL-20 subfamily, and are involved in host defence against bacteria and fungi, tissue remodelling and wound healing. Nevertheless, no previous studies have explored their expression in Mexican mestizo patients with inflammatory bowel disease (IBD). The aim of the study was to characterize and to enumerate peripheral and tissue IL-19- and IL-24-producing cells, as well as gene expression in patients with IBD with regard to its clinical activity. We studied a total of 77 patients with ulcerative colitis (UC), 36 Crohn's disease (CD) and 33 patients as control group (without endoscopic evidence of intestinal inflammation). Gene expression was measured by real-time–polymerase chain reaction (RT–PCR). Protein expression was detected in biopsies by immunohistochemistry and in freshly isolated peripheral blood mononuclear cells by flow cytometry. IL-19 and IL-24 gene expression was elevated significantly in patients with active IBD versus the inactive disease and non-inflammatory control groups (P < 0·05). However, IL-19- and IL-24-producing cells were only increased in active CD versus active UC and non-inflammatory tissues (P < 0·05). IL-19 was produced conspicuously by circulating B cells and monocytes in patients with inactive disease (P < 0·05). Conversely, IL-24 was noticeably synthesized by peripheral B cells, CD4⁺ T cells, CD8⁺ T cells and monocytes in patients with active disease. In conclusion, IL-19- and IL-24-producing cells in active CD patients were increased compared with active UC and non-inflammatory tissues. These cytokines could significantly shape and differentiate inflammatory process, severity and tolerance loss between UC and CD pathophysiology.

Introduction

The two major forms of inflammatory bowel disease (IBD) include ulcerative colitis (UC) and Crohn's disease (CD) 1. Currently, the pathogenesis of UC and CD is not completely understood. Chronic relapsing inflammation is thought to be the result of a proinflammatory microenvironment and an aberrant immune response to intestinal flora in a context of genetic predisposition. The loss of immune tolerance towards the enteric flora is mediated by different molecules. Several proinflammatory and immunoregulatory cytokines are up-regulated in the mucosa of patients with IBD 2. None the less, differences and similarities in the cytokine profiles among UC and CD have not been elucidated fully; i.e. the interleukin (IL)-10 family of cytokines and its involvement in IBD has not been completely understood. The IL-10 family consists of nine related molecules with ranging degrees of sequence homology, including IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B and IL-29, which play multiple roles in regulation of inflammation, host defence mechanisms against bacteria and fungi, anti-viral response, tissue remodelling, prevention of tissue damage and wound healing. The currently known facts regarding the effects of IL-10, IL-19, IL-20 and IL-24 play an important role in the pathogenesis of some chronic inflammatory diseases 3,4.

IL-19 was discovered in 2000. It has been implicated in some diseases and disorders, such as psoriasis, type I diabetes, endotoxic shock, periodontal disease, vascular disease and rheumatoid arthritis 5,6. IL-19 is produced primarily by keratinocytes, epithelial cells, myeloid cells and B cells 7, and its expression can be induced by lipopolysaccharide (LPS), granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-4, IL-6, IL-13, IL-17 and tumour necrosis factor (TNF)-α, while interferon (IFN)-γ down-regulates its expression. Moreover, epithelial cells produce IL-19 after stimulation with IL-17, IL-22 and IL-1β 8.

Binding of IL-19 to its heterodimeric receptor complex (IL-20Rα/IL-20Rβ) activates the signal transducers and activators of transcription (STAT) pathways, mainly STAT-1 and STAT-3 9. The IL-19 role has been investigated in patients with psoriasis; these patients showed an increase of IL-19 levels in keratinocytes from affected skin, suggesting that IL-19 contributes to the inflammatory response during the innate host defence mechanism and plays a role in tissue remodelling and wound healing 10.

IL-19 is capable of promoting T helper type 2 (Th2) immune polarization through a positive feedback loop 11,12. Serum IL-19 levels in asthmatic patients have been found to be twice those from healthy control subjects and correlated with higher levels of IL-15 and IL-13 13.

Nevertheless, it has been demonstrated recently that IL-19 regulates the inflammatory process in acute and chronic conditions as well as inducing the mucosa healing of the intestinal epithelium in a mouse model of IBD 14. IL-19 has also been implicated in the induction of endotoxin tolerance that ‘reprograms’ activated macrophages. This prevents the massive release of proinflammatory mediators, such as TNF-α and IL-12, which characterizes an excessive inflammatory response to infectious agents leading to septic shock and death 15.

