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Original Articles: Gastroenterology

Intestinal Expression of the Anti-Inflammatory Interleukin-1 Homologue IL-37 in Pediatric Inflammatory Bowel Disease

Weidlich, Simon*; Bulau, Ana-Maria*; Schwerd, Tobias*; Althans, Johanna; Kappler, Roland; Koletzko, Sibylle*; Mayr, Doris; Bufler, Philip*

Author Information
Journal of Pediatric Gastroenterology and Nutrition: August 2014 - Volume 59 - Issue 2 - p e18-e26
doi: 10.1097/MPG.0000000000000387

Abstract

Cytokines are key modulators of the intestinal immune response. An imbalance of pro- and anti-inflammatory cytokine release participates in the disruption of the otherwise tightly controlled immune system of the gut (1). The blockade of proinflammatory tumor necrosis factor-α is highly effective in treating both Crohn disease (CD) and ulcerative colitis (UC) (2). Alternatively, the inborn deficiency of anti-inflammatory interleukin (IL)-10 or its receptor was shown as the underlying cause of severe, early-onset pediatric inflammatory bowel disease (IBD) (3). Cytokines of the IL-1 family exhibit pro- and anti-inflammatory properties (4). IL-1β is a highly active proinflammatory cytokine, and IL-1 blocking monotherapies of autoinflammatory diseases are evolving in recent years (4). Although IL-1 family members IL-1α, IL-1β, IL-1Ra, and IL-18 have been well investigated, the functional properties of 7 additional members discovered from expressed sequence tags database searches are largely unraveled (IL-33, IL-36α, -36β, -36γ IL-36Ra, IL-37, IL-38) (5,6).

We identified IL-37b (formerly known as IL-1F7b) as a broad-spectrum inhibitor of innate immune responses (7). The IL-37 gene consists of 6 exons encoding 5 isoforms, of which isoform b is most abundantly expressed. IL-37 transcripts have been detected in a variety of tissues, including human gut (5,8–12). IL-37 protein localizes in the cytoplasm as well as the nucleus of peripheral blood mononuclear cell (PBMC) (11). Like other IL-1 family members IL-37 is processed by caspase-1 and caspase-4 (8). Nuclear translocation of IL-37 depends on caspase-1 cleavage (13); however, the functional impact of IL-37 maturation has not been resolved.

The anti-inflammatory properties of IL-37 were first described in murine RAW264.7 macrophage cells transfected with human IL-37, resulting in a reduced response to lipopolysaccharide (LPS) (13). Silencing of IL-37 in human PBMC induced increased levels of proinflammatory cytokines after LPS challenge (7). Transgenic mice expressing human IL-37b (IL-37tg) are protected against LPS-induced shock and have reduced dendritic cell activation (7).

Intracellular IL-37 binds Smad3, a core-signaling molecule of the TGFβ pathway (7,14). The inhibition of Smad3 activation reduces the anti-inflammatory properties of IL-37, indicating a functional interaction of both proteins (7). Extracellular IL-37b binds to the IL-18Rα, but there is no agonistic or antagonistic activity (8,11). IL-37 does not recruit the IL-18β chain. It is assumed that IL-37 recruits an accessory, anti-inflammatory receptor to the IL-18Rα complex (15).

Recently, we showed that IL-37tg mice are protected against acute dextran sodium sulfate (DSS)–induced colitis (16). IL-37tg mice showed less weight loss, lower disease activity scores, less reduction of colon length, and lower histological severity scores than wild-type (WT) mice. IL-37 expression in IL-37tg mice correlated with intestinal barrier breakdown, suppression of colonic proinflammatory cytokines, and an increase in anti-inflammatory IL-10. Interestingly, WT mice transplanted with hIL-37tg bone marrow were also protected from DSS-induced colitis, indicating that IL-37 expression in cells from hematopoetic origin is sufficient for the protective role of IL-37 in this model of disease. Prompted by these results we investigated the expression of IL-37 in pediatric IBD.

