Chromofungin Ameliorates the Progression of Colitis by Regulating Alternatively Activated Macrophages

Ulcerative colitis (UC) is characterized by a functional dysregulation of alternatively activated macrophage (AAM) and intestinal epithelial cells (IECs) homeostasis. Chromogranin-A (CHGA) secreted by neuroendocrine cells is implicated in intestinal inflammation and immune dysregulation. CHGA undergoes proteolytic processing to generate CHGA-derived peptides. Chromofungin (CHR: CHGA47–66) is a short CHGA-derived peptide encoded by CHGA Exon-IV and is involved in innate immune regulation, but the basis is poorly investigated. We investigated the expression of CHR in colonic tissue of patients with active UC and assessed the effects of the CHR in dextran sulfate sodium (DSS) colitis in mice and on macrophages and human colonic epithelial cells. We found that mRNA expression of CHR correlated positively with mRNA levels of AAM markers and gene expression of tight junction (TJ) proteins and negatively with mRNA levels of interleukin (IL)-8, IL-18, and collagen in patients with active UC. Moreover, AAM markers correlated positively with gene expression of TJ proteins and negatively with IL-8, IL-18, and collagen gene expression. Experimentally, intracolonic administration of CHR protected against DSS-induced colitis by priming macrophages into AAM, reducing colonic collagen deposition, and maintaining IECs homeostasis. This effect was associated with a significant increase of AAM markers, reduction of colonic IL-18 release and conservation of gene expression of TJ proteins. In vitro, CHR enhanced AAM polarization and increased the production of anti-inflammatory mediators. CHR-treated AAM conditioned medium increased Caco-2 cell migration, viability, proliferation, and mRNA levels of TJ proteins, and decreased oxidative stress-induced apoptosis and proinflammatory cytokines release. Direct CHR treatments had the same effect. In conclusion, CHR treatment reduces the severity of colitis and the inflammatory process via enhancing AAM functions and maintaining IECs homeostasis. CHR is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of colonic inflammation and could lead to new therapeutic strategies for UC.


