Abstract
It has been hypothesized that α2-adrenoceptors (α2-ARs) may be involved in the pathomechanism of colitis; however, the results are conflicting because both aggravation and amelioration of colonic inflammation have been described in response to α2-AR agonists. Therefore, we aimed to analyze the role of α2-ARs in acute murine colitis. The experiments were carried out in wild-type, α2A-, α2B-, and α2C-AR knockout (KO) C57BL/6 mice. Colitis was induced by dextran sulfate sodium (DSS, 2%); alpha2-AR ligands were injected i.p. The severity of colitis was determined both macroscopically and histologically. Colonic myeloperoxidase (MPO) and cytokine levels were measured by enzyme-linked immunosorbent assay and proteome profiler array, respectively. The nonselective α2-AR agonist clonidine induced a modest aggravation of DSS-induced colitis. It accelerated the disease development and markedly enhanced the weight loss of animals, but did not influence the colon shortening, tissue MPO levels, or histologic score. Clonidine induced similar changes in α2B- and α2C-AR KO mice, whereas it failed to affect the disease activity index scores and caused only minor weight loss in α2A-AR KO animals. In contrast, selective inhibition of α2A-ARs by BRL 44408 significantly delayed the development of colitis; reduced the colonic levels of MPO and chemokine (C-C motif) ligand 3, chemokine (C-X-C motif) ligand 2 (CXCL2), CXCL13, and granulocyte-colony stimulating factor; and elevated that of tissue inhibitor of metalloproteinases-1. In this work, we report that activation of α2-ARs aggravates murine colitis, an effect mediated by the α2A-AR subtype, and selective inhibition of these receptors reduces the severity of gut inflammation.
Introduction
Inflammatory bowel diseases (IBDs), such as Crohn’s disease and ulcerative colitis (UC), are chronic, relapsing inflammatory conditions of the gastrointestinal (GI) tract. Their pathogenesis is complex, involving various predisposing environmental and genetic factors, which together with the altered intestinal flora can induce mucosal disruption and result in penetration of luminal antigens into the gut wall, followed by an ungoverned immune response and a chronic inflammatory reaction (Xavier and Podolsky, 2007; Gyires et al., 2014; Neurath, 2014). Numerous agents are available to treat IBD patients (Bryant et al., 2015), but many of them cause severe adverse reactions, or fail to maintain long-term remission, and a lot of effort is currently put into finding new therapeutic approaches (Gyires et al., 2014).
There is increasing evidence that inflammation alters both cholinergic and noradrenergic neurotransmission in the enteric nervous system (Lomax et al., 2006; Moynes et al., 2014; Rahman et al., 2015). These changes occur at both inflamed and noninflamed sites of the gut and result in secretion and motor abnormalities, which contribute to the wide-ranging clinical symptoms such as abdominal discomfort, bloating, and diarrhea (Blandizzi et al., 2003; Hons et al., 2009). However, the communication between the autonomic nervous system and immune system is bidirectional, and not only inflammatory mediators induce neuronal responses, but also acetylcholine (ACh) and catecholamines modulate directly the functions of macrophages, neutrophils, and lymphocytes (recently reviewed by Di Giovangiulio et al., 2015; Kawashima et al., 2015; Pavlov and Tracey, 2015). Hence, pharmacological targeting of these neuro-immune cell interactions provides a novel approach to suppress intestinal inflammation.
Among the adrenergic receptors, α2-adrenoceptors (α2-ARs) may be particularly important targets in the pharmacotherapy of colitis. These receptors are localized both pre- and postsynaptically on enteric neurons and non-neuronal cells, such as enterocytes and smooth muscle cells, and play a pivotal role in the control of enteric neurotransmission, motility, electrolyte transport, and visceral sensation (Blandizzi, 2007). Moreover, α2-ARs are also expressed by various immune cells, such as neutrophils and macrophages, and their ligands are therefore able to directly modulate immune responses (Spengler et al., 1994; Flierl et al., 2007).
Surprisingly, literature data on the effect of α2-AR ligands on IBDs and experimental colitis models are sparse and contradictory. It was demonstrated that pharmacological blockade of α2-ARs suppressed the production of proinflammatory cytokines by lamina propria mononuclear cells and ameliorated acute murine colitis (Bai et al., 2009; Bai et al., 2015). In contrast, activation of α2-ARs by dexmedetomidine reduced the symptoms of 2,4,6-trinitrobenzenesulfonic acid (TNBS)–evoked colitis by inducing a versatile immunomodulatory effect (Erdogan Kayhan et al., 2013), and chronic administration of clonidine (CLO), another α2-AR agonist, induced clinical, endoscopic, as well as histologic improvement in patients with UC (Lechin et al., 1985; Furlan et al., 2006). Thus, it is still a matter of debate whether the activation of α2-ARs confers deleterious or protective effects on intestinal inflammation.
