The ICE Inhibitor Pralnacasan Prevents DSS-Induced Colitis in C57BL/6 Mice and Suppresses IP-10 mRNA but Not TNF-α mRNA Expression
- First Online:
- Cite this article as:
- Bauer, C., Loher, F., Dauer, M. et al. Dig Dis Sci (2007) 52: 1642. doi:10.1007/s10620-007-9802-8
- 376 Views
Previously we demonstrated an ameliorating effect of the interleukin-1ß converting enzyme (ICE) inhibitor pralnacasan on dextran sulfate sodium (DSS)-induced colitis. This study investigates the effects of pralnacasan on cytokine expression in DSS-induced colitis. Colitis was induced by oral administration of DSS. Mice were treated intraperitoneally with the ICE inhibitor pralnacasan (50 mg/kg body weight twice daily). Body weight as well as the presence of occult blood or diarrhea was monitored daily. Subgroups were sacrificed at days 4, 8, and 11 after the beginning of DSS application. Cytokine profiles in colonic tissue were analyzed on the protein level by ELISA and on the mRNA level by real time RT-PCR. Administration of DSS led to an increase in IL-18, IL-12, TNF-α, and IFN-γ protein as well as IP-10 and TNF-α mRNA. The increase in IL-18 and IFN-γ was reduced by ICE inhibition. Pralnacasan prevented DSS-induced colitis in C57BL/6 mice. In C57BL/6 mice, the DSS-induced increase in IP-10 mRNA, but not TNF-α mRNA, was completely prevented by ICE inhibition. In conclusion, prevention of colitis in C57BL/6 mice was associated with a suppresion of IP-10 mRNA, but not TNF-α mRNA expression, indicating that IL-18-mediated cytokine production is a key element in the pathogenesis of DSS-induced colitis.
KeywordsColitisCrohn's diseaseCytokinesDextran sulfate sodiumUlcerative colitisReal-time PCR
Inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are chronic inflammatory diseases of the gut that ultimately lead to the destruction of the mucosal barrier. Both disease entities are mediated by proinflammatory cytokines. Until recently it was assumed that Crohn's disease but not ulcerative colitis is due to an excessive activation of Th1 cells with elevated levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-18, and IL-12 in the intestinal tissue . Pizarro et al. have shown that activated macrophages secrete IL-12 and IL-18 in Crohn's disease . However, anti-TNF strategies, e.g., infliximab, have been demonstrated to be efficacious not only in Crohn's disease but also in ulcerative colitis . Thus, the strict differentiation of Crohn's disease and ulcerative colitis as Th1- and Th2-polarized disease entities has been modified in the light of a refined understanding of cytokine expression patterns. Apparently, an initial antigenic stimulus is necessary for the induction of inflammatory bowel disease. Normal colonic flora seems to be a decisive factor in the inflammatory cascade. Duchmann et al. have shown that only mucosal macrophages isolated from inflamed areas of the colon of patients with IBD, and not from disease-free areas, proliferate when brought into contact with autologous colonic bacteria . The epithelium is another important factor in mucosal immunity . It has been shown that mucosal permeability is increased in IBD .
Cytokines play a central role as mediators of inflammation. TNF-α is expressed mainly by intestinal macrophages, but also by epithelial cells, inducing an upregulation of MHCII molecules on macrophages. Various strategies have been applied in the past decade to prevent the destructive effect of this cytokine . The anti-TNF-α antibody infliximab has been used clinically for the therapy of Crohn's disease since 1998 [8, 9]. IL-18, initially described as IFN-γ-inducing factor, acts synergistically with IL-12 by inducing IFN-γ synthesis of T cells and NK cells . We and others have demonstrated that anti-IL-18 strategies were efficacious in animal models of IBD [11, 12].