METHODS

Patients

Pediatric patients with IBD were diagnosed according to clinical, endoscopic, and pathological criteria. Disease severity was classified using the Pediatric Ulcerative Colitis Activity Index (PUCAI) (17) or the mathematically weighted Pediatric Crohn's Disease Activity Index (wPCDAI) (18) for clinical assessment and the Paris classification for disease location (19). Standard serum inflammatory markers (CRP [C-reactive protein], leucocytes, hematocrit, thrombocytes, erythrocyte sedimentation rate [ESR], fibrinogen, and albumin) as well as weight and height were evaluated at the time of biopsy sampling. Colonic biopsies were taken from macroscopically inflamed areas of 18 children with CD and 14 children with UC. Control biopsies were taken from 11 pediatric patients with juvenile polyps of the colon or Hirschsprung disease. Tissue samples were either embedded in paraffin (4% buffered formalin overnight) for histology and immunohistochemistry or immediately frozen in liquid nitrogen until preparation for real-time polymerase chain reaction (PCR). Written informed consent was obtained from parents or children themselves if older than 12 years. This study was approved by the local ethics committee of the Ludwig-Maximilians-University Munich.

Chemicals

All reagents were purchased from Sigma-Aldrich GmbH (Munich, Germany) unless indicated.

Histology

Paraffin-embedded slides were stained for hematoxylin and eosin and assessed for histological inflammation by a standardized score as described by Fell et al (20). Histological severity of inflammation was divided into 4 groups: no inflammation (0); slight active inflammation (1); moderate active inflammation (2); and strong active inflammation (3). This classification was done according to epithelial damage and changes in the crypt architecture as well as the increasing presence of granulocytes and lymphocytes as reported by the pathologist.

Tissue sections were stained with the following antibodies: IL-17 (IL-17 affinity-purified polyclonal goat IgG; R&D Systems, Abingdon, UK), IL-18 (IL-18/IL-1F4 propeptide polyclonal goat IgG; R&D Systems), monoclonal IL-37 (IL-1F7 monoclonal mouse antibody (Ab; Abnova, Heidelberg, Germany), and polyclonal IL-37 (IL 1F7/FIL1 zeta Ab, polyclonal goat IgG, R&D Systems). For antigen retrieval, deparaffinized slides were incubated in Target Retrieval Solution Citrate for IL-37 staining with the R&D Ab (Dako, Hamburg, Germany) or Pro Taqs II Antigen-Enhancer for IL-37 detection with the Abnova Ab (Quartett GmbH, Berlin, Germany) and Target Unmasking Fluid for IL-17 and IL-18 staining (Pan Path, Budel, Holland) in a 750-W microwave followed by slow cooling. Endogenous peroxidase activity was blocked by subsequent exposure to 7.5% hydrogen peroxide. For Abnova IL-37 Ab, endogenous biotin was blocked using Biotin Blocking System (Dako). All washing steps were done in Tris-buffer pH 7.5. After blocking, primary antibodies (dissolved after manufacturer's instructions) were applied for 1 hour at RT with dilutions of 1:30 (R&D Abs) and 1:40 (Abnova Ab) in PBS. Staining was detected with ImmPRESS Reagent Kit (Vector, Burlingame, CA) for R&D Abs or Vectastain ABC-Kit (Vector) for the Abnova Ab according to the manufacturer's instructions. Slides were stained for 3 min with DAB+ (Dako) (for all IL-37 Abs) or 10 minutes with AEC+ (Dako) (IL-17 and IL-18). Counterstaining was performed for 10 seconds with hematoxylin (Leica, Wetzlar, Germany) before mounting in Kaisers glycerin gelatine (Merck, Darmstadt, Germany). Images were obtained with a Confocal Microscope FV1000 (Olympus, Hamburg, Germany) in the light transmission mode. To correlate the staining intensity against IL-37 with histological severity of inflammation we established a scoring system: intensity of epithelial IL-37 staining was scored with 1 (weak expression) or 2 (strong expression). The number of IL-37 positive subepithelial cells was scored as follows: <25% cells positive for IL-37 (0); >25% (1); >50% (2); >75% (3), leading to a maximum of 5 possible points for strong IL-37 staining.