Therefore, the present experimental study was undertaken to analyze the role of α2-ARs in the pathomechanism of colitis. Because these receptors can be divided into three distinct molecular subtypes (α2A, α2B, and α2C) (Bylund et al., 1994), we also aimed to identify which subtype is primarily involved in the modulation of intestinal inflammation. In this work, we report that activation of α2-ARs aggravates acute murine colitis, an effect mediated by the α2A-AR subtype, and selective inhibition of these receptors reduces the severity of gut inflammation.
Materials and Methods
Experimental Animals
Experiments were carried out in wild-type (WT), α2A-, α2B-, and α2C-AR knockout (KO) C57BL/6 mice (20–30 g, both sexes) obtained from the laboratory of L. Hein, University of Freiburg (Freiburg, Germany). The generation of the mouse lines lacking α2-AR subtypes has been described previously in detail (Link et al., 1995, 1996; Altman et al., 1999). The animals were housed in a temperature (22 ± 2°C)- and humidity-controlled room at a 12-hour light/dark cycle under conditions of animal housing and experimentation according to ethical guidelines issued by the Ethical Board of Semmelweis University, based on European Community Directive 86/609/European Economic Community. Food and water were available ad libitum.
All procedures conformed to the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes, and all efforts were made to minimize the suffering of animals. Number of animals in each experimental group was reduced to the minimum required for a statistical analysis, and pre-emptive euthanasia was performed if mice became moribund. The experiments were approved by the National Scientific Ethical Committee on Animal Experimentation and permitted by the government [Food Chain Safety and Animal Health Directorate of the Central Agricultural Office (PEI/001/1493-4/2015)].
Induction of Colitis
Colitis was induced by adding dextran sulfate sodium (DSS; 2 or 3%; molecular weight, 36,000–50,000 Da; MP Biomedicals, Illkirch, France) to the drinking water (tap water) for 7 days (Mitrovic et al., 2010). The DSS-containing drinking water was made up fresh every second day to avoid bacterial contamination, and the consumption of water and food was monitored in all groups. Body weight was measured daily during the course of the treatment. The development of colitis was evaluated by recording the disease activity index (DAI) covering fur appearance (0, normal; 1, disturbed), stool consistency (0, normal; 1, soft but formed stool; 2, loose stool), and presence of fecal blood (0, no trace of blood; 1; traces of blood on the stool; 2, bloody perianal region). The scores in each category were summed up, resulting in a total score between 0 and 5. On the morning of day 8, animals were killed and their whole colons were removed. The reduction of colon length was used as another parameter to assess colonic inflammation (Mitrovic et al., 2010). Care was taken not to stretch the colon. The length was expressed as percent of length in the control group. Afterward, full-thickness pieces of the distal colon were excised for further analysis.
Study Design
Study 1.
To characterize the concentration- and sex-dependent effect of DSS, 51 WT mice from both sexes (19 male, 32 female) were randomly divided into three groups, and they drank either normal tap water (control group, n = 20), 2% DSS solution (n = 17), or 3% DSS solution (n = 14) for 7 days.
Study 2.
The effect of CLO, a nonselective α2-AR agonist, on the development of colitis was first analyzed in WT mice. In this experiment, 36 mice were randomly divided into six groups (six mice in each group) and treated for 7 days, as follows: 1) normal tap water plus saline (SAL) i.p.; 2) normal tap water plus 3 mg/kg CLO i.p. (CLO 3); 3) 2% DSS plus SAL i.p. (DSS + SAL); 4) 2% DSS plus 0.3 mg/kg CLO i.p. (DSS + CLO 0.3); 5) 2% DSS plus 1 mg/kg CLO i.p. (DSS + CLO 1); 6) 2% DSS plus 3 mg/kg CLO i.p. (DSS + CLO 3). SAL and CLO were injected on each day, in a volume of 0.1 ml/10 g. The doses of CLO were selected based on literature data (Nguyen et al, 1997; Tank et al., 2004).
In an additional experiment, we also determined the sedative effect of CLO. Twelve WT mice were divided into two groups (six mice in each group) and treated for 7 days, as follows: 1) normal tap water plus SAL i.p.; 2) normal tap water plus 3 mg/kg CLO i.p. (CLO 3). The locomotor activity of animals was determined on 3 consecutive days (days 5–7) at three different time points (30 minutes, 3 hours, and 6 hours after the injection of CLO).
