Cardiotrophin-1 (CT-1) holds potent anti-inflammatory, cytoprotective, and anti-apoptotic effects in the liver, kidneys, and heart. In the present study, the role of endogenous CT-1 and the effect of exogenous CT-1 were evaluated in experimental ulcerative colitis. Colitis was induced in CT-1 knockout and wild-type (WT) mice by administration of dextran sulphate sodium (DSS) in the drinking water during 7 days. CT-1 knockout mice showed higher colon damage and disease severity than WT mice. In addition, CT-1 (200 µg/kg/day, iv) or vehicle (as control) was administered during 3 days to WT, colitic mice, starting on day 4 after initiation of DSS. Disease activity index (DAI), inflammatory markers (tumor necrosis factor α (TNF-α), INFγ, IL-17, IL-10, inducible nitric oxide synthase (iNOS)), colon damage, apoptosis (cleaved caspase 3), nuclear factor κB (NFκB) and STAT-3 activation, and bacterial translocation were measured. Compared with mice treated with DSS, mice also treated with exogenous CT-1 showed lower colon damage, DAI, plasma levels of TNFα, colon expression of TNF-α, INFγ, IL-17, iNOS and cleaved caspase 3, higher NFκB and signal transducer and activator of transcription 3 (STAT3) pathways activation, and absence of bacterial translocation. We conclude that endogenous CT-1 plays a role in the defense and repair response of the colon against ulcerative lesions through an anti-inflammatory and anti-apoptotic effect. Supplementation with exogenous CT-1 ameliorates disease symptoms, which opens a potentially new therapeutic strategy for ulcerative colitis.

Introduction

Cardiotrophin-1 (CT-1) is a 21.5-kDa member of the IL-6 family of cytokines that was named after its ability to stimulate the growth of cultured cardiac myocytes [1]. CT-1 acts by binding to a specific receptor complex containing gp130 and leukemia inhibitory factor receptor (LIFR) [2,3]. CT-1 is expressed in several cell types and tissues and exerts potent anti-apoptotic and cytoprotective effects on hepatocytes [4], cardiomyocytes [5], and neurons [6,7]. CT-1 exerts significant anti-inflammatory, anti-apoptotic, and cytoprotective effects in damaged tissues, including the heart [8,9], liver [10,11], kidneys [12,13] and nervous system [7,14], and induces nuclear factor κB (NFκB) activation [15]. The protective participation of endogenous CT-1 in diseases coursing with tissue damage and inflammation led us to hypothesize its potential role in inflammatory bowel diseases (IBD), such us ulcerative colitis (UC).

UC primarily involves disruption of colon mucosa integrity and loss of barrier function [16,17]. This disease is characterized by extensive, superficial mucosal ulceration with destruction of the epithelial architecture, loss of crypts and epithelial integrity, epithelial cell apoptosis [18–20], submucosal edema, and intense inflammatory cellular infiltration [21]. Although the precise inflammatory mechanisms involved in UC have not been fully described, several mediators, including NFκB [22–25], chemotactic peptides, and pro-inflammatory cytokines have been implicated in this disease [26–28]. Further knowledge of the underlying pathophysiological mechanisms and mediators will lead to the identification of new pharmacological targets and the development of new therapeutic strategies. UC affects mainly young patients and is characterized by the alternation of acute and remission phases. Together with the associated dysplasia and colorectal cancer, UC significantly impacts life quality and working ability [29–30]. The highest incidence of UC has been reported in northern Europe, especially the United Kingdom and Scandinavia [31,32], and in North America [33]. However, it is expected that over the next decade, Asian countries will have as many patients with IBD as the United States and European countries [34,35]. In addition, UC treatment is very expensive. For instance, in the United States, the total cost for UC prescriptions is greater than that of Crohn’s disease [36].

In this article, we showed that endogenous CT-1 participates in the physiopathology of experimental UC in mice with a protective and ameliorating role, and that supplementation with exogenous CT-1 further reduces the extent of damage, which may open new therapeutic opportunities.

Experimental

Unless otherwise indicated, all materials were purchased from Sigma–Aldrich (Madrid, Spain).

Animals

All protocols were reviewed and approved by the University of Salamanca Bioethics Committee. All studies were carried out in accordance with the Declaration of Helsinki Principles on the Advice on Care and Use of Animals referred to in: law 14/2007 (3 July) on Biomedical Research, Conseil de l’Europe (published in Official Daily N. L358/1-358/6, 18-12-1986), Government Spanish (Royal Decree 223/1 988, (14 March) and Order of 13-10-1989, and Official Bulletin of the State b. 256, pp. 31349–31362, 28-10-1990). Twenty-five 8-week-old male mice purchased from Charles River (Wilmington, MA, U.S.A.) were used. Additionally, four wild-type (WT) and four CT-1 null mice (CT-1−/−) backcrossed into a C57BL6 background were obtained from ‘Centro de Investigación Medica Aplicada’, University of Navarra, Pamplona, Spain. Only male littermates were used in order to minimize the possible effect of the physiological hormonal changes that occur in female due to estrus. Animals were group-housed under temperature- and humidity-controlled conditions, with a 12-h dark/12-h light cycle (University of Salamanca Animal Experimentation Service), and allowed free access to standard chow and tap water.