Ulcerative colitis (UC) is characterized by a functional dysregulation of alternatively activated macrophage (AAM) and intestinal epithelial cells (IECs) homeostasis.
Chromogranin-A (CHGA) secreted by neuroendocrine cells is implicated in intestinal inflammation and immune dysregulation. CHGA undergoes proteolytic processing to generate CHGA-derived peptides. Chromofungin (CHR: CHGA47-66) is a short CHGAderived peptide encoded by CHGA Exon-IV and is involved in innate immune regulation, but the basis is poorly investigated. We investigated the expression of CHR in colonic tissue of patients with active UC and assessed the effects of the CHR in dextran sulfate sodium (DSS) colitis in mice and on macrophages and human colonic epithelial cells. We found that mRNA expression of CHR correlated positively with mRNA levels of AAM markers and gene expression of tight junction (TJ) proteins and negatively with mRNA levels of interleukin (IL)-8, IL-18, and collagen in patients with active UC. Moreover, AAM markers correlated positively with gene expression of TJ proteins and negatively with IL-8, IL-18, and collagen gene expression. Experimentally, intracolonic administration of CHR protected against DSS-induced colitis by priming macrophages into AAM, reducing colonic collagen deposition, and maintaining IECs homeostasis. This effect was associated with a significant increase of AAM markers, reduction of colonic IL-18 release and conservation of gene expression of TJ proteins. In vitro, CHR enhanced AAM polarization and increased the production of anti-inflammatory mediators. CHR-treated AAM conditioned medium increased Caco-2 cell migration, viability, proliferation, and mRNA levels of TJ proteins, and decreased oxidative stress-induced apoptosis and proinflammatory cytokines release. Direct CHR treatments had the same effect. In conclusion, CHR treatment reduces the severity of colitis and the inflammatory process via enhancing AAM functions and maintaining IECs homeostasis. CHR is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of colonic inflammation and could lead to new therapeutic strategies for UC.
Keywords: chromogranin-a, gut-derived peptides, oxidative stress, mucosal drug action, epithelial homeostasis, macrophages switch, anti-inflammatory molecules, epithelial barrier inTrODUcTiOn Crohn's disease and ulcerative colitis (UC) are the two main forms of inflammatory bowel disease (IBD) in humans (1). The etiology of IBD is unknown, but evidence suggests that the abnormal immune response within the intestinal wall is directed against luminal bacterial antigens inducing intestinal tissue damage (2). Analysis of proinflammatory and anti-inflammatory pathways in IBD patients have demonstrated dysregulation in the immune responses associated with an altered balance between inflammatory, regulatory and anti-inflammatory cytokines (3). Macrophages implicated in presenting antigens to T and B cells are important cells regulating the host innate and adaptive immune responses (4). In IBD, macrophages play a crucial role in the resolution of tissue injury and promotion of tissue repair (5,6). Two main categories of macrophages are described, the classical-activated macrophages (CAMs) which generate Th1related cytokines (interferon-γ, tumor necrosis factor-α) response, and the alternatively activated macrophages (AAMs) linked to a Th2-related cytokines [interleukin (IL)-4 and IL-13] response (7). AAMs produce anti-inflammatory molecules (IL-10, arginase) and play a major role in the suppression of inflammation and tissue remodeling/repair (8). AAMs have been reported to attenuate experimental inflammation in the gut (9)(10)(11).
Intestinal epithelial cells (IECs) form a crucial first line of physical defense between the mucosa and the luminal milieu. Tight junctions (TJ) proteins are mainly responsible for the epithelial barrier function that includes selective transport of water, ions, and nutrients by forming an uninterrupted intercellular barrier between the epithelial cells (12). Thus, defects in intestinal epithelial TJ barrier are important contributing factors for the development of intestinal inflammation and lead to an amplified inflammatory response due to an increased passage of antigens into the colonic mucosa (13,14). Furthermore, in persons with IBD, IECs secrete a significant quantity of chemokines (i.e., IL-8) which cause excessive recruitment and transmigration of innate immune cells and proinflammatory cytokines, including IL-18 (15,16). Additionally, high collagen production by IECs, colonocytes, and fibroblasts favors intestinal fibrosis associated with stricture formation, which is a significant complication seen in persons with IBD (16). Furthermore, oxidative stress, and subsequent epithelial apoptosis, is a fundamental feature of colitis (17).
Chromogranin-A (CHGA), a member of the granin family of proteins (18), is an acidic protein distributed ubiquitously in vesicles of secretory cells of the enteric, endocrine, and immune systems (18). CHGA is the precursor of biologically active peptides implicated in several biological functions (19) by regulating the endocrine, the cardiovascular, and the immune systems (18,20). The protein is cleaved at multiple dibasic sites and exposed to an extensive degree of intracellular and extracellular proteolytic processing, particularly at the N-and C-terminal regions (21) to generate CHGA-derived peptides including chromofungin (CHR: CHGA47-66). CHR is an active short peptide encoded by the CHGA exon-IV (18) that possesses antimicrobial activity (22)(23)(24) and immune regulatory functions (25). Although CHR has antimicrobial activity, it is a non-hemolytic peptide, suggesting its non-toxicity (26). Moreover, CHGA47-57 peptide, which is a part of CHR, contains a cell adhesion site for fibroblasts and smooth muscle cells (26). Furthermore, CHR displays pronociceptive and antinociceptive effects in a model of somatovisceral pain through various mechanisms involving the corticotropin-releasing factor pathway, action on sensory neurotransmitter and direct or indirect regulation of inflammatory cells (27). Recently, CHR has been described as a post-conditioning agent against ischemia/ reperfusion (I/R) damages through the activation of prosurvival kinases and an increased miRNA-21 expression (28).
Although CHGA and its derived peptides are implicated in various inflammatory diseases including gut inflammation (29)(30)(31)(32)(33), there are no available data demonstrating the effects of CHR on AAM and IECs homeostasis during the progression of intestinal inflammation. Herein, we report on CHR expression in human colon tissue from persons with UC compared with unaffected controls. Further, we evaluated effects of CHR in dextran sulfate sodium (DSS) model of colitis and assessed its effects on AAM activities and human colonic cell line functions. We report that treatment with CHR significantly ameliorates disease severity and inhibits intestinal inflammation.