Study 3.
In this study, we analyzed the effect of CLO on the development of DSS-induced colitis in genetically engineered α2-AR subtype-deficient mice. In two separate experiments, 24 α2A- and 20 α2B-AR KO mice were divided randomly into 4 groups (with six and five mice/group, respectively), and treated as follows: 1) normal tap water plus SAL i.p.; 2) 2% DSS plus SAL i.p. (DSS + SAL); 3) 2% DSS plus 1 mg/kg CLO i.p. (DSS + CLO 1); 4) 2% DSS plus 3 mg/kg CLO i.p. (DSS + CLO 3). In a third experiment, 29 α2C-AR KO mice were divided into five groups (five to six mice/group). Experimental groups were the same, except that an additional group was included: 2% DSS plus 0.3 mg/kg CLO i.p. (DSS + CLO 0.3).
Study 4.
To assess the effect of BRL 44408, a selective α2A-AR subtype antagonist (Zádori et al., 2013), on the course of DSS-induced colitis, 30 WT mice were randomly allocated into five groups (six mice/group) and treated for 7 days, as follows: 1) normal tap water plus SAL i.p.; 2) normal tap water plus 3 mg/kg BRL 44408 i.p. (BRL 3); 3) 2% DSS plus SAL i.p. (DSS + SAL); 4) 2% DSS plus 3 mg/kg BRL 44408 i.p. (DSS + BRL 3); 5) 2% DSS plus 3 mg/kg BRL 44408 i.p. plus 3 mg/kg CLO i.p. (DSS + BRL 3 + CLO 3). The dose of BRL 44408 was selected based on literature data (Bhalla et al., 2013).
Histologic Analysis
For histologic examination, specimens of the distal colon were fixed in 10% formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin. The sections were graded in a blinded fashion by using a modified scoring system originally described by Dieleman et al. (1998). The following parameters were scored: degree of inflammation (0, none; 1, slight; 2, moderate; 3, severe), depth of inflammation (0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural), severity of crypt damage (0, none; 1, basal 1/3 damaged; 2, basal 2/3 damaged; 3, only surface epithelium intact; 4, entire crypt and epithelium lost), and percentage of mucosa involved (1, 1–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%). The total histologic score (ranging from 0 to 14) was calculated based on the sum of partial scores.
Measurement of Colonic Myeloperoxidase Levels
The tissue levels of myeloperoxidase (MPO) were determined to quantify inflammation-associated infiltration of neutrophils and monocytes into the tissue (Mitrovic et al., 2010). Full-thickness pieces of the distal colon were excised, shock-frozen in liquid nitrogen, and stored at −70°C until assay. After weighing, tissues were homogenized in lysis buffer containing 200 mM NaCl, 5 mM EDTA, 10 mM Tris, 10% glycerine, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4), supplemented with a protease inhibitor cocktail (cOmplete ULTRA Tablets; Roche, Basel, Switzerland). The MPO content of the supernatant was measured with an enzyme-linked immunosorbent assay kit specific for the mouse protein (Hycult Biotechnology, Uden, The Netherlands). Protein concentration of the homogenates was determined by bicinchoninic acid assay with bovine serum albumin as a standard, and the level of MPO was expressed as ng/mg total protein.
Measurement of Colonic Cytokine Levels
The cytokine profile of distal colonic segments was determined by a proteome profiler array (Mouse Cytokine Array Panel A; R&D Systems Europe, Abingdon, UK) (Szitter et al., 2014), according to the manufacturer’s instructions. Briefly, frozen tissues were thawed, weighed, and homogenized in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride and supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Then Triton X-100 was added to the samples to a final concentration of 1% and centrifuged at 10,000g for 5 minutes to remove cellular debris. Total protein concentrations were determined prior to cytokine analysis by bicinchoninic acid assay. Equal amount of protein (600 µg) from each sample was mixed with a cocktail of biotinylated detection antibodies and incubated overnight on nitrocellulose membranes containing 40 different anti-cytokine antibodies printed in duplicate. On the second day, unbound materials were removed by washing, and membrane-bound cytokines were detected by chemiluminescence (Chemidoc XRS+; Bio-Rad, Hercules, CA). The intensity of the emitted light at each spot was analyzed by densitometry (Image Lab Software, Bio-Rad, Hercules, CA) and normalized to the pixel density of reference spots (10,000) (Szitter et al., 2014).