Induction of dextran sulphate sodium colitis and CT-1 administration

The dextran sulphate sodium (DSS)-induced colitis model exhibits many symptoms of human UC, such as diarrhea, bloody feces, body weight loss, mucosal ulceration, and shortening of the colorectum [37–39]. DSS-induced colitis in mice has a number of advantages, including simple experimental methods, and reproducibility and uniformity of the time course and severity of colitis [40]. In order to induce ulcerative colitis, 20 mice received DSS (MW 36000–50000, MP Biomedical, Solon, OH) added at 5% concentration to the drinking water during 7 days [41]. Four days after starting DSS, animals were divided into two groups with parity criteria on disease severity symptoms (weight loss, diarrhea, and blood in feces) in order to obtain two groups with similar average severity of the disease. The first group (Group DSS + CT-1, n=10) received CT-1 (200 µg/kg, iv) daily between 10:00 and 11:00 h, from the fourth day of DSS treatment and for 3 additional days. The second group (Group DSS, n=10) only received the vehicle (i.e. CT-1 solvent, namely isotonic saline). An additional group of mice acting as control (Sham; n=5) did not received DSS or CT-1. At the end of the study, a sample of blood was obtained by heart puncture, mice were killed and the colons were removed, cleaned, and proximal and distal colon samples were fixed in buffered 4% formaldehyde for histological studies. Other colon samples were frozen in liquid nitrogen for other measurements. Mesenteric lymph nodes (MLNs), the lungs, and the spleen were dissected in sterile conditions for microbiological studies.

Evaluation of colitis

To assess the effects of DSS treatment, survival and changes in body weight were monitored daily over the course of colitis development. Mice were monitored throughout the experiment and any that showed extreme distress, became moribund, or lost more than 20% of their initial body weight were killed. Severity of colitis was quantitated using the disease activity index (DAI) that was the sum of scores given for body weight loss, stool consistency, and presence or absence of fecal blood as described previously [42].

Histology

Histological scores

Formaldehyde-fixed, colon tissue samples were embedded in paraffin, cut into 3-µm thick slices, stained with Hematoxylin-Eosin and examined in a blind manner by an experienced pathologist. Sections were scored blindly by two independent investigators within a range from 0 to 3 as to the extent and depth of acute inflammation, and the amount of crypt damage, as described by Dieleman et al. [40].

Immunohistochemistry

Additional 3 μm sections were processed for immunohistochemistry. In brief, sections were deparaffined in xylene and rehydrated in graded ethanol concentrations. Endogenous peroxidase was blocked with 3% hydrogen peroxide, followed by primary antibody incubation. Primary antibodies used were: anti-CD68 mouse monoclonal antibody, 1:100 dilution (Dako Diagnósticos, Barcelona, Spain), anti-iNOS mouse monoclonal antibody, 1:500 dilution (Santa Cruz Biotechnology Inc, CA, U.S.A.), and anti-cleaved caspase 3 rabbit polyclonal antibody, 1:50 dilution (Cell Signaling Technology, MA, U.S.A.). Then, sections were washed three times in PBS and incubated with the Novolink Polymer Detection System® (RE7140-K, Novocastra, MA, U.S.A.), using 3,3′-diaminobenzidine as chromogen. Negative slides were prepared without primary antibody. Ten images per slide were captured with an optical microscope. Then, the images were digitalized and the number of cells expressing the antibodies was quantitated by using the ImageJ software (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, Maryland, U.S.A.).

Western blot

Western blot (WB) was performed with colon tissue extracts prepared from frozen colon portions, as previously described [43–45]. Tissue extracts were obtained by homogenizing colon samples with a tissue mixer (Ultra-Turrax T8, IKA®-Werwe, Staufen, Germany) at 4°C in homogenization buffer (140 mM NaCl, 20 mM Tris/HCl pH = 7.5, 0.5 M EDTA, 10% glycerol, 1% Igepal CA-630, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM PMSF). Samples were separated by electrophoresis in 10–15% acrylamide gels (Mini Protean II system, Bio-Rad, Madrid, Spain). Immediately, proteins were electrically transferred to an Immobilon-P membrane (Millipore, Billerica, MA, U.S.A.). Membranes were probed with primary and secondary antibodies described in Supplementary Table S1. WB quantitation was performed with the Image Quant Software (GE Healthcare, Madrid, Spain) after scanning the films on an Office jet 8500 scanner (Hewlett Packard, Madrid, Spain).