MaTerials anD MeThODs human subjects
Patients who diagnosed with active UC and persons with no IBD were recruited from the University of Manitoba IBD Clinical and Research Centre. Endoscopic biopsies obtained from 10 patients with active UC and 10 healthy individuals. Patients were between 27 and 55 years and with a mean age of 40 years. Informed consent obtained from patients and control subjects before the study. This study approved by the University of Manitoba Health Research Ethics Board [HS14878 (E)]. animals Experiments were approved by the University of Manitoba Animal Ethics Committee (Protocol # 15-010) and conducted under the Canadian guidelines for animal research. Six-to eightweek-old male C57BL/6 mice (20-25 g body weight) purchased from Charles River (Sherbrook, Canada) were maintained in the animal care facility at the University of Manitoba under a specific pathogen-free barrier facility.
acute Dss-experimental colitis Intracolonic injection of CHR or sCHR or 1% PBS started 1-day before colitis induction and lasted for 5-days. The experimental design of these experiments is illustrated in Figure 3A. DSS (molecular weight, 40 kDa: MP Biomedicals, Soho, OH, USA) was added to the drinking water at a final concentration of 5% (wt/vol) for 5 days (36) to 6-to 8-week-old mice. DSS was freshly dissolved every 2 days. Controls were time-matched with mice receiving regular drinking water only. Mean DSS consumption was noted per cage each day. Weight loss, stool consistency, and bleeding were reported (37) from day 0 to day 5 during DSS treatment. Blood in the stool was evaluated using the Hemoccult II test (Beckman Coulter, Oakville, ON, Canada). Mice were sacrificed at day5. Collagen deposition and fibrosis scores were assessed as described previously (38). Samples were isolated from the splenic flexure, fixed in formalin, paraffin embedded, sectioned in 3-µm sections, and stained using Masson's Trichrome (Sigma, Mississauga, ON, Canada). Collagen deposition and fibrosis were scored based on a published scoring system that considers collagen deposition (score 0 = no increase, score 1 = increase in the submucosa, 2 = increase in the mucosa, 3 = increase in the muscularis mucosa and its thickening, 4 = increase in the muscularis propria, and 5 = gross disorganization in the muscularis propria) and the percent involvement (score 1 = 1-25%, score 2 = 26-50%, score 3 = 51-75%, and score 4 = 76-100%) (39).

colonic Protein assay
Colonic sample were homogenized mechanically in 700 µL of Tris HCl buffer containing protease inhibitors (Sigma, Mississauga, ON, Canada) then centrifuged for 30 min, and supernatants were frozen at 80°C until assay (32). Cytokines release and arginase activity measurements were performed on clarified full-thickness colon homogenates from mice and or supernatants from cell culture using enzyme-linked immunosorbent assays (ELISAs). Commercial ELISA kits for mouse IL-10, mouse IL-18, human IL-8 and human IL-18 (R&D Systems, Inc., MN, USA), and mouse arginase activity (Abnova, Walnut, CA, USA) were used.

Macrophage cell culture
Peritoneal macrophages were isolated from C57BL/6 male mice as described by Mosser and Zhang (40). Isolated macrophages were cultured in 2 mL Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% deactivated fetal bovine serum (FBS). Cell cultures were incubated in a humidified 5% CO2 incubator at 37°C. The overall cell viability of the adherent cell was greater than 95%. Ex vivo colitic macrophages isolation; 5 days after the beginning of the DSS treatment, resident peritoneal macrophages were isolated from all groups and subjected to further analysis. In vitro AAM activation; peritoneal macrophages were isolated from naive male C57BL/6 mice then serum starved overnight in DMEM with low FBS (0.5%). Macrophages were washed three times with 1% PBS solution and pretreated with CHR (200 ng/mL) for 2 h and then exposed for additional 6 h to 1% PBS in medium or IL-4/IL-13 (20 ng/mL) to induce AAM (40). Cell and supernatant medium were harvested for analysis. Eagle's Minimum Essential Medium (glutamine, high glucose) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 20% deactivated FBS was used. Cells were incubated in a humidified 5% CO2 incubator at 37°C. Caco-2 cells were detached by using 3 mL of trypsin (0.05% trypsin, 0.53 mM EDTA) and seeded at 3 × 10 5 cells/well onto tissue culture 24-well plates. Cells were counted using a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Inc., Mississauga, ON, Canada), and cell count was verified using a conventional hemocytometer cell counting method. The cell culture medium was changed every 3 days until the cells fully differentiated (80-90% confluent). For each experimental setup, three separate experiments were performed, and at least six wells per condition were assigned.