Measurement of Locomotor Activity
The locomotor activity of animals was registered by CONDUCTA System (Experimetria, Budapest, Hungary). The apparatus consisted of three individual black-painted testing boxes (40 × 50 × 50 cm each) set in an isolated room. In each box an animal was placed; thus, three animals were tested in parallel. Their movements were monitored by high-density arrays of infrared diodes. The time spent in ambulation (walking, running) was recorded individually for each box (Timár et al., 2010). The observation lasted 20 minutes, and the activity (ambulation time) was expressed in seconds.
Materials
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Statistical Analysis
Data are expressed as means ± S.E.M. Statistical analysis of the data was performed with Student t test (two treatment groups), one-way analysis of variance (many groups), or, in case of nonparametric values, Mann–Whitney test and Kruskal–Wallis test, followed by Holm–Sidak post hoc test. Two-way repeated measures analysis of variance and Friedman test were employed to compare the time course of weight losses and DAIs between different groups. A probability of P < 0.05 was considered statistically significant.
Results
Characterization of the Effect of DSS in WT Mice.
DSS at a concentration of 2% induced a modest inflammatory response, usually characterized by soft, formed stool with some blood traces, slight weight loss, and significant shrinkage of the colon (Fig. 1, A–C). When animals were treated with a higher concentration of DSS (3%), the symptoms of colitis started earlier and the weight loss and reduction of colon length were more pronounced (Fig. 1, A–C). Considering the potential aggravatory effect of α2-AR agonists on colonic inflammation, we chose the lower concentration of DSS for subsequent studies.
Comparison of the effect of DSS in two different concentrations, and in male and female WT mice. (A–C) DSS (2 and 3%) induced concentration-dependent inflammatory response in WT mice, characterized by elevation of DAI, weight loss, and reduction of colon length. The values represent means ± S.E.M. (control: n = 20; 2% DSS: n = 17; 3% DSS: n = 14). *P < 0.05 versus control group, +P < 0.05 versus both control and 2% DSS groups. Friedman test (A), two-way repeated measures analysis of variance (B), and one-way analysis of variance (C), followed by Holm–Sidak post hoc test. (D–F) Comparison of the effect of 2% DSS in male (M) and female (F) mice. There were no sex-dependent differences in the DSS-induced inflammatory parameters. The values represent means ± S.E.M. (control male: n = 7; control female: n = 13; 2% DSS male: n = 9; 2% DSS female: n = 8). *P < 0.05 versus control group. Friedman test (D), two-way repeated measures analysis of variance (E), and one-way analysis of variance (F), followed by Holm–Sidak post hoc test.
We did not find any sex-dependent difference in the DSS-induced inflammatory parameters. Figure 1, D and E, shows that the DAI scores and weight losses were similar in 2% DSS-treated male and female mice. Furthermore, the length of colon also did not differ significantly in the two groups (Fig. 1F). Therefore, we used animals of either sex, although care was taken to keep the same male/female ratio in all experimental groups.
The Effect of Clonidine on DSS-Induced Colonic Inflammation in WT Mice.
As Fig. 2 shows, daily administration of the nonselective α2-AR agonist CLO (0.3–3 mg/kg i.p.) aggravated some, but not all parameters of DSS-induced colitis. It accelerated the development of inflammation, which was reflected by earlier elevation of DAI and also enhanced DSS-induced weight loss in a nondose-dependent manner, but failed to influence the DSS-induced shortening of colon (Fig. 2, A–C).
The effect of CLO (0.3–3 mg/kg) on DSS-induced colitis in WT mice. CLO or SAL was injected i.p. on a daily basis. CLO accelerated the elevation of DAI and enhanced DSS-induced weight loss, but did not influence the other inflammatory parameters, such as colon length, colonic MPO level, or histologic score. The values represent means ± S.E.M. (n = 6/group). #P < 0.05 versus DSS + SAL, *P < 0.05 versus SAL, +P < 0.05 versus both SAL and DSS + SAL. Friedman test (A), two-way repeated measures analysis of variance (B), one-way analysis of variance (C and D), and Kruskal–Wallis test (E), followed by Holm–Sidak post hoc test.
Distal colon samples collected from mice treated with the highest dose of CLO were subjected to further analysis. As Fig. 2D demonstrates, DSS treatment increased the tissue level of MPO fivefold compared with absolute control group, which was not affected by CLO. Histologic analysis revealed only mild inflammation in DSS-treated mice (Figs. 2E and 3). Morphologic changes were almost entirely limited to the mucosa and included focal accumulation of inflammatory cells, irregular arrangement of crypts or the partial loss of their basal portion, and, in the most serious cases, focal superficial epithelial erosion. The majority of inflammatory cells were identified as lymphocytes, plasma cells, and macrophages, but some neutrophils were also present (Fig. 3). CLO at the highest dose tended to aggravate some inflammatory signs (e.g., epithelial erosion; lymphatic infiltration of lamina propria; widened, edematous submucosa), but the overall histologic score of these animals did not differ significantly from that of the DSS-control group (7.2 ± 0.7 versus 6.0 ± 1.0, P = 0.42; Figs. 2E and 3).