Reverse-transcription PCR

Total RNA was isolated from colon samples using Nucleospin RNAII (MACHEREY-NAGEL, Düren, Germany), according to the manufacturer’s instructions. Single-strand cDNA was generated from 2 μg of total RNA using poly-dT as primer with M-MLV reverse transcriptase (Promega, Barcelona, Spain). For reverse-transcription PCR (RT-PCR), 1 μl cDNA was used in a standard 50-μl PCR mixture with 400 nM of each primer and 2 U of FastStart Taq DNA polymerase (Roche, Barcelona, Spain). PCR products were separated by electrophoresis on 1% agarose gel and visualized by SybrSafe (Invitrogen, Barcelona, Spain) staining.

Quantitative RT-PCR was performed in triplicate. Each 20 μl reaction contained 1 μl cDNA, 400 nM of each primer, and 1× iQ SybrGreen Supermix (Bio-Rad, Madrid, Spain). Standard curves were run for each transcript to ensure exponential amplification and to rule out non-specific amplification. Gene expression was normalized to GAPDH expression. The reactions were run on an iQ5 Real-time PCR detection system (Bio-Rad, Madrid, Spain). The specific primers used for PCR are shown in Supplementary Table S2.

Plasma cytokines

Plasma levels of CT-1 (ELM-CT-1 RayBiotech, Norcross, GA, U.S.A.) and tumor necrosis factor α (TNFα; MTA00B, R&D Systems) were measured using commercial ELISAs, according to the manufacturers’ instructions.

Bacterial translocation

MLNs, spleens, and lungs removed under sterile conditions were weighed, homogenized in isotonic saline solution (10 ml/g of tissue) with a sterile grinder and then plated on to sheep’s blood agar, MacConkey agar, chocolate agar, and mannitol agar for the isolation of aerobic bacteria. Cultures were incubated at 37°C and examined at 24-h intervals for 3 days. For isolation of anaerobic bacteria, extracts were plated on 5% sheep’s blood Schaedler agar (Biomerieux, Madrid, Spain), and incubated in an anaerobic chamber at 37°C for 48 h. Blood was plated directly on to the medium. Any bacterial growth on the agar plates was quantitated and identified with standard microbiological methods [46].

Statistical analysis

Statistical analysis was performed using the NCSS software. The area under the curve (AUC) was calculated in each animal by the addition of the areas of the contiguous trapezes. Data were expressed as mean ± S.E.M. Differences between groups were assessed using one-way or two-way ANOVA as appropriate. Scheffe’s post-hoc test was used for multiple comparisons. P<0.05 was considered statistically significant. For data not conforming to a normal distribution, values were expressed as median and the Kruskal–Wallis Z-value test was used for multiple comparisons. Z > 1.96 was considered statistically significant.

Results

Lack of endogenous CT-1 impairs DSS-induced UC in mice

In order to study whether CT-1 participates in the pathophysiological process of UC in mice, we induced UC with DSS in CT-1 KO mice (CT-1−/−), and in WT mice as controls. Two in four of the CT-1−/- animals died at the fifth day of DSS treatment, whereas all the WT animals survived through the follow-up period. CT-1−/− animals lost more weight than WT animals (data not shown). Through the study, stool consistency decreased (i.e. stool consistency score increased) significantly more and earlier in CT-1−/− than in WT mice (Figure 1A). The AUC was 12.0 ± 0.8 arbitrary units (AU) in CT-1−/− and 4.1 ± 0.4 AU in WT mice (Z > 1.96). Fecal blood assessment yielded similar results: in CT-1−/− animals, the score increased progressively and was at all times higher than in WT animals (data not shown). The combined score (DAI) was also at all times higher in CT-1−/− than in WT mice (Figure 1B). Furthermore, the AUC was 11.7 ± 0.8 AU in CT-1−/− and 7.5 ± 0.6 in WT mice (Z > 1.96).The histological study evidenced severe, diffuse lesions in all specimens studied, occupying almost completely the colon mucosal surface. However, histological lesions observed in CT-1−/− animals were wider and more severe than those observed in WT mice (Figure 1C).

CT-1 gene knockout aggravates UC in mice

Figure 1
CT-1 gene knockout aggravates UC in mice

(A) Stool consistency score; (B) DAI; and (C) colon histology (Hematoxylin-Eosin staining), in WT and CT-1 knockout (CT-1−/−) mice. Bar: 200 µm. In (C), arrows indicate areas of mucosal damage in WT mice. In CT-1−/− mice, all the mucosal surface is severely damaged. Data are expressed as mean ± S.E.M. (n=4). *: P<0.05 compared with WT group.