Interleukin 24 (IL-24) was first identified in 1995. Since its discovery as a tumour suppressor in healthy melanocytes, it was named ‘melanoma differentiation-associated gene 7’ (MDA-7). This cytokine is synthesized mainly by immune cells, keratinocytes and colonic subepithelial myofibroblasts and acts upon non-haematopoietic tissues such as skin, lung and reproductive tissues 16. Its expression by human peripheral blood mononuclear cells can be induced by pathogen-associated molecules, including phytohaemagglutinin (PHA), LPS, IL-4 and the influenza A virus. In contrast to IL-19, IL-24 is also expressed by T lymphocytes (predominantly Th2) 17. However, regardless of the co-expression with IL-10 in Th2 cells, T lymphocyte derived IL-24 seems to lack anti-inflammatory or immunoregulatory functions. The major target tissues, based on expression patterns of its receptor, are the skin, gut, lungs and reproductive tissues. IL-24 interacts with two heterodimeric receptor complexes, IL-20R1/IL-20R2 and, preferentially, IL-10R2/IL-22R1. IL-24 binding to these receptors results in the activation of STAT-1 and STAT-3 signalling pathways. IL-24 acts through its cell-surface receptors as a classical cytokine, and intracellularly as a cytotoxic agent, in a non-receptor-mediated manner. Both these receptors are abundant in several tissues, such as those from the reproductive system, skin, gut, respiratory system and various glands. Furthermore, human IL-24 induces chemotaxis of CD11b-positive myeloid cells 18,19.

Little is known about the presence of IL-19 and IL-24 cytokines in Mexican mestizo patients with IBD. Thus, the aim of this study was to investigate and to enumerate at peripheral and tissue levels the gene expression and presence of IL-19 and IL-24 proteins with regard to clinical activity and compared with patients without endoscopic evidence of intestinal inflammation and/or healthy donors.

Patients and methods

Study subjects

The study population included 113 IBD patients (36 CD and 77 UC) during the period December 2009–July 2010, from the Inflammatory Bowel Disease Clinic at the Instituto Nacional de Ciencias Médicas y Nutrición, diagnosed based on well-established clinical, endoscopic and histopathological criteria for CD (70·8% male; 30·2% female, mean age 44 years, range 18–72) and UC (5% male; 95% female, mean age 43 years, range 20–75). The cohort studied also included non-inflammatory control subjects (77% male; 33% female, mean age 49 years, range 21–74), who underwent colonoscopy for colon cancer screening, evaluation of suspected irritable bowel syndrome (IBS), polyp screening or abdominal pain. The study was approved by the ethical committee at our institution, and biopsies were obtained from IBD patients and non-inflammatory control subjects after informed consent. Relevant clinical and demographic information in all IBD patients – gender, age at diagnosis, previous appendectomy, disease evolution, extension, etc. – were collected from medical records.

An independent gastrointestinal pathologist reviewed the slides and classified IBD histologically as being either active or inactive. Active disease was defined histologically by the presence of neutrophilic inflammation, including cryptitis and crypt abscesses. Uninvolved mucosa was defined as mucosa free of endoscopically and histologically active or chronic inflammation. In inactive disease, chronic inflammation, crypt distortion and/or lymphoid aggregates were common, although there was no neutrophilic inflammation.

Colonoscopy was performed in order to calculate the Mayo Score Activity Index and take colonic biopsies. Disease extension was defined by colonoscopy. The disease activity was determined by Mayo score and Riley criteria 20 for endoscopic and histological activity, respectively. CD was diagnosed by clinical, laboratory, endoscopic, radiological and/or histopathological findings 21,22. Disease activity was determined by Harvey–Bradshaw and the Crohn's Disease Activity Index (CDAI).

Human ileal and colonic mucosal biopsies ileal and rect-sigmoid pinch biopsies were obtained from IBD patients in areas with active disease or from uninvolved colon. In non-inflammatory control subjects, biopsies were obtained from the ileum and colon.

Exclusion criteria included patients with indeterminate colitis, post-radiation colitis, infectious colitis and others.