Direct CHR Treatment of LPS-and DSS-Stimulated Colonic Cell Line
Caco-2 cells were seeded at 3 × 10 5 cells/well onto tissue culture plates and treated with 2 mL of medium containing CHR, sCHR (100 ng/mL) or 1% PBS for 24 h. Then cells were challenged with LPS (1 µg/mL) or 5% DSS for an additional 24 h (41). Gene expression of TJ proteins and proinflammatory cytokines IL-8 and IL-18, migration, proliferation, viability, and oxidative stress survivability of Caco-2 cell line were evaluated.

Colonic Cell Line Migration Assessed by Using Wound-Healing Assay
Caco-2 cells were wounded using a sterile 100-µL pipette tip dragged perpendicular to a black line drawn on the underside of the plate for reference. Images were taken at wounding (0), 12, 24, and 48 h later using an Evos FL imaging system at 4× magnification. Wound widths were determined by averaging six measurements per image. Only scratches with edges that could be captured in one frame at the time point 0 h were included for final analysis. Measurements were taken from edge to edge at the time point 0 h and compared with measurements from 12, 24, and 48 h using ImageJ (National Institutes of Health, Bethesda, MD, USA) software (42). The reported values were the difference TCATTGCAGATGAACGGAT TTGGAGAGCGAGGTCTT between time point 0 and the other time points, with higher values representing increased cellular migration.

Colonic Cell Line Proliferation and Viability Assessed Using Cell Numbers and MTT Assay
Colonic cell line viability was studied in vitro by using the 3-(4, 5-dimethyl thiazolyl-2yl)-2, 5-diphenyl tetrazolium (MTT) assay. Briefly, Caco-2 cells were seeded into 96-well plates at a density of 5 × 10 5 cells/well and serum starved for 24 h. Cells were cultured for 3 days in 200 µL of medium containing CHR (100 nmol/ mL) or sCHR (

Data analysis
Group comparisons were determined using unpaired Mann-Whitney U test, and one-and two-way ANOVA followed by a post hoc test when appropriate. Spearman's correlation test was used. P values (two-tailed) below 0.05 were considered as significant. Data are presented as mean ± SEM and statistics were analyzed using GraphPad Prism software (version 6; GraphPad Software, Inc., La Jolla, CA, USA).   Two-way repeated measures or one-way ANOVA followed by multiple comparison tests. Each value represents the mean ± SEM, n = 8-10 mice/group. # refers to significance compared to control groups. Each experiment was repeated at least three times.  (Figures 3B-D). In colitic mice, intracolonic administration of CHR significantly reduced the collagen deposition and fibrosis scores (Figures 3E,F). Moreover, DSS administration increased Col1a2 mRNA colonic expression ( Figure 3G) and treatment with CHR decreased it significantly ( Figure 3G). Administration of the sCHR peptide did not modify the markers studied.
chr Decreases il-18 release and regulates colonic gene expression of TJ Proteins in Dss-induced colitis Tight junction proteins and IL-18 play critical roles during the progression of IBD (12,46). Compared with non-colitic mice, a significant decrease in Cldn1, Zo1, Cdh1, and Ocln colonic mRNA levels was detected in DSS-treated mice ( Figure 4B), however, CHR treatment abolished this effect (Figures 4A,B). We also observed that DSS treatment elevated colonic protein and mRNA expression levels of IL-18 ( Figure 4A) which was significantly decreased when mice were treated with CHR ( Figure 4A).
Administration of the sCHR peptide neither modified the control conditions nor the deleterious effect of the DSS treatment.