Isolation of RNA

Biopsies were immediately snap-frozen in liquid nitrogen. For RNA isolation frozen biopsies were homogenized for 30 seconds (Homogenizer T10 basic plus 5G dispersing element; IKA, Staufen, Germany) and RNA was isolated according to the provided protocol (RNeasy Mini Kit; Qiagen, Hilden, Germany). RNA quantity was measured with a NanoDrop photospectrometer. Only RNA with a 260 nm/280 nm ratio >1.8 was used for further analysis. RNA integrity was assessed with agarose gel electrophoresis.

Western Blot

Frozen tissue samples were lysated in RIPA-buffer (150 mmol/L NaCl, 1% Triton, 50 mmol/L Tris-Buffer, 0.5 mmol/L PMFS) with a homogenizer. Protein concentration was measured using Bradford method. Cell lysates were separated on a SDS-polyacrylamide gel (Any kD Resolving Gel; Bio-Rad, Munich, Germany) under reducing conditions and transferred onto a PVDF membrane (Immun-Blot PVDF Membrane; Bio-Rad). The membranes were stained by a mouse mAb against IL-37, as previously described (21), and developed with enhanced chemiluminescence reagent (Amersham ECL Prime Western Blotting Detection Reagent; GE Healthcare, Freiburg, Germany). Subsequently, membranes were stripped and stained against β-actin (monoclonal mouse IgG1; Santa Cruz Biotechnology, Heidelberg, Germany).

Quantitative Real-Time PCR

Two micrograms of DNase-digested RNA was transcribed into complementary DNA (cDNA) with random hexamers (20 ng/μL) (Qiagen) and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). PCR primers were designed using PrimerExpress (supplementary Table 1, http://links.lww.com/MPG/A319) and ordered from Metabion (Martinsried, Germany). Two microliters of cDNA and primers (0.4 pmol/μL) were added to iTaq SYBR Green Supermix with ROX (Bio-Rad). Each sample was run in triplicates on a 7900HT fast real-time PCR system (Applied Biosystems, Carlsbad, CA) for 40 cycles at 55°C annealing temperature. A melting curve was recorded. For standardization, TBP (TATA-box binding protein) was measured as a reference gene in each PCR run (22). PCR products were run on a 2% agarose gel to verify specificity. Fold changes of messenger RNA (mRNA) expression were calculated and normalized to TBP gene expression and to a healthy patient group using the ΔΔCt-method (23).

Statistical Analysis

Statistical analysis was performed with Microsoft Excel for Mac 2011 (Microsoft, Redmond, WA) and Prism GraphPad Version 5.0d for Mac OSX (GraphPad, La Jolla, CA). Statistical differences between clinical parameters and laboratory markers were compared using the Mann-Whitney U test. Statistical significance was assumed with a P < 0.05 (P < 0.05*, P < 0.01**, P < 0.001***). Correlation of real-time PCR data was done using linear regression and is indicated by the coefficient of determination R2 (range [0–1]).

RESULTS

Patient Characteristics

Pediatric patients with IBD were diagnosed according to clinical, endoscopic, and pathological criteria. Eighteen children with CD (median age 12.6 years), 14 children with UC (median age 13.3 years), and 11 control patients (median age 9.0 years) were investigated. In all, 3 groups, approximately one-third of the patients, were younger than 10 years. Disease severity was classified using the wPCDAI, with a mean score of 43 ± 26 (maximum number 125 points), or the PUCAI, with a mean score of 34 ± 12 (maximum number 85 points). Patients with CD had mainly ileocolonic or colonic inflammation, and involvement of the upper GI tract was found in 50% of patients. Most patients with UC (61%) had pancolitis (Table 1). Levels of CRP, ESR, leucocytes, and fibrinogen were elevated and hematocrit and albumin were lower in patients with IBD compared with healthy controls. ESR and CRP levels are higher in patients with CD than in those with UC (Table 1 and supplementary Figure 1, http://links.lww.com/MPG/A320 [Patient laboratory markers. Inflammatory markers such as CRP {A}, erythrocyte sedimentation rate {ESR} {B}, and numbers of leucocytes {C} were recorded for CD, UC, and healthy children as well as hematocrit {D}, fibrinogen {E}, and albumin {F}. The 3 groups were compared using the Mann-Whitney U test. Statistical significance was assumed with P < 0.05 (P < 0.05*, P < 0.01**, P < 0.001***.]). The majority of patients (26/32 were naïve to therapy and biopsies were obtained at time of diagnosis. Six patients received stable (>6 months) treatment with azathioprine, budesonide, 5-ASA, or infliximab (Table 1).