Representative histologic micrographs of colonic tissue sections. Full-thickness pieces of the distal colon were obtained from WT mice treated either with SAL i.p. + normal tap water (SAL), 3 mg/kg CLO i.p. + normal tap water (CLO), SAL i.p. + 2% DSS (DSS + SAL), or 3 mg/kg CLO i.p. + 2% DSS (DSS + CLO). Histologic signs of moderate inflammation were observed in the DSS + SAL group; focal lymphatic infiltration of the mucosa is marked with an asterisk. CLO tended to aggravate some inflammatory signs in DSS-treated animals (DSS + CLO), indicated by disintegration of the crypts and diffuse lymphatic infiltration of lamina propria (arrowheads), and widened, edematous submucosa. lp, lamina propria; TM, tunica muscularis; M, tunica muscularis; mm, muscularis mucosae; SM, tela submucosa. Scale bar, 250 µm.
Animals treated only with CLO, but not with DSS (drug control group), did not show any sign of inflammation and did not lose significant weight (Figs. 2 and 3).
The Effect of Clonidine on Food and Water Intake, and on Locomotor Activity of WT Mice.
Although the weight of mice consuming water did not change significantly in response to CLO, we addressed the question whether the marked weight loss in the DSS plus CLO–treated group is due to any direct or indirect (sedative) effect of CLO on the daily food and water consumption of animals. As Fig. 4 shows, cumulative food and water intakes were identical in all groups during the first days of treatment. Moreover, although we observed a marked reduction of animals’ locomotor activity in response to the highest dose of CLO (3 mg/kg), this effect was relatively short-lasting and disappeared after 6 hours (Fig. 4C). These results indicate that CLO has no major impact (via direct or indirect mechanism) on the food and water consumption at the tested dose range.
The effect of CLO on food and water intake, and on locomotor activity of WT mice. (A and B) CLO (0.3–3 mg/kg) had no effect on cumulative food and water consumption of WT mice. As colitis developed, daily food and water intake gradually declined in DSS-treated animals, which was slightly more pronounced in CLO-treated mice. CLO or SAL was injected i.p., and the total food and water intake of each group was assessed daily. The values represent the cumulative intake of each group (n = 6/group). *P < 0.05 versus SAL; the slopes of linear regression lines were analyzed by one-way analysis of variance. (C) CLO (3 mg/kg) markedly reduced the locomotor activity of WT mice, which effect was short-lasting and disappeared after 6 hours. Mice were treated with CLO or SAL i.p. for 7 days. They drank normal tap water. The locomotor activity of animals (time spent in ambulation during a 20-minute observation period) was determined on 3 consecutive days (on days 5–7) at different time points (30 minutes, 3 hours, and 6 hours after the injection of CLO). The values represent means ± S.E.M., n = 6/group. *P < 0.05 versus SAL, Student t test.
However, as colitis developed, daily food and water intake gradually declined in DSS-treated animals, as an indicator of disease severity, a phenomenon that was more pronounced (although statistically not significant) in CLO-treated mice (Fig. 4, A and B).
The Effect of Clonidine on DSS-Induced Colonic Inflammation in α2-AR Subtype-Deficient Mice.
In α2B- and α2C-KO animals CLO induced similar responses, as in WT animals, namely, it accelerated the DSS-induced elevation of DAI and caused a pronounced weight loss. In contrast, CLO failed to influence the DAI of DSS-treated α2A-KO mice and caused significantly less weight reduction than in WT animals (−5.8 ± 1.7% in mice treated with 3 mg/kg CLO, P < 0.001 compared with the −14.8 ± 1.5% weight loss measured in WT mice) (Fig. 5).