Sample processing and gene expression analysis

The 113 intestinal mucosal biopsies taken from colonoscopy were placed immediately in RNAlater (Ambion, Austin, TX, USA) and stored at −70°C (short-term; <6 months) until used. Then total RNA was isolated using high pure RNA tissue (Roche Diagnostics, Mannheim, Germany), following the manufacturer's guidelines. Two hundred nanograms of total RNA was reverse-transcribed into cDNA with random hexamer primers (Roche Diagnostics).

The IL-19 and IL-24 gene expressions were measured by real-time–polymerase chain reaction (RT–PCR) (IL19: Genebank NM_153758·1, oligonucleotides 3′-CGAGCTCTCCCAGGGATT, 5′-CAGAGTCATCCATGACAACTATGAT, probe no. 74; and IL24: Genebank NM_006850·3 oligonucleotides 3′-CAGGGTGTGGACAAGGTAACA, 5′-CTCAGGATAACATCACGAGTGC, probe no. 89). Expression of glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene, (GAPDH: Genebank NM_0020463, oligonucleotides 3′-AGCCACATCGCTCAGACAC, 5′-GCCCAATACGACCAAATCC, probe no. 60) was analysed for normalization purposes and quality controls.

PCR amplification of the above-mentioned genes was carried out with 20 ng of cDNA, 200 nM forward and reverse primers and Taqman Master Mix (Roche Diagnostics) in a final volume of 10 μl. PCR reactions were run in a Light Cycler 2·0 (Roche Diagnostics) for 45 cycles, each cycle consisting of denaturation for 15 s at 95°, primer annealing for 15 s at 55°, extension for 30 s at 72°C and cooling 30 s at 40°C.

Immunohistochemistry

In order to determine IL-19- and IL-24-expressing cells, 4-μm-thick sections of available formalin-fixed paraffin-embedded tissue were placed on positively charged slides. Sections were deparaffinized and rehydrated through a series of xylene and graded alcohols. Endogenous peroxidase was blocked with 3% H₂O₂ for 20 min. A 3% normal serum was employed for 30 min as protein blocker. Tissues were incubated for 18 h at 4°C with goat polyclonal anti-human IL-19 antibody (Sigma-Aldrich, St Louis, MO, USA) or mouse monoclonal anti-human IL-24 antibody (R&D Systems, Inc., Minneapolis, MN, USA) at 10 μg/ml. Binding was detected by incubating sections for 60 min at room temperature with biotinylated donkey anti-goat immunoglobulin (Ig)G antibody or goat anti-mouse IgG antibody (ABC Staining System; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Slides were incubated with horseradish peroxidase (HRP)–streptavidin for 45 min, followed by incubation with peroxidase substrate 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich) for 10 min. The sections were counterstained with haematoxylin, dehydrated with alcohol and xylene and mounted in resin. Negative control staining was performed with normal human serum diluted 1:100, instead of primary antibody. The reactive blank was incubated with phosphate-buffered saline–egg albumin (Sigma-Aldrich) instead of the primary antibody. Both controls excluded non-specific staining or endogenous enzymatic activities. At least two different sections and two fields of mucosa, submucosa, muscular and adventitia were examined for each biopsy.

Peripheral blood cell isolation

A 15-ml sample of venous blood was obtained from each subject. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation on Lymphoprep (Axis-Shield PoC AS, Oslo, Norway).

Flow cytometry

To determine IL-19- and IL-24-expressing cells, PBMCs were labelled with anti-human CD14-phycoerythrin (PE) and CD4-PE cyanin 5 (Cy5), CD14-PE and CD8-PECy5 or CD80-PE and CD19-Cy monoclonal antibodies (BD Biosciences, San José, CA, USA) in separate tubes at room temperature in the dark for 20 min at 37°C. Cells were washed and permeabilized with 200 μl of cytofix/cytoperm solution (BD Biosciences) at 4°C for 20 min. After two washes with permwash solution (BD Biosciences), PBMCs were stained with goat anti-human IL-19 (Sigma-Aldrich) or mouse monoclonal anti-human IL-24 antibodies (R&D Systems, Inc.) for 30 min at 4°C in the dark. Then, cells were stained with fluorescein isothiocyanate (FITC)-labelled rabbit anti-goat antibody or FITC-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 15 min at 4°C in the dark. After three washes with permwash solution, PBMCs subsets were analysed by flow cytometry with a fluorescence activated cell sorter (FACScan). As a control of FITC-labelled rabbit anti-goat and FITC-conjugated goat anti-mouse antibody specificity staining, PBMCs were incubated with surface antibodies and FITC-labelled rabbit anti-goat and FITC-conjugated goat anti-mouse antibody in the absence of goat anti-human IL-19 or mouse anti-human IL-24 antibodies. An electronic gate was made for each of the surface markers employed (Fig. 4e–h). A total of 100 000–500 000 events were recorded for each sample and analysed with the CellQuestPro software (BD Biosciences). Results are expressed as the relative percentage of CD4⁺/CD14⁻/IL-19⁺-, CD8⁺/CD14⁻/IL-19⁺-, CD4⁻/CD8⁻/CD14⁺/IL-19⁺-, CD19⁺/CD80⁺/IL-19⁺-expressing cells in each gate and CD4⁺/CD14⁻/IL-24⁺-, CD8⁺/CD14⁻/IL-24⁺-, CD4⁻/CD8⁻/CD14⁺/IL-24⁺-, CD19⁺/CD80⁺/IL-24⁺-expressing cells in each gate (see below).