chr increases aaM Polarization and increases anti-inflammatory Mediators in Dss-induced colitis
Alternatively activated macrophage plays a significant role in colonic tissue-repair through the high production of IL-10, arginase, and other extracellular molecules (47,48). Therefore, to further determine the role of CHR in modulating immune cells during the development of colitis, colonic AAM mediators and markers were investigated. Colitic CHR-treated mice displayed an increase in IL-10 and Arginase activity (Figure 5A), moreover, mRNA expression of Il10, arginase (Arg1), Ym1 Chitinase-like protein (Ym1), and found in inflammatory zone protein (Fizz1) were significantly upregulated ( Figure 5B). To confirm this effect, we next investigated the role of CHR in peritoneal macrophage isolated from colitic mice. Measurement of AAM mediators and markers revealed an increase in IL-10 and arginase activity in response to CHR (Figure 5C), along with increased mRNA expression of Il10, Arg1, Ym1, and Fizz1 ( Figure 5D), when compared to macrophages isolated from the colitic PBS-treated group. Administration of the sCHR peptide neither modified the control conditions nor the deleterious effect of the DSS treatment. One-way ANOVA followed by multiple comparison tests. Each value represents the mean ± SEM, n = 8-10 mice/group. # refers to significance compared to control groups. Each experiment was repeated at least three times. Considering the effect of other CHGA-derived peptides on macrophages and their contribution to macrophages polarization (32,33,(49)(50)(51)(52), we reasoned that CHR might be involved in AAM polarization. To determine whether CHR can directly affect the polarization of AAM, peritoneal macrophage of naive C57BL6 mice were isolated and pretreated with CHR and polarized toward AAM using IL-4/IL-13. CHR pretreatment increased mRNA expression levels of AAM markers, Il10, Arg1, Fizz1, and Ym1, and the release of IL-10 and Arginase activity (Figures 6A,B). Administration of the sCHR peptide neither modified the control conditions nor the deleterious effect of the IL-4/IL-13 treatment.

chr-Treated aaM conditioned Medium Maintains gene expression of TJ Proteins and Decreases il-8 and il-18 release in lPs-and Dss-stimulated colonic epithelial cells
The human Caco-2 IEC system has been commonly used as an in vitro model of the intestinal epithelium (53)(54)(55). Also, caco-2 cells have been used as in vitro model of IBD for potential drug testing and screening (56)(57)(58)(59)(60). Therefore, culture studies were performed using Caco-2 epithelial cells and AAM conditioned medium to assess whether CHR-treated AAM conditioned  , and Ym1) in the peritoneal macrophages. One-way ANOVA followed by multiple comparison tests. Each value represents the mean ± SEM, n = 8-10 mice/group. # refers to significance compared to control groups. Each experiment was repeated at least three times. TJ proteins (Figures 8A,B). Administration of the sCHR peptide neither modified the control conditions nor the deleterious effect of induced by LPS or DSS treatments.