Comparison of the effect of CLO (0.3–3 mg/kg) on DSS-induced colitis in α2A-, α2B-, and α2C-AR KO mice. CLO induced similar responses in α2B- and α2C-AR KO animals as in WT animals, but it failed to influence the DAI of DSS-treated α2A-AR KO mice, and caused significantly less weight reduction in this strain. CLO or SAL was injected i.p. on a daily basis. The severity of colitis was monitored daily by determining the DAI (A–C) and weight loss of animals (D–F). The values represent means ± S.E.M. (α2A-AR KO: n = 6/group; α2B-AR KO: n = 5/group; α2C-AR KO: n = 5 in control and 6 in all other groups). *P < 0.05 versus SAL, #P < 0.05 versus DSS + SAL, +P < 0.05 versus both SAL and DSS + SAL. Friedman test (A–C) and two-way repeated measures analysis of variance (D–F), followed by Holm–Sidak post hoc test.
The Effect of BRL 44408 on DSS-Induced Colonic Inflammation in WT Mice.
Because genetic deletion of α2A-ARs significantly reduced the aggravatory effect of CLO on colitis, we wondered whether pharmacological inhibition of this subtype results in any protection. As Fig. 6 shows, daily administration of BRL 44408 (3 mg/kg i.p.) significantly delayed the development of colitis and reduced the weight loss of animals (Fig. 6, A and B). These effects were lacking in mice treated with both BRL 44408 and CLO. Although we did not find any difference in colon lengths (Supplemental Fig. 1), we found that colonic MPO levels were significantly lower in BRL 44408–treated animals, similarly to that in α2A-AR KO mice (Fig. 6C). BRL 44408 alone had no effect on any of the investigated parameters (Fig. 6).
The effect of BRL 44408 (BRL, 3 mg/kg) on DSS-induced colitis in WT mice. (A and B) BRL 44408 alone, but not in combination with CLO (3 mg/kg), delayed the development of colitis and reduced the weight loss of DSS-treated WT animals. BRL 44408, CLO, or SAL was injected i.p. on a daily basis. The values represent means ± S.E.M. (n = 6/group). *P < 0.05 versus SAL, #P < 0.05 versus DSS + SAL, +P < 0.05 versus both SAL and DSS + SAL, $P < 0.05 versus DSS + BRL 3 + CLO 3. Friedman test (A) and two-way repeated measures analysis of variance (B), followed by Holm–Sidak post hoc test. (C) BRL 44408 (BRL, 3 mg/kg) reduced colonic MPO levels in WT mice to similar levels as those found in α2A-AR KO animals. The values represent means ± S.E.M. (n = 6/group). *P < 0.05 versus SAL (WT and α2A-AR KO mice consuming only tap water and being treated with SAL), #P < 0.05 versus WT mice treated with DSS + SAL. One-way analysis of variance, followed by Holm–Sidak post hoc test.
Analysis of the colonic cytokine profile revealed that DSS significantly increased the tissue level of various cytokines and chemokines (Fig. 7). Although a similar pattern was observed in the BRL 44408–treated group, we found a dramatic reduction in the tissue level of chemokine (C-X-C motif) ligand 13 (CXCL13). Moreover, BRL 44408 slightly suppressed the levels of chemokine (C-C motif) ligand 3 (CCL3), CXCL2, and granulocyte-colony stimulating factor (G-CSF), which were not significantly different from the expression levels measured in the absolute control group. Finally, DSS-induced elevation of tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was significantly increased in response to BRL 44408.
The effect of BRL 44408 (BRL, 3 mg/kg i.p.) on colonic cytokine and chemokine levels in WT mice. Local cytokine profile in distal colon homogenates determined by a proteome profiler array. Pixel density of each spot was normalized to that of reference spots (10,000). BRL 44408 reduced the colonic levels of CCL3, CXCL2, CXCL13, and G-CSF, and elevated that of TIMP-1. The values represent means ± S.E.M. (n = 6/group). *P < 0.05 versus SAL (mice consuming only tap water and being treated with SAL), #P < 0.05 versus DSS + SAL, +P < 0.05 versus both SAL and DSS + SAL. One-way analysis of variance, followed by Holm–Sidak post hoc test. IL, interleukin; TREM, triggering receptor expressed on myeloid cells.
Discussion
The pivotal role of α2-ARs in the control of enteric neurotransmission and in the regulation of GI functions has been recognized since decades (reviewed e.g., by Blandizzi, 2007). The idea that activation of these receptors may also be beneficial in the therapy of IBDs can be dated back to 1985, when Lechin et al. (1985) reported that chronic administration of CLO improved the symptoms as well as endoscopic and histologic changes in UC patients in a randomized, double-blind trial. These findings were later corroborated by another clinical study (Furlan et al., 2006), and more recently also by animal experiments (Erdogan Kayhan et al., 2013). In contrast, there is evidence that activation of α2-ARs localized on macrophages and neutrophils induces the release of proinflammatory cytokines (Spengler et al., 1994; Flierl et al., 2007) and enhances acute inflammatory lung injury in rats and mice (Flierl et al., 2007). These findings imply that stimulation of α2-AR signaling is rather detrimental than beneficial in colitis, which is also supported by the studies of Bai et al. (2009, 2015).