Fig. 4.

Interleukin (IL)-19-expressing peripheral blood cells. (a) Representative unstained and permeabilized peripheral blood mononuclear cells (PBMCs) sample (autofluorescence control) from an inflammatory bowel disease (IBD) patient analysed by flow cytometry. (b–d) Immunoglobulin (Ig)G₁-fluorescein isothiocyanate (FITC)/IgG₁-phycoerythrin (PE)/CD45-PE cyanin 5 (Cy5) mouse IgG₁, k isotype controls (BD Tritest™; BD Biosciences). (e) An electronic gate was made for CD14⁻ cells. CD4⁺/CD14⁻ single-positive T cells were determined from this gate. (f) CD4⁺/CD14⁻/IL-19⁺-expressing T cells were obtained. (g) An electronic gate was made for CD8⁺/CD14⁻ cells. (h) CD8⁺/CD14⁻/IL-19⁺ double-positive T cells were determined. (i) An electronic gate was made for CD19⁺/CD80⁺ double-positive B cells. (j) From the gate, (i) CD19⁺/CD80⁺/IL-19⁺ active B cells were determined. (k) An electronic gate was made for CD14⁺/CD4⁻/CD8⁻ monocytes. (l) From the gate (k), CD14⁺/CD4⁻/CD8⁻/IL-19⁺ double-positive monocytes were determined. The software employed was CellQuestPro (BD Biosciences). A total of 100 000–500 000 events are recorded for each sample before any gate setting.

As isotype controls, IgG1-FITC/IgG1-PE/CD45-PeCy mouse IgG1 k (BD Tritest™; BD Biosciences) (Fig. 4b–d) were used to set the threshold and gates in the cytometer.

In order to avoid a false positive and also for setting compensation for multi-colour flow cytometric analysis, we performed instrument calibration/standardization procedures each day according to the established protocols of our laboratory. Briefly, we run an unstained (autofluorescence control) and permeabilized PBMCs sample (Fig. 4a). Autofluorescence control (unstained cells) was compared with single-stained cell-positive controls to confirm that the stained cells were on scale for each parameter. Also, BD CaliBRITE™ 3 beads were used to adjust instrument settings, set fluorescence compensation and check instrument sensitivity (BD CaliBRITE™; BD Biosciences). Fluorescence minus one (FMO) controls were stained in parallel using the panel of antibodies with sequential omission of one antibody, with the exception of the anti-forkhead box protein 3 (FoxP3) antibody, which was replaced by an isotype control rather than simply omitted.

Ethical considerations

This work was performed according to the principles according to the Declaration of Helsinki. The study was approved by the ethical committee in our institution and a written informed consent was obtained from all patients.

Statistical analysis

Sample size was not determined for this study because it was merely observational.

Statistical analysis was performed using the SigmaStat version 11·2 program (Aspire Software International, Leesburg, VA, USA) by the Kruskal–Wallis one-way analysis of variance on ranks using the Holm–Sidak method for all pairwise multiple comparison procedures. Data were expressed as the median, range and mean ± standard deviation (s.d.)/standard error of the mean (s.e.m.). P-values smaller than or equal to 0·05 were considered significant. This study conforms to the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) statement, along with references to STROBE and the broader EQUATOR (Enhancing the QUAlity and Transparency Of health Research) guidelines 23.