chr-Treated aaM conditioned Medium Promotes epithelial Migration, Proliferation, Viability, and Oxidative stress Viability
The appropriate activation of AAM is crucial for tissue repair (48), and IBD involves functional impairment of IECs, associated with infiltration of macrophages in the lamina propria (6,61,62). Macrophages can mediate protective effects via a variety of mechanisms, including maintenance or reshaping of the epithelial homeostasis through cell proliferation and migration, and by promoting resistance to epithelial apoptosis induced by oxidative stress (5,63). Therefore, we investigated the potential consequences of CHR-treated AAM conditioned medium on the functions of colonic epithelial cells using Caco-2 cells. In the presence of CHR-treated AAM conditioned medium, migration, viability, and proliferation of the epithelial cells increased (Figures 9A-D). Administration of the sCHR peptide did not have any effect on cell proliferation or migration. Furthermore, oxidative stress is a feature of intestinal inflammation and initiates epithelial apoptosis (17,64). Therefore, Caco-2 cells were exposed to the free radical donor, H2O2, and cell survival was assessed. H2O2 caused a significant reduction in cell survival compared with untreated cells, and cell survival in H2O2-treated cultures significantly improved in the presence of CHR-treated AAM-conditioned medium ( Figure 9E). Administration of the sCHR peptide neither modified the control conditions nor the deleterious effect of induced by H2O2. . One-way ANOVA followed by multiple comparison tests. Each value represents the mean ± SEM, n = 3-5/group. # refers to significance compared to control groups, Each experiment repeated at least three times.  . One-way ANOVA was used to analyze the data followed by multiple comparison tests. Data represent mean ± SEM (n = 6). # refers to significance compared to control groups. Each experiment repeated at least three times. Finally, we investigated the direct interaction between human cell line and CHR in LPS-and DSS-stimulated cells. Exposing Caco-2 cells to LPS (1 µg/mL) or 5% DSS for 24 h led to a significant decrease in the cell migration, cell proliferation and viability (Figures 10A-D) and exogenous CHR treatment restored these properties (Figures 10A-D). Surprisingly, in the absence of stimuli, CHR alone induced a significant increase in migration, viability, and proliferation of the cells (Figures 10A-D). H2O2 caused a significant reduction in cell survival compared with untreated cells, and treatment with CHR significantly improved it ( Figure 10E). Administration of the sCHR peptide neither modified the control conditions nor the effect on cell proliferation or migration and the deleterious effect of induced by H2O2.

