INTIMP-03357; No of Pages 7 International Immunopharmacology xxx (2014) xxx–xxx
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Article history: Received 7 April 2014 Received in revised form 2 July 2014 Accepted 27 July 2014 Available online xxxx
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Mesenchymal stem cells (MSCs) were found to provide an effective therapeutic role in inflammatory diseases by modulating inflammatory responses and tissue regeneration by their differentiation ability. The present work sought to demonstrate the potential therapeutic use of MSCs in treating chronic inflammatory bowel disease (IBD) in mice. A new model to induce chronic IBD based on alternative administration periods of Dextran Sodium Sulfate (DSS). Mice were divided into 2 groups; one was treated with MSCs and the other was treated with phosphate-buffered saline (PBS). Assessment of therapeutic efficacy of MSCs was by measuring weight, stool scoring, histopathologic examination, and measuring the gene expression of inflammatory markers: Interleukin-23 (IL-23), Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), and Intercellular adhesion molecule-1 (ICAM-1). The results showed that DSS administration causes bloody and watery stool, weight loss, and altered histopathologic picture. MSC treated mice showed a significant improvement in stool condition, weight gain, and normal histopathologic picture compared to the PBS treated mice. Moreover, gene expressions of inflammatory markers in the intestines of the MSC treated mice were also significantly lower than those of the PBS treated mice. In conclusion, the data here showed that MSCs have a clear potential efficacy in the treatment for IBD, as their immune modulation effects include inhibition in the expression of key inflammatory markers that each plays an important role in the pathogenesis of IBD. © 2014 Elsevier B.V. All rights reserved.
Keywords: DSS induced colitis Interleukin Tumor necrosis factors Interferon Adhesion molecules Bone marrow derived mesenchymal stem cell transplantation Immunomodulation
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Department of Biochemistry, Faculty of Pharmacy, Misr International University, Cairo, Egypt Department of Biochemistry, Faculty of Medicine, Cairo University, Egypt Department of Biochemistry, Faculty of Pharmacy, Al Azhar University, Cairo, Egypt
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Ahmed Gouda Abdel Rahman Abdel Salam a,⁎, Hazem M. Ata b, Tarek M. Salman c, Laila A. Rashed b, Dina Sabry b, Mona F. Schaalan a
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Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice
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1. Introduction
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Inflammatory bowel disease (IBD) is a chronic idiopathic disease 45 mainly represented by two major forms, ulcerative colitis (UC) and 46 Crohn's disease (CD) [1]. While the precise etiology of IBD is still 47 Q14 unknown, possible etiologies include the role of environmental factors 48 [2], genetics [3], microbial factors [4], and mucosal immune defects 49 [5]. Based on these etiologies, many treatment lines were developed. 50 The current range of treatments for IBD covers both conventional and 51 biological therapies. Conventional therapy includes the use of anti52 inflammatory drugs, immunosuppressive agents, antibiotics, and 53 probiotics; biological therapies mainly include the use of different 54 anti-TNF-α agents, and a plethora of other novel biological agents [6]. 55 The mechanism of DSS-induced colitis is mainly due to the direct 56 toxicity to the colonic epithelial cells, subsequently increasing the 57 permeability of the intestinal mucosa and allowing the transport of 58 luminal bacterial products from the bowel lumen to the submucosal 59 tissue [7]. This hypothesis is bolstered by the work of Leung et al. [8], ⁎ Corresponding author. Tel.: +20 1 589095. E-mail address: [email protected] (A.G.A.R.A. Salam).
who demonstrated that activated T cells in the colonic mucosa play a role in modulating the immunological reaction in DSS induced colitis in mice by interacting with lysed bacterial cell wall lipopolysaccharide and activated macrophages. It has been shown that CD4+ T cells play an important role in the development of DSS induced experimental colitis [9]. According to Yan et al. [10], DSS administration increases the gene expression of TNF-α, IFN-γ and IL-12 in the area of the distal colon due to massive infiltration of macrophages, T cells, B cells and neutrophils. Moreover, DSS causes an increase in the level of ICAM-1 that leads to the migration of leukocytes to the site of inflammation [7]. According to Dieleman et al. [11], acute DSS induced colitis is predominantly macrophage-driven colitis, while, Dieleman et al. [12] reported that chronic DSS induced colitis is mediated by Th1 and Th2 activation. Thus, it appears that activated macrophages alone lead to an acute colitis but that only activated macrophages interacting with activated T-cells lead to chronic disease [13]. Bone marrow transplantation (BMT) has improved not only hematological diseases such as leukemia, but also autoimmune diseases. However, BMT significantly affects the systemic immune system of recipients; whether it is effective for IBD remains controversial [14]. Bone marrow contains hematopoietic stem cells and non-hematopoietic
http://dx.doi.org/10.1016/j.intimp.2014.07.030 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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2.1. Animals
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Eight week female albino mice with an average weight of 18–25 g (provided from The Nile Company for Pharmaceutical Industries) were housed in the animal house facility at Misr International University and kept at 25 °C with normal light/dark cycle with free access to food and water for 7 days. 8 week male mice were housed in the same conditions and used for flushing of the bone marrow for isolation and culture of MSCs.
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2.2. Development of DSS-induced colitis model in mice (pilot study)
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According to Wirtz et al. [21], the appropriate cycle for the induction of chronic IBD was 5 ml DSS/day/mouse for 7 days, followed by replacing DSS on Day 8 with DSS-free drinking water for another 14 days, for three cycles. This approach was recommended because prolonged administration of DSS causes high mortality rates, and DSS-induced colitis (being a reversible model) requires administration of DSS and normal water alternatively for a specific period of time. Thus, a pilot study was undertaken in two parts: the first to determine the appropriate concentration of DSS to induce IBD, and the second to determine the proper DSS/ water cycle for our experimental purposes.
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2.4. Administration of MSCs
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2.2.1. Part A: assessment of determination of appropriate DSS concentration To determine the appropriate DSS concentration, 150 mice were placed in individual cages and randomly divided into three groups. In the first group, each mouse was given 5 ml of 2% DSS, in the second group 5 ml of 4% DSS, and in the third group 5 ml of 5% DSS for 7 days. 2.2.2. Part B: determination of appropriate DSS/water cycle To determine the appropriate DSS/water cycle to be used, 90 mice were placed in individual cages and randomly divided into three groups. In the first group, mice were administered 5 ml of 4% DSS for 7 days followed by 14 days of water administration. In the second group, mice were administered 5 ml of 4% DSS for 7 days followed by 10 days of water administration. In the third group, mice were administered 5 ml of 4% DSS for 7 days followed by 5 days of water administration. The suitable model to induce chronic colitis in mice using DSS is by administration of 4% DSS for 7 days followed by administration of water for 5 days for 3 consecutive cycles.