Results

Demographic and clinical characteristics

A total of 53 female and 60 male patients with IBD were analysed. The mean age at diagnosis was 40 ± 2 years. The extent of disease was evaluated by using total colonoscopy and biopsies were taken from different segments of intestine in all cases. The demographic and clinical characteristics of the IBD patients and controls are shown in Tables 1 and 2.

Table 1.

Demographic and clinical characteristics of ulcerative colitis and Crohn's disease patients included in gene and protein expression analysis.

	Non-inflammatory control subjects (n = 23)	Active UC patients (n = 35)	Inactive UC patients (n = 18)	Active CD patients (n = 11)	Inactive CD patients (n = 15)
Variable
Age, years
Mean ± s.d.	49 ± 16	39 ± 11·5	47 ± 15	40 ± 2	37 ± 13·9
Median	50·0	38·0	42·0	38·0	30·0
Range	21–74	20–60	28–75	18–42	28–53
Sex
Female/male	12/11	18/17	12/6	3/8	4/11
Disease duration, years
<3		13%	20%	0	0%
>3		87%	80%	100%	100%
Treatment
Mesalazine		31	16	0	0
Azathioprine		7	7	10	13
Prednisone		4	4	5	9
Azulfidine		0	0	4	3
Mercaptopurine		0	0	8	8
Extra-intestinal manifestations
Absent		28	14	11	15
Present		7	4	0	0

CD = Crohn's disease patient group; UC = ulcerative colitis patient group; s.d. = standard deviation.

Table 2.

Demographic and clinical characteristics of ulcerative colitis and Crohn's disease patients included in flow cytometry analysis.

	Healthy donors (n = 14)	Active UC patients (n = 12)	Inactive UC patients (n = 12)	Active CD patients (n = 5)	Inactive CD patients (n = 5)
Variable
Age, years
Mean ± s.d.	47·0 ± 17·1	37·3 ± 9·5	40·3 ± 12·1	52·4 ± 21·2	47·5 ± 17·1
Median	36·0	39·0	40·0	58·0	36·0
Range	33–69	21–49	23–63	22–72	33–69
Sex
Female/male	7/7	7/5	5/7	2/3	2/3
Disease duration, years
<3		0%	25%	0%	20%
>3		100%	75%	100%	80%
Treatment
Mesalazine		11	9	3	0
Azathioprine		3	1	4	1
Prednisone		4	0	3	0
Azulfidine		1	2	0	0
Mercaptopurine		0	0	0	1
Extra-intestinal manifestations
Absent		10	8	5	5
Present		2	4	0	0
ESR, mm Hg
Mean ± s.d.		38·2 ± 24·7	7·7 ± 5·8†	29·6 ± 18·1	8·4 ± 2·9‡
Median		28·0	6·5	30·0	7·0
Range		18–90	2–17	10–50	6–12
CRP, mg/dl
Mean ± s.d.		1·3 ± 0·7	0·4 ± 0·3§	2·1 ± 0·5	0·4 ± 0·1¶
Median		0·9	0·4	1·7	0·4
Range		0·7–2·3	0·1–0·9	1·3–4·2	0·2–0·7

CD = Crohn's disease patient group; UC = ulcerative colitis patient group; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; s.d. = standard deviation.

^†aUC versus iUC, P = 0·005.

^‡aCD versus iCD, P = 0·032.

^§aUC versus iUC, P = 0·010.

^¶aCD versus iCD, P = 0·031.

IL-19 and IL-24 mRNA expression is increased in colonic mucosa from active IBD patients

IL-19 and IL-24 mRNAs were detected and quantitated by RT–quantitative PCR (qPCR) in colonic biopsies from UC patients, CD patients and non-inflammatory control tissues. Results showed that IL-19 mRNA expression was increased in colonic mucosa from patients with active UC when compared with non-inflammatory control group (Fig. 1a, P < 0·05). We also determined a significant difference among active CD versus non-inflammatory control tissues (Fig. 1a, P < 0·001). Lastly, higher levels of IL-19 mRNA were detected in active CD compared with inactive CD (Fig. 1a, P < 0·001). The IL-19 expression was associated significantly with a mild clinical course of UC characterized by one relapse within a year (P = 0·03, r² = 0·585). No significant differences were found in relation to IL-19 gene expression and other demographic and clinical characteristics such as age at diagnosis, gender and extent of disease, extra-intestinal manifestations, medical treatment and the need for surgery.