Figure 1
CT-1 gene knockout aggravates UC in mice

(A) Stool consistency score; (B) DAI; and (C) colon histology (Hematoxylin-Eosin staining), in WT and CT-1 knockout (CT-1−/−) mice. Bar: 200 µm. In (C), arrows indicate areas of mucosal damage in WT mice. In CT-1−/− mice, all the mucosal surface is severely damaged. Data are expressed as mean ± S.E.M. (n=4). *: P<0.05 compared with WT group.

Colitic WT mice have higher plasma levels of CT-1 than controls

We then studied whether CT-1 was overexpressed in colitic mice. CT-1 content in colonic tissue (assessed by WB) tended to be lower in DSS-treated and control mice (Figure 2A). Similar results were observed for CT-1 gene expression in the colon (Figure 2B). However, plasma levels of CT-1 were higher in animals that received DSS than in control (sham) animals (Figure 2C).

Colon and plasma levels of CT-1 in control and colitic mice

Figure 2
Colon and plasma levels of CT-1 in control and colitic mice

(A) WB analysis of CT-1 and β-actin levels in colon tissue homogenates. (B) PCR analysis of CT-1 and GAPDH expression levels in colon tissue homogenates, and CT-1/GAPDH quantitation. (C) Plasma CT-1 levels. Data are expressed as mean ± S.E.M. (A) Sham, n=6; DSS, n=9; DSS + CT-1, n=8. (B) Sham, n=4; DSS, n=5; DSS + CT-1, n=5. (C) Sham, n=4; DSS, n=5; DSS + CT-1, n=5. *: P<0.05 compared with Sham group.

Figure 2
Colon and plasma levels of CT-1 in control and colitic mice

(A) WB analysis of CT-1 and β-actin levels in colon tissue homogenates. (B) PCR analysis of CT-1 and GAPDH expression levels in colon tissue homogenates, and CT-1/GAPDH quantitation. (C) Plasma CT-1 levels. Data are expressed as mean ± S.E.M. (A) Sham, n=6; DSS, n=9; DSS + CT-1, n=8. (B) Sham, n=4; DSS, n=5; DSS + CT-1, n=5. (C) Sham, n=4; DSS, n=5; DSS + CT-1, n=5. *: P<0.05 compared with Sham group.

Supplementation with exogenous CT-1 reduces the extent of colon damage

The previous results suggest that CT-1 is induced in experimental colitis and that, although not originated in the colon, it attenuates the extent of colon damage. We then reasoned that administration of exogenous CT-1 might further attenuate colitic damage. For this purpose, we treated colitic mice (i.e. DSS-treated mice) with CT-1 or with vehicle (as controls). No animals died during the study. Stool consistency and fecal blood scores increased during the period of DSS administration, and this effect was attenuated by co-administration of CT-1 (Figure 3A,B). AUC for stool consistency score was 8.95 ± 0.7 in DSS-treated mice and 3.9 ± 0.4 in DSS + CT-1 treated mice (P<0.01). AUC for fecal blood score was 7.2 ± 0.6 in DSS-treated mice and 2.8 ± 0.3 in DSS + CT-1 treated mice (P<0.01).The composed DAI score, markedly increased in DSS-treated mice, was also attenuated by co-treatment with CT-1 (Figure 3C). AUC for DAI score was 11.7 ± 0.8 in DSS-treated mice and 7.5 ± 0.6 in DSS + CT-1 treated mice (P<0.01).

Exogenous CT-1 ameliorates colitis

Figure 3
Exogenous CT-1 ameliorates colitis

(A) Stool consistency score; (B) fecal blood score; (C) DAI; and (D) colon histology (Hematoxylin-Eosin staining), in control mice (Sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). In (D), bar: 200 µm, arrows: areas of mucosal damage, asterisks: areas of massive infiltration. Data are expressed as mean ± S.E.M.; n=3 for sham and n=10 for DSS and DSS + CT-1 in (A–C), and n=60 (i.e. ten fields per mouse, six mice in each group) in (D). *: P<0.05 compared with WT group. #: P<0.05 compared with DSS group

Figure 3
Exogenous CT-1 ameliorates colitis

(A) Stool consistency score; (B) fecal blood score; (C) DAI; and (D) colon histology (Hematoxylin-Eosin staining), in control mice (Sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). In (D), bar: 200 µm, arrows: areas of mucosal damage, asterisks: areas of massive infiltration. Data are expressed as mean ± S.E.M.; n=3 for sham and n=10 for DSS and DSS + CT-1 in (A–C), and n=60 (i.e. ten fields per mouse, six mice in each group) in (D). *: P<0.05 compared with WT group. #: P<0.05 compared with DSS group