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After 7 days of 4% DSS administration, mice were divided into two 155 groups; the first group, injected with PBS, and the second group, 156 injected intraperitoneally with MSCs [20] . 157 158
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The treatment efficacy of the MSC was evaluated by three methods: 159 physical examination, histopathological examination, and measuring 160 the gene expression of inflammatory markers. 161 2.5.1. Macroscopic/microscopic evaluation of therapeutic efficacy of MSC on DSS-colitis The following indices of experimental colitis were monitored daily among the various experimental mice: (a) body weight, (b) food and water intake, and (c) stool condition. Daily, the mice were removed from their individual cages and weighed. The amount of food present and the level of water in their cage bottle were also noted. Loss of food, due to powdering, and water, due to any seeping/forced loss (i.e., if bedding was pushed up at the bottle), was noted when apparent. Stool condition was subjectively scored from grades 0 to 3 as described: grade 0 indicates a normal-to-semi-solid stool and no blood; grade 1 indicates a normal-to-semi-solid stool and blood-tinged; grade 2 indicates a semi-solid-to-fluid stool with definite evidence of blood; and, grade 3 indicates a bloody fluid stool [20].
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2.5.2. Histopathological examination of intestine tissue Necropsy samples were taken from the intestine of mice in different groups and fixed in 10% formalin for 24 h. After rinsing in tap water, the samples were serially dehydrated in alcohol (methanol, ethanol, and then absolute ethanol). Specimens were then cleared in xylene, and embedded in paraffin. Samples were then sectioned at 4-μm thickness using a microtome. The tissue sections were collected on glass slides, de-paraffinized, stained with hematoxylin and eosin, and then subjected to blinded examination under a light microscope [20].
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2.5.3. Inflammation by measuring the gene expression of inflammatory markers The mRNA expression of inflammatory mediators such as TNF-α, IFN-γ, IL-23, and ICAM-1 in the colon was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). Local mRNA expression was measured in the rectum and distal colon (slightly orally to the rectum) samples. Total RNA was extracted from the tissues using QIAamp RNA mini kit (Qiagen, Cairo, Egypt) according to the manufacturer's instructions. PCR primers and Taqman probes for each mediator were used. Real-time quantitative RT-PCR analyses were performed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
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Bone marrow was harvested by flushing the tibiae and femurs of 8-wk-old male albino mice (18–25 g) with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (each from Gibco, Grand Island, NY). Nucleated cells were isolated with a density gradient [Ficoll/Histopaque (Pharmacia, Uppsala, Sweden)] and re-suspended in a complete culture medium supplemented with 1% penicillin–streptomycin (Gibco). Cells were incubated at 37 °C in 5% humidified CO2 for 12–14 days; media were changed every 2–3 days. When large colonies developed (80–90% confluence), cultures were washed twice with PBS (pH 7.4) and cells were trypsinized with 0.25% trypsin/1 mM EDTA solution for 5 min at 37 °C. After centrifugation, cell pellets were re-suspended with serumsupplemented medium and incubated in a 50-cm2 culture flask (Falcon, Cairo, Egypt). The resulting cultures were referred to as first-passage cultures [19].
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MSCs. These MSCs are isolated from the adult bone marrow and expanded ex vivo in order to differentiate into several types of cells, such as osteocytes, chondrocytes, adipocytes, hematopoietic supporting stroma, and endothelial cells [15]. Therefore, these MSCs may be useful in tissue regeneration [16]. Moreover, MSCs have shown to modulate allogeneic immune cell responses from a pro-inflammatory toward an antiinflammatory state by affecting dendritic cells, T-lymphocytes, and natural killer (NK) cells [17]. MSCs also appear to regulate the immune function in inflamed tissues by affecting the formation and secretion of pro-inflammatory cytokines and chemokines. In fact, the in vivo exogenous administration of MSCs imparts an anti-inflammatory action and improves the health of tissues that had become inflamed in response to injury induction [18]. Although, MSCs have shown an anti-inflammatory effect, their cellular mechanisms to pursue such function should be examined. In the present study, we sought to examine the anti-inflammatory potential of MSC exogenously administered to mice that were suffering from DSS induced colitis. Assessment of therapeutic efficacy of MSCs was by measuring weight, stool scoring, histopathologic examination, and measuring the gene expression of inflammatory markers: IL-23, TNF-α, IFN-γ, and ICAM-1 in colonic tissues recovered from the mice.
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Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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Data are expressed as mean ± SE. Significant differences were determined using independent t-test and SPSS for Windows Version 15.0 Computer Software (SPSS, Chicago, IL, USA). Non-parametric analyses were used for evaluating all the colitis indices. Results were considered significant at P-values b 0.01. 3. Results
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3.1.1. Part A: determination of appropriate DSS concentration To determine the appropriate model of induction of IBD by DSS, different DSS concentrations (2% [21], 4% and 5% [20]) were administered for 1 week. Assessment of IBD degree was performed using stool scoring, weight loss, and histopathologic examination.
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3.1.1.2. Average body weight. Fig. 2 shows that through one week of DSS administration, 2% DSS administered mice gain weight as an indication to normal bowel condition, 4% DSS administered mice gradually lose weight as an indication to deterioration of bowel condition, and 5% DSS administered mice lose weight with 100% mortality rate.
3.1.1.3. Histopathologic examination. Fig. 3(a, b, c) reveals that the administration of 2% DSS did not cause any harm to the intestinal mucosa, while, the administration of 4% and 5% DSS causes almost the same degree of inflammation. 3.1.2. Part B: determination of appropriate DSS/water cycle To determine the appropriate DSS/water cycle, three cycles were examined. Assessment of IBD degree was performed using stool scoring, weight loss, and histopathologic examination.
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3.1.2.2. Average body weight. Fig. 5 shows that the administration of 4% DSS for 7 days followed by 5 days of normal drinking water leads to mild improvement in the body weight.
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3.1.3. Assessment of MSC potential therapeutic effect on IBD in tested groups Efficacy of MSCs in treating IBD in the tested groups was assessed by stool scoring, average body weight, histopathologic examination, and measuring the gene expression of the inflammatory markers.
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3.1.2.3. Histopathologic examination. As per previous observations, histopathologic examination was performed for three cycles (7 days 4% DSS + 5 days water) in mice. Fig. 6 reveals a continuous inflammation state even after 5 days of normal drinking water.
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3.1.3.1. Assessment of stool scoring in the studied groups. Fig. 7 shows that 244 stool condition was significantly improved by the administration of 245 MSCs. 246 3.1.4. Average body weight Fig. 8 shows that the average body weight of the mice in the studied groups in the indicated cycle intervals and revealed a significant increase in the body weight of treated group.