While anti-TNF-α antibodies have been successfully introduced into clinical practice, there are immunological drawbacks of protein-based therapies. Repeated application of infliximab leads to the formation of antibodies against the therapeutic antibody, making therapy ineffective . Moreover, infliximab has to be administered intravenously. One strategy to overcome these drawbacks has led to the development of novel antibody-based therapeutics such as adalimumab and certolizumab, which have a reduced risk of antibody-antibody development [14, 15]. In addition, these substances can be administered subcutaneously. Another therapeutic strategy is based on small molecules designed to act on the molecular level and inhibit the formation or biological activation of proinflammatory cytokines. The IL-1β converting enzyme (ICE), which activates IL-18 and IL-1β by molecular cleavage, represents a promising target for this strategy .
ICE is a member of the family of aspartate-specific cysteine proteases (caspases). Also known as caspase-1, the enzyme is expressed at low levels in most tissues, but it is expressed constitutively at high levels in monocytes, macrophages, and, to a lesser extent, neutrophiles [17, 18]. Active ICE cleaves the pro-proteins of IL-18 and IL-1β, thereby releasing soluble IL-18 and IL-1β. In contrast to IL-1β, IL-18 mRNA and precursor protein are constitutively expressed in a wide panel of cell types. Siegmund et al. have demonstrated a key role of ICE in the pathophysiology of DSS-induced colitis by showing that ICE-deficient mice were less susceptible to DSS-induced colitis, thereby suggesting a Th1 pathway in this model . Based on these findings, targeted inhibition of ICE may represent a successful rationale in the treatment of DSS-induced colitis.
The ICE inhibitor pralnacasan exerts anti-inflammatory effects by reducing the activation of IL-1β and IL-18. Pralnacasan reduces inflammation and joint destruction in models of murine arthritis [20, 21]. In a phase II clinical trial pralnacasan was effective in the treatment of rheumatoid arthritis . Recently we have reported that pralnacasan ameliorates DSS-induced colitis . The present study further delineates the effect of pralnacasan in different mouse strains. Different from our prior work, which demonstrated therapeutic efficacy of pralnacasan in a murine model of inflammatory bowel disease, here we use pralnacasan as a pharmacologic tool to investigate the pathophysiology of DSS-induced colitis. The effect of ICE inhibition on the induction of the inflammatory response during DSS administration is characterized by studying cytokine profiles in the gut of DSS-treated animals on the mRNA and on the protein level. In particular, we studied the effect of pralnacasan on IL-18-mediated IFN-γ protein and IP-10 mRNA levels as well as TNF-α mRNA levels in the time course of DSS-induced colitis.
Materials and Methods
Female, 8-week-old Balb/c and C57BL/6 mice (Harlan Winkelmann, Borchen, Germany) weighing 20 to 22 g were housed in rooms at a controlled temperature and a 12-hr day-night rhythm. They were fed standard mice chow pellets, had access to tap water supplied in bottles, and were acclimatized to the environmental conditions for at least 7 days before they were studied in experiments. Mice were killed by cervical dislocation under isoflurane anesthesia (Forene; Abbott GmbH, Wiesbaden, Germany). All experiments were approved by the regional animal study committee and are in agreement with the guidelines for the proper use of animals in biomedical research. Both animal handling and clinical and histological scoring of colitis were performed in a blinded experimental design.
For i.p. use, pralnacasan was dissolved in 25% Cremophor EL solution. The substance was filtered through syringe filters (0.2 μm) purchased from Gelman Sciences (Ann Arbor, MI, USA). As demonstrated in previous experiments, Cremophor EL alone had no effect on the clinical course of DSS-induced colitis and did not alter cytokine profiles.
Induction of Colitis and Treatment
DSS (ICN, Aurora, OH, USA) was dissolved in tap water. Balb/c mice were fed DSS at a concentration of 3.5% ad libitum (days 1 to 10) as described ; C57BL/6 mice received 1.5% DSS. Control mice were fed with tap water. Pralnacasan was administered intraperitoneally at a dosage of 50 mg/kg body weight twice daily. Control animals received Cremophor EL i.p. twice daily. The volume for i.p. application was 200 μl.