Histological examination revealed that typical ulcerative colonic alterations were observed in colitic mice, compared with sham mice, which did not show any structural alterations. Ulcers were focally distributed by the inner surface of the colon and invaded the entire thickness of the colon wall, penetrating even the lamina propria. In ulcerated areas, a total destruction of the crypts was seen with disappearance of epithelium, including goblet cells (arrows). Instead, granulation tissue and a large transmural inflammatory infiltrate occupied the ulcerated zones (asterisks). Peripheral tissue surrounded the colonic ulcers was normal, without alterations (Figure 3D). In colitic mice treated with CT-1, even though ulcers were observed mainly in distal colon, these were fewer than those found in untreated colitic mice, and their extent was also smaller. In many areas, although an infiltration of inflammatory cells in the lamina propria was occasionally detected (asterisks), the epithelium of the colon mucosa appears fairly well preserved, including goblet cells, and most of the colon has no structural alterations (Figure 3D).

As an index of tissue destruction, colonic apoptosis was assessed by the number of cells immunostained for cleaved caspase 3, a hallmark of apoptotic execution. It can be observed in the representative images (Figure 4A) and the corresponding quantitation (Figure 4B), that the number of cleaved caspase 3 positive cells in colon was very low in control animals and significantly higher in mice receiving DSS. Those animals receiving DSS + CT1showed a number of cells positive for cleaved caspase 3 in colon much lower than those receiving DSS alone (Figure 4A,B).

CT-1 reduces colon apoptosis in colitic mice

Figure 4
CT-1 reduces colon apoptosis in colitic mice

(A) Representative images of cleaved caspase 3 (cC3) staining in the colon, in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS+CT-1); Bar: 200 µm. (B) Number of cells positive for cleaved caspase 3 staining. Data are expressed as mean ± S.E.M. (A,B) n=60 fields quantitated; ten fields per mouse, six mice in each group. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

Figure 4
CT-1 reduces colon apoptosis in colitic mice

(A) Representative images of cleaved caspase 3 (cC3) staining in the colon, in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS+CT-1); Bar: 200 µm. (B) Number of cells positive for cleaved caspase 3 staining. Data are expressed as mean ± S.E.M. (A,B) n=60 fields quantitated; ten fields per mouse, six mice in each group. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

Consistent with a better preserved colonic wall, bacterial translocation was dramatically minimized by treatment with CT-1. Whereas no germs were isolated in any of the studied organs in the Sham group, in DSS-treated animals four amongst five animals presented bacterial translocation, mainly to the lungs, with isolation of Enterobacter cloacae (the most abundant bacteria found), Escherichia coli, Escherichia faecalis, Lactobacillus murinus, and less frequently Lactobacillus reuteri and Bacterioides vulgatus. E. cloacae, E. coli, and E. faecalis were isolated in mesenteric ganglia, and E. coli in the spleen, but in not all the animals were isolated germs neither in mesenteric ganglia nor in the spleen (Supplementary Table S3). In DSS-treated animals that also received CT-1, no germs were isolated, except for one animal suspicious of procedural sample contamination.

CT-1 reduces colonic inflammation

A key pathological event in colitis, i.e. inflammation, was clearly and significantly reduced by CT-1. The three histological scores used in the present study to evaluate inflammation (i.e. inflammation severity score (Figure 5A), inflammation extent score (Figure 5B), and crypt damage score (Figure 5C)) were lower in CT-1-treated than in untreated colitic animals. Both parameters of inflammation reached statistical differences, whereas crypt damage showed the same tendency. In general, the effect of CT-1 was more evident in proximal than in distal colon. Mucosal monocyte/macrophage infiltration (estimated by CD68+ cells) was increased in colitic mice, and this increment was clearly reduced by CT-1 (Figure 6A,B). The number of inducible nitric oxide synthase (iNOS)+ cells was also increased in colitic mice, and this number was also significantly lower in colitic mice treated with CT-1 (Figure 7A,B). iNOS expression was also confirmed by WB, which yielded similar results (Figure 7C).

CT-1 reduces colon inflammation in colitic mice

Figure 5
CT-1 reduces colon inflammation in colitic mice

(A) Inflammation severity. (B) Inflammation extent. (C) Crypt damage. Data are expressed as mean ± S.E.M. Sham, n=3; DSS, n=10; DSS + CT-1, n=10. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

Figure 5
CT-1 reduces colon inflammation in colitic mice

(A) Inflammation severity. (B) Inflammation extent. (C) Crypt damage. Data are expressed as mean ± S.E.M. Sham, n=3; DSS, n=10; DSS + CT-1, n=10. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

CT-1 mildens colon infiltration in colitic mice

Figure 6
CT-1 mildens colon infiltration in colitic mice

(A) Representative images of CD68+ cells in colonic tissue sections; and (B) quantitation of CD68+ cells, in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Bar: 200 µm. Data are expressed as mean ± S.E.M. n=60 fields quantitated; ten fields per mouse, six mice in each group. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