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3.1.5. Histopathological examination of tested groups' intestine The gross picture of control group intestine revealed that circumscribed round ulceration was observed in the inner mucosa associated with congestion and redness all over the wall and that there was no gross finding observed in the mucosa and outer surface all over the intestine wall. Moreover, focal desquamation was observed in the mucosal lining epithelial cells associated with a massive number of inflammatory cell infiltration mainly neutrophils, macrophages and lymphocytes with congestion of the blood vessels in the underlying lamina propria, and the muscularis showed that oedema with inflammatory cell infiltration was observed in both caecum and colon, (Fig. 9a) which shows histopathological confirmation of IBD. On the other hand, the gross picture of treated group intestine revealed that there was no gross finding observed in the mucosa and outer surface all over the intestine wall. Moreover, no histopathological alteration was observed in the mucosal layer with the lamina propria as well as in the submucosal and muscular layers, (Fig. 9b), which shows a histopathologically normal intestine.
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3.1.6. Assessment of body weight in the studied groups As illustrated in Fig. 10, the average body weights of the treated groups are significantly higher (P b 0.01), compared with the control group. This significant weight elevation is obvious starting from the second DSS administration cycle on days 19, 24 and 31.
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3.1.7. The inflammatory markers in control and treated groups Fig. 11 illustrates the relative gene expression of pro-inflammatory markers TNF-α, and IL-23, IFN-γ, and ICAM-1 were measured in both control and treated groups, showing that, the relative gene expression
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was used as an internal standard with Taqman GAPDH control reagents (PE Applied Biosystems, CA, USA). Levels of mRNA expression were expressed as percentages of controls.
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Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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The application of MSC therapy has opened a new horizon for the treatment of IBD, as most of the pharmacological approaches haven't 283 yet reached the desired efficacy and safety outcomes. MSCs are 284 multipotent cells found in various species, including mice, and are 285 often used for cellular transplantation therapy [22]. Most previous 286 studies have focused on these attractive regenerative properties 287 of MSCs [22,23], indicating that MSCs can promote regeneration 288 of wounded or injured tissues by a differentiation into vascular 289 smooth muscle cells, endothelial cells, pericytes, or epithelial cells 290 [24]. 291 Chemical induction of IBD by DSS was considered an efficient and 292 easily applied model, however, choosing the correct dose to induce 293 colitis is a variable [21]. In the present study, the 4% DSS model was 294 used as administration of 2% and 5% DSS did not give the desired 295 outcome. In the first part of the pilot study the 2% DSS failed to induce 296 inflammation, which is in contrast to Wirtz et al. [21], who showed 297 that the administration of 2% DSS for 7 days had produced acute colitis. 298 However, using 5% DSS showed 100% mortality in accordance to Wirtz 299 et al. [21]. Concerning the second part of the pilot study, our results 300 showed a cycle of 7 days of 4% DSS followed by 5 days of normal drink301 Q22 ing water which is optimum for the induction of chronic IBD, in contrast 302 to Wirtz et al. [21] who induced a chronic model of colitis by the admin303 istration of DSS for 7 days followed by 14 days of water.
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Experiments done to investigate the therapeutic effect of MSCs in IBD are very few; the first attempt was done by Tanaka et al. [20] that induced DSS acute colitis and proved that the exogenous administration of MSCs ameliorates acute DSS induced colitis. Tanaka et al. [20] assessed the MSC therapeutic effect by measuring the RNA expression of inflammatory mediators such as TNF-α, IL-1β, COX-2, basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), all of which significantly decreased in MSC treated mice. The second attempt was done by Liang et al. [25] in which human umbilical cord mesenchymal stem cells (hUC-MSCs) were used to treat acute trinitrobenzene sulfonic acid (TNBS) induced IBD. The inflammatory markers such as IL-17, IL-23, IFN-γ, and IL-6 were measured to assess the therapeutic efficacy of hUC-MSCs and were shown to be significantly decreased in the treated mice. Another attempt was done by Gonzalez et al. [26] in which acute colitis was induced by TNBS and adipose tissue derived MSCs were injected to the mice. Gonzalez et al. assessed the therapeutic effect of MSCs by measuring inflammatory cytokines such as TNF-α, IFN-γ, IL-6, and IL-1β, and chemokines such as macrophage inhibitory protein II which were significantly decreased in treated mice. In the current work, the MSCs were injected once after the first cycle of chronic DSS-induced colitis. We assessed the gene expression of pro-inflammatory cytokine levels (TNF-α, IFN-γ, IL-23 and ICAM-1) in an attempt to investigate their association with MSC therapy in IBD. The results of this work indicated that the level of TNF-α gene expression was significantly decreased after MSC administration. This result is supported by recent studies that have shown that MSCs can also modulate the systemic immune systems [17,18]. Ortiz et al. [18]
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of the four inflammatory markers is significantly decreased (P b 0.01) in the treated group compared to the control group.
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Fig. 3. (a) shows that that through one week of DSS administration, 2% DSS administration causes a colon of mice with intact histological structure of mucosal layer (mu), lamina propria (Lp), submucosa (s), and muscularis (m) mice. (b) revealed that 4% DSS administration causes caecum with the massive number of inflammatory cell infiltration in the lamina propria (f) with congestion in blood vessels (v) and desquamation of the lining mucosal epithelium (m). (c) 5% DSS administration causes acute colitis extended to the muscular layer revealing caecum of the mice with oedema and inflammatory cell infiltration in the muscularis (m).