Fig. 1.

Interleukin (IL)-19 and IL-24 mRNA levels in colonic mucosa from patients with inflammatory bowel disease and controls. (a) IL-19 gene expression. (b) IL-24 gene expression. Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) was performed to assess mRNA levels in colonic mucosa biopsies from inflammatory bowel disease (IBD) patients. Results are expressed as mean ± standard error of the mean (s.e.m.) of IL-19 and IL-24 transcript levels with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene determined by 2^{Δ ΔCt}; differences among groups were assessed by Kruskal–Wallis test. aUC: ulcerative colitis patients with active disease, iUC: ulcerative colitis patients with inactive disease, aCD: patients with active Crohn's disease, iCD: patients with inactive Crohn's disease.

IL-24 mRNA expression were detected clearly in the samples from active and inactive IBD patients compared with non-inflammatory control tissues (P < 0·05, Fig. 1b). Analysis of the whole samples showed that IL-24 mRNA levels were higher in rectal mucosa from patients with active UC when compared with inactive UC (P < 0·05, Fig. 1b). An increase of IL-24 mRNA expression was determined in active CD patients versus inactive CD patients (P < 0·001, Fig. 1b).

IL-19 and IL-24 protein expression in biopsies from active IBD patients

In order to determine in-situ IL-19 and IL-24 protein expression in intestinal biopsies from active UC and active CD patients, tissues were immunostained and compared with non-inflammatory control tissue. The percentage of IL-19 and IL-24 immunoreactive cells was higher in active CD compared with UC patients and non-inflammatory control tissues. IL-19-producing cells were found mainly in mucosa, submucosa, adventitia and perivascular inflammatory infiltrates. IL-19 was expressed largely by myeloid cells, epithelial cells, fibroblasts, endothelial cells and lymphocytes, according to morphological identification (Fig. 2a,b).

Fig. 2.

Interleukin (IL)-19-expressing cells in biopsies from patients with ulcerative colitis or Crohn's disease. (a) Representative immunoperoxidase analysis in non-inflammatory control tissue (n = 5) (left panel), active Crohn's disease (CD, n = 5) tissue (middle panel) and active ulcerative colitis (UC, n = 6) tissue (right panel). Arrows depict immunoreactive cells in mucosa, submucosa, muscular and adventitia. Original magnification was ×320. (b) Percentage of IL-19-expressing cells in active inflammatory bowel disease (IBD) (CD and UC) patients. Results are expressed as mean ± standard deviation (s.d.).

In the same vein, IL-24 protein expression from intestinal biopsies from active CD patients was plentiful compared with active UC patients and non-inflammatory colonic tissue. IL-24-producing cells were localized mainly in mucosa, submucosa, adventitia and perivascular inflammatory infiltrates. It was determined morphologically that IL-24 was produced by lymphocytes, monocytes/macrophages, fibroblasts and endothelial cells (Fig. 3a,b).

Fig. 3.

Interleukin (IL)-24-expressing cells in biopsies from patients with ulcerative colitis or Crohn's disease. (a) Representative immunoperoxidase analysis in non-inflammatory control tissue (n = 5) (left panel), active Crohn's disease (CD, n = 5) tissue (middle panel) and active ulcerative colitis (UC, n = 6) tissue (right panel). Arrows depict immunoreactive cells in mucosa, submucosa, muscular and adventitia. Original magnification was ×320. (b) Percentage of IL-24-expressing cells in active inflammatory bowel disease (IBD) (CD and UC) patients. Results are expressed as mean ± standard deviation (s.d.).

IL-19-expressing peripheral cells in patients with UC or CD

Dysregulation of IL-20 subfamily cytokines results in inflammation and autoimmune disease. In order to determine the different subpopulations and frequency of circulating IL-19⁺-producing cells, CD4⁺ T cells, CD8⁺ T cells, CD14⁺ monocytes and CD19⁺ B cells were phenotyped (Fig. 4e–l).