TABLE 1
TABLE 1:
Patient characteristics

Immunohistochemical Staining Against IL-37, IL-18, and IL-17

In our previous study we used a polyclonal goat IL-37 Ab (R&D Systems) for staining of mouse liver sections (24). This Ab was now tested for immunohistochemical staining of bowel tissue (Fig. 1A, E). A mouse IL-37 mAb (Abnova) showed a similar staining pattern (Fig. 1B). Control stainings with an IgG isotype were negative (Fig. 1C, D). Specificity of staining against IL-37 was confirmed by overnight preabsorption of the polyclonal goat IL-37 Ab with a 50-fold molar excess of recombinant IL-37 protein (21), which resulted in a significantly decreased intensity of IL-37 staining (Fig. 1F).

FIGURE 1
FIGURE 1:
Establishment of immunohistochemical staining against IL-37. A polyclonal goat antibody (R&D Systems, Minneapolis, MN) (A) and a monoclonal mouse antibody (Abnova) (B) show a similar staining pattern against IL-37 in healthy human colon. Isotype control (C) and system control (D) were negative. To test antibody specificity, the R&D antibody was preincubated overnight with 50-fold molar excess of recombinant IL-37 protein. Unblocked antibody (E) and preincubated antibody (F). IL = interleukin.

We next compared immunostainings against IL-37, IL-18, and IL-17 in ileal and colonic sections of 10 patients with CD, 8 patients with UC, and 6 healthy patients. IL-37 is strongly expressed in the ileal und colonic epithelium as well as in infiltrating subepithelial cells with a gradient of positive staining from luminal to basal (Fig. 2A). In addition to the strong cytoplasmic staining, few single lamina propria mononuclear cells also show nuclear expression of IL-37 (Fig. 2B). The majority of lymph follicle cells of the colon express IL-37 in the germinal center (Fig. 2C).

FIGURE 2
FIGURE 2:
Expression of IL-37 in healthy human colon. A, IL-37 is strongly expressed in the colonic epithelium as well as in infiltrating subepithelial cells with a gradient of positive staining from luminal to basal. B, Single cells show IL-37 staining of the nucleus. C, In lymph follicles of the colon a gradient can be seen showing that not all lymphocytes are expressing IL-37. The line on the bottom right indicates a distance of 100 μm. 100-fold magnification. IL = interleukin.

The pattern of ileal and colonic IL-37 staining is similar in CD, UC, and healthy bowel (Fig. 3A–D). The mean IL-37 expression score tended to be higher in inflamed tissues, ranging from a median score of 3.3 in samples without inflammation to 4.4 in samples with severe inflammation (Fig. 3E), but was not statistically significant.

FIGURE 3
FIGURE 3:
Expression of IL-37 in the ileum and colon of CD and UC patients. Expression of IL-37 in the ileum (A) and the colon (B) of a patient with CD and in the ileum (C) and colon (D) of a patient with UC. 100-fold magnification. The scale bar on the bottom right indicates a distance of 100 μm. A score for IL-37 staining was developed and plotted against a score of histologic disease severity (E). The mean score is shown as a line for every column. Western blot with mouse mAb antibody against IL-37 (F). Lysates of colonic tissue samples were isolated from CD, UC, and healthy control patients. CD = Crohn disease; IL = interleukin; UC = ulcerative colitis.

Lysates of 3 healthy patient biopsies, 2 UC patient samples, 8 CD patient samples, and recombinant IL-37 protein were separated on a reducing SDS-polyacrylamide gel. On Western blot 1 predominant band at 25 kDa corresponds to monomeric IL-37 and a second band at 50 kDa corresponds to IL-37 dimer. The intensity of IL-37 specific signals on the Western blot did not correlate with the pathological score of inflammation (Fig. 3F).