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Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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et al. [26] provided further confirmation to our findings as in their experiment AMSC injected mice showed reduced levels of TNF-α in comparison to untreated mice. The results revealed that the expression of IFN-γ was significantly decreased in MSC treated mice. This finding was confirmed by Gonzalez et al. [26] who showed reduced levels of IFN-γ in AMSC injected mice in comparison to untreated mice and related the effect of AMSCs to their ability to suppress Th1 responses and induce T regulatory cells. Liang et al. [25] also confirmed our results when they found that hU-MSC significantly decreased the level of IFN-γ. Recent findings reported that IFN-γ has a dual role in the immunosuppressive effect of MSCs; the first is stated before as MSCs suppress Th1 and induce T regulatory cells. The second effect is that MSCs inhibited the proliferation of B cells only in the presence of IFN-γ, and the capacity of IFN-γ to induce the suppressive activity of MSCs is not dependent on indoleamine 2,3 deoxygenase (IDO) but is dependent on cell to cell contact mediated by the interaction between programmed death 1 (PD-1) and PD ligand 1 (PDL-1) [28]. The most possible mechanism supporting this theory is that the expression of PD-1 and PDL-1 from both MSCs and B cells displays a significant increase in the presence of IFN-γ. These results suggest that PD-1/PDL-1 interaction between MSCs and B lymphocytes inhibits the signal transduction pathways triggered by productive B cell stimulation by antigen and affects B cell expansion. Another mechanism was supported by Krampera et al. [29] who supposed that MSCs inhibit the proliferation of B cells only in the presence of IFN-γ which implies that IFN-γ causes MSCs to produce IDO, which catalyzes the conversion of tryptophan to kynurenine which in turn suppressed the proliferation of B cells. Spaggiari et al. [30] highlighted another role of IFN-γ in MSC
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indicated that MSCs have an anti-inflammatory property and reduce inflammation and lung injury induced by bleomycin. In addition, Aggarwal and Pittenger [17] showed that human MSCs can modulate anti-inflammatory effects in co-cultures with purified subpopulations of immune-associated cells, such as dendritic cells (DCs), naive and effector T cells (T helper 1, Th1 and T helper 2, Th2), regulatory T cells (Tregs), and natural killer (NK) cells. MSCs can down-regulate inappropriate inflammatory responses by inhibiting pro-inflammatory Th1, DC1, and NK signaling and by promoting anti-inflammatory Th2 and/or suppressive Treg signaling. MSCs may thus orchestrate a shift from Th1 (pro-inflammatory) toward Th2 (anti-inflammatory) [17]. Accordingly, it is possible that MSCs could prove efficacy in the treatment of Th1-mediated inflammatory diseases. As for the pathogenesis of CD, a systemic and/or local inflammatory bowel disease, Th1 cytokines are known to have a more important role in pathogenesis than Th2 cytokines in local inflammatory tissues [27]. Recently, infliximab (anti-TNF-α antibody) as well as conventional anti-inflammatory agents are often used clinically to treat CD by attenuating inflammation. As for experimental colitis, chronic DSS-induced mucosal injury has the cytokine profile of Th1 responses [13]. Furthermore, Tanaka et al. [20] have reported that the exogenous administration of MSCs significantly decreased the level of TNF-α, IL-1β, and COX-2. Moreover, Gonzalez
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Fig. 6. The figure shows a colon of mice with desquamation of the lining of the epithelium (m) with inflammatory cell infiltration in the underlying lamina propria (f).
Fig. 8. Studied groups: average body weight.
Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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Regarding the decreased the gene expression of ICAM-1 in MSC treated mice, this is the first attempt to prove that MSC immunosuppressive effect includes the suppression of ICAM-1 expression. In contrast to our findings, Ren et al. [35] showed that MSCs do express ICAM-1 in order to perform the immunosuppressive effect. Generally, adhesion molecules play a dual role in the immunosuppressive effect of MSCs; first, Chamberlain et al. [36] stated that inflamed endothelial tissue releases proinflammatory cytokines, chemokines, and adhesion molecules in order to attract macrophages, lymphocytes, and other inflammatory cells. Second, the release of pro-inflammatory cytokines, chemokines, and adhesion molecule plays a major role in the homing of MSCs to the site of injury. Interestingly, Segers et al. [37] stated that ICAM-1 and VCAM-1 are considered markers for stem cells; the observation that confirms that MSCs can express adhesion molecules. According to Ren et al. [35] T cells secrete several inflammatory cytokines, including IFN-γ, TNF-α, and IL-1 that leads to striking upregulation in the expression of two adhesion molecules: ICAM-1 and VCAM-1 that are responsible for the increased adhesion between MSCs and T cells. Importantly, blocking of the function of adhesion molecules significantly reversed the immunosuppressive effect of MSCs in vitro and in vivo. Summing up, ICAM-1 plays a dual role as it facilitates the homing of MSCs to the site and allows the cell to cell contact, the cornerstone MSC immunosuppressive effect. To correlate these observations with our results we can conclude that in the chronic model of IBD the administration of MSCs will significantly increase the expression of ICAM-1 in the first few days of injection. However, after 26 days of administration and the severity of inflammation has decreased and most of the ICAM-1 has been consumed during the
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cell-mediated lyses upon incubation of MSCs with IFN-γ. Moreover, in this work, the gene expression of IL-23 was significantly decreased in MSC treated mice. Our result is supported by the study of Liang et al. [25] in which the level of IL-23 was significantly lower in the colon tissues of hUC-MSC treated mice. IL-23 performs its antiinflammatory function mainly by induction of Th17 that releases IL-17 [31]. Although the precise mechanism by which IL-23 maintains Th17 responses in vivo is still not well understood, recent studies have shown that Th17 cell lineage commitment is driven by TGF-β in the presence of pro-inflammatory cytokines, whereas IL-23 seems to expand or maintain Th17 cell populations [32]. IL-23 is a type 1 heterodimeric IL-12 like cytokine, comprising of a 19-kD 4-fold helical core α subunit (IL-23p19), and a disulfide linked to an additional 40-kD distinct β subunit (IL-12p40). IL-23 and IL-12 share a common subunit in their receptor complex due to the common p40 subunit [33]. Gonzalez et al. [26] reported that colons of AMSC treated mice contained a reduced level of IL-12 in comparison with untreated IBD induced colitis mice. Moreover, MSCs inhibit the differentiation of monocytes into dendritic cells by inhibiting the response of the former to maturation signals, reducing the expression of co-stimulatory molecules and hampering the ability of the former to stimulate naïve T cell proliferation and IL-12 secretion [34]. Furthermore, the level of IL-17 was significantly decreased in the colon tissue of hU-MSC treated mice [25].
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Fig. 9. (a) reveals histopathologic findings in the control group; this figure shows the inflammatory cell infiltration (f), oedema (o), and dilated blood vessels (v) in the submucosal layer. (H&E ×80). (b) reveals histopathological findings in the treated group. this figure shows a normal histological structure of the mucosa (m) and muscularis (ml) with serosa (s) (H&E ×64).
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anti-inflammatory process of MSCs, resulting in a significant decrease of ICAM-1 gene expression at the end of experiment. In conclusion, induction of chronic IBD in mice showed an increase in the gene expression of proinflammatory markers such as TNF-α, IFN-γ, IL-23, and ICAM-1. The control group (IBD-induced mice injected by sterile PBS) showed no decrease in the gene expression level of proinflammatory markers. Upon treating IBD induced mice with bone marrow derived MSCs, the gene expression of proinflammatory marker was significantly decreased. These conclusions highlighted many of the pathophysiological cascades of inflammatory bowel disease. The inflammation induced by DSS was significantly associated with the production of ICAM-1 that leads to migration and infiltration of macrophages and Th1, producing thereby TNF-α, IFN-γ and IL-23. Interestingly, ICAM-1 has a dual role in stem cell immunomodulatory effect; the first role is that high levels of ICAM-1 in inflammation site lead to the migration of MSCs because stem cells have surface receptors similar to those on the surface of macrophages, T cells, and B cells. The second role is that the high levels of TNF-α and IFN-γ in the inflamed tissue provoke MSCs to produce ICAM-1 that attracts inflammatory cells to adhere to the stem cell surface to exert its immunomodulatory effect. Finally, we can conclude that bone marrow derived MSCs can be used as an effective treatment of IBD.