Thus, both aggravation and amelioration of colitis have been described in response to α2-AR agonists in different preclinical and clinical settings, and the reasons for these discrepancies remain uncertain. In the case of animal experiments, they may be at least partly due to the use of various colitis models induced by different chemical inducers (DSS, TNBS, or 2,4-dinitrobenzenesulfonic acid). These models differ in several aspects, such as pathomechanism, clinical/histologic picture, and cytokine profile (Strober et al., 2002; Alex et al., 2009); therefore, it is possible that a drug is able to influence the development of inflammation in one, but not in another model (Park et al., 2004). Nevertheless, it has to be emphasized that, regarding the role of α2-ARs, controversial results were reported by using the same colitis model (TNBS) as well (Bai et al., 2009; Erdogan Kayhan et al., 2013). Thus, other factors, such as using different α2-AR ligands with potential additional receptorial actions (dexmedetomidine, UK14304), different amounts of the chemical inducer (2.5 and 3.75 mg TNBS), or different ages of experimental animals (5–6 and 7–8 weeks), may also contribute to the diverging findings.
Because the role of α2-ARs in the pathomechanism of colitis is still insufficiently understood, we aimed to investigate the effect of CLO, a prototypical nonselective α2-AR agonist, on the development of colonic inflammation. In addition, because these receptors are divided into three genetically distinct subtypes, all of which are expressed by phagocytes (Flierl et al., 2007) and take part in the regulation of GI functions (Blandizzi et al., 1995; Scheibner et al., 2002; Zádori et al., 2011), we aimed to characterize the subtype(s) involved in the modulation of colitis. Gut inflammation was induced by DSS, which is one of the most commonly used animal models of colitis, and is also relevant for the translation of animal data to human disease (Melgar et al., 2008).
We found that CLO induced only mild changes in DSS-induced inflammation. It promoted the development of colitis, as reflected by an earlier increase of DAI scores compared with the DSS-control group, but the majority of inflammatory parameters remained unchanged at the time of final evaluation. The most prominent effect of CLO was the marked weight loss of DSS-treated mice. It might be argued that this effect is either due to direct modulation of animals’ eating and drinking behavior, which could be mediated by central α2-ARs and/or imidazoline receptors (Sugawara et al., 2001; Chung et al., 2012), or may result from the sedative effect of CLO, which is mediated by central α2A-ARs (Hunter et al., 1997). However, our results demonstrate that CLO induced only short-term sedation (up to a few hours) and had no significant effect on daily food and water intake in the first days of treatment. Moreover, it did not induce significant weight loss in mice consuming only water. Thus, the marked wasting of DSS plus CLO–treated mice rather reflects the aggravation of disease severity, which was accompanied by pronounced decrease of food and water intake in the later phase of colitis development.
Our experiments with mice lacking either α2A-, α2B-, or α2C-ARs suggested that the deleterious effect of CLO on colitis is likely to be mediated by the α2A-AR subtype because CLO-induced responses were only marginal in α2A-AR–deficient mice and were substantially different from those observed in WT mice and in the other two genetically modified strains. To support this assumption, we investigated the effect of BRL 44408, a selective α2A-AR antagonist, in WT mice and found that it significantly delayed the development of colitis, reduced the weight loss, and lowered the colonic MPO content to similar levels observed in control animals. Notably, the protective effect of BRL 44408 was diminished when it was coinjected with CLO, which competed with it for binding to α2A-ARs.
To better understand the anti-inflammatory action of BRL 44408, we analyzed the expression of numerous cytokines and chemokines in the gut, and found that BRL 44408 prevented the DSS-induced elevation of CXCL13, which recruits CXCR5+ follicular helper T cells to the B cell follicles, leads to the formation of antibody-secreting plasma cells and memory B cells (Moser, 2015), and has been proposed to be a novel target for IBD therapy (Nagy-Szakal et al., 2015). BRL 44408 also suppressed the levels of the neutrophil chemoattractants CCL3, CXCL2, and G-CSF, which all have been implicated in the pathomechanism of colitis (Melgar et al., 2005; McDermott et al., 2016). Although the changes in their individual expression were only mild, the marked reduction in the level of MPO suggests that the net effect resulted in a pronounced inhibition of neutrophil infiltration into the colonic tissue.