The immunohistochemical staining pattern of IL-18 is similar to that of IL-37. Mean IL-18 score was slightly higher in more inflamed tissue areas (supplementary Fig. 2A, http://links.lww.com/MPG/A321 [Evaluation of IL-18 and IL-17 immunohistochemistry. A score for IL-18 staining was developed in analogy to the IL-37 score and plotted against a score of histologic disease severity. The mean score is shown as a line for every column. {P < 0.01**}.]). As for IL-37, IL-18 staining was positive in the epithelium and in lamina propria cells but more limited to distinct cells. IL-18 staining was weaker in intestinal crypts than in villi. Epithelial IL-37 expression is equal in crypts and villi (Fig. 4C–F). IL-17 staining is limited to single mononuclear cells of the lamina propria (Fig. 4G, H). The total number of IL-17–positive cells was counted in 1 representative high-power field of vision of each patient, ranging from a mean of 18 cells in the control group to 46 in the CD group (P = 0.002) and 52 in the UC group (P = 0.002) (supplementary Fig. 2B, http://links.lww.com/MPG/A321 [Evaluation of IL-18 and IL-17 immunohistochemistry. The total number of IL-17–positive cells was counted in 1 representative high-power field of vision for each patient {P < 0.01**}.]).

FIGURE 4
FIGURE 4:
Staining pattern of IL-37 in comparison with IL-18 and IL-17 in the colon of patients with CD and UC. Upper row: (A) Hematoxylin and eosin staining and stainings against IL-37 (C), IL-18 (E), IL-17 (G) for a patient with CD. Lower row: (B) Hematoxylin and eosin staining, IL-37 (D), IL-18 (F), IL-17 (H) staining for a patient with UC. The scale bar on the bottom right indicates a distance of 100 μm, 100-fold magnification. CD = Crohn disease; IL = interleukin; UC = ulcerative colitis.

Expression of interleukin (IL)-37 messenger RNA (mRNA) in Comparison With Other Cytokines

Colonic biopsy samples of 5 healthy, 12 CD, and 8 UC patients were analyzed for IL-37b mRNA expression. Additionally, mRNA levels of IL-8, IL-18, IL-17, and anti-inflammatory IL-10 were investigated. In the CD group, mRNA levels of IL-37b (Fig. 5A, B) and IL-18 were not significantly elevated compared with control patients (IL-37, 2.7-fold, with P = 0.43; IL-18, 1.8-fold, with P = 0.42) (Fig. 5B). Levels of IL-8–, IL-17–, and IL-10–specific mRNA showed a significant increase in CD biopsies (IL-8, 172.0-fold, with P = 0.0002***; IL-17, 31.3-fold, with P = 0.002**; IL-10, 3.1-fold, with P = 0.05*). Similar results were observed in patients with UC (Fig. 5A, C). No difference was found in IL-37b and IL-18 expression (IL-37, 1.8-fold, with P = 0.65; IL-18, 1.3-fold with P = 0.65), but expression of IL-8, IL-17, and IL-10 was increased (IL-8, 182.7-fold, with P = 0.0001***; IL-17, 43.5-fold, with P = 0.0003***; IL-10, 4.2-fold, with P = 0.03*) (Fig. 5C).

FIGURE 5
FIGURE 5:
Expression of interleukin (IL)-37 messenger RNA (mRNA) in comparison with other cytokines using quantitative polymerase chain reaction (qPCR). mRNA was isolated from colonic tissue samples, complementary DNA was transcribed for qPCR. Logarithmic scale of fold change expression in comparison to a pool of healthy control patients is shown on the y-axis. A, Overview of IL-37 expression, each column represents one patient. A fold change of 1 indicates no difference in mRNA expression in comparison with the healthy patients. B, Fold changes of IL-37, IL-8, IL-17, IL-18, and IL-10 mRNA in the CD group. C, Fold change in the UC group. Significance levels were marked with *(P < 0.05), ***(P < 0.01), or ***(P < 0.001). CD = Crohn disease; UC = ulcerative colitis.

A significant correlation was observed in IL-37 and IL-18 mRNA fold changes (P = 0.0001, R2 = 0.856) (Fig. 6). Expression of IL-8–, IL-10–, and IL-17–specific mRNA did not correlate with IL-37 mRNA levels.

FIGURE 6
FIGURE 6:
Interleukin (IL)-37 and IL-18 quantitative polymerase chain reaction (qPCR) correlation. For qPCR, messenger RNA (mRNA) was isolated from colonic tissue samples and transcribed into complementary DNA. Both axes show a logarithmic scale of fold change expression. Correlation of IL-37 and IL-18 mRNA analyzed using linear regression (R 2 = 0.856).