Body weight, the presence of occult or gross blood per rectum, and stool consistency were determined by two investigators blinded to the treatment groups. A scoring system described was applied to assess diarrhea and the presence of occult or overt blood in the stool. Changes of body weight are indicated as percentage loss of baseline body weight.
Synthesis of Colonic Cytokines
mRNA Extraction and RT-PCR
Colonic tissue was cleaned in phosphate-buffered saline (PBS), snap-frozen in liquid nitrogen, and stored at −70°C. Total cellular RNA was isolated by homogenizing tissue with an Ultra Turrax instrument (Janke und Kunkel, Staufen im Breisgau, Germany) and using the Roche Total RNA Tissue Extraction Kit (Roche, Mannheim, Germany). RNA was stored with an equal volume of ethanol at −80°C until use. Total RNA was collected by centrifugation for 15 min at 12,000 g at 4°C. The pellet was washed with 70% ethanol before resuspension in water. The yield and purity of the RNA were determined by spectroscopic analysis and the concentration of total RNA was equilibrated. For reverse transcription M-MLV Reverse Transcriptase (Gibco Life Technologies, Paisley, UK), RNase Inhibitor (Roche), oligo(dT) Primer for cDNA synthesis (Roche), and dNTP (Promega, Madison, WI, USA) were used. Conventional PCR was performed with TaqDNA polymerase, under conditions recommended by the supplier (Roche). Primers were manufactured by Applied Biosystem (Weiterstadt, Germany) based on sequences from previous publications [25, 26]: murine β-actin (348 bp; sense, 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′; antisense, 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′), murine TNF-α (307 bp; sense, 5′-GGC AGG TCT ACT TTG GAG TCA TTG C-3′; antisense, 5′-ACA TTC GAG GCT CCA GTG AAT TCG G-3′), murine MCP-1 (274 bp; sense, 5′-ACT GAA GCC AGC TCT CTC TTC CTC-3′; antisense, 5-TTC CTT CTT GGG GTC AGC ACA GAC-3′), and murine KC (454 bp; sense, 5′-GGA TTC ACC TCA AGA ACA TCC AGA G-3′; antisense, 5′-CAC CCT TCT ACT AGC ACA GTG GTT G-3′).
For real-time PCR, we used the Light Cycler Instrument (Roche) and the Light Cycler Fast Start DNA Master SYBR Green I Kit (Roche) according to the manufacturers recommendations. Primers for murine GAPDH, IP-10 (CXCL-10), TNF-α, and IL-18 were purchased as Light Cycler Primer Sets including standard DNA from Search-LC (Heidelberg, Germany). Cycle-by-cycle monitoring allowed exact quantification of cDNA. After each Light Cycler run, melting curve analysis was performed to control for specificity of the amplification products. Melting curve analysis of each primer was reconfirmed by examining the amplified product under UV light in an agarose gel. The number of copies in each sample was correlated with the number of GAPDH copies as measured by Light Cycler PCR.
Data are expressed as means ± SE. Statistical significance of differences between treatment and control groups was determined by factorial ANOVA. Differences were considered statistically significant at P < 0.05. Statistical analyses were performed using Stat-View 4.51 software (Abacus Concepts, Calabasas, CA, USA).