An intriguing finding of our study is that DSS-induced elevation of TIMP-1 was further increased by BRL 44408. It is well-established that matrix metalloproteinases (MMPs) are major contributors to the breakdown of extracellular matrix components in a variety of physiologic and pathologic processes, including colitis (Naito and Yoshikawa, 2005). Although the activity of MMPs is tightly controlled by TIMPs, whose level also increases in the course of inflammation, it is proposed that the overly increased elevation of MMPs and the consequent imbalance between MMPs and TIMPs play a crucial role in tissue injury (von Lampe et al., 2000; Jakubowska et al., 2016). Thus, it is conceivable that the strong elevation of TIMP-1 induced by BRL 44408 tips the imbalance in favor of TIMPs, which results in reduced tissue damage and improved healing processes. A similar TIMP-1–mediated protection has been suggested in the therapeutic action of infliximab in Crohn’s disease (Di Sabatino et al., 2007).
Our results showing that activation of α2-ARs induces mild aggravation of colitis, whereas inhibition of these receptors evokes a pronounced anti-inflammatory action, are in good accordance with previous findings (Bai et al., 2009) and suggest the following: 1) α2-ARs are overactive in the acute phase of colitis and 2) contribute to a proinflammatory milieu. The increased colonic activity of α2-ARs may rely on several factors, including an enhanced catecholamine release from both sympathetic nerves (Furlan et al., 2006) and local immune cells (Bai et al., 2009), and an increased neuronal expression of these receptors (Blandizzi et al., 2003). The proinflammatory action of α2-ARs may also arise from at least two causes. First, they inhibit the release of ACh from cholinergic enteric nerves, which otherwise is anti-inflammatory by activating α7 nicotinic ACh receptors on macrophages (Wang et al., 2003). Second, they induce directly the release of various proinflammatory cytokines and chemokines from immune cells, including tumor necrosis factor-α, interleukin-1β, interleukin-6, cytokine-induced neutrophil chemoattractant-1 (Spengler et al., 1990, 1994; Flierl et al., 2007; Bai et al., 2009), and, as our results indicate, CCL3, CXCL2, CXCL13, and G-CSF.
To our knowledge, we demonstrate for the first time that these proinflammatory α2-ARs belong to the α2A-AR subtype, which is in line with previous studies showing that this subtype is predominantly expressed throughout the GI tract and regulates most of the digestive functions (Blandizzi et al., 1995; Scheibner et al., 2002; Shujaa et al., 2011), expressed also by macrophages and polymorphonuclear cells (Flierl et al., 2007), and that its neuromuscular expression is upregulated in response to colitis (Blandizzi et al., 2003).
In summary, our results show that activation of α2-ARs induces mild aggravation of colitis, whereas inhibition of these receptors evokes a pronounced anti-inflammatory action. To our knowledge, we demonstrate for the first time that, among the three α2-AR subtypes, the α2A one is primarily involved in the modulation of DSS-induced colonic inflammation. Our findings imply that selective pharmacological blockade of this adrenoceptor subtype may be beneficial in the treatment of colitis, but further studies using other colitis models are required to assess whether these results can be successfully translated into clinical practice.
Authorship Contributions
Participated in research design: Zádori, Helyes, Holzer, Gyires.
Conducted experiments: Zádori, Tóth, Fehér, Al-Khrasani, Puskár, Kozsurek, Timár, Tábi.
Contributed new reagents or analytic tools: Hein.
Performed data analysis: Zádori, Tóth, Fehér.
Wrote or contributed to the writing of the manuscript: Zádori, Helyes, Holzer, Gyires.
Footnotes
- Received May 11, 2016.
- Accepted July 8, 2016.
This work was supported by the Austrian-Hungarian Action Foundation [84öu6 Project] and the Hungarian Scientific Research Fund [OTKA PD 109602].
↵
This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- ACh
- acetylcholine
- α2-AR
- α2-adrenoceptor
- CCL
- chemokine (C-C motif) ligand
- CLO
- clonidine
- CXCL
- chemokine (C-X-C motif) ligand
- DAI
- disease activity index
- DSS
- dextran sulfate sodium
- G-CSF
- granulocyte-colony stimulating factor
- GI
- gastrointestinal
- IBD
- inflammatory bowel disease
- KO
- knockout
- MMP
- matrix metalloproteinase
- MPO
- myeloperoxidase
- SAL
- saline
- TIMP
- tissue inhibitor of metalloproteinases
- TNBS
- 2,4,6-trinitrobenzenesulfonic acid
- UC
- ulcerative colitis
- WT
- wild-type
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
References
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Inhibition of α2A-Adrenoceptors Alleviates Colitis