DISCUSSION

IL-37 downregulates innate immune responses (7,13) and suppresses intestinal inflammation in mice (16). Here, we investigated the expression of IL-37 in the intestines of children with active CD and UC. IL-37 is expressed in epithelial and infiltrating immune cells of ileal and colonic biopsies from control patients as well as pediatric patients with CD and UC. The expression pattern of IL-37 and IL-18 was similar and differed from IL-17 expression that is restricted to single positive lymphoid cells of the subepithelial layer. IL-37 protein expression is increased with histological severity of inflammation.

The 18 CD and 14 UC patients of our study presented with clinical and inflammatory serum markers suggestive for active IBD (25,26). Systemic inflammatory parameters were worse in patients with CD considering transmural inflammation in CD but not UC. Disease localization and clinical assessment match well what has been described for children with IBD (27). Therefore, our cohort of pediatric patients to study intestinal IL-37 expression was characteristic for active CD and UC.

IL-37 shares critical amino acid sequence with IL-18 and binds to the IL-18BP and IL-18Rα (28). IL-18 expression is detected at increased levels in the colonic epithelium and inflammatory cells of the lamina propria in adult patients with active CD and UC (29). IL-17 plays a pivotal role in chemokine regulation and IL-17–positive cells are detected at the site of intestinal inflammation (30). We therefore investigated the expression of IL-37 along with IL-17 and IL-18 in pediatric patients with CD and UC.

Here, we established immunohistochemical staining against IL-37 by using 5 different antibodies. Two commercially available antibodies revealed the most specific and comparable staining pattern. The specificity of staining was proven by preabsorption of the antibodies by recombinant IL-37b protein that we previously expressed in Escherichia coli(11) and by Western blotting.

IL-37 protein is strongly expressed by intestinal epithelial cells as well as lamina propria lymphoid cells of control patients. In inflamed ileal and colonic tissue of children with CD and UC, IL-37 expression was increased with higher numbers of infiltrating IL-37–positive lymphoid cells compared with control biopsies; however, epithelial and lymphoid cells express IL-37 even in the absence of inflammation. Constitutive epithelial expression was also described for IL-1α (31). In the gut, constitutive epithelial expression of both inflammatory and anti-inflammatory immune mediators such as IL-1α (32) and IL-37 may be mandatory to maintain the homeostasis of the local immune response against commensal bacteria. We also speculate that increased IL-37, which is delivered by infiltrating lymphocytes, counteracts mucosal inflammation in IBD.

Single lamina propria cells show strong nuclear expression of IL-37. Previously we have reported that caspase-1 processing is required for nuclear translocation of IL-37 (13). Because the antibodies against IL-37 used for immunohistochemistry do not differentiate between pro- and mature IL-37b, we analyzed protein extracts of intestinal biopsies by Western blotting; however, we were not able to detect a minor band corresponding to mature IL-37b, indicating that the amount of mature IL-37 in the gut epithelium is low.

We next compared the expression pattern of IL-37 with IL-18. IL-18 is expressed in intestinal epithelial cells and in lamina propria cells that were previously identified as macrophages and dendritic cells (29). IL-18 and IL-37–positive cells are detected in corresponding tissue areas, and both IL-37 and IL-18 are expressed in the epithelium. Although IL-18 staining was weaker in intestinal crypts than in villi, epithelial IL-37 expression is equal in crypts and villi. In our stainings, the main source for IL-18 is the epithelium, whereas IL-37 is more abundant in local and infiltrating immune cells.

Bone marrow transfer from human IL-37tg mice protected WT mice from DSS-induced acute colitis (16). This indicates that IL-37 expression from infiltrating immune cells is more relevant than IL-37 released from epithelial cells to control intestinal inflammation in acute experimental colitis.

In contrast to IL-37 and IL-18, IL-17 staining is limited to single cells located in subepithelial layers or infiltrating epithelial lymphocytes as previously shown (30). IL-17–positive cells are not linked to areas of strong IL-37 staining.