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Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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Article history: Received 7 April 2014 Received in revised form 2 July 2014 Accepted 27 July 2014 Available online xxxx
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Mesenchymal stem cells (MSCs) were found to provide an effective therapeutic role in inflammatory diseases by modulating inflammatory responses and tissue regeneration by their differentiation ability. The present work sought to demonstrate the potential therapeutic use of MSCs in treating chronic inflammatory bowel disease (IBD) in mice. A new model to induce chronic IBD based on alternative administration periods of Dextran Sodium Sulfate (DSS). Mice were divided into 2 groups; one was treated with MSCs and the other was treated with phosphate-buffered saline (PBS). Assessment of therapeutic efficacy of MSCs was by measuring weight, stool scoring, histopathologic examination, and measuring the gene expression of inflammatory markers: Interleukin-23 (IL-23), Tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), and Intercellular adhesion molecule-1 (ICAM-1). The results showed that DSS administration causes bloody and watery stool, weight loss, and altered histopathologic picture. MSC treated mice showed a significant improvement in stool condition, weight gain, and normal histopathologic picture compared to the PBS treated mice. Moreover, gene expressions of inflammatory markers in the intestines of the MSC treated mice were also significantly lower than those of the PBS treated mice. In conclusion, the data here showed that MSCs have a clear potential efficacy in the treatment for IBD, as their immune modulation effects include inhibition in the expression of key inflammatory markers that each plays an important role in the pathogenesis of IBD. © 2014 Elsevier B.V. All rights reserved.
Keywords: DSS induced colitis Interleukin Tumor necrosis factors Interferon Adhesion molecules Bone marrow derived mesenchymal stem cell transplantation Immunomodulation
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Department of Biochemistry, Faculty of Pharmacy, Misr International University, Cairo, Egypt Department of Biochemistry, Faculty of Medicine, Cairo University, Egypt Department of Biochemistry, Faculty of Pharmacy, Al Azhar University, Cairo, Egypt
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Ahmed Gouda Abdel Rahman Abdel Salam a,⁎, Hazem M. Ata b, Tarek M. Salman c, Laila A. Rashed b, Dina Sabry b, Mona F. Schaalan a
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Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice
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1. Introduction
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Inflammatory bowel disease (IBD) is a chronic idiopathic disease 45 mainly represented by two major forms, ulcerative colitis (UC) and 46 Crohn's disease (CD) [1]. While the precise etiology of IBD is still 47 Q14 unknown, possible etiologies include the role of environmental factors 48 [2], genetics [3], microbial factors [4], and mucosal immune defects 49 [5]. Based on these etiologies, many treatment lines were developed. 50 The current range of treatments for IBD covers both conventional and 51 biological therapies. Conventional therapy includes the use of anti52 inflammatory drugs, immunosuppressive agents, antibiotics, and 53 probiotics; biological therapies mainly include the use of different 54 anti-TNF-α agents, and a plethora of other novel biological agents [6]. 55 The mechanism of DSS-induced colitis is mainly due to the direct 56 toxicity to the colonic epithelial cells, subsequently increasing the 57 permeability of the intestinal mucosa and allowing the transport of 58 luminal bacterial products from the bowel lumen to the submucosal 59 tissue [7]. This hypothesis is bolstered by the work of Leung et al. [8], ⁎ Corresponding author. Tel.: +20 1 589095. E-mail address: [email protected] (A.G.A.R.A. Salam).
who demonstrated that activated T cells in the colonic mucosa play a role in modulating the immunological reaction in DSS induced colitis in mice by interacting with lysed bacterial cell wall lipopolysaccharide and activated macrophages. It has been shown that CD4+ T cells play an important role in the development of DSS induced experimental colitis [9]. According to Yan et al. [10], DSS administration increases the gene expression of TNF-α, IFN-γ and IL-12 in the area of the distal colon due to massive infiltration of macrophages, T cells, B cells and neutrophils. Moreover, DSS causes an increase in the level of ICAM-1 that leads to the migration of leukocytes to the site of inflammation [7]. According to Dieleman et al. [11], acute DSS induced colitis is predominantly macrophage-driven colitis, while, Dieleman et al. [12] reported that chronic DSS induced colitis is mediated by Th1 and Th2 activation. Thus, it appears that activated macrophages alone lead to an acute colitis but that only activated macrophages interacting with activated T-cells lead to chronic disease [13]. Bone marrow transplantation (BMT) has improved not only hematological diseases such as leukemia, but also autoimmune diseases. However, BMT significantly affects the systemic immune system of recipients; whether it is effective for IBD remains controversial [14]. Bone marrow contains hematopoietic stem cells and non-hematopoietic
http://dx.doi.org/10.1016/j.intimp.2014.07.030 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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Eight week female albino mice with an average weight of 18–25 g (provided from The Nile Company for Pharmaceutical Industries) were housed in the animal house facility at Misr International University and kept at 25 °C with normal light/dark cycle with free access to food and water for 7 days. 8 week male mice were housed in the same conditions and used for flushing of the bone marrow for isolation and culture of MSCs.
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2.2. Development of DSS-induced colitis model in mice (pilot study)
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According to Wirtz et al. [21], the appropriate cycle for the induction of chronic IBD was 5 ml DSS/day/mouse for 7 days, followed by replacing DSS on Day 8 with DSS-free drinking water for another 14 days, for three cycles. This approach was recommended because prolonged administration of DSS causes high mortality rates, and DSS-induced colitis (being a reversible model) requires administration of DSS and normal water alternatively for a specific period of time. Thus, a pilot study was undertaken in two parts: the first to determine the appropriate concentration of DSS to induce IBD, and the second to determine the proper DSS/ water cycle for our experimental purposes.
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2.2.1. Part A: assessment of determination of appropriate DSS concentration To determine the appropriate DSS concentration, 150 mice were placed in individual cages and randomly divided into three groups. In the first group, each mouse was given 5 ml of 2% DSS, in the second group 5 ml of 4% DSS, and in the third group 5 ml of 5% DSS for 7 days. 2.2.2. Part B: determination of appropriate DSS/water cycle To determine the appropriate DSS/water cycle to be used, 90 mice were placed in individual cages and randomly divided into three groups. In the first group, mice were administered 5 ml of 4% DSS for 7 days followed by 14 days of water administration. In the second group, mice were administered 5 ml of 4% DSS for 7 days followed by 10 days of water administration. In the third group, mice were administered 5 ml of 4% DSS for 7 days followed by 5 days of water administration. The suitable model to induce chronic colitis in mice using DSS is by administration of 4% DSS for 7 days followed by administration of water for 5 days for 3 consecutive cycles.