Attenuation of DSS-Induced Colitis by Pralnacasan Is Associated with Decreased Levels of IL-18
Female Balb/c mice received 3.5% dextran sulfate sodium for a maximum of 11 days. Animals were separated into three groups: 10 mice received 3.5% DSS plus Cremophor EL (subsequently termed placebo), 9 mice received 3.5% DSS plus 50 mg/kg pralnacasan i.p., and 8 mice served as a control group, receiving tap water and Cremophor EL. Predefined subgroups of mice were sacrificed 4, 8, and 11 days after the start of the experimental protocol. Application of DSS resulted in clinical signs of colitis, with diarrhea and blood in the stool, beginning at the third day of DSS administration (Fig. 1a). Mice in the placebo group reached a score of 1.75 ± 0.18 at day 11 (P = 0.005). Pralnacasan reduced diarrhea and the presence of occult or overt blood in the stool significantly (P = 0.026). However, weight loss was not influenced by treatment with pralnacasan (Fig. 1b): mice in the placebo group had lost 13% of their basic body weight at the end of the experiment; animals in the pralnacasan-treated group, 8%. At no time during the course of the experiment was there a difference in weight loss between the placebo group and the pralnacasan-treated group. Colon length was reduced by DSS administration, with the reduction being inhibited by pralnacasan treatment (Fig. 1c): on day 11 colon length was 9.8 ± 0.9 cm in the placebo group, compared to 14.3 ± 0.3 cm in the control group (P = 0.002). Pralnacasan-treated animals showed colon lengths of 12.3 ± 0.5 cm on day 11.
To determine the effects of pralnacasan on the expression of inflammatory cytokines, the content of colonic TNF-α, IL-18, IFN-γ, IL-1β, and IL-12p70 was determined by ELISA (Fig. 2). After 4 days of DSS application, IL-18 had increased significantly, to a level of 1977 ± 400 pg/mg protein, compared to 923 ± 136 pg/mg protein in the control group (P = 0.009). Pralnacasan prevented this increase (P = 0.049). After 8 and 11 days of DSS administration colonic IL-18 had fallen to the levels in the control group. Similarly, the highest levels of TNF-α were found in the placebo group sacrificed after 4 days of DSS administration, with 89 ± 40 pg/mg protein, compared to 35 ± 5 pg/mg protein in the control group (P = 0.050). In contrast to its effect on IL-18 levels, pralnacasan showed no influence on TNF-α levels (98 ± 36 pg/mg protein; P = 0.018 compared to control group). Eight days after the beginning of the treatment protocol, the TNF-α content in the placebo group had decreased to 71 ± 27 pg/mg protein, and after 11 days it reached 23 ± 7 pg/mg protein, being paralleled by a similar decrease in the pralnacasan-treated group. IL-12p70 showed the same peak as IL-18 and TNF-α at day 4 of DSS application. Again, pralnacasan had only a moderate effect on IL-12p70 production. Interestingly, IFN-γ content in the colonic homogenate was only moderately influenced by DSS application. In contrast to IL-18, IL-1β levels increased throughout the course of DSS administration and reached 486 ± 248 pg/mg protein at day 11. Pralnacasan reduced IL-1β levels to 101 ± 16 pg/mg on day 11. In the control group 39 ± 9 pg/mg of IL-1β protein was found (P < 0.001 compared to placebo group on day 11).
Pralnacasan Reduces the Increase in IP-10 and TNF-α mRNA Expression
Pralnacasan Prevents Colitis in Th1-Biased C57/BL6 Mice and Suppresses the IFN-γ Increase Induced by DSS Application
Two groups of C57BL/6 mice (n = 8 each) received 1.5% DSS dissolved in tap water and were treated with 50 mg/kg bid pralnacasan i.p. and 25% Cremophor EL, respectively. A control group of eight mice received tap water and no treatment. Predefined subgroups (n = 4 in each group) were sacrificed at day 6 of DSS administration; the surviving animals were sacrificed at day 11. Mice of the placebo group started to show severe signs of colitis beginning at day 6 of DSS application (Fig. 4a): C57BL/6 mice in the placebo group had severe diarrhea and hematochezia, reaching a mean score of 3.00 ± 0.34 after 10 days of DSS administration. As with Balb/c mice, pralnacasan reduced symptoms. Until day 9 diarrhea and presence of occult blood in the stool were almost completely suppressed. In contrast to Balb/c mice, C57BL/6 mice were protected against weight loss by treatment with pralnacasan (Fig. 4b). Mice in the placebo group started to lose weight beginning at day 7 of DSS application, losing 21% of their basic body weight after 10 days (P < 0.001 compared to control). Mice in the pralnacasan-treated group had lost only 3% of their basic body weight at that time point. The difference in weight loss between the pralnacasan-treated group and the group receiving only Cremophor EL was highly significant (P = 0.004). In summary, pralnacasan was even more effective in C57/BL6 mice than in Balb/c mice and prevented the induction of colitis significantly.