Different isoforms of IL-37 have been described in various human tissues (28). We therefore tested by real-time PCR which isoform of IL-37 is most abundantly expressed in human bowel, liver, and PBMC. By using isotype-specific primers we only detected the isoform IL-37b in healthy and inflamed human bowel and liver samples as well as PBMC (data not shown). Hence, in our real-time, quantitative PCR experiments, only isoform b was measured. In contrast to IL-8 and IL-17 mRNA (30,33), steady-state levels of IL-37b and IL-18 mRNA were not significantly upregulated in colonic biopsies of children with CD and UC compared with healthy intestinal tissue even if a subgroup of more severely affected children with CD and UC (PCDAI and PUCAI >40) showed a trend toward higher IL-37b mRNA levels (supplementary Fig. 3, http://links.lww.com/MPG/A322 [Expression of IL-37 mRNA according to clinical IBD manifestation. IL-37 mRNA fold changes were plotted against clinical severity of disease, distinguishing 2 subgroups of slight {score < 40} and severe {score >40} clinical inflammation. The bars indicate mean and standard error of mean within each subgroup. Results were not statistically significant.]). We previously demonstrated that IL-37 mRNA contains a coding region instability element inducing a rapid decay of inflammation-induced mRNA levels (21). This explains the lack of elevated steady state levels of IL-37b mRNA in intestinal biopsies of our cohort of patients with IBD despite elevated IL-37 protein expression by infiltrating immune cells.

IL-10 acts as a key mediator in the maintenance of gut immune homeostasis (34). As expected, anti-inflammatory IL-10 mRNA was increased with bowel inflammation. IL-37 does not directly induce IL-10 because IL-10 production is not altered in IL-37 expressing RAW macrophages (13). Although IL-37tg mice also express higher levels of IL-10 in DSS-induced colitis, the protective role of IL-37 was attributed to an IL-37-driven downregulation of tumor necrosis factor-α and IL-1β because blocking of IL-10R did not alter the severity of colitis (16).

Although both IL-37 and IL-18 mRNA levels were not significantly upregulated in the colonic tissue of children with CD and UC, we observed a positive correlation of quantitative mRNA levels between both cytokines. This indicates similar regulatory mechanisms to control expression of IL-18 and IL-37 in vivo. Similar mechanisms to regulate expression of pro- and anti-inflammatory immune mediators such as IL-18 and IL-37 may protect the organism of an exaggerated, potentially deleterious immune response.

With the writing of this manuscript, a research group from Japan published the intestinal expression of IL-37 in the inflamed bowels of adult patients with IBD (12). This group established immunohistochemistry against IL-37 using the same polyclonal goat anti–IL-37 Ab as we used in our study and found a similar expression pattern of IL-37. In contrast to our results, adult patients with high activity scores for CD and UC showed stronger IL-37 protein expression than healthy subjects. All of the patients of the Japanese study were treated with salicylates and steroids, but we did not see differences in IL-37 expression in patients with UC treated with steroids. It is possible that our patients had only moderate or an early stage of disease and therefore lower levels of IL-37 expression in the inflamed gut. Another explanation could be that the developing intestinal immune system of children needs higher levels of immunomodulatory cytokines to maintain mucosal homeostasis than the mature immune system of adults.

Single-nucleotide polymorphisms resulting in a decrease in NLRP3 expression contribute to CD susceptibility (35). This was an unexpected finding because the NLRP3 inflammasome activates caspase-1 and caspase-1 is needed to activate proinflammatory IL-1β and IL-18. In accordance with an increased CD susceptibility in humans, NLRP3-deficient mice are hyperresponsive to DSS-induced colitis (36). Because caspase-1 is also needed to process anti-inflammatory IL-37, it may be speculated that the increased susceptibility to CD in patients with single-nucleotide polymorphisms within the NLRP3 gene is in part to be attributed to the impaired activation of anti-inflammatory IL-37. Future studies are warranted to delineate the specific contribution of IL-37 to modulate chronic bowel inflammation in humans.

Acknowledgments

The authors thank Andrea Sendelhofert and Anja Heier for excellent technical assistance establishing immunohistochemical stainings.

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Keywords:

cytokines; interleukin-37; immunohistochemistry; inflammatory bowel disease; pediatric

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© 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,