Therefore, in active UC and CD patients, the relative percentage of IL-19⁺-producing CD4 T cells, IL-19⁺-producing CD8 T cells, active B cells and monocytes was decreased compared to the relative percentage of healthy donor cells (P < 0·05, Fig. 5). Interestingly, in remission the CD patient cell percentage of CD4 T cells, B cells and monocytes reached similar proportions to those found in healthy donors, with the exception of CD8 T cells (Fig. 5). Meanwhile IL-19-expressing cells from inactive UC patients had a statistically significant increase compared with active disease (P < 0·05, Fig. 5). None the less, cell frequency was lower compared with healthy donors (P < 0·05, Fig. 5). It is important to highlight that inactive CD patients had higher levels of IL-19-producing B cells and monocytes compared with inactive UC patients (P < 0·001).

Fig. 5.

Interleukin (IL)-19- and IL-24-expressing peripheral blood cells in patients with ulcerative colitis or Crohn's disease. Bar graphs show percentage of (a) CD4⁺/CD14⁻/IL-19⁺- and CD4⁺/CD14⁻/IL-24⁺-expressing T cells, (b) CD8⁺/CD14⁻/IL-19⁺- and CD8⁺/CD14⁻/IL-24⁺-producing T cells, (c) CD4⁻/CD8⁻/CD14⁺/IL-19⁺- and CD4⁻/CD8⁻/CD14⁺/IL-24⁺-expressing monocytes and (d) CD19⁺/CD80⁺/IL-19⁺- and CD19⁺/CD80⁺/IL-24⁺-producing active B cells. Results are expressed as mean (yellow bar), median (black bar), 10th, 25th, 75th, and 90th percentiles. *P < 0·05. HD: healthy donors, aUC: ulcerative colitis patients with active disease, iUC: ulcerative colitis patients with inactive disease, aCD: patients with active Crohn's disease, iCD: patients with inactive Crohn's disease.

Frequency of IL-24 cells circulating in patients with UC or CD

Interleukin-24 or MDA-7 regulates cell survival and proliferation by inducing rapid activation of STAT-1 and STAT-3. It has important roles in wound healing, psoriasis and cancer. For these reasons, IL-24-producing cell subpopulations were immunophenotyped and peripheral cell frequency was determined.

IL-24-producing CD8 T cells, CD19 B cells and CD14 monocytes frequency was increased conspicuously in UC and CD patients with clinical activity compared with inactive UC and CD patients and healthy donors (P < 0·05, Fig. 5). Conversely, peripheral cell frequency of CD4 and CD8 T cells, monocytes and B cells from inactive UC and inactive CD patients was lower compared with healthy donors and patients with clinically active disease (P < 0·05, Fig. 5). It is noteworthy that clinically active or inactive CD patients had higher levels of IL-24-expressing cells compared with clinically active or inactive UC patients, respectively.

Discussion

The IL-10 cytokine family has nine members, four of which are located in the IL10 cluster on chromosome 1q32. These cytokines are the immune regulatory cytokine IL-10 itself, and the IL-20 subfamily members IL-19 IL-20, and IL-24 24,25. IL-10 initiates innate and adaptive immune response and limits proinflammatory responses in order to prevent tissue damage. The IL-20 subfamily members are involved in host defence mechanisms, particularly from epithelial cells, and seem essential for tissue integrity. Dysregulation of IL-10 family cytokines results in inflammation and autoimmune disease 25–27. Azuma et al. have demonstrated that IL-19 is a negative regulator of TRL signalling, particularly controlling cytokines in macrophages, that it may play a function in endotoxin tolerance and that IL-19^−/− mice increases susceptibility to dextran sodium sulphate (DSS)-induced colitis, resulting in severe weight loss as well as death 14,16. These observations show that IL-19 has a crucial negative regulatory role in the inflammatory process during the innate response to pathogenic microbial stimuli, as well as inducing mucosa healing in IBD intestinal animal models 15. Conversely, it has been demonstrated that IL-19 is related to the development of T helper type 2 (Th2) responses in the pathogenesis of psoriasis 12,13.

IL-24 has also been demonstrated to play a role in the pathogenesis of IBD. IL-24 mRNA expression is elevated significantly in active lesions from patients with UC and CD. Furthermore, IL-24 derived from human colonic subepithelial myofibroblasts acts on colonic epithelial cells to elicit Janus kinase 1 (JAK-1)/STAT-3 activation and the expression of suppressor of cytokine signalling 3 (SOCS3) and membrane-bound mucins (MUC1, MUC3 and MUC4). Thus, properties of IL-24 suggest that it plays a mainly protective and suppressive role on mucosal inflammation in IBD mediating the innate immune response 17.