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After 7 days of 4% DSS administration, mice were divided into two 155 groups; the first group, injected with PBS, and the second group, 156 injected intraperitoneally with MSCs [20] . 157 158
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The treatment efficacy of the MSC was evaluated by three methods: 159 physical examination, histopathological examination, and measuring 160 the gene expression of inflammatory markers. 161 2.5.1. Macroscopic/microscopic evaluation of therapeutic efficacy of MSC on DSS-colitis The following indices of experimental colitis were monitored daily among the various experimental mice: (a) body weight, (b) food and water intake, and (c) stool condition. Daily, the mice were removed from their individual cages and weighed. The amount of food present and the level of water in their cage bottle were also noted. Loss of food, due to powdering, and water, due to any seeping/forced loss (i.e., if bedding was pushed up at the bottle), was noted when apparent. Stool condition was subjectively scored from grades 0 to 3 as described: grade 0 indicates a normal-to-semi-solid stool and no blood; grade 1 indicates a normal-to-semi-solid stool and blood-tinged; grade 2 indicates a semi-solid-to-fluid stool with definite evidence of blood; and, grade 3 indicates a bloody fluid stool [20].
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2.5.2. Histopathological examination of intestine tissue Necropsy samples were taken from the intestine of mice in different groups and fixed in 10% formalin for 24 h. After rinsing in tap water, the samples were serially dehydrated in alcohol (methanol, ethanol, and then absolute ethanol). Specimens were then cleared in xylene, and embedded in paraffin. Samples were then sectioned at 4-μm thickness using a microtome. The tissue sections were collected on glass slides, de-paraffinized, stained with hematoxylin and eosin, and then subjected to blinded examination under a light microscope [20].
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2.5.3. Inflammation by measuring the gene expression of inflammatory markers The mRNA expression of inflammatory mediators such as TNF-α, IFN-γ, IL-23, and ICAM-1 in the colon was measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). Local mRNA expression was measured in the rectum and distal colon (slightly orally to the rectum) samples. Total RNA was extracted from the tissues using QIAamp RNA mini kit (Qiagen, Cairo, Egypt) according to the manufacturer's instructions. PCR primers and Taqman probes for each mediator were used. Real-time quantitative RT-PCR analyses were performed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
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Bone marrow was harvested by flushing the tibiae and femurs of 8-wk-old male albino mice (18–25 g) with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (each from Gibco, Grand Island, NY). Nucleated cells were isolated with a density gradient [Ficoll/Histopaque (Pharmacia, Uppsala, Sweden)] and re-suspended in a complete culture medium supplemented with 1% penicillin–streptomycin (Gibco). Cells were incubated at 37 °C in 5% humidified CO2 for 12–14 days; media were changed every 2–3 days. When large colonies developed (80–90% confluence), cultures were washed twice with PBS (pH 7.4) and cells were trypsinized with 0.25% trypsin/1 mM EDTA solution for 5 min at 37 °C. After centrifugation, cell pellets were re-suspended with serumsupplemented medium and incubated in a 50-cm2 culture flask (Falcon, Cairo, Egypt). The resulting cultures were referred to as first-passage cultures [19].
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MSCs. These MSCs are isolated from the adult bone marrow and expanded ex vivo in order to differentiate into several types of cells, such as osteocytes, chondrocytes, adipocytes, hematopoietic supporting stroma, and endothelial cells [15]. Therefore, these MSCs may be useful in tissue regeneration [16]. Moreover, MSCs have shown to modulate allogeneic immune cell responses from a pro-inflammatory toward an antiinflammatory state by affecting dendritic cells, T-lymphocytes, and natural killer (NK) cells [17]. MSCs also appear to regulate the immune function in inflamed tissues by affecting the formation and secretion of pro-inflammatory cytokines and chemokines. In fact, the in vivo exogenous administration of MSCs imparts an anti-inflammatory action and improves the health of tissues that had become inflamed in response to injury induction [18]. Although, MSCs have shown an anti-inflammatory effect, their cellular mechanisms to pursue such function should be examined. In the present study, we sought to examine the anti-inflammatory potential of MSC exogenously administered to mice that were suffering from DSS induced colitis. Assessment of therapeutic efficacy of MSCs was by measuring weight, stool scoring, histopathologic examination, and measuring the gene expression of inflammatory markers: IL-23, TNF-α, IFN-γ, and ICAM-1 in colonic tissues recovered from the mice.
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Data are expressed as mean ± SE. Significant differences were determined using independent t-test and SPSS for Windows Version 15.0 Computer Software (SPSS, Chicago, IL, USA). Non-parametric analyses were used for evaluating all the colitis indices. Results were considered significant at P-values b 0.01. 3. Results
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3.1.1. Part A: determination of appropriate DSS concentration To determine the appropriate model of induction of IBD by DSS, different DSS concentrations (2% [21], 4% and 5% [20]) were administered for 1 week. Assessment of IBD degree was performed using stool scoring, weight loss, and histopathologic examination.
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3.1.1.2. Average body weight. Fig. 2 shows that through one week of DSS administration, 2% DSS administered mice gain weight as an indication to normal bowel condition, 4% DSS administered mice gradually lose weight as an indication to deterioration of bowel condition, and 5% DSS administered mice lose weight with 100% mortality rate.
3.1.1.3. Histopathologic examination. Fig. 3(a, b, c) reveals that the administration of 2% DSS did not cause any harm to the intestinal mucosa, while, the administration of 4% and 5% DSS causes almost the same degree of inflammation. 3.1.2. Part B: determination of appropriate DSS/water cycle To determine the appropriate DSS/water cycle, three cycles were examined. Assessment of IBD degree was performed using stool scoring, weight loss, and histopathologic examination.
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3.1.3. Assessment of MSC potential therapeutic effect on IBD in tested groups Efficacy of MSCs in treating IBD in the tested groups was assessed by stool scoring, average body weight, histopathologic examination, and measuring the gene expression of the inflammatory markers.
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3.1.2.3. Histopathologic examination. As per previous observations, histopathologic examination was performed for three cycles (7 days 4% DSS + 5 days water) in mice. Fig. 6 reveals a continuous inflammation state even after 5 days of normal drinking water.
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3.1.3.1. Assessment of stool scoring in the studied groups. Fig. 7 shows that 244 stool condition was significantly improved by the administration of 245 MSCs. 246 3.1.4. Average body weight Fig. 8 shows that the average body weight of the mice in the studied groups in the indicated cycle intervals and revealed a significant increase in the body weight of treated group.