In contrast to Balb/c mice, C57BL/6 mice treated with placebo showed a significant increase in IFN-γ content in colon homogenate after 6 days of DSS administration (134 ± 18 pg/mg protein, compared to 52 ± 4 pg/mg protein in the control group; P = 0.010). As demonstrated in Fig. 4c, pralnacasan prevented this increase in IFN-γ (97 ± 10 pg/mg; P = 0.010). At the end of the experimental protocol the colonic IFN-γ content (61 ± 5 pg/mg protein in the placebo group) was significantly reduced compared to the peak at day 6 (P = 0.017).
Increase in IP-10 mRNA, but not TNF-α mRNA Expression Is Prevented by Pralnacasan in C57/BL6 Mice and Correlates with Amelioration of Colitis
Destruction of the Epithelial Barrier in the Late Phase of DSS-Induced Colitis
TNF-α, MCP-1, and KC mRNA Increase During DSS Application and Correlate with Colitic Symptoms
In addition, we quantified secondary mediators of inflammatory cytokines on the mRNA level. To confirm findings obtained by real-time PCR, we used conventional PCR to determine levels of TNF-α mRNA, calculated as optical densities of TNF-α per β-actin (Fig. 7). TNF-α mRNA expression was highest at the end of the experiment (1.3 ± 0.1 in the control group, compared to 2.0 ± 0.1 on day 11 in nontreated DSS-receiving animals; P = 0.013). In the pralnacasan-treated group a value of 2.0 ± 0.1 on day 11 was determined, approving that pralnacasan treatment had no effect on the mean TNF-α mRNA expression.
MCP-1 mRNA expression showed a pattern similar to that of TNF-α. Again, there was no influence of pralnacasan on MCP-1 mRNA expression. However, the expression of the inflammatory chemokine KC was influenced by treatment with pralnacasan. Control animals revealed an expression index of 0.1 ± 0.0, and animals receiving DSS had even lower levels on day 6 of DSS application. After 11 days, elevated levels of KC mRNA were found (0.3 ± 0.1; P < 0.001). This increase was significantly diminished by pralnacasan, to an index of 0.1 ± 0.0 (P = 0.028).
Pralnacasan significantly ameliorated the clinical signs of DSS-induced colitis, diarrhea and the presence of occult or overt blood in the stool, in both Balb/c and C57BL/6 mice. However, weight loss was prevented by treatment with pralnacasan only in Th1-biased C57BL/6 mice. IFN-γ and IL-18 protein levels in colonic homogenate were reduced by pralnacasan, with only a slight and nonsignificant effect of pralnacasan on TNF-α and IL-12 levels. The efficacy of pralnacasan correlated with a suppression of IFN-γ in Th1-biased C57BL/6 mice. Consistently, IP-10 mRNA, but not TNF-α mRNA, expression was significantly suppressed by ICE inhibition in C57/BL6 mice. Therefore, we suggest that pralnacasan specifically inhibits IL-18-mediated cytokine production, leading to reduced colonic inflammation and clinical symptoms. Recently, we reported that pralnacasan ameliorates DSS-induced colitis . Here we describe in detail the cytokine expression during the experimental time course of DSS administration. Our data support the concept that ICE/caspase-1 plays a key role in the model of DSS-induced colitis.
The present study used the ICE inhibitor pralnacasan as an instrument to elucidate the immunological mechanisms in DSS-induced colitis. Colitis was induced in mice of two different strains by administration of DSS. Pralnacasan efficiently ameliorated colitic symptoms in both mouse strains. Balb/c mice showed signs of colitis after 4 days of administration of 3.5% DSS. As a Th1-biased mouse strain [36–38], C57BL/6 mice received only 1.5% DSS and developed severe colitis beginning at the sixth day of DSS application. In both mouse strains pralnacasan prevented the induction of symptoms for several days.