This is the first study to our knowledge in Mexican mestizo patients with inflammatory bowel disease (IBD) where IL-19 and IL-24 were evaluated at gene and protein expression levels in tissue and peripheral cells with regard to clinical activity. Thus, we found an increase of IL-19 and IL-24 mRNA levels in active UC and CD patients compared with healthy donors, as described previously 13,16. The mRNA levels of IL-19 and IL-24 do not show differences between treatment groups. None the less, the percentage of IL-19 and IL-24 immunoreactive cells in UC patients was comparable to non-inflammatory tissues. Meanwhile, IL-19- and IL-24-producing cells in CD patients were increased conspicuously in colonic mucosa. We suggest that the increase of IL-19 and IL-24 in active CD patients could be a compensatory mechanism in the anti-inflammatory response in order to regulate the acute inflammatory process. The overexpression of IL-19/IL-24 shows less severity of disease in Mexican mestizo CD patients compared with UC patients.

IL-19 expression was associated significantly with a mild clinical course of UC characterized by one relapse within a year (P = 0·03), suggesting a protective role of IL-19 in patients with UC due to its anti-inflammatory activity. We found no significant differences in relation to IL-19 gene expression and other demographic and clinical characteristics.

We also identified the different subpopulations and frequency of circulating IL-19⁺-producing cells, CD4⁺ T cells, CD8⁺ T cells, CD14⁺ monocytes and CD19⁺ B cells, and the results show that the relative percentage of CD8⁺/CD14⁻/IL-19⁺ T cells, CD19⁺/CD80⁺/IL-19⁺ active B cells and CD14⁺/CD4⁻/CD8⁻/IL-19⁺ monocytes was remarkably decreased in active UC and active CD patients compared with inactive disease and healthy controls. It would not be unreasonable to suggest that IL-19 may act at a local level and its regulation and synthesis depends upon tissue cell activation and cell migration (IL-19-producing B cells, CD8⁺ T cells and CD14⁺ monocytes) from periphery into the tissue; the latter correlates with the decrease of circulating IL-19⁺ cells. Meanwhile, IL-24 could act at local and systemic levels with regard to disease activity, as suggested by the conspicuous increase of circulating and tissue IL-24-producing cells.

It is important to highlight that IL-19 and IL-24 suggest a role as a cytokine in tissue repair processes rather than inflammation.

A clearer understanding of the mediators involved in intestinal inflammation will open new lines of research based on manipulation of the immune response for therapeutic purposes, such as administration of IL-10 (anti-inflammatory cytokine).

To date, there are no studies related to the clinical efficacy of recombinant IL-19 or IL-24 in IBD. None the less, basic research and data obtained from animal models suggest that these cytokines could be therapeutically useful for the down-regulation of IBD inflammation, as reported previously in IBD, atherosclerosis and cancer 14,16,17,28,29. Azuma et al. have shown that IL-19-deficient mice are more susceptible to experimental acute colitis induced by DSS, and this increased susceptibility is correlated with the accumulation of macrophages and the increased production of IFN-γ, IL-1β, IL-6, IL-12 and TNF-α. The finding that IL-19 drives pathogenic innate immune responses in the colon suggests that the selective targeting of IL-19 may be an effective therapeutic approach in the treatment of human IBD 14,16. In addition, there are other studies concerning the capability of IL-24 to suppress multiple signalling pathways, such as Src kinase in angiogenesis and up-regulating lysosomal proteases in autophagy and caspases 3 and 9 in apoptosis 29. It is important to highlight that IL-24 receptors (IL-22R1, IL-20R1 and IL-20R2) are expressed mainly in human epithelial colonic mucosa. These suggest that IL-24 is involved in the innate immune response, due to IL-24-induced selective activation of STAT-3 in colonic epithelial cells, but not in acquired immune cells. Moreover, it has been demonstrated that IL-24 stimulates MUC gene expression via JAK-1/STAT-3 activation, contributing to a protective role in the mucosa from IBD patients 17.

Even though this is a descriptive study, our findings are of interest because, as far as we know, this is the first depiction of the presence of IL-19 and IL-24 in IBD. Additional studies concerning IL-19 and IL-24 in the gut mucosal immune response and epithelial restitution can begin to support the immunoregulatory role of the IL-10 family in patients with inflammatory bowel disease.

Disclosures

The authors declare that they have no competing interests.