DiscUssiOn
This study, for the first time, shows possible novel mechanisms by which CHR ameliorated intestinal inflammation by regulating IECs homeostasis and enhancing the activity of AAM in preclinical models. In patients with active UC, CHR showed a positive correlation with AAM markers and gene expression of TJ proteins and a negative correlation with IL-8, IL-18, and collagen gene expression. Experimentally, CHR treatment reduced the onset and severity of colitis, decreased colonic collagen deposition, promoted AAM mediators, and ultimately maintained the homeostasis of IECs during the development of DSS-induced colitis. Although CHR alone had no apparent effect on the AAM, CHR significantly expanded the polarization of AAM in the presence of IL-4/IL-13. Moreover, CHR indirectly and directly regulated colonic gene expression of TJ proteins, decreased IL-8 and IL-18 release in LPS-and DSS-stimulated human colonic epithelial cell line, and exhibited a protective effect in regulating epithelial cell migration, proliferation, viability, and oxidative stress survivability. Taken together, these findings extend the influence of CHGA-derived peptides to intestinal inflammation. A complex network of events at molecular, cellular, and tissue levels underlie inflammation and remodeling that are tightly regulated by various mediators and mechanisms and that eventually contribute to the development of IBD. One of these molecules is the CHGA and its derived peptides, which, have emerged as an essential axis in immune cells migration and immune responses in IBD (32,33). Recently, it has been described that CHR can affect neutrophils (25,65). In our study, we demonstrated a positive correlation between the expression of CHR and AAM markers in patients with active UC. Experimentally, intracolonic administration of CHR reduced colitis severity through the production of IL-10 and arginase activities and the promotion of AAMassociated gene expression (Ym1, Fizz1) in the colonic mucosa and peritoneal macrophages. Although peritoneal macrophages are present at a distance from the mucosal inflammatory site, several studies have implicated these cells in the progression of colitis Two-way or one-way ANOVA was used to analyze the data followed by multiple comparison tests. Data represent mean ± SEM (n = 6). # refers to significance compared to control groups. Each experiment was repeated at least three times. and in the unbalanced proinflammatory and anti-inflammatory axis (66)(67)(68). Previously, we reported that intracolonic administration of CHGA-derived peptides reduced the clinical sequelae of colitis and modulated the functional activity of peritoneal macrophages (32,69). In addition to that, CHR can penetrate the cells and interfere with some intracellular pathways (70)(71)(72). It is therefore possible that intrarectal administration not only has local effects on the colonic mucosa but may also exert effects in the surrounding and adjacent tissues and cavities. Activation of anti-inflammatory AAM by stimulatory signals (IL-4, IL-13, or TGFβ1) (40) can restrain the proinflammatory immune responses through the release of anti-inflammatory molecules and various components affecting the extracellular matrix and tissue repair (8). Over the past decade, several studies have demonstrated an excessive production of proinflammatory Th1-and Th17related cytokines (73) and a reduced AAM number in the gut of patients with IBD (9) and experimental studies confirmed these observations. DSS-induced colitis is mainly driven by an activation of CAMs and treatments with drugs interfering with their proinflammatory function result in amelioration of the intestinal inflammation (32). Conversely, it has been demonstrated that AAM can decrease the onset and severity of murine colitis (9). AAM not only protect against colitis directly but also can support the directionally concordant expansion of the Treg/Th17 cell axis associated with a restoration of the gastrointestinal immune tolerance and the repair of mucosal injuries (62). For example, Lupeol™ can mitigate intestinal inflammation by inducing and increasing survival from lethal DSS-induced colitis by upregulating AAM-related genes and downregulating CAMs-related genes (74). Furthermore, worm infections have been associated with a reduced progression of colitis through the increase of IL-4/IL-13 and the upregulation of AAM (9). Intestinal injury and inflammation can induce excessive transmural extracellular matrix collagen deposition accompanied by an alteration of normal tissue architecture leading ultimately to fibrosis (39). Here, we reported that CHR negatively correlated with collagen expression in patients with active UC and that exogenous CHR treatment decreased significantly colonic collagen expression and deposition and protected against DSS-induced colitis. In that context it has been described that the arginase activity by murine AAM can facilitate the assembly of proline, which is critical for collagen production (75). AAM-fibroblast interaction is imperative for wound healing, but a dysregulated interaction can result in fibrosis and possibly stricture formation in the gastrointestinal tract. Although the role of AAM in the pathophysiology of fibrosis is not clear, some studies suggest that AAM display a profibrotic profile and stimulate collagen deposition (75)(76)(77)(78)(79), conversely other reports demonstrate that AAM can protect against fibrosis (80,81). Here, CHR displayed a unique feature by reducing colitis severity and maintaining the IECs homeostasis without promoting collagen deposition and fibrosis. Therefore, it can be postulated that the role of AAM in collagen synthesis and deposition can be influenced by the surrounding microenvironment and the type of tissue. Our data confirm the non-deleterious effect of AAM activation in the context of colonic inflammation as demonstrated previously by other groups (9,82).