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3.1.5. Histopathological examination of tested groups' intestine The gross picture of control group intestine revealed that circumscribed round ulceration was observed in the inner mucosa associated with congestion and redness all over the wall and that there was no gross finding observed in the mucosa and outer surface all over the intestine wall. Moreover, focal desquamation was observed in the mucosal lining epithelial cells associated with a massive number of inflammatory cell infiltration mainly neutrophils, macrophages and lymphocytes with congestion of the blood vessels in the underlying lamina propria, and the muscularis showed that oedema with inflammatory cell infiltration was observed in both caecum and colon, (Fig. 9a) which shows histopathological confirmation of IBD. On the other hand, the gross picture of treated group intestine revealed that there was no gross finding observed in the mucosa and outer surface all over the intestine wall. Moreover, no histopathological alteration was observed in the mucosal layer with the lamina propria as well as in the submucosal and muscular layers, (Fig. 9b), which shows a histopathologically normal intestine.
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3.1.6. Assessment of body weight in the studied groups As illustrated in Fig. 10, the average body weights of the treated groups are significantly higher (P b 0.01), compared with the control group. This significant weight elevation is obvious starting from the second DSS administration cycle on days 19, 24 and 31.
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3.1.7. The inflammatory markers in control and treated groups Fig. 11 illustrates the relative gene expression of pro-inflammatory markers TNF-α, and IL-23, IFN-γ, and ICAM-1 were measured in both control and treated groups, showing that, the relative gene expression
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3.1.1.1. Stool scoring. Fig. 1 shows that through one week of administration; 2% DSS causes no effect on the stool, 4% DSS causes gradual deterioration in stool condition, and 5% DSS causes severe deterioration in stool condition with 100% mortality.
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Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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The application of MSC therapy has opened a new horizon for the treatment of IBD, as most of the pharmacological approaches haven't 283 yet reached the desired efficacy and safety outcomes. MSCs are 284 multipotent cells found in various species, including mice, and are 285 often used for cellular transplantation therapy [22]. Most previous 286 studies have focused on these attractive regenerative properties 287 of MSCs [22,23], indicating that MSCs can promote regeneration 288 of wounded or injured tissues by a differentiation into vascular 289 smooth muscle cells, endothelial cells, pericytes, or epithelial cells 290 [24]. 291 Chemical induction of IBD by DSS was considered an efficient and 292 easily applied model, however, choosing the correct dose to induce 293 colitis is a variable [21]. In the present study, the 4% DSS model was 294 used as administration of 2% and 5% DSS did not give the desired 295 outcome. In the first part of the pilot study the 2% DSS failed to induce 296 inflammation, which is in contrast to Wirtz et al. [21], who showed 297 that the administration of 2% DSS for 7 days had produced acute colitis. 298 However, using 5% DSS showed 100% mortality in accordance to Wirtz 299 et al. [21]. Concerning the second part of the pilot study, our results 300 showed a cycle of 7 days of 4% DSS followed by 5 days of normal drink301 Q22 ing water which is optimum for the induction of chronic IBD, in contrast 302 to Wirtz et al. [21] who induced a chronic model of colitis by the admin303 istration of DSS for 7 days followed by 14 days of water.
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Experiments done to investigate the therapeutic effect of MSCs in IBD are very few; the first attempt was done by Tanaka et al. [20] that induced DSS acute colitis and proved that the exogenous administration of MSCs ameliorates acute DSS induced colitis. Tanaka et al. [20] assessed the MSC therapeutic effect by measuring the RNA expression of inflammatory mediators such as TNF-α, IL-1β, COX-2, basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), all of which significantly decreased in MSC treated mice. The second attempt was done by Liang et al. [25] in which human umbilical cord mesenchymal stem cells (hUC-MSCs) were used to treat acute trinitrobenzene sulfonic acid (TNBS) induced IBD. The inflammatory markers such as IL-17, IL-23, IFN-γ, and IL-6 were measured to assess the therapeutic efficacy of hUC-MSCs and were shown to be significantly decreased in the treated mice. Another attempt was done by Gonzalez et al. [26] in which acute colitis was induced by TNBS and adipose tissue derived MSCs were injected to the mice. Gonzalez et al. assessed the therapeutic effect of MSCs by measuring inflammatory cytokines such as TNF-α, IFN-γ, IL-6, and IL-1β, and chemokines such as macrophage inhibitory protein II which were significantly decreased in treated mice. In the current work, the MSCs were injected once after the first cycle of chronic DSS-induced colitis. We assessed the gene expression of pro-inflammatory cytokine levels (TNF-α, IFN-γ, IL-23 and ICAM-1) in an attempt to investigate their association with MSC therapy in IBD. The results of this work indicated that the level of TNF-α gene expression was significantly decreased after MSC administration. This result is supported by recent studies that have shown that MSCs can also modulate the systemic immune systems [17,18]. Ortiz et al. [18]
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Fig. 3. (a) shows that that through one week of DSS administration, 2% DSS administration causes a colon of mice with intact histological structure of mucosal layer (mu), lamina propria (Lp), submucosa (s), and muscularis (m) mice. (b) revealed that 4% DSS administration causes caecum with the massive number of inflammatory cell infiltration in the lamina propria (f) with congestion in blood vessels (v) and desquamation of the lining mucosal epithelium (m). (c) 5% DSS administration causes acute colitis extended to the muscular layer revealing caecum of the mice with oedema and inflammatory cell infiltration in the muscularis (m).
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Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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et al. [26] provided further confirmation to our findings as in their experiment AMSC injected mice showed reduced levels of TNF-α in comparison to untreated mice. The results revealed that the expression of IFN-γ was significantly decreased in MSC treated mice. This finding was confirmed by Gonzalez et al. [26] who showed reduced levels of IFN-γ in AMSC injected mice in comparison to untreated mice and related the effect of AMSCs to their ability to suppress Th1 responses and induce T regulatory cells. Liang et al. [25] also confirmed our results when they found that hU-MSC significantly decreased the level of IFN-γ. Recent findings reported that IFN-γ has a dual role in the immunosuppressive effect of MSCs; the first is stated before as MSCs suppress Th1 and induce T regulatory cells. The second effect is that MSCs inhibited the proliferation of B cells only in the presence of IFN-γ, and the capacity of IFN-γ to induce the suppressive activity of MSCs is not dependent on indoleamine 2,3 deoxygenase (IDO) but is dependent on cell to cell contact mediated by the interaction between programmed death 1 (PD-1) and PD ligand 1 (PDL-1) [28]. The most possible mechanism supporting this theory is that the expression of PD-1 and PDL-1 from both MSCs and B cells displays a significant increase in the presence of IFN-γ. These results suggest that PD-1/PDL-1 interaction between MSCs and B lymphocytes inhibits the signal transduction pathways triggered by productive B cell stimulation by antigen and affects B cell expansion. Another mechanism was supported by Krampera et al. [29] who supposed that MSCs inhibit the proliferation of B cells only in the presence of IFN-γ which implies that IFN-γ causes MSCs to produce IDO, which catalyzes the conversion of tryptophan to kynurenine which in turn suppressed the proliferation of B cells. Spaggiari et al. [30] highlighted another role of IFN-γ in MSC
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indicated that MSCs have an anti-inflammatory property and reduce inflammation and lung injury induced by bleomycin. In addition, Aggarwal and Pittenger [17] showed that human MSCs can modulate anti-inflammatory effects in co-cultures with purified subpopulations of immune-associated cells, such as dendritic cells (DCs), naive and effector T cells (T helper 1, Th1 and T helper 2, Th2), regulatory T cells (Tregs), and natural killer (NK) cells. MSCs can down-regulate inappropriate inflammatory responses by inhibiting pro-inflammatory Th1, DC1, and NK signaling and by promoting anti-inflammatory Th2 and/or suppressive Treg signaling. MSCs may thus orchestrate a shift from Th1 (pro-inflammatory) toward Th2 (anti-inflammatory) [17]. Accordingly, it is possible that MSCs could prove efficacy in the treatment of Th1-mediated inflammatory diseases. As for the pathogenesis of CD, a systemic and/or local inflammatory bowel disease, Th1 cytokines are known to have a more important role in pathogenesis than Th2 cytokines in local inflammatory tissues [27]. Recently, infliximab (anti-TNF-α antibody) as well as conventional anti-inflammatory agents are often used clinically to treat CD by attenuating inflammation. As for experimental colitis, chronic DSS-induced mucosal injury has the cytokine profile of Th1 responses [13]. Furthermore, Tanaka et al. [20] have reported that the exogenous administration of MSCs significantly decreased the level of TNF-α, IL-1β, and COX-2. Moreover, Gonzalez
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Fig. 6. The figure shows a colon of mice with desquamation of the lining of the epithelium (m) with inflammatory cell infiltration in the underlying lamina propria (f).