Figure 6
CT-1 mildens colon infiltration in colitic mice

(A) Representative images of CD68+ cells in colonic tissue sections; and (B) quantitation of CD68+ cells, in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Bar: 200 µm. Data are expressed as mean ± S.E.M. n=60 fields quantitated; ten fields per mouse, six mice in each group. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

CT-1 reduces colonic iNOS tissue level in colitic mice

Figure 7
CT-1 reduces colonic iNOS tissue level in colitic mice

(A) Representative images of iNOS+ cells in colonic tissue sections; (B) quantitation of iNOS+ cells; and (C) WB analysis of iNOS in colon tissue homogenates, from control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Bar: 200 µm. Data are expressed as mean ± S.E.M. (A,B) (n=60 fields quantitated; ten fields per mouse, six mice in each group). In (C), Sham, n=3; DSS, n=5; DSS + CT-1, n=5. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

Figure 7
CT-1 reduces colonic iNOS tissue level in colitic mice

(A) Representative images of iNOS+ cells in colonic tissue sections; (B) quantitation of iNOS+ cells; and (C) WB analysis of iNOS in colon tissue homogenates, from control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Bar: 200 µm. Data are expressed as mean ± S.E.M. (A,B) (n=60 fields quantitated; ten fields per mouse, six mice in each group). In (C), Sham, n=3; DSS, n=5; DSS + CT-1, n=5. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

A number of inflammatory mediators were increased in the colon of colitic mice, compared with those in control (sham) mice, indicating the occurrence of a manifesting inflammatory process. These included NFκB activation (assessed as the ratio p-p65/total p65 (RelA)) (Figure 8A), signal transducer and activator of transcription 3 (STAT3) activation (measured as p-STAT3/total STAT3) (Figure 8B), as well as INFγ, interleukin (IL) 17 (IL17) and IL10 gene expression (Figure 9A and B). Treatment with CT-1 produced a non-statistically significant increase in NFκB and STAT-3 activation (Figure 8A and B), and in IL10 gene expression (Figure 9A and B) and a non-statistically significant reduction in INFγ and IL17 gene expression (Figure 9A and B. Finally, plasma TNFα levels were much higher in colitic than in sham mice. Animals treated with CT-1 had lower plasma TNFα levels than those of untreated colitic mice, and not significantly different to those of the sham group (Figure 9C).

CT-1 increases NFκB and STAT3 activation in the colon of colitic mice

Figure 8
CT-1 increases NFκB and STAT3 activation in the colon of colitic mice

(A) WB analysis of NFkB and NFkB activation (pNFkB) levels in colon tissue homogenates, and densitometric quantitation. (B) WB analysis of STAT3 activation (pSTAT3) levels in colon tissue homogenates and densitometric quantitation. Experiments were carried out in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Data are expressed as mean ± S.E.M. (A) Sham, n=3; DSS, n=5; DSS + CT-1, n=5. (B) Sham, n=3; DSS, n=5; DSS + CT-1, n=4. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

Figure 8
CT-1 increases NFκB and STAT3 activation in the colon of colitic mice

(A) WB analysis of NFkB and NFkB activation (pNFkB) levels in colon tissue homogenates, and densitometric quantitation. (B) WB analysis of STAT3 activation (pSTAT3) levels in colon tissue homogenates and densitometric quantitation. Experiments were carried out in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Data are expressed as mean ± S.E.M. (A) Sham, n=3; DSS, n=5; DSS + CT-1, n=5. (B) Sham, n=3; DSS, n=5; DSS + CT-1, n=4. *: P<0.05 compared with Sham group; #: P<0.05 compared with DSS group.

Effect of the CT-1 on colon cytokine production in colitic mice

Figure 9
Effect of the CT-1 on colon cytokine production in colitic mice

(A) Representative images of agarose electrophoresis of PCR analysis of IL17, INFγ, IL10, and GAPDH levels in colon tissue homogenates. (B) Densitometric quantitations of IL-17/GAPDH, INFγ/GAPDH, and IL-10/GAPDH. (C) Plasma levels of TNFα. Experiments were carried out in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Data are expressed as mean ± S.E.M. Sham, n=3; DSS, n=4; DSS + CT-1, n=4. *: P<0.05 compared with Sham group.

Figure 9
Effect of the CT-1 on colon cytokine production in colitic mice

(A) Representative images of agarose electrophoresis of PCR analysis of IL17, INFγ, IL10, and GAPDH levels in colon tissue homogenates. (B) Densitometric quantitations of IL-17/GAPDH, INFγ/GAPDH, and IL-10/GAPDH. (C) Plasma levels of TNFα. Experiments were carried out in control mice (sham), colitic mice (DSS), and colitic mice treated with CT-1 (DSS + CT-1). Data are expressed as mean ± S.E.M. Sham, n=3; DSS, n=4; DSS + CT-1, n=4. *: P<0.05 compared with Sham group.