Tight junction-deficient mouse models revealed pathophysiologic features of mucosal inflammation compatible with human UC (83). As intestinal epithelial barrier is regulated by TJ proteins (12) and as regulation of TJ proteins is correlated with intestinal inflammation (13,14), we quantified the gene expression of TJ proteins in colonic tissue. In patients with active UC, we demonstrated a strong positive correlation between CHR and gene expression of TJ proteins and AAM. In our animal model, we showed that CHR treatment ameliorated the disease severity by maintaining colonic gene expression of TJ proteins and enhancing polarization of AAM. Several studies have reported that the activity of AAM can promote tissue-repair functions such as cell proliferation or matrix remodeling through the expression of different molecules such as arginase, IL-10, TGFβ1, Ym1, and Fizz1 (47,48,84). In our study, using an in vitro culture system, we demonstrated that CHR-treated AAM conditioned medium preserved gene expression of TJ proteins in LPS-and DSS-stimulated epithelial cells and improved the functional capacities of epithelial cells by regulating migration, proliferation, and viability. This is supported by previous data demonstrating that a reduction of intestinal inflammation is associated with an enhancement of AAM activity, limitation of the proinflammatory signals, and maintenance of IECs functions (85,86). Furthermore, we demonstrated that CHR can directly restore the epithelial homeostasis by maintaining gene expression of TJ proteins and by improving the epithelial cells functional abilities to migrate, proliferate, and survive in response to LPS, DSS, or oxidative stress stimuli. Similar study demonstrated a protective effects of 5-hydroxytryptamine receptor 4 agonist against DSSinduced colitis, involving resistance of caco-2 epithelial cells to the detrimental effects of oxidative stress by the free radical donor (H2O2) (64).
Our study also described the ability of CHR to improve proliferation and viability of Caco-2 epithelial cells. Receptors for CHGA-derived peptides seem not to exist, but the sequence similarity of these peptides with cell penetrating abilities (70-72) may explain the ability of CHR to enter the cell and interact with the Two-way or one-way ANOVA was used to analyze the data followed by multiple comparison tests. Data represent mean ± SEM (n = 6). # refers to significance compared to control groups. Each experiment was repeated at least three times. intracellular pathways. We speculate that CHR might affect some specific intracellular pathways including the p38 MAP kinase or the activator of transcription 1 (STAT1), which are well known to enhance the functional abilities of epithelial cells to proliferate and migrate (87,88). Supporting this idea, recent studies have demonstrated the importance of these two pathways. Treatment of Caco-2 with pregnane X receptor agonists or IL-28 significantly increased wound healing activity and proliferation, and in both context, when give to mice, a significant decrease of colitis was determined (88,89). Other pathways are also for consideration as CHR induces calcium entry in human neutrophils through a calmodulin-regulated calcium independent phospholipase A2 (70,72), as this enzyme seems to play a role in the regulation of the integrity of epithelial TJ proteins and the pathogenesis of colitis (90).
Our findings also revealed that CHR is negatively correlated with IL-8 and IL-18 in colonic biopsies from patients with active UC. In parallel using our mouse model of colitis, we demonstrated that exogenous CHR treatment reduced the weight loss and colonic IL-18 release. Moreover, CHR peptide directly and indirectly through CHR-treated AAM conditioned medium decreased IL-8 and IL-18 release in LPS-and DSS-stimulated epithelial cell line. Studies have reported that a deletion of IL-18 protected against experimental colitis and minimized the mucosal damage through maintenance of the epithelium equilibrium (46,91), demonstrating the importance of IL-18. The overall effect of CHR on IL-18 can also explain indirectly the impact of fibrosis described above, as transmural intestinal inflammation favors colitis-associated fibrosis through the promotion and the expression of collagen and IL-18 (92). Downregulation of IL-18 expression results in a decreased inflammatory process (92). IL-8 known as CXCL-8, is a potent chemoattractant secreted by IECs, and mediates polymorphonuclear leukocytes recruitment from the lamina propria to the epithelium and is increased during IBD (93,94). The close relation between IL-8 and CHGA-derived peptides is supported by previous data demonstrating that vasostatin-1, another CHGA-derived peptide, can decrease the onset and severity of experimental colitis via an inhibition of human IECs IL-8 production (95). As in mice, the homolog of human IL-8 is completely absent from their genome IL-8 was not quantified (93).
In this study, we assessed only the mRNA level considering the main concept of molecular biology, which states that "DNA makes RNA makes proteins, " suggesting a direct association between mRNA and protein levels (96). Although, several studies have found significant correlations between mRNA levels and protein levels (96)(97)(98), in some conditions the mRNA levels do not correlate with protein expression levels or even with the protein function. Therefore, the gene expression presented in our study can only provide an idea of understanding the potential action of CHR involved in the protection against colitis, and further studies are warranted to investigate the precise effects of CHR on the protein expression and localization of the proteins studied.
We cannot rule out the possibility that other mechanisms, including gut microbiota dysbiosis, apoptosis, and permeability, can also contribute to the changes seen post-treatment. Several studies have highlighted the importance of gut microbiota in IBD pathophysiology, innate immunity, and epithelial homeostasis (99)(100)(101). Previous studies have demonstrated that CHR features antimicrobial activity (25,26) that may affect the gut microbiota and their associated metabolites. Consequently, future studies are required to investigate the potential effect of CHR on gut microbiota and also to confirm its role on intestinal permeability and apoptosis.

cOnclUsiOn
Here, we report a protective effect of CHR during the development of colonic inflammation (Figure 11). The results of this study have clinical relevance. First, they prompt close consideration of the relationship between CHR and disease activity in patients with IBD. Second, if this correlation is confirmed, then patients with IBD might be an appropriate group to target with novel treatment strategies involving CHR.

eThics sTaTeMenT
Human subjects: patients diagnosed with active UC and persons with no IBD who were undergoing colonoscopy were recruited from the University of Manitoba IBD Clinical and Research Centre. Informed consent was obtained from patients and control subjects before the study. This study was approved by the