Fig. 8. Studied groups: average body weight.
Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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Regarding the decreased the gene expression of ICAM-1 in MSC treated mice, this is the first attempt to prove that MSC immunosuppressive effect includes the suppression of ICAM-1 expression. In contrast to our findings, Ren et al. [35] showed that MSCs do express ICAM-1 in order to perform the immunosuppressive effect. Generally, adhesion molecules play a dual role in the immunosuppressive effect of MSCs; first, Chamberlain et al. [36] stated that inflamed endothelial tissue releases proinflammatory cytokines, chemokines, and adhesion molecules in order to attract macrophages, lymphocytes, and other inflammatory cells. Second, the release of pro-inflammatory cytokines, chemokines, and adhesion molecule plays a major role in the homing of MSCs to the site of injury. Interestingly, Segers et al. [37] stated that ICAM-1 and VCAM-1 are considered markers for stem cells; the observation that confirms that MSCs can express adhesion molecules. According to Ren et al. [35] T cells secrete several inflammatory cytokines, including IFN-γ, TNF-α, and IL-1 that leads to striking upregulation in the expression of two adhesion molecules: ICAM-1 and VCAM-1 that are responsible for the increased adhesion between MSCs and T cells. Importantly, blocking of the function of adhesion molecules significantly reversed the immunosuppressive effect of MSCs in vitro and in vivo. Summing up, ICAM-1 plays a dual role as it facilitates the homing of MSCs to the site and allows the cell to cell contact, the cornerstone MSC immunosuppressive effect. To correlate these observations with our results we can conclude that in the chronic model of IBD the administration of MSCs will significantly increase the expression of ICAM-1 in the first few days of injection. However, after 26 days of administration and the severity of inflammation has decreased and most of the ICAM-1 has been consumed during the
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cell-mediated lyses upon incubation of MSCs with IFN-γ. Moreover, in this work, the gene expression of IL-23 was significantly decreased in MSC treated mice. Our result is supported by the study of Liang et al. [25] in which the level of IL-23 was significantly lower in the colon tissues of hUC-MSC treated mice. IL-23 performs its antiinflammatory function mainly by induction of Th17 that releases IL-17 [31]. Although the precise mechanism by which IL-23 maintains Th17 responses in vivo is still not well understood, recent studies have shown that Th17 cell lineage commitment is driven by TGF-β in the presence of pro-inflammatory cytokines, whereas IL-23 seems to expand or maintain Th17 cell populations [32]. IL-23 is a type 1 heterodimeric IL-12 like cytokine, comprising of a 19-kD 4-fold helical core α subunit (IL-23p19), and a disulfide linked to an additional 40-kD distinct β subunit (IL-12p40). IL-23 and IL-12 share a common subunit in their receptor complex due to the common p40 subunit [33]. Gonzalez et al. [26] reported that colons of AMSC treated mice contained a reduced level of IL-12 in comparison with untreated IBD induced colitis mice. Moreover, MSCs inhibit the differentiation of monocytes into dendritic cells by inhibiting the response of the former to maturation signals, reducing the expression of co-stimulatory molecules and hampering the ability of the former to stimulate naïve T cell proliferation and IL-12 secretion [34]. Furthermore, the level of IL-17 was significantly decreased in the colon tissue of hU-MSC treated mice [25].
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Fig. 9. (a) reveals histopathologic findings in the control group; this figure shows the inflammatory cell infiltration (f), oedema (o), and dilated blood vessels (v) in the submucosal layer. (H&E ×80). (b) reveals histopathological findings in the treated group. this figure shows a normal histological structure of the mucosa (m) and muscularis (ml) with serosa (s) (H&E ×64).
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Please cite this article as: Salam AGARA, et al, Potential therapeutic utility of mesenchymal stem cells in inflammatory bowel disease in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.07.030
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anti-inflammatory process of MSCs, resulting in a significant decrease of ICAM-1 gene expression at the end of experiment. In conclusion, induction of chronic IBD in mice showed an increase in the gene expression of proinflammatory markers such as TNF-α, IFN-γ, IL-23, and ICAM-1. The control group (IBD-induced mice injected by sterile PBS) showed no decrease in the gene expression level of proinflammatory markers. Upon treating IBD induced mice with bone marrow derived MSCs, the gene expression of proinflammatory marker was significantly decreased. These conclusions highlighted many of the pathophysiological cascades of inflammatory bowel disease. The inflammation induced by DSS was significantly associated with the production of ICAM-1 that leads to migration and infiltration of macrophages and Th1, producing thereby TNF-α, IFN-γ and IL-23. Interestingly, ICAM-1 has a dual role in stem cell immunomodulatory effect; the first role is that high levels of ICAM-1 in inflammation site lead to the migration of MSCs because stem cells have surface receptors similar to those on the surface of macrophages, T cells, and B cells. The second role is that the high levels of TNF-α and IFN-γ in the inflamed tissue provoke MSCs to produce ICAM-1 that attracts inflammatory cells to adhere to the stem cell surface to exert its immunomodulatory effect. Finally, we can conclude that bone marrow derived MSCs can be used as an effective treatment of IBD.
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