Discussion

Overall, the present study reveals that CT-1 participates in the pathophysiology of experimental UC in mice, with a protective and anti-inflammatory role. This is based on the higher severity of the disease in mice lacking CT-1, and in a less severe course of the disease (including fecal consistency, bleeding, and bacterial translocation), partial preservation of colonic wall structure, and less inflammation, in the colitic mice treated with CT-1, compared with the same parameters in untreated mice.

On the basis of our results, we can hypothesize that CT-1 is a mediator of the defense mechanism, and of the protective and repair response triggered in order to minimize and reverse colon injury. Our hypothesis is also congruent with a more general protective role of CT-1 in the organism, because CT-1 deficient mice are more susceptible than WT mice to ischemia/reperfusion liver damage and other pro-apoptotic insults, thus suggesting that CT-1 also functions as an endogenous protective molecule against liver injury [10]. Curiously, CT-1 is not overexpressed by the damaged colon. Because both control and colitic mice have a certain (and equal) basal expression of CT-1 in the colon, it may be speculated that CT-1 poses a permanently activated, housekeeping protective defense that mitigates damage until its buffering capacity is overtaken. This is also congruent with an additional protective effect exerted by extra CT-1 provided by exogenous administration. The protective and anti-inflammatory effect of exogenous CT-1 has been demonstrated in other pathologies involving tissue injury, such as the lungs [47], heart [48], liver [49], and kidneys [13]. However, in our study CT-1 appears elevated in the blood of colitic mice, suggesting that undetermined extracolonic sources of CT-1 might sustain or contribute to the protective effect of this cytokine. Further research is necessary to answer this question.

Inflammation is in the core of colitis pathophysiology. Colon inflammation is more likely a secondary response to tissue destruction than a primary response to DSS, and CT-1 has a strong anti-apoptotic effect in colitis. A theoretical argument might thus link reduction in apoptosis to reduction in inflammation, and it would pose the anti-apoptotic effect in the core of CT-1 action. These anti-apoptotic and cell surviving properties of CT-1 have been already described in several damage models in other organs, such as isolated myocardiocytes [50,51], in the liver [49,52,53], pancreatic β cells [54], neurons [55], and the kidneys [13]. Different IL-6 family members including CT-1 share the gp130 membrane receptor, which activates the JAK/STAT pathway [2,56,57]. The STAT-3 pathway is activated in colitic patients [58,59], but it seems to play opposing roles in UC. While STAT-3 favors colitis by enhancing the survival of pathogenic T cells participating in the acquired immune response and by disrupting immune tolerance [60], STAT-3 also activates the innate response to counteract colitis. In fact, activation of STAT3 potentiates and maintains the barrier function of epithelial cells and activates macrophages to protect the colon [61–64]. In addition, STAT3 is required for the induction of IL-10 [65,66], and of suppressor of cytokine signaling (SOCS) 3 [67–69], both of which are known to oppose colitic inflammation. All in all, the effect of STAT-3 activation in our model needs further investigation, although a net protective effect appears to prevail in our model, according to our overall results.

In addition to decreasing pro-inflammatory mediators (Figures 8 and 9), CT-1 further activates NFκB and IL-10. NFκB activation plays a role in both the onset and resolution of acute inflammation. NF-κB regulates immune responses and promotes cellular proliferation and survival [70], and mediates the protection afforded by CT-1 on cardiomyocytes [71], neurones [55], or kidney cells [13] ischemic damage. NFκB activation plays an anti-apoptotic role in many cell types and contributes to damage resolution [72,73]. In colitis, the anti-apoptotic functions of NFκB might be thought to improve epithelial cell survival and thus mucosal barrier integrity. And it has also anti-inflammatory effects by inducing apoptosis of inflammatory cells during the resolution phase of inflammation [74,75]. In fact, inhibition of NFκB during the resolution of inflammation is associated with an extended inflammatory response [72,73]. CT-1 also induces the synthesis and release of IL10, an important immunoregulatory cytokine [76] produced by many cell populations, with immunomodulatory as well as anti-inflammatory properties [77]. IL10 limits and terminates inflammatory responses and regulates differentiation and proliferation of several immune cells [78]. IL10-deficient mice develop lethal inflammation of the intestine, associated with increased concentrations of INFγ, TNFα, and IL1β, and these effects are reversed by administration of IL10 [79]. Administration of IL10 in several animal models of colitis reduces intestinal inflammation when administered before the initiation of colitis, but it is ineffective at reversing established inflammation [80]. In addition, IL10 gene transfer to the intestine prevented experimental colitis in rats [81] and in mice [82].