However, efficacy of pralnacasan was more pronounced in C57BL/6 mice. A high therapeutic efficacy of pralnacasan in Th1-biased C57BL/6 mice is concordant with the suggested mechanism of action of the ICE inhibitor pralnacasan. Cytokines were measured on the mRNA level by conventional and real-time PCR and on the protein level by ELISA. The increase in IP-10 mRNA, but not TNF-α mRNA, expression following DSS application was significantly suppressed by pralnacasan. The expression of IP-10 (also known as IFN-γ-inducible protein-10) is regulated by IFN-γ. Therefore, reduced levels of IP-10 mRNA reflect reduced activity of IFN-γ in tissue. IFN-γ itself lies downstream of IL-18. As this cytokine is activated by ICE, our finding of reduced IP-10 mRNA expression following pralnacasan treatment provides evidence for effective in vivo suppression of IL-18 activity by pralnacasan. Absence of suppression of TNF-α mRNA expression by pralnacasan reflects a specific effect of pralnacasan on cytokine expression.
Measurement of TNF-α, IL-12, and IL-18 protein showed a significant peak of detectable protein in the early phase of DSS administration. These cytokines are typically produced by macrophages. Thus the present data argue for a role of activated macrophages in the pathogenesis of DSS-induced colitis. Pralnacasan reduced this initial peak of IL-18, but not of TNF-α and IL-12, significantly. Tomoyose et al. found reduced levels of proinflammatory cytokines at the end of an experimental protocol administering 4% DSS for 7 days . It was concluded that the loss of soluble cytokines into the lumen of the gut after destruction of colonic integrity might be a possible mechanism for the reduction of proinflammatory protein in the late phase of DSS administration. A similar effect could contribute to our observation of reduced TNF-α, IL-12, and IL-18 protein levels in the late phase of DSS administration. Beginning at day 8, the nontreated group showed high epithelial damage scores, which reached a maximum at the end of the experimental protocol. Colon length was reduced by 31% in the untreated group at day 11 (data not shown) and macroscopic evaluation showed a markedly damaged and hemorrhagic colon at the end of the experiment.
Levels of proinflammatory cytokines as measured in the colon homogenate inadequately reflected the clinical score of the experimental animals. Therefore, we examined whether mRNA levels correlated better with intestinal inflammation. We suggest mRNA measurement as a better tool to evaluate immune activation in the gut than ELISA protein measurement of colon homogenate samples. IL-18 mRNA was constitutively expressed at a high level (especially in C57BL/6 mice) and expression was not affected by administration of DSS. Sivakumar et al. reported elevated IL-18 mRNA levels after application of 2% DSS to C57BL/6 mice, but the increase was discrete compared to the increase with other measured cytokines . As we found elevated levels of IL-18 protein which were reduced by ICE inhibition, IL-18 is—according to our data—regulated in a posttranscriptional way in DSS-induced colitis.
TNF-α mRNA expression increased significantly during DSS administration and correlated with the severity of inflammatory symptoms, but was not affected by ICE inhibition. Therefore a combined approach of TNF-α-reducing substances with the ICE inhibitor pralnacasan appears promising . Our data address the issue of alternative therapeutical strategies in the treatment of human IBD. As demonstrated in the DSS model, pralnacasan has a mechanistic effect on IL-18, IFN-γ, and IP-10. These cytokines are thought to play key roles in human inflammatory bowel disease as well. IP-10, which is induced by IFN-γ, has been demonstrated to be elevated in the serum of patients with ulcerative colitis . Therefore, patients with Crohn's disease and with ulcerative colitis might benefit from treatment with the caspase-1/ICE inhibitor pralnacasan. A phase II clinical trial suggests the safety and effectiveness of this compound in the treatment of rheumatoid arthritis . A clinical pilot study on pralnacasan in patients with IBD seems warranted.