Microbiota plays a major role in the development of UC, and it could be hypothesized that CT-1 could modify the microbiota. However, there is no published evidence that CT-1 could have any effects on microbiota.

An important practical consequence of the present study is that CT-1 might be used for the treatment of colitis, specifically to acutely minimize incipient relapses as, although chronic exposure to CT-1 has been associated with organ fibrosis and functional impairment of the heart and kidneys [83], none of these effects have been observed when CT-1 is administered in low doses or for short periods [15]. Furthermore, CT-1 has already been approved for human use in several conditions by the U.S. Food and Drug Administration (FDA) as an orphan drug for the treatment of acute liver failure (designation request 11-3507) and for liver protection from ischemia/reperfusion injury inherent to the transplantation procedure (designation request 07-2449). The European Medicines Agency (EMA) has also granted this status for the prevention of ischemic/reperfusion injury associated with solid organ transplantation (EU/3/06/396). A clinical trial to assess the safety, tolerability, and pharmacokinetics of CT-1 in healthy volunteers has been registered in the United States at ClinicalTrials.gov (https://clinicaltrials.gov/; identifier NCT01334697, checked on 5 April, 2018).

In conclusion, our data demonstrate that endogenous CT-1 shows a limited protective capacity to oppose tissue damage during colitis. Supplementation with exogenous CT-1 provides additional protection, which might be potentially exploited in the clinical handling of acute relapses of colitis.

Clinical perspectives

  • UC is a disease with high prevalence in the developed countries, characterized by a high extent of colonic inflammation. CT-1 is a cytokine of the IL-6 superfamily with anti-inflammatory, anti-apoptotic, and cell surviving properties in several damage models in other organs, such as the heart and isolated myocardiocytes, liver, pancreatic β cells, neurons, and kidneys.

  • Our results demonstrate that endogenous CT-1 participates in the pathophysiology of experimental UC induced by DSS in mice with a protective and ameliorating role. In animals lacking CT-1, the disease is more severe than in control animals; and supplementation with exogenous CT-1 further reduces the extent of damage.

  • These results suggest that CT-1 administration might be potentially exploited in the clinical handling of acute relapses of colitis.

We thank Angustias Pérez, Marta Ortiz, Annette Düwell and Jose I. Sanchez-Gonzalez for technical help.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the Digna Biotech [grant number Digna 003/14]; the Fundación Salmantina para el Estudio de las Enfermedades Hepáticas [grant number FSEEH/14/02]; the Junta de Castilla y Leon [grant numbers GRS 1217/A/15 (to A.R.-P.), GR-100 (to J.M.L.-N.)]; and the Biomedical Research Institute of Salamanca (IBSAL) [grant number IBSAL15/04 (to A.R.-P.)].

Author contribution

A.I.S.-G., V.P.-V., A.R.-P., and J.M.L.-N. developed the study concept and design. A.I.S.-G., V.P.-V., V.B.-G., M.A., D.L.-M., and E.G.-S. performed acquisition of data. A.I.S.-G., V.P.-V., V.B.-G., D.L.-M., M.A., Y.Q., F.J.L.-H., A.R.-P., and J.M.L.-N. performed analysis and interpretation of data. J.M.L.-N. wrote the manuscript. V.P.-V., A.I.S.-G., V.B.-G., F.J.L.-H., A.R.-P., M.A., Y.Q., and E.G.-S. performed critical revision of the manuscript for important intellectual content. A.R.-P. and J.M.L.-N. obtained funding, technical and material support, and performed study supervision. All the authors approved the final version of the manuscript.

Abbreviations

     
  • AU

    arbitrary unit

  •  
  • AUC

    area under the curve

  •  
  • CT-1

    cardiotrophin-1

  •  
  • DAI

    disease activity index

  •  
  • DSS

    dextran sulphate sodium

  •  
  • GAPDH

    glyceraldehyde-3-phosphate-dehydorgenase

  •  
  • gp130

    glycoprotein 130

  •  
  • IBD

    inflammatory bowel disease

  •  
  • IFN-γ

    interferon-γ

  •  
  • IL

    interleukin

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • iv

    intravenous

  •  
  • JAK

    janus kinase

  •  
  • KO

    knock-out

  •  
  • M-MLV

    Moloney murine leukemia virus

  •  
  • MLN

    mesenteric lymph node

  •  
  • NFκB

    nuclear factor κB

  •  
  • RT-PCR

    reverse-transcription PCR

  •  
  • STAT3

    signal transducer and activator of transcription 3

  •  
  • TNFα

    tumor necrosis factor α

  •  
  • UC

    ulcerative colitis

  •  
  • WB

    Western blot

  •  
  • WT

    wild-type

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Author notes

*

These authors share first authorship.

Supplementary data